Thomas' Hematopoietic Cell Transplantation

Thomas’ Hematopoietic Cell Transplantation Stem Cell Transplantation Fourth Edition

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

Dedication This book is dedicated to our patients and their families whose courage and trust enabled them to choose a difficult, dangerous, and sometimes unproven therapy which offered the only chance in their fight against a fatal disease. Today, many thousands of patients are alive after a successful hematopoietic cell transplantation which would have been impossible without the fortitude of our patients, who took risks and thereby paved the way for the increasing application of this therapy. The progress in the area of clinical transplantation of hematopoietic cells would not have been possible without the dedicated, tireless work of the nurses and the supporting staff in the many transplant units around the world.

Thomas’

Hematopoietic Cell Transplantation Stem Cell Transplantation

Edited by

Frederick R. Appelbaum,

MD

Member and Director, Clinical Research Division Fred Hutchinson Cancer Research Center Professor and Head, Division of Medical Oncology University of Washington School of Medicine Seattle, Washington

Stephen J. Forman,

MD

Director, Department of Hematology and Hematopoietic Cell Transplantation Staff Physician, Department of Medical Oncology and Therapeutics Research City of Hope Comprehensive Cancer Center Duarte, California

Robert S. Negrin,

MD

Professor of Medicine Chief, Division of Blood and Marrow Transplantation Stanford University Stanford, California

Karl G. Blume,

MD

Emeritus Professor of Medicine Division of Blood and Marrow Transplantation Stanford University Stanford, California

Fourth Edition

A John Wiley & Sons, Ltd., Publication

This edition first published 1994, © 1994, 1999, 2004 by Blackwell Publishing Ltd Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing program has been merged with Wiley’s global Scientific, Technical and Medical business to form WileyBlackwell. Registered office: John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial offices: 9600 Garsington Road, Oxford, OX4 2DQ, UK The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK 111 River Street, Hoboken, NJ 07030-5774, USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wileyblackwell The right of the author to be identified as the author of this work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. The contents of this work are intended to further general scientific research, understanding, and discussion only and are not intended and should not be relied upon as recommending or promoting a specific method, diagnosis, or treatment by physicians for any particular patient. The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of fitness for a particular purpose. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of medicines, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each medicine, equipment, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. Readers should consult with a specialist where appropriate. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising herefrom. Library of Congress Cataloging-in-Publication Data Thomas’ hematopoietic cell transplantation / edited by Frederick R. Appelbaum . . . [et al.]. – 4th ed. p. ; cm. Includes bibliographical references and index. 1. Hematopoietic stem cells–Transplantation. I. Appelbaum, Frederick R. II. Thomas, E. Donnall. III. Title: Hematopoietic cell transplantation. [DNLM: 1. Hematopoietic Stem Cell Transplantation. 2. Transplantation Immunology. WH 380 T457 2008] RD123.5.B652 2008 617.4′4 – dc22 2008030325 ISBN: 978-1-4051-5348-5 A catalogue record for this book is available from the British Library. Set in 8.75 on 11 pt Times by SNP Best-set Typesetter Ltd., Hong Kong Printed & bound in Singapore by Fabulous Printers Pte Ltd 1

2009

Contents

Contributors, ix Preface to the First Edition, xvii Preface to the Second Edition, xviii Preface to the Third Edition, xix Preface to the Fourth Edition, xx Thomas HCT Fourth Edition, xxi List of Abbreviations, xxiii

9 Mesenchymal Stromal Cells and Hematopoietic Cell Transplantation, 102 Edwin M. Horwitz 10 Genetic Manipulation of Hematopoietic Stem Cells, 116 Grant D. Trobridge & Hans-Peter Kiem Section 2b Immunology

SECTION 1 History and Use of Hematopoietic Cell Transplantation 1 A History of Allogeneic Hematopoietic Cell Transplantation, 3 E. Donnall Thomas 2 The History of Autologous Hematopoietic Cell Transplantation, 8 James O. Armitage 3 Uses and Growth of Hematopoietic Cell Transplantation, 15 Mary M. Horowitz SECTION 2 Scientific Basis for Hematopoietic Cell Transplantation Section 2a Hematopoiesis and Stem Cell Biology

4 Generation of Definitive Engraftable Hematopoietic Stem Cells from Human Embryonic Stem Cells, 27 Laurence Dahéron & David T. Scadden 5 Biology of Hematopoietic Stem and Progenitor Cells, 36 Susan Prohaska & Irving Weissman 6 Molecular Biology of Stem Cell Renewal, 64 Peter M. Lansdorp 7 Cellular Biology of Hematopoiesis, 72 Catherine M. Flynn & Catherine M. Verfaillie 8 Expansion of Hematopoietic Stem Cells, 88 Colleen Delaney & Irwin Bernstein

11 Overview of Hematopoietic Cell Transplantation Immunology, 131 Paul J. Martin 12 Histocompatibility, 145 Eric Mickelson & Effie W. Petersdorf 13 Natural Killer Cells and Allogeneic Hematopoietic Cell Transplantation, 163 Michael R. Verneris & Jeffrey S. Miller 14 Murine Models of Graft-versus-Host Disease and Graft-versus-Tumor Effect, 176 Robert Korngold & Thea M. Friedman 15 Mechanisms of Tolerance, 188 Megan Sykes 16 The Pathophysiology of Graft-Versus-Host Disease, 208 James L.M. Ferrara & Joseph H. Antin 17 Immune Reconstitution Following Hematopoietic Cell Transplantation, 222 Robertson Parkman & Kenneth I. Weinberg 18 The Human Graft-versus-Tumor Response – and How to Exploit It, 232 Edus H. Warren 19 Dendritic Cells in Hematopoietic Cell Transplantation, 248 Miriam Merad, Matthew P. Collin & Edgar G. Engleman 20 The Experimental Basis for Hematopoietic Cell Transplantation for Autoimmune Diseases, 264 Judith A. Shizuru Section 2c Technical Aspects

21 Pharmacologic Basis for High-dose Chemotherapy, 289 James H. Doroshow & Timothy W. Synold v

vi

Contents

22 High-dose Preparatory Regimens, 316 William I. Bensinger 23 Radiotherapeutic Principles of Hematopoietic Cell Transplantation, 333 Jeffrey Y.C. Wong & Timothy Schultheiss 24 Radioimmunotherapy and Hematopoietic Cell Transplantation, 351 Damian J. Green & Oliver W. Press 25 Documentation of Engraftment and Characterization of Chimerism Following Hematopoietic Cell Transplantation, 365 Paul J. Martin 26 The Detection and Significance of Minimal Residual Disease, 376 Jerald P. Radich & Marilyn L. Slovak 27 Pathology of Hematopoietic Cell Transplantation, 390 Howard M. Shulman, Robert C. Hackman & George E. Sale 28 Biostatistical Methods in Hematopoietic Cell Transplantation, 406 Joyce C. Niland & Paul Frankel 29 Outcomes Research in Hematopoietic Cell Transplantation, 428 Stephanie J. Lee

SECTION 3 Patient-oriented Issues in Hematopoietic Cell Transplantation 30 The Evaluation and Counseling of Candidates for Hematopoietic Cell Transplantation, 445 Karl G. Blume & Robert A. Krance 31 Nursing Role in Hematopoietic Cell Transplantation, 461 Rosemary C. Ford & Mihkaila M. Wickline 32 Ethical Issues in Hematopoietic Cell Transplantation, 478 David S. Snyder 33 Psychosocial Issues in Hematopoietic Cell Transplantation, 488 Richard P. McQuellon & Michael Andrykowski 34 Assessment of Quality of Life in Hematopoietic Cell Transplantation Recipients, 502 Karen L. Syrjala & Samantha Burns Artherholt 35 Sexuality Following Hematopoietic Cell Transplantation: An Important Health-related Quality of Life Issue, 515 D. Kathryn Tierney 36 Hematopoietic Cell Transplantation: The Patient’s Perspective, 526 Susan K. Stewart

SECTION 4 Sources of Hematopoietic Cells for Hematopoietic Cell Transplantation 37 Hematopoietic Cell Procurement, Processing, and Transplantation: Standards, Accreditation, and Regulation, 535 Phyllis I. Warkentin & Elizabeth J. Shpall 38 Bone Marrow and Peripheral Blood Cell Donors and Donor Registries, 544 Dennis L. Confer, John P. Miller & Jeffrey W. Chell 39 Cord Blood Hematopoietic Cell Transplantation, 559 Hal E. Broxmeyer & Franklin O. Smith 40 In Utero Transplantation, 577 Alan W. Flake & Esmail D. Zanjani 41 Mobilization of Autologous Peripheral Blood Hematopoietic Cells for Cellular Therapy, 590 Thomas C. Shea & John F. DiPersio 42 Removal of Tumor Cells from the Hematopoietic Graft, 605 John G. Gribben 43 Peripheral Blood Hematopoietic Cells for Allogeneic Transplantation, 618 Norbert Schmitz 44 Cryopreservation of Hematopoietic Cells, 631 Scott D. Rowley 45 Use of Recombinant Growth Factors After Hematopoietic Cell Transplantation, 645 Jürgen Finke & Roland Mertelsmann 46 Hematopoietic Cell Transplantation from Human Leukocyte Antigen Partially Matched Related Donors, 657 Claudio Anasetti, Franco Aversa & Andrea Velardi 47 Hematopoietic Cell Transplantation from Unrelated Donors, 675 Effie W. Petersdorf 48 Donor Selection for Hematopoietic Cell Transplantation, 692 Ann E. Woolfrey SECTION 5 Hematopoietic Cell Transplantation for Acquired Diseases 49 Hematopoietic Cell Transplantation for Aplastic Anemia, 707 George E. Georges & Rainer Storb 50 Hematopoietic Cell Transplantation for Paroxysmal Nocturnal Hemoglobinuria, 727 Robert P. Witherspoon 51 Allogeneic and Autologous Transplantation for Chronic Myeloid Leukemia, 734 Jerald P. Radich & Ravi Bhatia

Contents

52 Hematopoietic Cell Transplantation for Juvenile Myelomonocytic Leukemia, 751 Charlotte M. Niemeyer & Franco Locatelli 53 Hematopoietic Cell Transplantation for Adult Acute Myeloid Leukemia, 761 Frederick R. Appelbaum 54 Hematopoietic Cell Transplantation for Childhood Acute Myeloid Leukemia, 775 Julie-An M. Talano, James T. Casper & David A. Margolis 55 Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Adults, 791 Stephen J. Forman 56 Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Children, 806 Parinda A. Mehta & Stella M. Davies 57 Hematopoietic Cell Transplantation for Myelodysplastic Syndrome and Myeloproliferative Disorders, 827 H. Joachim Deeg 58 Hematopoietic Cell Transplantation for Multiple Myeloma, 845 Muzaffar H. Qazilbash & Sergio A. Giralt 59 Hematopoietic Cell Transplantation for Hodgkin’s Disease, 860 Philip J. Bierman & Auayporn Nademanee 60 Non-Hodgkin’s Lymphoma, 878 Laura J. Johnston & Sandra J. Horning 61 Hematopoietic Cell Transplantation for Chronic Lymphocytic Leukemia, 897 David B. Miklos 62 Autologous Hematopoietic Cell Transplantation for Systemic Light Chain (AL-) Amyloidosis, 914 Raymond L. Comenzo & Morie A. Gertz 63 Hematopoietic Cell Transplantation for Breast Cancer, 931 Yago Nieto & Elizabeth J. Shpall 64 Hematopoietic Cell Transplantation in Germ Cell Tumors, 948 Christie J. Moore, Brandon Hayes-Lattin & Craig R. Nichols 65 Hematopoietic Cell Transplantation for Renal Cell and other Solid Tumors, 958 Richard W. Childs & Ramaprasad Srinivasan 66 Hematopoietic Cell Transplantation for Neuroblastoma, 970 Jason Law & Katherine K. Matthay 67 Hematopoietic Cell Transplantation for Other Pediatric Solid Tumors, 985 David M. Loeb & Allen R. Chen 68 Hematopoietic Cell Transplantation for Patients with Human Immunodeficiency Virus Infection, 1001 John A. Zaia, Amrita Krishnan, & John J. Rossi 69 Hematopoietic Cell Transplantation for Autoimmune Diseases, 1014 Richard A. Nash

vii

70 Hematopoietic Cell Transplantation for Rare Hematologic Malignancies, 1030 Vinod Pullarkat & Stephen J. Forman 71 Reduced-intensity Conditioning Followed by Hematopoietic Cell Transplantation for Hematologic Malignancies, 1043 Brenda M. Sandmaier & Rainer Storb 72 Management of Relapse after Hematopoietic Cell Transplantation, 1059 Ginna G. Laport & Robert S. Negrin

SECTION 6 Hematopoietic Cell Transplantation for Inherited Diseases 73 Hematopoietic Cell Transplantation for Thalassemia, 1079 Guido Lucarelli & Javid Gaziev 74 Hematopoietic Cell Transplantation for Sickle Cell Disease, 1090 Mark C. Walters 75 Hematopoietic Cell Transplantation for Immunodeficiency Diseases, 1105 Trudy N. Small, Wilhelm Friedrich, & Richard J. O’Reilly 76 Hematopoietic Cell Transplantation for Osteopetrosis, 1125 Peter F. Coccia 77 Hematopoietic Cell Transplantation for Storage Diseases, 1136 Charles Peters 78 Hematopoietic Cell Transplantation for Macrophage and Granulocyte Disorders, 1163 Rajni Agarwal 79 Hematopoietic Cell Transplantation for Fanconi’s Anemia, 1178 John E. Wagner, Margaret L. MacMillan, & Arleen D. Auerbach SECTION 7 Complications of Hematopoietic Cell Transplantation 80 Mechanisms and Treatment of Graft Failure, 1203 Robert Lowsky & Hans Messner 81 Blood Group Incompatibilities and Hemolytic Complications of Hematopoietic Cell Transplantation, 1219 Margaret R. O’Donnell 82 Principles of Transfusion Support Before and After Hematopoietic Cell Transplantation, 1226 Jeffrey McCullough

viii

Contents

83 Vascular Access and Complications, 1244 I. Benjamin Paz 84 Pharmacologic Prevention of Acute Graft-Versus-Host Disease, 1257 Nelson J. Chao & Keith M. Sullivan 85 T-Cell Depletion to Prevent Graft-versus-Host Disease, 1275 Robert J. Soiffer 86 Manifestations and Treatment of Acute Graft-Versus-Host Disease, 1287 Corey Cutler & Joseph H. Antin 87 Chronic Graft-versus-Host Disease: Clinical Manifestations and Therapy, 1304 Steven Z. Pavletic & Georgia B. Vogelsang 88 Bacterial Infections, 1325 Helen L. Leather & John R. Wingard 89 Fungal Infections after Hematopoietic Cell Transplantation, 1346 Janice (Wes) M.Y. Brown 90 Cytomegalovirus Infection, 1367 John A. Zaia 91 Herpes Simplex Virus Infections, 1382 James I. Ito 92 Varicella-zoster Virus Infections, 1388 Dora Y. Ho & Ann M. Arvin 93 Epstein–Barr Virus Infection, 1410 Wen-son Hsieh & Richard F. Ambinder 94 Adenoviruses, Respiratory Viruses, HHV-6, HHV-7, HHV-8, Papovaviruses and Other Viruses After Hematopoietic Cell Transplantation, 1419 Michael Boeckh 95 Gastrointestinal and Hepatic Complications, 1434 Simone I. Strasser & George B. McDonald 96 Lung Injury Following Hematopoietic Cell Transplantation, 1456 Kenneth R. Cooke & Gregory A. Yanik 97 Kidney and Bladder Complications of Hematopoietic Cell Transplantation, 1473 Sangeeta Hingorani 98 Endocrine Complications Following Hematopoietic Cell Transplantation, 1487 Fouad R. Kandeel

99 Common Potential Drug Interactions Following Hematopoietic Cell Transplantation, 1523 Anne Poon & Lowan Ly 100 Critical Care of the Hematopoietic Cell Transplant Recipient, 1539 Gundeep S. Dhillon & Norman W. Rizk 101 Nutrition Support of the Hematopoietic Cell Transplant Recipient, 1551 Polly Lenssen & Saundra Aker 102 Pain Management, 1570 Noelle V. Frey & Jonathan R. Gavrin 103 Oral Complications of Hematopoietic Cell Transplantation, 1589 Mark M. Schubert & Douglas E. Peterson 104 Growth and Development after Hematopoietic Cell Transplantation, 1608 Jean E. Sanders 105 Delayed Nonmalignant Complications after Hematopoietic Cell Transplantation, 1620 Mary E.D. Flowers & H. Joachim Deeg 106 Secondary Malignancies after Hematopoietic Cell Transplantation, 1638 Smita Bhatia & Ravi Bhatia 107 Neurologic Complications of Hematopoietic Cell Transplantation, 1653 Harry Openshaw 108 Vaccination of Allogeneic and Autologous Hematopoietic Cell Recipients, 1664 Trudy N. Small

SECTION 8 Looking Forward 109 Hematopoietic Cell Transplantation in the Future, 1673 Ernest Beutler Colorplates, facing page 514 Robert C. Hackman Index, 1677

Companion CD-ROM A companion CD-ROM is included with the book, containing: • The complete text • A database of figures from the book for downloading • A Search feature The CD-ROM is suitable for PC and Mac computers.

Contributor list

Rajni Agarwal, MD Assistant Professor of Pediatrics, Division of Stem Cell Transplantation, Department of Pediatrics, Stanford University School of Medicine, Stanford, California Saundra Aker, RD, CD Consultant, Nutrition and Patient Food Services, Seattle Cancer Care Alliance, Seattle, Washington Richard F. Ambinder, MD, PhD Professor in the Departments of Oncology, Pathology, Pharmacology and Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland

Samantha Burns Artherholt, PhD Research Fellow, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington Ann M. Arvin, MD Professor of Pediatrics, Microbiology and Immunology, Department of Pediatrics, Infectious Disease Division, Stanford University School of Medicine, Stanford, California Arleen D. Auerbach, PhD Director of the Laboratory of Human Genetics and Hematology, The Rockefeller University New York, New York

Claudio Anasetti, MD Chair, Department of Blood and Marrow Transplantation, Moffitt Cancer Center Professor of Medicine and Oncology, University of South Florida, Tampa, Florida

Franco Aversa, MD Head, Hematopoietic Stem Cell Transplant Unit Professor, Department of Clinical and Experimental Medicine Sections of Hematology and Immunology Perugia University, School of Medicine, Perugia, Italy

Michael A. Andrykowski, PhD Department of Behavioural Science, University of Kentucky College of Medicine, Seattle, Washington

William I. Bensinger, MD Professor of Medicine, University of Washington Fred Hutchinson Cancer Research Center, Seattle, Washington

Joseph H. Antin, MD Professor of Medicine, Harvard Medical School, Dana Farber Cancer Institute, Boston, MA

Irwin Bernstein, MD Member, Fred Hutchinson Cancer Research Center; and Professor of Medicine, University of Washington School of Medicine, Seattle, Washington

Frederick R. Appelbaum, MD Member and Director, Clinical Research Division, Fred Hutchinson Cancer Research Center, Professor and Head, Division of Medical Oncology, University of Washington School of Medicine, Seattle, Washington James O. Armitage, MD The Joe Shapiro Professor of Medicine Section of Oncology/ Hematology, University of Nebraska, Medical Center, Nebraska Medical Center, Omaha, Nebraska

Ernest Beutler, MD Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California Ravi Bhatia, MD Director, Stem Cell Biology Program, Division of Hematology and Bone Marrow Transplantation, City of Hope National Medical Center, Duarte, California ix

x

Contributor list

Smita Bhatia, MD, MPH Staff Physician; Director, Epidemiology and Outcomes Research, Division of Pediatric Hematology/Oncology and Bone Marrow Transplantation, City of Hope National Medical Center, Duarte, California Philip J. Bierman, MD Associate Professor of Medicine, Department of Internal Medicine, Section of Oncology and Department Hematology, University of Nebraska Medical Center, Omaha, Nebraska Karl G. Blume, MD, FACP Emeritus Professor of Medicine, Division of Bone Marrow Transplantation, Department of Medicine, Stanford University, School of Medicine, Stanford, California Michael Boeckh, MD Assistant Member, Program in Infectious Diseases Fred Hutchinson Cancer Research Center, Assistant Professor, University of Washington School of Medicine, Seattle, Washington Janice M. Y. Brown, MD Associate Professor Divisions of Blood and Marrow Transplantation and Infectious Diseases, Stanford University School of Medicine, Stanford, California Hal E. Broxmeyer, PhD Chairman and Mary Margaret Walther Professor of Microbiology and Immunology, Professor of Medicine Scientific Director, the Walther Oncology Center Indiana University School of Medicine Cancer Research Institute, Indianapolis, Indiana James A. Casper, MD Professor of Pediatrics, Section of Pediatric HematologyOncology-Transplantation Department of Pediatrics Medical College of Wisconsin Children’s Hospital of Wisconsin, Milwaukee, Wisconsin Nelson J. Chao, MD Professor of Medicine and Imunology, Duke University Medical Center, Durham, North Carolina Jeffrey W. Chell, MD Chief Executive Officer, National Marrow Donor Program, Minneapolis, Minnesota Allen R. Chen, MD, PhD, MHS Associate Professor, Oncology and Pediatrics, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Department of Pediatrics, Baltimore, Maryland

Richard Childs, MD Senior Investigator, National Heart, Lung and Blood Institute, National Institutes of Health, Bethseda, Maryland Peter F. Coccia, MD Ittner Professor of Pediatrics and Chief Section Pediatric Hematology/Oncology; and Director, Pediatric Bone Marrow Transplantation Program; and Vice-Chairperson, Department of Pediatrics, University of Nebraska Medical Center, Omaha, Nebraska Matthew Collin, MD, PhD Department of Gene and Cell Medicine, Mount Sinai Medical School, New York, New York Raymond L. Comenzo, MD Associate Attending, Hematology Service Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York Dennis L. Confer, MD Clinical Professor of Medicine, University of Minnesota; and Chief Medical Officer, National Marrow Donor Program, Minneapolis, Minnesota Kenneth Robert Cooke, MD Associate Professor of Pediatrics, Division of Hematology/ Oncology, Blood and Marrow Transplant Program, Case Western Reserve School of Medicine, Rainbow Babies and Children’s Hospital, Cleveland, Ohio Corey S. Cutler, MD MPH FRCPC Assistant Professor of Medicine, Harvard Medical School, Dana Farber Cancer Institute, Boston, Massachusetts Laurence Daheron, PhD Massachusetts General Hospital, Center for Regenerative Medicine, Boston, Massachusetts Stella Davies, MB, BS, PhD, MRCP Jacob G. Schmirdlapp Endowed Chair and Professor of Pediatrics; and Director, Blood and Marrow Transplantation Program, Division of Hematology/Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio H. Joachim Deeg, MD Member, FHCRC Professor of Medicine, University of Washington School of Medicine, Seattle, Washington Colleen Delaney, MD, MSc Assistant Member, Clinical Research Division, Fred Hutchinson Cancer Research Center, Assistant Professor, Department of Pediatrics, University of Washington, School of Medicine, Seattle, Washington

Contributor list

Gundeep S. Dhillon, MD, MPH Assistant Professor of Medicine, Stanford University School of Medicine, Stanford, California John Dipersio, MD, PhD Chief, Division of Oncology, Deputy Director, Siteman Cancer Research, Washington University School of Medicine, St Louis, Missouri

xi

Stephen J. Forman, MD Director, Department of Hematology and Hematopoietic Cell Transplantation, Staff Physician, Department of Medical Oncology and Therapeutics Research, City of Hope Comprehensive Cancer Center, Duarte, California Paul Frankel, PhD Statistician, City of Hope National Medical Center, Duarte, California

James H. Doroshow, MD, FACP Chairman, Department of Medical Oncology and Therapeutics Research, Associate Director for Clinical Investigation, City of Hope Comprehensive Cancer Care Center, Duarte, California

Noelle V. Frey, MD Instructor of Medicine Division of Hematology-Oncology Abramson Cancer Center University of Pennsylvania, Philadelphia, Pennsylvania

Edgar G. Engleman, MD Professor of Pathology and Medicine, Stanford University, School of Medicine, Stanford Blood Center, Stanford, California

Thea M. Friedman, PhD Director of Research Laboratory Services, The Cancer Center at Hackensack Medical University, Hackensack, New Jersey

James L. M. Ferrara, MD Professor of Medicine and Pediatrics; and Director, Blood and Marrow Transplantation Program, University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan

Wilhelm Frierich, MD Professor of Pediatrics, Department of Pediatrics, University Children’s Hospital Ulm, University of Ulm, Ulm, Germany

Jürgen Finke, MD Head, Division of Allogeneic Stem Cell Transplantation, Dept Hematology and Oncology, Medizinische Klinik Univesity, Medical Center, Frieberg, Germany Alan W. Flake, MD Professor of Surgery and Obstetrics and Gynecology, University of Pennsylvania; and Ruth and Tristram Colket Professor of Pediatric Surgery, Director, Institute for Surgical Science, Children’s Hospital of Philadelphia Abramson Research Center, Philadelphia, Pennsylvania Mary E. D. Flowers, MD Assistant Member, Fred Hutchinson Cancer Research Center; and Assistant Professor, University of Washington School of Medicine, Seattle, Washington Catherine Flynn, MB BCH BAO Faculty of Medicine, Stem Cell Institute Leuven, Catholic University Leuven, Belgium Rosemary C. Ford, RN, BSN, OCN Nurse Manager, Transplant Clinic, Seattle Cancer Care Alliance, Seattle, Washington

Jonathan R. Gavrin, MD Director, Symptom Management and Palliative Care Interim Director, Pain Management Services, Physician Co-chair HUP Ethics Committee Department of Anesthesiology and Critical Care Department of Medicine Hospital of the University of Pennsylvania Javid Gaziev, MD Senior Hematologist, Mediterranean Institute of Hematology International Centre for Transplantation in Thalassemia and Sickle Cell Anaemia, Rome, Italy George Earl Georges, MD Associate Member, Fred Hutchinson Cancer Center Associate Professor of Medicine, University of Washington Seattle, Washington Morie Gertz, MD Professor of Medicine, Mayo Medical School; and Chair, Division of Hematology, Mayo Clinic, Rochester, Minnesota Sergio A. Giralt, MD Professor of Medicine, Department of Stem Cell Transplantation and Cellular Therapy, University of Texas, MD Anderson Cancer Center, Houston, Texas Damian J. Green, MD Research Associate, Fred Hutchinson Cancer Research Center and The University of Washington, Seattle, Washington

xii

Contributor list

John G. Gribben MD, DSc Professor of Experimental Cancer Medicine, St. Bartholomew’s Hospital, Barts and The London NHS Trust, London, UK Robert C. Hackman, MD Member and Director of Pathology, Fred Hutchinson Cancer Research Center, Seattle, Washington Brandon Hayes-Lattin, MD Associate Professor of Medicine, Division of Hematology/ Medical Oncology Director, Adolescent and Young Adult Oncology Program, Oregon Health and Science University Portland, Oregon

Fouad R. Kandeel, MD, PhD Director, Department of Diabetes, Endocrinology and Metabolism, City of Hope National Medical Center, Associate Clinical Professor, UCLA School of Medicine, Duarte, California Hans Peter Kiem, MD Member, Fred Hutchinson Cancer Research Center Professor of Medicine, University of Washington, Seattle, Washington Robert Korngold, PhD Chief, Division of Research, The Cancer Center Hackensack University Medical Center, Hackensack, New Jersey

Sangeeta Ram Hingorani, MD, MPH Assistant Professor, Pediatrics University of Washington Children’s Hospital and Regional Medical Center, Division of Nephrology, Seattle, Washington

Robert A. Krance, MD Professor of Pediatric and Medicine, Baylor College of Medicine; and Director of Pediatric Stem Cell Transplantation Program, Texas Children’s Hospital, Houston, Texas

Dora Y. Ho, MD Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California

Peter M. Lansdorp, MD, PhD Senior Scientist, Terry Fox Laboratory, BC Cancer Research Centre; Professor, Division of Hematology, University of British Columbia, Vancouver, BC

Sandra J. Horning, MD Professor of Medicine, Stanford University Medical Center, Stanford Cancer Center, Stanford, California

Ginna G. Laport, MD Assistant Professor of Medicine, Stanford University Medical Center, Division of Blood and Marrow Transplantation, Stanford, California

Mary M. Horowitz, MD, MS Chief Scientific Director, Center for Blood and Marrow Transplant Registry (CIBMTR), Medical College of Wisconsin, Milwaukee, Wisconsin Edwin M. Horwitz, MD, MS Children’s Hospital of Philadelphia, Abramson Research Center, Philadelphia, Pennsylvania Wen-son Hsieh, MD Assistant Professor in the Department of Oncology, Johns Hopkins School of Medicine, Baltimore, Maryland James I. Ito, MD Director, Department of Infectious Diseases, City of Hope National Medical Center, Duarte, California Laura J. Johnston, MD Assistant Professor of Medicine, Stanford University Medical Center, Division of Bone Marrow Transplantation, Stanford, California

Helen L. Leather, B. Pharm, BCPS Clinical Pharmacy Specialist BMT/Leukemia, Shands at the University of Florida; and Clinical Associate Professor, College of Pharmacy, University of Florida, Gainesville, Florida Stephanie J. Lee, MD, MPH Associate Member, Fred Hutchinson Cancer Research Center, Seattle, Washington Polly Lenssen, MD Manager, Clinical Nutrition Children’s Hospital and Regional Medical Center, Seattle, Washington David M. Loeb, MD, PhD Assistant Professor, Oncology and Pediatrics, Director, Musculoskeletal Tumor Program, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University Robert Lowsky, MD Assistant Professor of Medicine, Stanford University Medical Center, Stanford, California

Contributor list

Guido Lucarelli, MD Director, Mediteranean Institute of Hematology, Policlinic of the University of Roma Tor Vergata, Roma, Italy Lowan L. Ly, Pharm D Clinical Pharmacist, Hematopoietic Stem Cell Transplant, University of Washington Medical Center/Seattle Cancer Care Alliance, Seattle, Washington David A. Margolis, MD Associate Professor, Medical College of Wisconsin, Milwaukee, Wisconsin Paul J. Martin, MD Member, Fred Hutchinson Cancer Research Center, Professor of Medicine, University of Washington, School of Medicine, Seattle, Washington Kathryn K. Matthay, MD Professor of Pediatrics, University of California School of Medicine, Chief, Pediatric Hematology-Oncology Department UCSF, San Francisco, CA Jeffrey McCullough, MD Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota George B. McDonald, MD Professor of Medicine, University of Washington; and Head Gastroenterology/Hepatology Section, Fred Hutchinson Cancer Research Center, Seattle, Washington Margaret L. MacMillan, MD Assistant Professor of Pediatrics, Division of Hematology/ Oncology and Blood and Marrow Transplantation, University of Minnesota Medical School, Minneapolis, Minnesota Richard P. McQuellon, PhD Comprehensive Cancer Center of Wake Forrest University, Bowman Gray School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina Parinda A. Mehta, MD Director in the Blood Disease Center, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio Miraim Merad, MD Department of Gene and Cell Medicine, Mount Sinai Medical School New York, New York Roland Mertelsmann, MD, PhD Medical Director, University Medical Center, Department Medicine I Hematology/Oncology, Frieberg, Germany

xiii

Hans Messner, MD, PhD Director of Bone Marrow Transplant Program, Princess Margaret Hospital, and Senior Scientist, Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, Canada Eric Mickelson, BSc Associate Staff Scientist, Fred Hutchinson Cancer Research Center, Seattle, Washington David B. Miklos, MD, PhD Assistant Professor of Medicine, Division of Blood and Marrow Transplantation Department of Medicine, Stanford University, Stanford, California Jeffrey S. Miller, MD Professor of Medicine, Division of Hematology, Oncology & Transplantation, University of Minnesota, Minneapolis, Minnesota Christie J. Moore, DO Providence Cancer Center Division of Medical Oncology Robert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, Portland, Oregon Auayporn P. Nademanee, MD Associate Clinical Director, Division of Hematology and Bone Marrow Transplantation, Department of Medicine, Stanford University School of Medicine, Stanford, California Richard A. Nash, MD Member, Program in Transplantation Biology Clinical Research Division Fred Hutchinson Cancer Research Center; and Associate Professor, University of Washington, Seattle, Washington Robert S. Negrin, MD Professor of Medicine and Chief, Division of Blood and Marrow Transplantation, Stanford University, Stanford, CA Craig R. Nichols, MD Director, Oregon Testicular Cancer Program, Division of Medical Oncology, Robert W. Franz Cancer Research Center Earle A. Chiles Research Institute, Portland, Oregon Lewis Nick, RN, BSN, MT(ASCP) Technical Director, Accreditation Program, Foundation for the Accreditation of Cellular Therapy, University of Nebraska Medical Center, Omaha, Nebraska Charlotte Niemeyer, MD Professor of Pediatrics, Medicical Director, Division of Hematology and Oncology, University Children’s Hospital Frieberg, Germany

xiv

Contributor list

Yago Nieto, MD, PhD Department of Stem Cell Transplantation and Cellular Therapy, the University of Texas, M. D. Anderson Cancer Center, Houston, Texas

Anne Poon, Pharm D Clinical Pharmacist, Hematopoietic Stem Cell Transplant University of Washington Medical Center/Seattle Cancer Care Alliance, Seattle, Washington

Joyce C. Niland, PhD Chair, Division of Information Sciences Edward and Estelle Alexander Chaired Professor, Beckman Research Center Associate Director for Informtation Sciences, City of Hope Cancer Center, Duarte, California

Oliver W. Press, MD, PhD Member, Fred Hutchinson Cancer Research Center, Penney E. Petersen Chair for Lymphoma, Research, Professor of Medicine and Biological Structure, University of Washington, Seattle, Washington

Margaret R. O’Donnell, MD, FRCPC Associate Clinical Director Division of Hematology/ Hematopoietic Cell Transplantation, City of Hope, Duarte, California

Susan Prohaska, PhD Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford, California

Richard O’Reilly, MD Chairman, Department of Pediatrics, Chief, Marrow Transplantation Service, Memorial Sloan-Kettering Center, New York, New York Harry Openshaw, MD City of Hope National Medical Center, Duarte, California Robertson Parkman, MD Professor of Pediatrics, Children’s Hospital of Los Angeles Division of Research Immunology and Bone Marrow Transplantation, Keck School of Medicine, University of Southern California, Children’s Hospital of Los Angeles, Los Angeles, California Steven Z. Pavletic, MD Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland I. Ben Paz, MD Associate Professor, Division of Surgery, Director, Department of General Oncologic Surgery, City of Hope, Duarte, California Charles Peters, MD Professor of Pediatrics, University of Missouri – Kansas City, School of Medicine, The Children’s Mercy Hospital, Kansas City, Missouri Effie W. Petersdorf, MD Member, Fred Hutchinson Cancer Research Center, Professor of Medicine, University of Washington School of Medicine, Seattle, WA Douglas E. Peterson, DMD, PhD Professor and Interim Head, University of Connecticut Health Center, Department of Oral Health and Diagnostic Sciences Farmington, Connecticut

Vinod Pullarkat, MB, BS, MRCP Assistant Professor Division of Hematology and Hematopoietic Cell Transplantation, City of Hope Medical Center, Duarte, California Muzaffar H. Qazilbash, MD Associate Professor of Medicine, Department of Stem Cell Transplantation and Cellular Therapy, M.D. Anderson Cancer Center, Houston, Texas Jerald P. Radich, MD Member, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington Norman Rizk, MD Guggenhime Professor of Medicine, Stanford University School of Medicine, Stanford, California Scott D. Rowley, MD, FACP Chief, Adult Blood and Marrow Transplant Program Hackensack University Medical Center, Hackensack, NJ; Clinical Associate Professor of Medicine, University of New Jersey School of Medicine, Newark, NJ George E. Sale, MD Member, Fred Hutchinson Cancer Research Center, Seattle, Washington Jean E. Sanders, MD Full Member, Clinical Research Division Director, Pediatric Hematopoietic Stem Cell Transplantation, Fred Hutchinson Cancer Research Center, Professor of Pediatrics, University of Washington, Seattle, Washington Brenda Sandmaier, MD Member, Fred Hutchinson Cancer Center, Professor of Medicine, University of Washington, Seattle, Washington

Contributor list

Dave Scadden, MD Gerald and Darlene Jordan Professor of Medicine, CoChair, Department of Stem Cell and Regenerative Biology, Co-Director, Harvard Stem Cell Institute, Harvard University Director, MGH Center for Regenerative Medicine Chief, Hematologic Malignancies, MGH Cancer Center Massachusetts General Hospital, Boston, Massachusetts Norbert Schmitz, MD Director, Department of Hematology and Stem Cell Transplantation, Lohmiehlenstr. Germany

David Snyder, MD Chair, City of Hope Bioethics Committee Associate Director, Division of Hematology and Hematopoietic Cell Transplantation, City of Hope, Duarte, Calitornia Robert J. Soiffer, MD Dana Farber Cancer Institute, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA Rampprasad Srinivasan, MD, PhD Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Mark M. Schubert, DDS, MSD Professor of Oral Medicine School of Dentistry, University of Washington; and Director of Oral Medicine, Seattle Cancer Care Alliance and Fred Hutchinson Cancer Research Center, Seattle, Washington

Susan K. Stewart Blood & Marrow Transplant Information Network (BMT InfoNet), Highland Park, Illinois

Timothy Schulteiss, PhD Department Director and Professor, Division of Radiation Oncology, City of Hope Cancer Center, Duarte, California

Simone I. Strasser, MBBS, MD, FRACP A.W. Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia

Thomas Shea, MD University of North Carolina at Chapel Hill, School of Medicine, Division of Hematology/Oncology, Chapel Hill, North Carolina Judith A. Shizuru, PhD, MD Associate Professor of Medicine, Division of Blood and Marrow Transplantation, Department of Medicine, Stanford University, School of Medicine, Stanford, California E. J. Shpall, MD Professor of Medicine, Baylor College of Medicine, Houston, Texas Howard M. Shulman, MD Member, Fred Hutchinson Cancer Research Center, Seattle, Washington Marilyn L. Slovak, PhD, FACMG Director, Department of Cytogenics, City of Hope, Duarte, California Trudy N. Small, MD Associate Attending, Department of Pediatrics Bone Marrow Transplantation Service, Memorial Sloan-Kettering Cancer Center, New York, New York Franklin O. Smith, MD Director, Division of Hematology/Oncology Professor of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio

xv

Rainer Storb, MD Head & Member, Transplantation Biology Program, Fred Hutchinson Cancer Research Center, Professor of Medicine University of Washington School of Medicine, Seattle, Washington Simone I. Strasser, MBBS, MD, FRACP Clinical Associate Professor, Senior Staff Specialist, A.W. Morrow Gastroenterology and Liver Center, Royal Prince Alfred Hospital, Australia Keith M. Sullivan, MD James B Wingaarden Professor of Medicine, Director, LongTerm Follow-up and Information Research Division of Cellular Therapy, Department of Medicine, Durham, North Carolina Megan Sykes, MD Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts Timothy W. Synold, Pharm.D. Associate Professor, Department of Clinical and Molecular Pharmacology, Co-Director, Analytical Pharmacology Core Facility, City of Hope Comprehensive Cancer Center, Duarte, California Karen Syrjala, PhD Director of Biobehavioral Sciences and Member, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington

xvi

Contributor list

Julie Talano, MD Assistant Professor of Pediatrics, Department of Pediatric Hematology and Oncology, Medical College of Wisconsin, MaccFund Research Center, Milwaukee, Wisconsin E. Donnall Thomas, MD Member, Fred Hutchinson Cancer Research Center, Professor Emeritus, University of Washington School of Medicine, Nobel Laureate Medicine/Physiology, 1990, Seattle, WA D. Kathryn Tierney, RN, PhD Oncology Clinical Nurse Specialist, Division of Blood and Marrow Transplantation, Stanford University Medical Center, Stanford, California Grant D. Trobridge, PhD Fred Hutchinson Cancer Research Center, Seattle, WA Andrea Velardi, MD Professor, Department of Clinical and Experimental Medicine, Sections of Hematology and Clinical Immunology Perugia University, School of Medicine, Perugia, Italy Catherine M. Verfaillie, MD Professor of Medicine, Director, Stem Cell Institute, Director, Stamcelinstituut, Leuven, Belgium Michael R. Verneris, MD Assistant Professor Department of Pediatrics – BMT University of Minnesota, Minneapolis, Minnesota Georgia B. Vogelsang, MD The Johns Hopkins University, Sidney Kimmel Cancer Center, Baltimore, Maryland John E. Wagner, MD Professor of Pediatrics, Director, Division of Hematology/ Oncology and Blood and Marrow Transplantation CoDirector, Center for Translational Medicine, University of Minnesota Medical School, Minneapolis, Minnesota Mark C. Walters, MD Blood and Marrow Transplantation Program, Children’s Hospital & Research Center, Oakland, CA and the Dept. of Pediatrics, University of California, San Francisco School of Medicine, San Francisco, California Phyllis I. Warkentin, MD Professor of Pathology and Pediatrics, University of Nebraska Medical Center, Department of Pediatrics, Omaha, Nebraska

Edus H. Warren, III, MD, PhD Associate Member, Fred Hutchinson Cancer Research Center, Seattle, Washington Kenneth I. Weinberg, MD Department of Pediatrics, Division of Pediatric Stem Cell Transplantation, Anne T. and Robert M. Bass Professor in Pediatric Cancer and Blood Diseases, Stanford University, Stanford, California Irving Weissman, MD Director, Stanford Institute for Stem Cell Biology and Regenerative Medicine Director, Director, Ludwig Center at Stanford, University of Stanford, California Mihkaila Wickline, MN, RN, AOCN HSCT Clinical Nurse Specialist, Seattle Cancer Care Alliance, Seattle, Washington John R. Wingard, MD Price Eminent Scholar and Professor of Medicine, Director, Bone Marrow Transplant Program, Division of Hematology/ Oncology, Deputy Director of the University of Florida, Gainesville, Florida Robert R. Witherspoon, MD Member, Clinical Research Division, Fred Hutchinson Cancer Research Center, Professor of Medicine, University of Washington, Medical Director, Transplant Clinic, Seattle Cancer Care Alliance, Seattle, Washington Jeffrey Y. C. Wong, MD Professor & Chair, Division of Radiation, Oncology, City of Hope Cancer Center, Duarte, California Ann E. Woolfrey, MD Director, Unrelated Donor Program, Associate Member, Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington Gregory A. Yanik, MD Clinical Associate Professor of Pediatrics and Communicable Diseases, Division of Hematology/Oncology, Blood and Marrow Transplantation Program, University of Michigan Cancer Center, Ann Arbor, Michigan John A. Zaia, MD Chair, Division of Virology, Beckman Research Institute of City of Hope, Duarte, California Esmail D. Zanjani, PhD Professor, Department of Medicine, University of NevadaReno, Reno, Nevada

Preface to the First Edition

The widespread application of bone marrow transplantation (BMT) to the treatment of a steadily increasing number of life-threatening hematological, oncological, hereditary, and immunological disorders is the culmination of more than four decades of research by many investigators. Early attempts in the 1950s to transplant living cells from one individual to another were carried out in the face of considerable skepticism. It was generally accepted as axiomatic that the immunological barrier to “foreign tissue” could never be overcome. The horrors of Nagasaki and Hiroshima spurred interest in studies of the lethal effects of irradiation. It was discovered that mice given total body irradiation in doses lethal to the marrow could be protected from death by shielding the spleen or by an infusion of marrow, and that the marrow of such animals contained living cells of donor origin. These observations suggested that patients with leukemia might be given a lethal exposure of total body irradiation, which would destroy the malignant cells along with remaining normal marrow. The exposure would also destroy the immune system, making it possible to protect against lethality by a transplant of normal marrow cells. The theory was correct, but results were disappointing. Because the procedure was both unproved and dangerous, only those patients who had no other options were considered. Except for a few patients with an identical twin donor, there were no survivors beyond a few months. Understanding of the human leukocyte antigen (HLA) system was not yet available, and little was known about the complication we now call graft-versus-host disease (GVHD). Thus, after a brief period of enthusiasm, most investigators abandoned this seemingly hopeless pursuit. Fortunately, work in animal models continued. Studies in inbred rodents defined the genetics of the major histocompatibility system and the fundamental rules of transplantation biology. Immunosuppressive drugs were developed to limit the severity of the immune reactions between donor and host. Demonstration of successful marrow transplants in the canine model using littermates matched for the major histocompatibility complex set the stage for successful transplantation of marrow between human siblings. Thus, it is clear that a long series of experimental studies in animals ultimately made human marrow transplantation possible. By the late 1960s, much was known about the HLA system, more effective antibiotics were available, and platelet transfusions were becoming routine. Thus began the modern era of human BMT. THe past 25 years have witnessed an almost exponential growth in the number of transplants being performed and the number of diseases being considered for BMT. Initially, most grafts employed marrow from an HLAidentical sibling. Autologous marrow, long known to be effective in animal systems, is now being used with increasing frequency following intensive cancer chemotherapy. Hematopoietic progenitor cells from the peripheral blood are now being used for BMT, either alone or to supplement marrow. As a result of increasing national and international cooperation, large panels of volunteer marrow donors of known HLA type are becoming available to patients whose own marrow cannot be used or who do not have a family donor.

Currently, thousands of transplants are being performed each year world-wide. With the demonstration that marrow could be transplanted and that the cure rate would be substantial, the logical step was taken to treat patients early during the course of their respective disease (i.e. in leukemia when the burden of malignant cells was relatively low and when the patient was in excellent clinical condition). With improved patient selection, development of improved tissue typing methods, availability of potent antimicrobial agents, advances in supportive care, and improved prevention of GVHD, the results of BMT have continued to improve. Marrow transplantation is now being applied to a long list of diseases with a wide range of results depending on the disease, the type of transplant, and the stage of the disease. For some of the diseases, BMT has already proven to be the most effective therapy (e.g., some leukemias and severe aplastic anemia), whereas for others it is the only available curative treatment (e.g., thalassemia). In very rare genetic disorders, one successful BMT may establish the success of the treatment. For other more common disorders, controlled trials are necessary to define the proper role of allogeneic or autologous BMT, or therapy not involving BMT. Only through rigorous study and long-term follow-up can novel approaches be confirmed as effective (or ineffective). For those working in the field of marrow transplantation, a source of intellectual satisfaction has been the interdisciplinary nature of the studies. A view of the wide-ranging disciplines involved can be gleaned by reading the chapter titles for this book. A successful BMT program is always a team effort. There must be cooperation between blood banks, referring physicians, radiation oncologists, immunologists, and physicians from many subspecialties. A dedicated support staff of technicians, data managers, and, above all, nurses, is crucial. The nursing team in particular is responsible for the day-to-day care of patients. Nurses not only provide the bedside management of complex protocol studies, but also bear the burden of emotional support through the difficult hospital period. They are the most readily available source of information for the patients and families day and night. Without a strong nursing team, the entire BMT program is jeopardized. Most important are the patients who come to the transplant center with the courage to accept days, weeks, and sometimes months of discomfort in the hope of surviving a fatal disease. We must ensure that we acknowledge and respect the dignity and individuality of each patient, that we provide adequate information for informed decision making and then include patients and families in the decision process. The greatest reward for clinical investigators is to see patients reintegrated into their personal, social, and professional lives, free of their disease and its complications. Stephen J. Forman Karl G. Blume E. Donnall Thomas Summer, 1993

xvii

Preface to the Second Edition

During the five-year period since the first edition of this book was published, an enormous amount of new scientific information has been generated through experimental research and through clinical trials. This growth in knowledge is reflected by the doubling in the volume of this book and a 50 percent increase in the number of new chapters. The selection of a new title Hematopoietic Cell Transplantation became necessary because bone marrow is no longer the major source of allogeneic and autologous hematopoietic cells. Hematopoietic stem cells from peripheral blood collections and from umbilical cord blood are rapidly gaining clinical importance for hematologic and immunologic reconstitution following myeloablative, high-dose therapies for malignant and non-malignant, hereditary or acquired life-threatening disease. It would be impossible to identify a single scientific observation as the leading contribution to the progress made during the past five years. Exciting new data have been reported in all areas of preclinical and clinical research: New histocompatibility antigens for optimal donor selection have been described, novel concepts have been developed to eradicate the underlying malignancies, engineering of allogeneic and autologous grfts has been further developed, previously unavailable drugs for post-transplant immunosuppression and for the prevention or therapy of infectious complications have been tested. The indication list

xviii

for high-dose therapy and hematopoietic cell transplantation has been extended and the long-term evaluation and management of transplant recipients has been further defined. Still, the editors would caution the young investigator that “breakthroughs” do not occur. Each step towards a cure has to be earned through thoughtfully designed and carefully conducted pre-clinical experiments and clinical trials. Single institution studies require confirmation through prospective randomized trials, each needing several years before a new concept can be fully accepted as a proven advance. The editors hope that the new edition will serve as a resource for physicians and many other members of health care teams at transplant centers and for new and established investigators in the area of hematopoietic cell transplantation and related fields of research; in brief, for everyone who wishes to learn about this exciting field which is now at the beginning of its fifth decade of development as a curative treatment modality for disorders with an otherwise poor prognosis. E. Donnall Thomas Karl G. Blume Stephen J. Forman Summer, 1998

Preface to the Third Edition

Ten years after the first and five years following the publication of the second edition of this textbook, the editors and the publisher, Blackwell Publishing, present now the third edition. Because of the continued impressive growth of knowledge in the field of hematopoietic cell transplantation, a new and completely revised book was needed to document and critically review the scientific progress made during the past five years. The editors wish to emphasize several changes in the third edition compared to the second edition. First, the title of the book has been changed to honor the pioneering work of E. Donnall Thomas, the 1990 Nobel Laureate in Physiology or Medicine (see Tribute). Second, the reader will find ten new chapters which deal with new experimental or clinical research observations while four prior chapters have been deleted. Topics for the new chapters include stem cell expansion, the theory and application of non-myeloablative regimens followed by allogeneic hematopoietic cell transplantation, the experimental basis for transplantation for autoimmune diseases, long-term complications and

others. Third, we have again tried to avoid overlap and contradiction between chapters, however, following academic principles, we have allowed the expression of opposing views and opinions in certain areas, for example, on the topic of plasticity of embryonic and adult stem cells. The editors are indebted to the publishing house which again has produced a high quality product in a timely fashion for the international community of investigators at all professional levels and disciplines in the field of hematopoietic cell transplantation. Finally, this book would not have been possible without the dedicated work of our assistants Sara E. Clark, Stephen J. Loy and Kristine A. Logan. Karl G. Blume Stephen J. Forman Frederick R. Appelbaum Spring 2003

xix

Preface to the Fourth Edition

Fifteen years after the first, and five years following the third edition of this textbook, the editors and the publisher, Blackwell Publishing, are proud to present the fourth edition of Thomas’ Hematopoietic Cell Transplantation. The impetus for the development of this fourth edition came from the rapid expansion in our understanding of the biologic basis of hematopoietic cell transplantation coupled with its rapidly expanding clinical application. When compared to the last edition, readers will find fourteen entirely new chapters. These new chapters focus on the scientific basis of transplantation (including chapters on embryonic stem cells, mesenchymal stem cells, NK cell biology, and dendritic cell biology), on emerging applications of hematopoietic cell transplantation (including chapters on hematopoietic cell transplantation for chronic lymphocytic leukemia and other less common indications), and on complications of transplantation (including chapters on graft failure, vascular access and complications, interstitial pneumonia, nephro-urologic complications, endocrine complications and common potential drug interactions). While new chapters have been added, others have been dropped or their materials combined in an effort to avoid redundancies. The remaining chapters have been revised and updated to reflect the current state of the science.

xx

For the first time, a CD-ROM accompanies this textbook. The CDROM contains the entire text of the book in easily searchable form, as well as all figures in a convenient downloadable format. We are hopeful that our readers will find this addition useful. The editors greatly appreciate the assistance of Blackwell Publishing, which has been extremely helpful in the process of creating this volume and in producing a finished product of the highest quality. Also, this book would not have been possible without the dedicated work of our assistants, Kimberly Laramie, Sara E. Clark-Fuentes, and especially, Tanya Chiatovich. Finally, this edition honors the memory of Dr. Ernest Beutler whose laboratory and clinical contributions helped begin the field of transplantation as a curative therapy for patients suffering from leukemia. He was a colleague and mentor for many people who have made significant contributions to the field of hematopoietic cell transplantation. Frederick R. Appelbaum, MD Stephen J. Forman, MD Robert S. Negrin, MD Karl G. Blume, MD

Tribute

Thomas HCT Fourth Edition

Dr. E. Donnall Thomas and King Carl Gustaf of Sweden. Dr. Thomas and Dr. Joseph Murray (not shown in the picture) shared the Nobel Prize for Physiology or Medicine, 1990, recognizing the field of organ transplantation. Dr. Thomas was honored for endeavors in experimental and clinical bone marrow transplantation. Their prizes emphasize the importance of patient-related research.

Our textbook title honors Dr. E. Donnall Thomas, the single individual most responsible for creating the subject of this book. Don was born on March 15, 1920, the son of a solo general practitioner in a small Texas village. He recalls as a child accompanying his father to his small office and to patients’ homes. As Don has mentioned, between he and his father, they span the period from horse and buggy house calls to our modern high-tech medicine. He received his B.A. and M.D. from the University of Texas and that is where he met his wife Dottie. Besides raising three children and eight grandchildren together, Dottie has been Don’s partner in every aspect of his professional life, from working in the laboratory, to editing manuscripts and administering grants. Anyone who has been lucky enough to work with Don knows that if he is the father of marrow transplantation, then Dottie is, without question, the mother.

Don graduated from Harvard Medical School in 1947 and completed his internship and residency at Peter Brent Brigham Hospital in Boston. It was while in medical school that Don first became interested in normal and malignant hematopoiesis. During those years, Sydney Farber was initiating his first studies of the use of antifolates to treat children with acute leukemia and Don witnessed the very first patient to achieve a remission with this approach. He was exposed to the pioneering work of Allan Erslev and his search for erythropoietin. Most importantly, he learned of Leon Jacobsen and his studies showing that shielding the spleen protected mice from the otherwise lethal effects of total body irradiation. As data emerged that a similar irradiation protection effect could be achieved by transferring bone marrow from a non-irradiated to an irradiated mouse, Don became convinced of the clinical potential of marrow transplantation.

xxi

xxii

Thomas HCT Fourth Edition

In 1955, Don moved to Cooperstown, New York and the Imogene Basset Hospital, a Columbia University affiliate, where he began working on marrow transplantation both in the canine model and in humans with Dr. Joseph Farrebee. In 1957, Don published the first report in human patients showing that complete remissions of leukemia could be achieved using total body irradiation followed by infusion of marrow from an identical twin. At that time, there was little understanding of the principles of human histocompatibility and so attempts to expand these studies to patients without identical twins were uniformly unsuccessful. These failures were the stimulus for a long series of experiments conducted by Don in the canine model showing it was possible to expose dogs to supralethal doses of irradiation and rescue them by reinfusing their own marrow, that the marrow could be cryopreserved, and that large doses of peripheral blood could substitute for marrow. But attempts at allogeneic transplantation in this outbred species continued to fail because of graft-versus-host disease or graft rejection. In 1963, Don moved to Seattle and the University of Washington to become the first head of the Division of Oncology. There he developed techniques for rudimentary histocompatibility typing in the dog and by the mid-1960s showed that by selecting matched donors and using methotrexate post-transplant, it was possible to successfully transplant marrow between matched littermates in almost every case. At the same time, based on the work of Dausset, Payne, Amos and others, the understanding of human histocompatibility also dramatically increased and so, in the late 1960s, Don made the decision to return to the subject of allogeneic transplantation in humans. He began to assemble a team of physicians, nurses and support personnel (many of whom are still part of the current Seattle Transplant Program) and obtained a program project grant from the National Cancer Institute. In November 1968, Dr. Robert Good and his colleagues carried out the first marrow transplant from a matched sibling for an infant with immunodeficiency and in March 1969, Don performed the first matched sibling donor transplant for a patient with leukemia. The Seattle Transplant Program was originally housed at the Seattle Public Health Hospital but in 1972, when the hospital was faced with closure by the federal government, the Program moved to Providence Hospital. In 1975, Don and his team moved to the newly created Fred Hutchinson Cancer Research Center, a move that provided Don with increased space, resources and scientific collaborations. That same year, he and his colleagues published their classic New England Journal of Medicine paper summarizing the field of allogeneic transplantation and particularly the early Seattle experience. These results demonstrated not

only the feasibility of the procedure, but also that there was a plateau on the survival curves following transplantation, suggesting that some of these patients were cured with this novel technique. Don continued to lead the Clinical Research Division of the Center and its transplant program until his partial retirement in 1989. Yet he continues to work writing manuscripts, delivering lectures and participating in research discussions at the Center. Appropriately, Don has received almost every possible award for his work, including the American Society of Hematology’s Henry M. Stratton Award, the General Motor’s Kettering Prize, the American Society of Oncology’s Karnofsky Award, the Presidential Medal of Science, and of course, the 1990 Nobel Prize in Medicine which he shared with Joseph Murray. With each award, Don has always emphasized how much his work was a team effort. He invariably mentions the contributions of Rainer Storb, Dean Buckner, Reg Clift, Paul Neiman, Alex Fefer, and Bob Epstein, who helped form the original Seattle Transplant Team, and of Ted Graham, who moved with Don from Cooperstown to help in the animal research. Don never fails to credit the nursing and support staff who played such a critical role in these efforts, and he always acknowledges the patients and their families who have been true partners in his work. Although Don is most noted for his pioneering scientific achievements, for those of us who have been fortunate enough to work with him, Don is equally admired for the way in which he achieved his success. He has always been focused, hardworking and uncompromising in his laboratory and clinical research. He can be a demanding critic and holds others accountable for their actions, yet at the same time he has always been generous with his ideas, loyal to his employees and quick to deflect praise to his coworkers. Although he has dedicated an enormous amount of himself to a life in medical science, he has managed to maintain a great deal of balance. He has traveled widely as a visiting lecturer, donated time to professional organizations and supported local and international charities. He and Dottie are avid and expert hunters and fishers and have a close and loving extended family. It is difficult to think of many other fields of modern medicine that are so much the result of a single man’s work. Don’s vision and dedication have changed the lives of literally hundreds of thousands of patients. By naming this textbook for him, we are joining those patients, and the many nurses, physicians and staff privileged to work in the field he created, in thanking Don both for what he has accomplished and for the way he has done it.

List of Abbreviations

AABB AAV ABMTR ABW ACC ACEI ACIF ACIP ACP ACTH ACV AD ADA ADCC ADL ADP ADR AEL AF AFO AFP AGM AGU AH AHCT AHRQ AIBW AICD AIDS AIHA AITD AITL AKI ALC ALCL ALD ALG ALI ALL ALT AMKL AML AMM

= American Association of Blood Banks = Adeno-Associated Virus = Autologous Blood and Marrow Transplant Registry – North America = Actual Body Weight = Acute Chest Syndrome = Angiotensin Converting Enzyme Inhibitor = Anticomplement Immunofluorescence = Advisory Committee on Immunization Practices = Advanced Care Planning = Adrenocorticotropic Hormone = Acyclovir = Autoimmune Disease = Adenosine Deaminase = Antibody Dependent Cell Mediated Cytotoxicity = Activities of Daily Living = Adenosine Diphosphate = Adverse Drug Reaction = Acute Erythroleukemia = Amniotic Fluid = Air Flow Obstruction = α-Feto Protein = Aorta-Gonad-Mesonephros (Region) = Aspartylglucosaminuria = Ancestral Haplotype = Autologous Hematopoietic Cell Transplantation = Agency for Health Care Research and Quality = Adjusted Ideal Body Weight = Activation Induced Cell Death = Acquired Immune Deficiency Syndrome = Auto-Immune Hemolytic Anemia = Autoimmune Thyroid Disease = Angioimmunoblastic T-Cell Lymphoma = Acute Kidney Injury = Absolute Lymphocyte Count = Anaplastic Large Cell Lymphoma = Adrenoleukodystrophy = Antilymphocyte Globulin = Acute Lung Injury = Acute Lymphoblastic Leukemia = Alanine Aminotransferase = Acute Megakaryoblastic Leukemia = Acute Myeloid Leukemia = Agnogenic Myeloid Metaplasia

AMN ANA ANC ANRC AP APC APL ARAC ARB ARDS ARF ARL ARSA ASBMT ASHI AST ATG ATL ATP ATRA ATTL AUC AVN AYA AZT B2M BACT BAL BAVC BCAA BCMA BCNU BCG BCR BCR BCRP BDNF BDP BEAC BEAM

= Adrenomyeloneuropathy = Anti-Nuclear Antibody = Absolute Neutrophil Count = Anthony Nolan Research Center = Alkaline Phosphatase = Antigen Presenting Cell = Acute Promyelocytic Leukemia = Cytosine Arabinoside = Angiotensin Receptor Blocker = Adult Respiratory Distress Syndrome = Acute Renal Failure = AIDS Related Lymphoma = Arylsulfatase A = American Society for Blood and Marrow Transplantation = American Society for Histocompatibility and Immunogenetics = Aspartate Aminotransferase = Antithymocyte Globulin = Adult T-Cell Leukemia = Autoimmune Thrombocytopenia = All-Transretinoic Acid = Acute T-Cell Leukemia/Lymphoma = Area under the Curve = Avascular Necrosis = Adolescent and Young Adult = Zidovudine = Beta-2-Microglobulin = BCNU, Cytosine-Arabinoside, Cyclophosphamide, Thioguanine = Bronchoalveolar Lavage = BCNU, Amsacrine, Etoposide, Cytosine-Arabinoside = Branched Chain Amino Acid = B Cell Maturation Antigen = 1,3-Bis(2-Chloroethyl)-1-Nitrosourea (Carmustine) = Bacillus Calmette-Guerin = B Cell Receptor = Breakpoint Cluster Region = Breast Cancer Resistance Protein = Brain Derived Neurotropic Factor = Beclomethasine-17, 21-Dipropionate = BCNU, Etoposide, ARA-C, Cyclophosphamide = BCNU, Etoposide, Cytosine Arabinoside, Melphalan

xxiii

xxiv

List of Abbreviations

BED BEE BEP BES BFU-E BL-CFC BLS BM BMC BMD BMDW BMI BMP BMSC BMT BMTSS BNLI BO BOOP BOS BSA BSE BSI BU BUN

= = = = = = = = = = = = = = = = = = = = = = = = =

CA11 CAFC CALGB CA-MRSA CAR CARES CBC CBER CBSC CBV CC CCI CCI CCNU CCR CCSG CD CDAD CDC CDC CDK CDRH CEH CFU-B CFU-BLAST CFU-F CFU-GEMM

= Carbonic Anhydrase Isoenzyme 11 = Cobblestone Area Forming Colony = Cancer and Acute Leukemia Group B = Community Acquired MRSA = Chimeric Antigen Receptor = Cancer Rehabilitation Evaluation System = Cord Blood Cell = Center for Biologics Evaluation and Research = Cord Blood Stem Cell = Cyclophosphamide, BCNU, Etoposide = Complete Chimerism = Charlson Comorbidity Index = Corrected Count Increment = 1-(2-Chloroethyl)-3-Cyclohexyl-1-Nitrosurea = Complete Cytogenetic Response = Children’s Cancer Study Group = Cluster Designation = Clostridium Difficile Associated Diarrhea = Complement Dependent Cytotoxicity = Center for Disease Control = Cyclin/Cyclin Dependent Kinase = Center for Devices and Radiological Health = Conserved Extended Haplotype = Colony Forming Units-B-Lymphocyte = Blast Forming Colony Units = Colony Forming Unit – Fibrolast = Colony Forming Unit – Granulocyte/Erythroid/ Macrophage/Megakaryocytic = Colony Forming Unit – Granulocyte Macrophage = Colony Forming Unit – Megakaryocytic = Colony Forming Unit – Mixed = Colony Forming Unit – Spleen = Colony Forming Unit-T-Lymphocyte = Chronic Granulomatous Disease = Comparative Genomic Hybridization = Current Good Tissue Practice

CFU-GM CFU-MEG CFU-Mix CFU-S CFU-T CGD CGH CGTP

Biologically Effective Dose Basic Energy Expenditure Bleomycin, Etoposide, Cisplatin BMT Events Scale Burst Forming Units – Erythroid Blast-Colony-Forming Cell Bare Lymphocyte Syndrome Bone Marrow Bone Marrow Cell Bone Mineral Density Bone Marrow Donors Worldwide Body Mass Index Bone Morphogenic Protein Bone Marrow Stem Cell Bone Marrow Transplantation Bone Marrow Transplant Survivor Study British National Lymphoma Investigation Bronchiolitis Obliterans Bronchiolitis Obliterans Organizing Pneumonia Bronchiolitis Obliterans Syndrome Body Surface Area Bovine Spongiform Encephalopathy Brief Symptom Inventory Busulfan (Myleran) Blood Urea Nitrogen

CMV-IP CNI CNL CNS CNS CNV COG COHNMC Con A CONSORT COX-2 CP CPR CR CREG CRH CSF CSP CSSCD CT CTC CTL CTLP CTN CTZ CVB CVC CVS CY CYP

= Coronary Heart Disease = Congestive Heart Failure = Cyclophosphamide, Hydroxydaunomycin, Vincristin, Prednisone = Chediak-Higashi Syndrome = Confidence Interval = Center for International Blood and Marrow Transplant Research = Chlorine Channel Pump 7 = Chronic Idiopathic Myelofibrosis = Combined Immunodeficiency Syndrome = Chemical Inducers of Dimerization = Critical Illness Polyneuropathy = Ciprofloxacin = Creutzfeldt-Jakob Disease = Chronic Kidney Disease = Cutaneous Lymphocyte Antigen = Common Lymphocyte Progenitor = Clinical and Laboratory Standards Institute = Chronic Myelogenous Leukemia = Chronic Myelomonocytic Leukemia = Common Myeloid Progenitor = Cytomegalovirus = CMV-Antibody Enriched Intravenous Immunoglobulin = Cytomegalovirus-Associated Interstitial Pneumonia = Calcineurin Inhibitor = Chronic Neutrophilic Leukemia = Central Nervous System = Clinical Nurse Specialist = Copy Number Variation = Children’s Oncology Group = City of Hope National Medical Center = Concanavilin A = Consolidated Standards of Reporting Trials = Cyclo-oxygenase-2 (inhibitor) = Chronic Phase = Cardio-Pulmonary Resuscitation = Complete Remission = Cross-Reactive Group = Corticotropin Releasing Hormone = Colony Stimulating Factor = Cyclosporine = Cooperative Study of Sickle Cell Disease = Computerized Tomography = Cyclophosphamide, Thiotepa, Carboplatin = Cytotoxic T-Lymphocyte = Cytotoxic T-Lymphocyte Precursor = Clinical Transplant Network = Chemotactic Trigger Zone = Cyclophosphamide, Etoposide, BCNU = Central Venous Catheter = Chorionic Villous Sampling = Cyclophosphamide = Cytochrome P

DAD DAG DAH DAT DC DCC

= Diffuse Alveolar Damage = Diacylglycerol = Diffuse Alveolar Hemorrhage = Direct Agglutinin Test = Dendritic Cells = Data Coordinating Center

CHD CHF CHOP CHS CI CIBMTR CICN7 CIMF CID CID CIPN CIPRO CJD CKD CLA CLP CLSI CML CMML CMP CMV CMV-IG

List of Abbreviations

DEB DEXA DEXA DFA DFCI DFO DFP DFS DHAP DHFR DHTR DIC DISF DKA DLA DLCL DLCO DLI DLT DM DMSO DNA DNAX DNR DPC DPT DRG DRS DSMB DSRCT DST DTH DTPA

= Diepoxybutane = Dual Energy X-ray Absorptiometry = Dual Energy X-ray Absorptiometry = Direct Fluorescent Antibody (Test) = Dana-Farber Cancer Institute = Desferrioxamine = Deferiprone = Disease-Free Survival = Decadron, High dose Cytarabine, Cisplatinum = Dihydrofolate Reductase = Delayed Hemolytic Transfusion Reaction = Disseminated Intravascular Coagulation = Derogatis Interview of Sexual Functioning = Diabetic Ketoacidosis = Dog Leukocyte Antigen = Diffuse Large Cell Lymphoma = Diffusion Capacity = Donor Lymphocyte Infusion = Dose Limiting Toxicity = Diabetes Mellitus = Dimethylsulfoxide = Deoxyribonucleic Acid = DNA Accessory Molecule = Do Not Resuscitate = Days Post Conception = Diphtheria/Pertussis/Tetanus = Dorsal Root Ganglion = Disability Rating Scale = Data and Safety Monitoring Board = Desmoplastic Small Round Cell Tumor = Donor Specific Transfusion = Delayed Type Hypersensitivity = Diethylenetriaminepentaacetic Acid

EACA EAE EB EBMT

= Epsilon Amino Caproic Acid = Experimental Autoimmune Encephalomyelitis = Embryoid Body = European Group for Blood and Marrow Transplantation = Epstein-Barr (Virus) Nuclear Antigen = Epstein-Barr Virus = Epstein-Barr Virus Associated Lymphoproliferative Disorder = Extracorporal Adsorption Therapy = Electrocardiogram = Extracellular Matrix = Eastern Cooperative Oncology Group = Extra Corporal Phototherapy = Early Death = Erectile Dysfunction = Etoposide, Dexamethasone, Cytosine Arabinoside, Cisplatin = Expanded Disability Status Scale = European Federation for Immunogenetics = Event-Free Survival = Enzyme-Linked Immunosorbent Assay = Eutectic Mixture of Local Anesthetic = European Neuroblastoma Study Group = European Organization for Research and Treatment of Cancer = Erythropoietin = Endoplasmic Reticulum

EBNA EBV EBV-LDP ECAT ECG ECM ECOG ECP ED ED EDAP EDSS EFI EFS ELISA EMLA ENSG EORTC EPO ER

xxv

ERCP ERT ESA ESBL ESC ESFT ESRD ET ETP EWOG

= Endoscopic Retrograde Cholangiopancreatography = Enzyme Replacement Therapy = Erythropoiesis Stimulating Agent = Extended-Spectrum beta-Lactamase = Embryonic Stem Cell = Ewing Sarcoma Family of Tumors = End Stage Renal Disease = Essential Thrombocythemia = Early Thymic Progenitor = European Working Group

FA FAB FACS FACT

FLC FLIC FLIPI FLU FLV FMF FNA FSCL FSH 5-FU FUO FVC

= Fanconi Anemia = French American British Classification = Fluorescence Activated Cell Sorter = Foundation for the Accreditation of Cellular Therapy = Foundation for the Accreditation of Hematopoietic Cell Therapy = Fluorescent Antibody to Membrane Antigen = Fas Ligand = Facilitator Cell = Famciclovir = Food and Drug Administration = Forced Expiratory Ventilation in 1 Second = Fresh Frozen Plasma = Fred Hutchinson Cancer Research Center = Familial Hemophagocytic Lymphohistiocytosis = Fluorescence in-situ Hybridization = Tacrolimus = Tacrolimus (FK) Binding Protein = Follicular Lymphoma = Face, Legs, Activity, Chest X-ray, Consolability (Pain Assessment Tool) = Free Light Chain = Functional Living Index – Cancer = Follicular Lymphoma International Prognostic Index = Fludarabine = Friend Murine Leukemia Virus = Flow Microfluorometry = Fine Needle Aspiration = Follicular Small Cleaved Cell Lymphoma = Follicle Stimulating Hormone = 5-Fluorouracil = Fever of Unknown Origin = Forced Volume Capacity

GAD GAD65 GAG GALC GALT GALV GAP GAVE G-CSF GCV GDP GELA GEP GF GFJ GFP

= Glutamic Acid Decarboxylase = Glutamic Acid Decarboxylase 65 = Glycosaminoglycan = Galactoceribrosidase = Gut Associated Lymphoid Tissue = Gibbon Ape Leukemia Virus = GTP-ase Activating Protein = Gastric Antral Vascular Ectasia = Granulocyte-Colony Stimulating Factor = Ganciclovir = Guanosine Diphosphate = Groupe d’Etude des Lymphomes d’Adulte = Gene Expression Profiling = Graft Failure = Grapefruit Juice = Green Fluorescent Protein

FAHCT FAMA FasL FC FCV FDA FEV1 FFP FHCRC FHL FISH FK506 FKBP FL FLACC

xxvi

List of Abbreviations

GITMO GLD GLR GLSG GMBP GM-CSF GMP GMP GnRH GOG G-6-PD GRE GRH GSH GSSG GST GTP GTP GU GvA GVHD GVLE

= Glomerular Filtration Rate = Growth Hormone = Growth Hormone Releasing Hormone = German Hodgkin’s Lymphoma Study Group = Gastro-Intestinal (Tract) = Gruppo Italiano Malattie Ematologiche Maligne dell’ Adulto = Gruppo Italiano Trapianti di Midolio Osseo = Globoid Cell Leukodystrophy = Glomerular Filtration Rate = German Lymphoma Study Group = Guinea Pig Myelin Basic Protein = Granulocyte/Macrophage-Colony Stimulating Factor = Good Manufacturing Practices = Granulocyte Macrophage Progenitor = Gonadotropin Releasing Hormone = Gynecologic Oncology Group = Glucose-6-Phosphate Dehydrogenase = Glucocorticoid Response Elements = Gonadotropin-Releasing Hormone = Glutathione (Reduced) = Glutathione (Oxidized) = Glutathione-S-Transferase = Good Tissue Practice = Guanosine Triphosphate = Genitourinary = Graft-versus-Autoimmunity = Graft-versus-Host Disease = Graft-versus-Leukemia Effect

4-HC HA HAART HAMA HBV HC HC HCFA HCG HCoV HCT HCV HD HD-AC HDC HDC HD-CY HDIT HDR HDT HES HES HHV6 HIPAA HIV HL HLA HLH HMPV HPC HPLC HPP

= 4-Hydroperoxycyclophosphamide = Hemagglutinins = Highly Active Anti-Retroviral Therapy = Human Antimouse Antibody = Hepatitis B Virus = Hematopoietic Cell = Hemorrhagic Cystitis = Health Care Financing Administration = Human Chorion Gonadotropin = Human Coronavirus = Hematopoietic Cell Transplantation = Hepatitis C Virus = Hodgkin Disease = High Dose ARAC = High Dose Chemotherapy = High Dose Conditioning = High Dose Cyclophosphamide = High Dose Immunosuppressive Therapy = Hypersensitivity Delayed Reaction = High Dose Therapy = Hydroxyethyl Starch = Hypereosinophilic Syndrome = Human Herpes Virus 6 = Health Insurance Portability and Accountability Act = Human Immunodeficiency Virus = Hodgkin Lymphoma = Human Leukocyte Antigen = Hemophagocytic Lymphohistiocytosis = Human Metapneumovirus = Hematopoietic Progenitor Cells = High-Pressure Liquid Chromatography = High Proliferative Potential

GFR GH GHRH GHSG GI GIGEMA

HPRT HPFH HPV HRPBC HRQOL HRSA HRT HSA HSC HSP HSV HSV-tk HTC HTLP HTLV HU HUVEC HUS HVEM HVG HVR

= Hypoxanthine-Guanine Phosphoribosyltransferase = Hereditary Persistence of Fetal Hemoglobin = Human Papillomavirus = High Risk Primary Breast Cancer = Health Related Quality of Life = Health Resources and Services Administration = Hormone Replacement Therapy = Heat Stable Antigen = Hematopoietic Stem Cell = Heat Shock Protein = Herpes Simplex Virus = Herpes Simplex Virus Thymidine Kinase = Homozygous Typing Cells = Helper T-Lymphocyte Precursor = Human T-cell Lymphotropic Virus = Hydroxyurea = Human Umbilical Cord Endothelial Cells = Hemolytic Uremic Syndrome = Herpesvirus Entry Mediator = Host-versus-Graft (Reaction) = Hyper-Variable Region

IA IAA IBD IBMTR IBW ICA ICAM ICH ICL 670 ICOS ICU iDC IDDM IDE IDSA IE IEF IFAR IFI IFN IFRT IG IGF-1 IHC IIP IL IL-1 IM IMPDH IMUST

= Invasive Aspergillus (Infection) = Insulin Autoantibody = Inflammatory Bowel Disease = International Bone Marrow Transplant Registry = Ideal Body Weight = Islet Cell Antibody = Intracellular Adhesion Molecule = International Conference on Harmonization = Deferasirox = Inducible Costimulator (Protein) = Intensive Care Unit = Immature Dendritic Cells = Insulin Dependent Diabetes Mellitus = Investigational Device Exemption = Infectious Diseases Society of America = Immediate Early (Antigen Expression) = Isoelectric Focusing = International Fanconi Anemia Registry = Invasive Fungal Infection = Interferon = Involved Field Radiotherapy = Immunoglobulin = Insulinlike Growth Factor-1 = International Conference on Harmonization = Idiopathic Interstitial Pneumonia = Interleukin = Interleukin-1 = Infectious Mononucleosis = Inosine Monophosphate Dehydrogenase = International Marrow Unrelated Search and Transplant = Investigational New Drug = International Neuroblastoma Research Group = International Neuroblastoma Staging System = Intraoperative Radiation Therapy = Interstitial Pneumonia = Intraperitoneal = Inositol-l, 4, 5-Triphosphate = Inherited Paternal Antigen = Invasive Pulmonary Aspergillosis

IND INRG INSS IORT IP IP IP3 IPA IPA

List of Abbreviations

IPI IPS IPS IPSS IRB IS IS ISCT ISO ISS IST IT ITD ITIM ITR IU-HCT IV IVF IVIG

= = = = = = = = = = = = = = = = = = =

JACIE

= Joint Accreditation Committee of ISCT-Europe and EBMT = Joint Commission of Health Care Organizations = Juvenile Idiopathic Arthritis = Juvenile Myelomonocytic Leukemia

JAHCO JIA JMML

International Prognostic Index Idiopathic Pneumonia Syndrome Interstitial Pneumonia Syndrome International Prognostic Scoring System Institutional Review Board Immunosuppressive Immune Synapse International Society for Cellular Therapy Isohemagglutinin International Staging System Immunosuppressive Therapy Intrinsic Timetable (Model) Internal Tandem Duplication Immunoreceptor Tyrosine Binding Inhibition Motif Inverted Terminal Repeats Intrauterine-Hematopoietic Cell Transplantation Intravenous In Vitro Fertilization Intravenous Immunoglobulin

KLH KPC KPS

= Keratoconjunctivitis Sicca = Killer-Cell Inhibitory Receptor or Killer Ig-like Receptor = Keyhole Limpet Hemocyanin = Klebsiella Pneumoniae Carbapenemase = Karnofsky Performance Score

LA LAD LAK LAMP LAT LC LCH LCL LCR LCT LD LDA LDH LET LFA LFR LFS LH LHRH LIF LIN LMP LMWH LOH L-PAM LPC LPD LPHD LPR

= Latex Agglutination = Leukocyte Adhesion Deficiency = Lymphokine Activated Killer (Cells) = Loop Mediated Isothermal Amplification = Latency Associated Factor = Langerhans Cell = Langerhans Cell Histiocytosis = Lymphoblastic Cell Lines = Locus Control Region = Long Chain Triglyceride = Linkage Disequilibrium = Limiting Dilution Assay = Lactate Dehydrogenase = Linear Energy Transfer = Leukocyte Function-Associated Antigen = Limited Field Radiation = Leukemia-free Survival = Luteinizing Hormone = Luteinizing Hormone Releasing Hormone = Leukemia Inhibitory Factor = Lineage = Latency Membrane Protein = Low Molecular Weight Heparin = Loss of Heterozygosity = L-Phenylalanine Mustard = Leukemic Progenitor Cell = Lymphoproliferative Disease = Lymphocyte Predominant Hodgkin Disease = Lipoprotein Receptor-Related Protein

KCS KiR

xxvii

LPS LTA LTC-IC LTR LUTS LV LVEF LY

= = = = = = = =

MAB MACS MALT MAOI MAPC MAPC MAPK MBC MBL MBP MCA MCD MCL MCL MCP MCR M-CSF MCT mDC MDR MDS MEAC MEC MEG-CSA MEG-GPA MEL MEP mESC MF MG MGDF MGF MGUS

= Monoclonal Antibody = Multipotent Adult Stem Cell = Mucosa Associated Lymphoid Tissue = Monoamine Oxidase Inhibitor = Mesenchyme Associated Progenitor Cell = Multipotent Adult Progenitor Cell = Mitogen Activated Protein Kinase = Metastatic Breast Cancer = Metallo-beta-Lactamase = Myelin Basic Protein = Middle Cerebral Artery = Minimal Change Disease = Mantle Cell Lymphoma = Mast Cell Leukemia = Monocyte Chemoattractant Protein = Major Cytogenetic Response = Macrophage-Colony Stimulating Factor = Median Chain Triglyceride = Mature Dendritic Cells = Multi Drug Resistance = Myelodysplastic Syndrome = Minimum Effective Analgesic Concentration = Medullary Epithelial Cell = Megakaryocyte-Colony Stimulating Activity = Megakaryocyte-Growth Promoting Activity = Melphalan = Megakaryocytic Erythrocyte Progenitor = Mouse Embryonic Stem Cell = Mycosis Fungoides = Myasthenia Gravis = Megakaryocyte Growth and Development Factor = Mast Cell Growth Factor = Monoclonal Gammopathy of Undetermined Significance = Minor Histocompatibility Antigen = Modified Health Assessment Questionnaire = Major Histocompatibility Complex = Metaiodobenzylguanidine = Minor Histocompatibility Antigens = Medical Internal Radiation Dose = Mucolipidosis = Mixed Leukocyte Culture = Metachromatic Leukodystrophy = Myeloid-Lymphoid-Initiating Cell = Mixed Lineage Leukemia = Mixed Leukocyte Reaction = Maroteaux-Lamy Syndrome = Moloney Leukemia Virus = Multiple Myeloma = Mitomycin C = Mycophenolate Mofetil = Matrix Metalloprotease = Measles/Mumps/Rubella

mHA MHAQ MHC MIBG miHA MIRD ML MLC MLD ML-IC MLL MLR MLS MLV MM MMC MMF MMP MMR

Lipopolysaccharide Latency Associated Transcripts Long Term Culture Initiating Cell Long Terminal Repeat Lower Urinary Tract Syndrome Lenti Virus Left Ventricular Ejection Fraction Life Years

xxviii

List of Abbreviations

MMR MN MODS MOG MOPP MP MP MPA MPB MPO MPP MPS MPSV MPT MPV MR MRC MRD MRI m-RNA MRP MRS MRSA mRSS MS MSC MSC MSCH MSD MSFC MSI MSKCC MS-LCH MSOF MTD MTOR MTX MUGA

= Mannose Macrophage Receptor = Membranous Nephropathy = Multiple Organ Dysfunction Syndrome = Myelin Oligodendrocyte Glycoprotein = Mustargen, Vincristine, Prednisone, Procarbazine = Melphalan Prednisone = Methylprednisolone = Mycophenolic Acid = Mobilized Peripheral Blood (Cells) = Myeloperoxidase = Multipotent Progenitor = Mucopolysaccharidosis = Myeloproliferative Sarcoma Virus = Melphalan, Prednisone, Thalidomide = Metapneumovirus = Molecular Remission = Medical Research Council (United Kingdom) = Minimal Residual Disease = Magnetic Resonance Imaging = Messenger RNA = Multidrug Resistance-Associated Protein = Magnetic Resonance Spectroscopy = Methicillin Resistant Staphylococcus Aureus = Modified Rodman Skin Score = Multiple Sclerosis = Mesenchymal Stem Cell = Mesenchymal Stromal Cells = Mouse Spinal Cord Homogenate = Multiple Sulfatase Deficiency = Multiple Sclerosis Functional Composite = Microsatellite Instability = Memorial Sloan Kettering Cancer Center = Multisystem Langerhans Cell Histiocytosis = Multi-System Organ Failure = Maximum Tolerated Dose = Mammalian Target of Rapamycin = Methotrexate = Multiple Gated Acquisition (Scan)

NAB NANT NAPI NASBA NAT NB NCCN NCI NCIC NCM nCR NCR NEO Nf1 NF-AT NGFR NGVL NHL NIH NIMA NIPA NK NK-T

= Natural Antibody = New Approaches to Neuroblastoma Therapy = North American Pulsed Field Type 1 = Nucleic Acid Sequence-Based Amplification = Nucleic Acid Amplification Test = Neuroblastoma = National Comprehensive Cancer Network = National Cancer Institute = National Cancer Institute of Canada = Nurse Case Manager = Near Complete Response = Natural Cytotoxicity Receptor = Neomycin = Neurofibromatosis type 1 = Nuclear Factor of Activated T Cells = Nerve Growth Factor Receptor = National Gene Vector Laboratory = Non-Hodgkin Lymphoma = National Institutes of Health = Non-Inherited Maternal Antigen = Non-Inherited Paternal Antigen = Natural Killer (Cells) = NK-T cells

NO NOD NPC NRH NRM NS NS NSAID NYBC NZW

= Non-Myeloablative = National Marrow Donor Program = Non-Myeloablative Hematopoietic Cell Transplantation = Nitric Oxide = Non-Obese Diabetic (Mouse) = Non-Protein Calories = Nodular Regenerative Hyperplasia = Non-Relapse Mortality = Nephrotic Syndrome = Natural Suppressor (Cells) = Non-Steroidal Anti-Inflammatory Drug = New York Blood Center = New Zealand White (Mouse)

OCTGT ODN OLD OMIM ONJ ONS OPA OPG OPM OR OS OSTM1

= = = = = = = = = = = =

PACT PAIgG PAIS PB PBHC PBHCT PBMC PBP PBPC PBS PCA PCNSL PCR PD PD PDC PDQ PDT PEC PET PFA PFS PFT PGD PGF PGK Ph PH PHA PHN PHQ PHS PIC PICC

= Psychological Assessment for Transplantation Scale = Platelet Associated Immunoglobulin = Psychological Adjustment to Illness Scale = Peripheral Blood = Peripheral Blood Hematopoietic Cells = Peripheral Blood Hematopoietic Cell Transplantation = Peripheral Blood Mononuclear Cells = Penicillin-Binding Protein = Peripheral Blood Progenitor Cells = Primer Binding Site = Patient Controlled Analgesia = Primary Central Nervous System Lymphoma = Polymerase Chain Reaction = Programmed Death = Protective Dose = Plasmacytoid Dendritic Cell = Physician Data Query = Photodynamic Therapy = Cisplatin, Etoposide, Cyclophosphamide = Positron Emission Tomography = Phosphonoformic Acid (Foscarnet) = Progression-Free Survival = Pulmonary Function Test = Preimplantation Genetic Diagnosis = Poor Graft Function = Phosphoglycerokinase = Philadelphia (Chromosome) = Pulmonary Hypertension = Phytohemagglutinin A = Post-Herpetic Neuralgia = Perceived Health Questionnaire = Public Health Service = Pre-Integration Complex = Peripherally Inserted Central Venous Catheter

NM NMDP NMHCT

Office of Cellular, Tissue and Gene Therapies Oligodeoxynucleotide Obstructive Lung Disease Online Mendelian Inheritance in Man Osteonecrosis of the Jaw Oncology Nursing Society Office of Patient Advocacy Osteoprotegerin Oropharyngeal Mucositis Odds Ratio Overall Survival Osteopetrosis Membrane Protein 1

List of Abbreviations

PIG-A PIH PIQ PIXY 321 P-gp PK PKC PLL PLP PLPHA PLS PLT PMC PML PMR PMRD PN PNET PNH POG POMS PORN PORT POV PR PRA PRCA PRO PRP PSA PSE PTA PTCL PTH PTLD PTT PTX PUVA PV PVP PVR

= Phosphatidyl Inositol Glycan-A = Prolactin Inhibiting Hormone = Performance IQ = Fusion Protein: IL-3/GM-CSF = P-Glycoprotein = Pharmacokinetic = Protein Kinase C = Prolymphocytic Leukemia = Proteolipid Protein = Post-Lumbar Puncture Headache = Psychosocial Level System = Platelet = Persistent Mixed Chimerism = Progressive Multifocal Leukoencephalopathy = Progressive Muscle Relaxation = Partially Matched Related Donor = Parenteral Nutrition = Primitive Neuroectodermal Tumor = Paroxysmal Nocturnal Hemoglobinuria = Pediatric Oncology Group = Profile of Mood Status = Progressive Outer Retinal Necrosis = Patient Outcomes Assessment Research Team = Premature Ovarian Failure = Partial Remission or Partial Response = Panel Reactive Antibody = Pure Red Cell Aplasia = Patient Reported Outcome = Polyribosylphosphate = Prostate Specific Antigen = Prednisone = Peripheral Tissue Antigen = Peripheral T Cell Lymphoma = Parathyroid Hormone = Post-Transplant Lymphoproliferative Disorder = Partial Thromboplastin Time = Pentoxifylline = Psoralen-Ultraviolet A Irradiation (Treatment) = Polycythemia Vera = Polyvinylpyrrolidone = Polio Virus Receptor

QALY QLQ QOL QTWIST

= = = =

RA RA RAC RAEB RAEB-T

= Refractory Anemia = Rheumatoid Arthritis = Recombinant DNA Advisory Committee = Refractory Anemia with Excess Blasts = Refractory Anemia with Excess Blasts in Transformation = Radioimmunotherapy = Receptor Activator of NF-Kb Ligand = Refractory Anemia with Ringed Sideroblasts = Red Blood Cell = Relative Biological Effectiveness = Renal Cell Cancer = Ratio of Cost and Charges = Replication-Competent Lentivirus = Refractory Cytopenia with Multilineage Dysplasia

RAIT RANKL RARS RBC RBE RCC RCC RCL RCMD

Quality-Adjusted Life Years Quality of Life Questionnaire Quality of Life Quality Time Without Symptoms of Toxicity

xxix

RCT RDA REE RFS RHC RIA RIC RIFLE RIR RISC RIT RFLP RLD RNA ROI ROS RPM RQ RR RRT RRT RSCH RSV RT RT3U RTOG

= Randomized Controlled Trial = Recommended Daily Allowance = Resting Energy Expenditure = Relapse-Free Survival = Residual Host Cells = Radioimmune Assay = Reduced Intensity Conditioning = Risk, Injury, Failure, Loss, Endstage = Reduced Intensity Regimen = RNAi Induced Silencing Complex = Radioimmunotherapy = Restriction Frequent Length Polymorphism = Restrictive Lung Disease = Ribonucleic Acid = Region of Interest = Reactive Oxygen Species = Rapamycin (Sirolimus) = Respiratory Quotient = Relative Risk = Renal Replacement Therapy = Regimen-Related Toxicity = Rat Spinal Cord Homogenate = Respiratory Syncytial Virus = Reverse Transcriptase = Resin T3 Uptake = Radiation Therapy Oncology Group

SAA SAE SAP SAS-SR SBA SCC SCD SCF SCID SCID-hu

= Severe Aplastic Anemia = Severe Adverse Event = Serum Amyloid Protein P = Social Adjustment Scale – Self Report = Soy Bean Agglutinin = Squamous Cell Carcinoma = Sickle Cell Disease = Stem Cell Factor = Severe Combined Immunodeficiency Syndrome = Severe Combined Immunodeficiency Syndrome/ Human (Mouse) = Symptoms Check List = Stem Cell Therapeutics Outcomes Database = Standard Deviation = Standard Dose Chemotherapy = Stroma Cell Derived Factor = Shwachman Diamond Syndrome = Standard Error = Sleep, Energy, Appetite Scale = Surveillance Epidemiology and End Result = Symptom Experience Report = Serological Identification of Antigens by Recombinant Expression Cloning = Spleen Focus-Forming Virus = Societe Francaise de Greffe de Moelle = French Society of Pediatric Oncology = Sex Hormone Binding Globulin = Stanford Hospital and Clinics = Sonic Hedgehog = Sickness Impact Profile = Systemic Inflammatory Response Syndrome = Signaling Lymphocyte Activation Molecule = Systemic Lupus Erythematosus = SLE Disease Activity Index = Small Lymphocytic Leukemia

SCL SCTOD SD SDC SDF SDS SE SEAS SEER SER SEREX SFFV SFGM SFOP SHBG SHC Shh SIP SIRS SLAM SLE SLEDAI SLL

xxx

List of Abbreviations

SM SMP SNP SNRI SOP SOS SPA SPECT sPLA2 SRBC SRC SS SSC SSOP SSP SSRI STAMP-I STAMP-V SUMC SVC SWOG

= Systemic Mastocytosis = Sympathetically Mediated Pain = Single Nucleotide Polymorphism = Serotonin-Norepinephrine Reuptake Inhibitor = Standard Operating Procedure = Sinusoidal Obstruction Syndrome = Staphylococcal Protein A = Single Photon Emission Computed Tomography = Secretory Phospholipase A2 = Sheep Red Blood Cell = SCID Repopulating Cells = Sézary Syndrome = Systemic Sclerosis = Sequence Specific Oligonucleotide Probes = Sequence Specific Primer = Selective Serotonin Reuptake Inhibitor = BCNU-Cisplatinum-Cyclophosphamide = Thiotepa-Carboplatin-Cyclophosphamide = Stanford University Medical Center = Superior Vena Cava = Southwest Oncology Group

α-TIF T1DM T3 T4 TAA TAI TAM TAP TBI TC TCA TCD TCD TCIRG1 TCR TDM TdT TERS TERT TFO TGF-β TGN T-GVHD TH TIL TIP TIPS TK TLC TLI TLR TM TMA TMC TMD TMP-SMX

= α-Transinducing Factor = Type 1 Diabetes Mellitus = Triiodothyronine = Thyroxine = Tumor Associated Antigen = Thoraco Abdominal Irradiation = Transplant-Associated Microangiopathy = Transporter-Associated Antigen Processing = Total Body Irradiation = Cytotoxic T cells = Tricyclic Antidepressant = T Cell Depletion = Transcranial Doppler (Ultra-sonography) = T Cell Immune Regulator 1 = T Cell Receptor = Therapeutic Drug Monitoring = Terminal Deoxynucleotidyl Transferase = Transplant Evaluation Rating Scale = Telomerase Reverse Transcriptase = Triple Helix Forming Oligodeoxynucleotide = Transforming Growth Factor Beta = Transgolgi Network = Transfusion Associated Graft-versus-Host Disease = Helper T Lymphocytes = Tumor Infiltrating Lymphocytes = Paclitaxel, Ifosfamide, Cisplatin = Transjugular Intrahepatic Portosystemic Shunt = Thymidine Kinase = Total Lung Capacity = Total Lymphoid Irradiation = Toll Like Receptor = Thrombotic Microangiopathy = Thrombotic Microangiopathy = Transient Mixed Chimerism = Temporomandibular Dysfunction = Trimethoprim-Sulfamethoxazole

TNC TNF TNF-α TNFR TPA TPMT TPN TPO TPO TRAIL TRALJ TREC TREG TRH TRIM TRM TSH TSP TTP TTR

= Total Nucleated Cells = Tumor Necrosis Factor = Tumor Necrosis Factor α = Tumor Necrosis Factor Receptor = Tissue Plasminogen Activator = Thiopurine S-Methyltransferase = Total Parenteral Nutrition = Thrombopoietin = Thyroid Peroxidase = TNF-Related Apoptosis-Inducing Ligand = Transfusion-Related Acute Lung Injury = T Cell Receptor Excision Circles = Regulatory T Cell = Thyrotropin-Releasing Hormone = Transfusion-Related Immune Modulation = Treatment Related Mortality = Thyroid Stimulating Hormone = Tropical Spastic Paraparesis = Thrombotic Thrombocytopenic Purpura = Transthyretin

UAMS UCB UCB UGD URD URI UV

= = = = = = =

VAD VATS VCAM VEGF VGPR VIP VLA VLCFA VNTR VOD VP-16 VRE VSV VSV-G VWF VWMD VZIG VZV

= Vincristine, Adriamycin, Decadron = Video-Assisted Thoracoscopic Surgery = Vascular Cell Adhesion Molecule = Vascular Endothelial Growth Factor = Very Good Partial Remission = Etoposide, Ifosfamide, Cisplatin = Very Late Activation Antigen = Very Long Chain Fatty Acid = Variable Number of Tandem Repeat = Venocclusive Disease = Etoposide = Vancomycin Resistant Enterococcus = Vesicular Stomatitis Virus = Vesicular Stomatitis Rhabdovirus = von Willebrand Factor = Vanishing White Matter Disease = Varicella Zoster Immune Globulin = Varicella Zoster Virus

WAS WBC WBM WBRT WHI WMDA WNV

= Wiskott-Aldrich Syndrome = White Blood Cell = Whole Bone Marrow = Whole Brain Radiotherapy = Women’s Health Initiative = World Marrow Donor Association = West Nile Virus

X-AND

X-Linked Adrenoleukodystrophy

University of Arkansas Medical Science Center Umbilical Cord Blood Unrelated Cord Blood Uridine 5’ Diphosphate Glucoronosyl Transferase Unrelated Donor Upper Respiratory Infection Ultraviolet (Light)

Plate 5.1 Chimeric analysis of yolk sac blood islands with fluorescent embryonic stem cell clones. Tetrachimeric mice were generated by the injection of four distinctly colored single cells into developing blastocysts. The resulting embryos were then analyzed for color combinations observed in the yolk sac, and revealed endothelial and hematopoietic cells of different colors in all individual blood islands, indicating that they developed from more than one cell. (a) A chimeric embryo at the early neural plate stage (EB). A white rectangle indicates an immature blood island. The arrow indicates the direction of tissue extension. al, allantois; am, amnion; ch, chorion; ec, embryonic ectoderm; me, intraembryonic mesoderm; ve, visceral endoderm. (b) Experimental scheme indicating the possible derivations of blood islands and the percentage of each type observed. (c) A type IV chimeric blood island with EGFP, ECFP, and mRFP1 endothelial cells, and ECFP, EGFP, and nonfluorescent hematopoietic cells. (Adapted from [172].)

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

Progression to acute myelogenous leukemia (AML) or myeloid blast crisis, CML LT-HSC

ST-HSC

MPP

CLP

Progenitors (GMP)

CMP Blood cells

Self-renewal (regulated)

EVENTS ACCUMULATED: 1. Bcr-Abl or Aml-1–Eto, or jun-b, etc. 2. Antisenescence (e.g. tert) 3. Antiapoptosis 1 3. Antiapoptosis 2 5. Evasion of immune cells (1–4 events) 6. Activation/overexpression self-renewal genes

Self-renewal (Unregulaed)

blasts

blasts

blasts Leukemic Stem Cell

Plate 5.2 Comparison of stem cell self-renewal during normal hematopoiesis and leukemic transformation. During normal hematopoiesis, the signaling pathways that regulate self-renewal are tightly controlled, allowing proper differentiation to each of the mature blood cell lineages (top). Accumulation of mutations in the self-renewing hematopoietic stem cells (HSCs) over time until dysregulated self-renewal ability is attained, and leukemia propagation is initiated. Dysregulation of self-renewal mechanisms in transformed cancer stem cells leads to uncontrolled self-renewal and the production of leukemic blast cells. The leukemic population capable of sustaining disease is often distinct from the blast population, and may reside in the stem cell or progenitor cell compartment. Importantly, if the transformation event occurs in a progenitor cell, it must endow the progenitor cell with the self-renewal, because the progenitors would otherwise differentiate. See text for abbreviations.

5‘ 3‘

W W‘ C

-

W C C‘

+

a1A2 b 1b 2 c 1c 2

a

-

A

c A a

D a A

b B

c C

3‘ 5‘

+

b B

A1a2 B1B2 C1C2

C c

S asymmetric division

maintenance

A1a2 b 1B2 C1c 2

a1A2 B1b 2 c 1C2

S symmetric division

S self-renewal

stem cell niche

Cell Counts

Plate 6.1 The “silent sister” hypothesis [32]. Top panel: According to this hypothesis the two sister chromatids in metaphase chromosomes carry distinct epigenetic marks (indicated by + and − signs) at centromeric DNA and at certain “stem cell” genes (shown here is a single gene expressed as “A” or silent as “a”). Epigenetic differences at centromeres between sister chromatids are proposed to enable chromatid-specific segregation of chromosomes during mitosis. Such nonrandom partition of chromatids is proposed to regulate the expression of certain genes present on those chromatids in the daughter cells following mitosis. Bottom panel: Selective attachment of microtubules (MT) coming from the “mother” centrosome [96] (indicated by the red dot close to the stem cell niche) to sister chromatids containing the Watson (W) template strand (here defined as the 3′ to 5′ template strand indicated by the solid red line) during a polar, asymmetric stem cell division. Shown are three metaphase chromosomes (e.g. out of the 46 chromosomes in a human cell). The preference for one of the chromatids could result from a gradient of MT guiding proteins (red) that regulate preferential MT binding to the kinetochore at the sister chromatid containing the Watson template strand. Selective partition of sister chromatids results in one stem cell (S) with two active copies of “self-renewal” genes (the two parental copies of these genes are indicated by 1 and 2) ready to be transcribed (A, B, and C) and one cell committed to differentiate (D) in which expression of the two copies of the “stem cell” genes is suppressed (a, b, and c). Note that an occasional sister chromatid exchange event between a centromere and a relevant “stem cell” gene (illustrated as a2 in cell S and A2 in cell D) is not incompatible with the “silent sister” hypothesis. In stem cells (S) expression of silenced “stem cell” genes is predicted to be restored by default (bottom panel, right-hand side), and aberrant expression of a single “stem cell” gene in a differentiating cell (A2 in cell D) is not predicted to prevent differentiation altogether. However, inappropriate expression of multiple “self-renewal” genes (perhaps in combination with inappropriate suppression of tumor suppressor genes) in differentiating cells could result in abnormal cell proliferation. Such defects could result from high levels of sister chromatid exchange, defects in cell polarity, failure to establish or recognize epigenetic marks at centromeres or other factors involved in the proposed strand-specific chromatid segregation pathways. (c) Random segregation of sister chromatids restores expression of “silent” stem cell genes and results in self-renewal of stem cells. Without a polar distribution of guiding proteins (even red color), both daughter cells inherit a random mixture of active and inactive “stem cell” genes, which is predicted to restore the expression of the “stem cell” genes in both daughter cells if both cells are in the proper microenvironment (the stem cell “niche”). Epigenetic differences between sister chromatids are restored following expression during replication. Note that only one out of eight possible expression patterns for the three genes in the two daughter cells following random sister chromatid segregation is shown.

MSCs

Plate 9.1 A panel of data demonstrating the defining characteristics of mesenchymal stromal cells (MSCs): adherence, immunophenotype, and in vitro differentiation. Top left: Photomicrograph of undifferentiated MSCs showing the characteristic spindle shape and adherent properties of the cells (original magnification ×40). Top right: Flow cytometry histograms demonstrating the typical expression pattern of surface antigens (—) and isotype control (- - -), as indicated. Bottom: Immunocytochemical staining demonstrating the differentiation of MSCs into osteoblasts (Alizarin red stain), adipocytes (oil red O stain), and chondroblasts (Alcian blue stain). (Reproduced from [43], with permission.)

Osteoblasts

CD105

CD45

CD34

CD73

CD11b

CD19

CD90

CD3

HLA-DR

Log Fluoresence Intensity

Adipocytes

Chondroblasts

(a)

(b)

(c)

(d)

(e)

(f)

(g)

Plate 14.1 Evaluation of murine graft-versus-host disease (GVHD) lingual tissue for apoptosis, T-cell infiltration, and cytokeratin 15 expression. (a) Light microscopy of hematoxylin and eosin-stained tissue from GVHD at day 11 after bone marrow transplantation reveals lymphocytic infiltration and apparent apoptosis in the rete-like prominences (RLPs) (arrow). (b) Apoptosis restricted to RLPs was confirmed by TUNEL immunohistochemistry (arrows). (c) Transmission electron microscopy demonstrates RLP infiltrated by a lymphocyte (left arrow) in apposition to an epithelial cell undergoing mitotic division (right arrow). (d,e) Immunohistochemistry for CD4 at day 4 (d) and day 7 (e) after bone marrow transplantation with progressive infiltration of RLPs by CD4+ T cells. Note the focus where CD4+ T cells ((e); arrow) surround a centrally located nonreactive cell ((e) inset). (f,g) K15 expression in sections adjacent to (d) and (e), respectively, reveals focal loss of K15 immunoreactivity ((f); arrow). Note the cell strongly positive for K15 and surrounded by nonreactive cells ((g); arrow and inset), a pattern reciprocal to the inset in (e) and suggestive of satellitosis. (Adapted from [98], with permission.)

Plate 21.1 Theoretical models of drug resistance. In the conventional model of drug resistance (a) a subpopulation of cells (yellow) have pre-existing genetic variations that confer multidrug resistance (MDR). Following chemotherapy, the resistant cells survive and proliferate, forming a recurrent tumour that is composed of the drug-resistant clone. In the cancer stem-cell model (b), tumors have a small population of tumor stem cells (red) and their differentiated progeny (blue). Following chemotherapy, the differentiated cells are killed, but the stem cells survive. In the “acquired resistance” stem-cell model (c), the tumor stem cells (red) survive therapy, and differentiated cells are killed. Mutations in the surviving stem cells (yellow) and their progeny (purple) may also display the drug-resistant phenotype. In the “intrinsic resistance” model (d), both the stem cells (yellow) and the differentiated cells (purple) are inherently drug resistant. (From [34], with permission.)

Plate 23.1 Dose distribution through T5–6 showing 6, 8, 10, 12, and 13.2 Gy isodose lines. The prescription dose was 12 Gy, with 50% transmission lung blocks.

Plate 23.2 Color wash demonstrating the typical dose distribution of a patient treated with targeted total body irradiation using tomotherapy. The target structure is skeletal bone. A, anterior; L, left; R, right. (Reproduced from [4], with permission from the American Society for Blood and Marrow Transplantation.)

Plate 23.3 Color wash demonstrating dose distribution of 30 Gy to the site of a chloroma in the maxillary sinus and high neck, combined with 12 Gy total marrow irradiation, in a patient with acute myeloid leukemia.

(a)

(b)

Plate 25.1 Assessment of chimerism in sorted granulocytes (a) and leukemic blasts (b) by dual-color fluorescence in situ hybridization (FISH). Hybridization was carried out with a biotin-labeled Y chromosome-specific DNA probe and a digoxigenin-labeled X chromosome-specific DNA probe. Hybridization was detected with Texas Red-conjugated avidin and fluorescein-conjugated antibody against digoxigenin. Cells were counterstained with 4′,6-diamidino-2phenylindole API. Male cells contain a single red fluorescent spot and a single green fluorescent spot, while female cells contain two green fluorescent spots and no red fluorescent spots. In this case, granulocytes were derived from a male donor, and leukemic blasts were derived from the female recipient.

14 14

der(4) der ?

4

(b)

(a)

(c)

(d)

(e)

Targeted scanning with CD19 monoclonal antibody CD19

+

lymphoblast

der(9)

9

22 BCR/ABL (f)

(g)

CD10 positive

BCR–ABL positive

(i)

(h) CD10 negative

BCR–ABL negative

Plate 26.1 Plasma cell-specific fluorescence in situ hybridization (FISH) analysis. (a–e) Cytospin slides are made from the bone marrow aliquot and stained with May-Grünwald stain. After the cells have been mapped to the slide, the slide is destained and hybridized with the selected DNA probe. For details, see [5]. (a) A plasma cell that has been mapped. This same cell is seen in panel (b) showing a variant immunoglobulin H (IGH) pattern using a dual-color 5′IGH/3′IGH break apart probe. Once an IGH gene rearrangement is found in a patient with multiple myeloma, the same slide is rehybridized to determine its partner chromosome in the IGH translocation. (c) A fusion signal is detected with the FGFR3 (fibroblast growth factor receptor-3)/IGH probe indicating the derivative chromosome 4 of the t(4;14) or der(4)t(4;14). The second fusion that would have indicated the der(14)t(4;14) is not present in this patient, the result of loss of the derivative 14 chromosome. In another slide from this patient (d and e), monosomy 13 is shown in the neoplastic plasma cells, but the segmented neutrophil adjacent to the plasma cells shows both chromosome 13 signals (one each in each of the segments of the nucleus). The chromosome 13 probe set used was composed of D13S319 probe (red), which maps to 13q14.3, and the LAMP1(lysosomal-associated membrane protein1)/13q34 reference probe labeled in green. Sequential immunohistochemistry (IHC)/FISH is shown in (f–i). (f) A CD19+ lymphoblast that is BCR/ABL1 fusion gene positive (g), indicating residual disease and not a hematogone. Similarly, (h) and (i) show the cell that is CD10+ is positive for the BCR/ABL1 fusion gene, and the CD− cell is negative for the BCR/ABL1 fusion gene. The BCR/ ABL1 probe used for the sequential IHC/FISH studies was the single fusion probe spiked with 9q34/argininosuccinate synthetase gene probe labeled in Spectrum aqua. All probes were obtained from Abbott Molecular Inc. (Des Plaines, IL).

Plate 26.2 Flow cytometry assays for detection of minimal residual disease. Leukemia samples can be labeled with multiple fluorescent antibodies, and cells can be distinguished by the size of the cell and the types of antibody that bind the cell surface. Normal hematopoiesis has very stereotypical cell antigen expression linked to differentiation; leukemia cells are distinguished by inappropriate expression patterns of these antibodies. In the example, a residual acute monocytic leukemia is analyzed with multiple antigens. In the bottom left panel, the sample shows an inappropriate expression of CD56 and CD4 consistent with residual disease. In this example, these cells made up 0.9% of the total nucleated cells. (Courtesy of Dr Brent Wood, University of Washington.)

Plate 26.3 Quantitative polymerase chain reaction (PCR). The cartoon shows the principle behind the two most popular quantitative approaches: the Taqman (ABI) and the LightCycler (Roche). In the Taqman (I) reaction, the probe 5′ reporter dye and a 3′ quencher molecule. When the probe is intact, the quencher blocks laser excitation of the reporter. During PCR, the 5′ exonuclease activity of Taq polymerase releases the reporter, which, free from the quencher, is excited by the laser detection system. In the LightCycler system (II), annealing of juxtaposition probes during PCR synthesis allows for the transference of resonance energy that can be stimulated by the laser and detected. In both systems, a standard curve is generated that plots dilutions of the standards versus the PCR cycle number at which a threshold signal is obtained (middle panel). The starting amount of target in unknown samples is thus detected by a backcalculation of cycle threshold in the unknown (Ct) against the standard curve. (Courtesy of Dr John Goldman.)

Plate 27.1 Fatal Mylotarg-associated sinusoidal obstruction syndrome. The obliterated central venule is surrounded by focal sinusoidal fibrosis. (Trichrome stain)

Plate 27.2 Markedly hypocellular marrow without evidence of engraftment, day 7. The interstitial fluid accumulation, fat necrosis, and iron deposition are consistent with chemoirradiation damage.

Plate 27.3 Post-transplant Epstein–Barr virus-associated lymphoma infiltrating the intestinal mucosa in a boy treated for graft-versus-host disease (GVHD) with cyclosporine and a monoclonal antibody to T lymphocytes. Although the GVHD improved, large bizarre cells with atypical nuclei characteristic of a high-grade post-transplant lymphoproliferative disorder rapidly formed tumor nodules in liver, spleen, lymph nodes, and mesentery.

(a)

(b)

Plate 27.4 (a) Unsuspected Epstein–Barr virus post-transplant lymphoproliferative disorder. After the first hematopoietic cell transplantation (HCT), the patient developed severe liver graft-versus-host disease. Donor lymphocyte infusion was followed by loss of engraftment. A second HCT was carried out following conditioning with antithymocyte globulin, Campath, and steroids. Jaundice developed 35 days later. At low power, the portal space contains a prominent infiltrate that extends outward into the periportal zone. (b) In the same case, higher magnification shows the large atypical immunoblastic cells that were positive for CD20 and EBER staining.

Plate 27.5 Skin biopsy from the recipient of identical twin marrow who had received a single 15 mg/kg dose of dimethylbusulfan 15 days previously. The epidermal separation, keratinocyte atypia, and scanty lymphoid dermal infiltrate can also be seen in graft-versus-host disease.

Plate 27.7 Severe mucositis of the lips and tongue with pseudomembranous exudate 20 days after hematopoietic cell transplantation following conditioning with total body irradiation and chemotherapy. Similar changes may be present with severe acute graft-versus-host disease or secondary infection such as with a herpes virus. Plate 27.6 Acute glossitis 1 day post hematopoietic cell transplantation after a conditioning regime of 60 mg/kg of cyclophosphamide followed by 12 Gy of fractionated total body irradiation. There is early mucosal damage with severe erythema and focal atrophy.

Plate 27.8 Effect of high-dose therapy on colonic mucosa, day 7. This biopsy specimen was taken following therapy with busulfan, cyclophosphamide, and total body irradiation (12 Gy). There has been obliteration of almost all epithelial cells, leaving cystic crypt remnants. (Hematoxylin and eosin with alcian blue counterstain)

Plate 27.9 Recovery from the effects of high-dose therapy on colonic mucosa, day 16. This repeat biopsy from the same patient as in Plate 27.8 indicates that regeneration is occurring, although some cystic crypts remain. (Hematoxylin and eosin with alcian blue counterstain)

Plate 27.10 Hepatic sinusoidal obstructive syndrome (SOS) 95 days post hematopoietic cell transplantation for malignancy. The trichrome stain shows the blue outline of an hepatic venule with marked luminal narrowing (arrow shows lumen diameter) caused by subendothelial trapping of red cells. The hepatocytes of acinar zone 3 are severely disrupted. When the clinical diagnosis of hepatic SOS is uncertain, it may be confirmed by a transvenous liver biopsy with measurement of an elevated gradient between wedged and free hepatic venous pressures.

Plate 27.13 Fatal hepatic sinusoidal obstructive syndrome (SOS) at autopsy 12 days post bone marrow transplantation for leukemia. At this low magnification, the hematoxylin and eosin-stained section demonstrates striking congestion, hemorrhage, and hepatocyte disruption in zone 3 (centrilobular) of the liver acinus toward the right. The portal space and surrounding acinar zone 1 on the left are well preserved. These early severe hepatic SOS lesions at times can be identified with the hematoxylin and eosin stain after tissue fixation with B5 or methyl Carnoy’s. However, stains for connective tissue are more effective.

Plate 27.11 Early hepatic sinusoidal obstructive syndrome (SOS) showing perivenular staining for procoagulants. Immunohistochemical staining for factor VIII (von Willebrand) demonstrates postsinusoidal obstruction corresponding to the endothelium-lined pores through which the sinusoids drain into the venules. Immunostaining for fibrin localizes in the same regions. These observations provide some of the rationale for using anticoagulation and antithrombotic therapy for hepatic SOS; however, these therapies are ineffective.

Plate 27.14 CD31 immunostaining of liver with fatal sinusoidal obstructive syndrome. There has been partial to complete loss of sinusoidal endothelial staining of the hepatic cords in zone 3 (arrows) which surround the subtotally occluded venule lacking an endothelium (arrowhead).

Plate 27.12 Late hepatic sinusoidal obstructive syndrome, day 63. The trichrome stain demonstrates fibrotic obliteration of the venous lumen with extensive fibrosis in the surrounding sinusoids. The resulting vascular obstruction produced intractable ascites. Immunohistochemical stains at this stage reveal extensive deposition of collagen, while fibrin and other blood procoagulants are no longer identifiable.

Plate 27.15 Hepatic sinusoidal obstructive syndrome (SOS) and coexistent graft-versus-host disease (GVHD), day 26. On this trichrome stain, the two most common liver problems after hematopoietic cell transplantation can be differentiated by the zone of the liver acinus they affect. The expanded portal space in the lower right demonstrates changes from GVHD, including fibrosis, proliferation of atypical cholangioles along the limiting plate, and destruction of small interlobular bile ducts. The SOS lesion along the left margin shows striking collections of embolized hepatocytes beneath the endothelial basement membrane.

Plate 27.16 Fatal sinusoidal obstructive syndrome. (a) Hematoxylin and eosin staining demonstrates a large perivenular zone of hemorrhagic necrosis with destruction of hepatocyte cords surrounding the damaged venule (V); the areas around the portal spaces (PS) are spared. (b) Trichrome staining demonstrates the subtotal occlusion of the venular lumen by collagen and extracellular matrix. (c) Immunostain Mac 387 for macrophage/ monocytes demonstrates a zone of cells at the interface of the viable and necrotic hepatocytes. (d) Smooth muscle actin immunostain demonstrates marked expansion of hepatic stellate cell processes within the damaged sinusoids surrounding the venule.

Plate 27.18 Acute skin graft-versus-host disease, day 28 post mismatched allogeneic hematopoietic cell transplantation. The epidermis demonstrates a prominent lichenoid reaction involving the tip of the rete ridge in the center of the specimen, a location of epithelial stem cells within the epidermis.

Plate 27.17 Hepatic phlebosclerosis, day 96. Eccentric thickening of the venular wall without striking luminal narrowing was associated with an early liver toxicity syndrome. The patient had received a 5-day infusion of highdose arabinoside-C followed by cyclophosphamide and total body irradiation. Liver enzyme and bilirubin elevations developed prior to the infusion of marrow. Changes of phlebosclerosis were widespread, but there were no identifiable intravenular hepatic sinusoidal obstructive syndrome lesions at autopsy. (Trichrome stain)

Plate 27.19 Early skin graft-versus-host disease involving the parafollicular bulge region of the pilar unit. Note the small numbers of mononuclear inflammatory cells which infiltrate the parafollicular bulge area (*) near the arrector pilorum muscle in association with apoptotic bodies.

Plate 27.20 Gastric biopsy with graft-versus-host disease (GVHD), day 35. Several studies have shown that of all upper endoscopic biopsy sites, the stomach most frequently shows histologic changes of GVHD. In the area shown here, large numbers of lymphocytes infiltrate the lamina propria and the basilar portions of the gastric crypts. There is apoptosis of crypt epithelial cells and frank early crypt destruction. Inflammation and apoptosis of this intensity are not required for the diagnosis of gastric GVHD. More subtle yet still diagnostic alterations are illustrated in Plates 27.27 and 27.28.

Plate 27.21 Erythema and desquamation before treatment for acute graftversus-host disease, day 27 (see also Plate 27.24).

Plate 27.22 Maculopapular rash of skin graft-versus-host disease without a notable decrease in inflammation, 1 week after institution of PUVA therapy (see also Plate 27.25).

Plate 27.23 Painful red-to-violaceous maculopapular rash involving the sole of the foot consistent with acute graft-versus-host disease in a 2-year-old girl 11 days after human leukocyte antigen-nonidentical hematopoietic cell transplantation.

Plate 27.25 Marked improvement in skin graft-versus-host disease following 6 weeks of PUVA treatment (same patient as in Plate 27.22).

Plate 27.24 Virtually normal skin with focal depigmentation after treatment for acute graft-versus-host disease, day 74 (same patient as in Plate 27.21).

Plate 27.26 Endoscopic appearance of acute graft-versus-host disease in the stomach, day 32. The pyloric channel is in the center of the picture. The mucosa of the gastric antrum is edematous, reddened, and friable.

Plate 27.27 Gastric graft-versus-host disease in which the severe mucosal erythema, edema, and erosion seen endoscopically on the left are more striking than the focal, mild, epithelial cell apoptosis (arrow) in the histologic section on the right. Although a lymphocytic infiltrate is absent, apoptosis in multiple crypts is consistent with graft-versus-host activity. Inflammation may have been partially controlled by immunosuppressive therapy. (Reproduced from [42], with permission.)

Plate 27.28 Gastric graft-versus-host disease (GVHD) in which the antral mucosa is endoscopically normal aside from edema (left). With air insufflation through the endoscope, the antrum did not distend and there was no motor activity. Biopsy of such a bland area can be diagnostic. In this case, as shown in the photomicrograph on the right, there was epithelial cell apoptosis (arrow) and moderate lymphocytic infiltration diagnostic for GVHD. (Reproduced from [42], with permission.)

Plate 27.29 Gastric graft-versus-host disease. Within the edematous antral mucosa, numerous lymphocytes infiltrate the glands. Apoptosis is widespread, along with segmental disruption of the glands.

Plate 27.30 Rectal biopsy with graft-versus-host disease, day 53. Three mucosal crypts display varying degrees of damage. The crypt on the right shows many apoptotic cells. More extensive cell damage is present in the crypt on the lower left. The upper crypt has lost all enterocytes. Note infiltrating lymphocytes in the lower left crypt associated with nuclear debris characteristic of apoptosis.

Plate 27.31 Surgical resection of severe longstanding intestinal graft-versushost disease (GVHD), day 207. Scarred, ulcerated mucosa alternates with dilated areas of mucosal regeneration. The ulcerated portions demonstrate complete loss of epithelium and severe submucosal fibrosis. Very few patients have responded to intestinal resection for localized GVHD.

Plate 27.34 Fatal pseudomembranous colitis. An acute abdomen developed 15 days post transplant. The degenerative colonic crypts were covered by a cap of mucin admixed with sloughed cellular debris containing large colonies of bacteria consistent with Clostridium spp.

Plate 27.32 Severe graft-versus-host disease (GVHD) of ileum and cecum, day 80. The mucosal lining and folds are replaced by a friable, beefy red, diffusely ulcerated lamina propria. The numerous exposed capillaries ooze blood and serum, a major cause of morbidity from intestinal GVHD. The endothelial cells and fibroblasts within the lamina propria are often infected with cytomegalovirus, as was true in this patient.

Plate 27.35 Endoscopic appearance of cytomegalovirus esophagitis, day 61. A large shallow ulcer with serpiginous, reddened borders is seen in the distal esophagus. The ulcer base (arrows) has a yellow, reticulated appearance.

Plate 27.33 Collagen deposition in the colon with longstanding refractory extensive chronic graft-versus-host disease. The thickened bowel has extensive deposition of collagen in the submucosa, serosa, and lamina propria of the mucosa.

Plate 27.36 Esophageal biopsy showing cytomegalovirus (CMV) infection, day 61. Beneath the epithelium are two cytomegalic cells (large arrows), each containing a single, large, oval intranuclear inclusion surrounded by a clear halo (Cowdry type A). A third cell (small arrow) is probably also infected but is morphologically nondiagnostic. It would presumably be positive by immunocytochemistry and in situ hybridization techniques. Studies of cell morphology as well as viral DNA and antigen expression indicate that the esophageal squamous epithelium is never infected with CMV.

Plate 27.37 Autologous gastric graft-versus-host disease (GVHD) or lymphocytic gastritis. The focal mononuclear inflammatory cell infiltration of the lamina propria and mucosal crypt associated with crypt epithelial cell apoptosis is histologically indistinguishable from GVHD in an allograft recipient.

Plate 27.38 Hepatic graft-versus-host disease. Periodic acid–Schiff-stained section highlights a damaged interlobular bile duct that is infiltrated by lymphocytes. The nuclei vary in size and are irregularly arranged. The syncytium-like segment reflects nuclear loss.

Plate 27.39 Acute hepatitic onset of graft-versus-host disease at 10 months post transplant. Three months after tapering off immunosuppression, the aminotransferases rose to 2000 U, and alkaline phosphatase was three times normal, with normal bilirubin. This biopsy (1) was done 26 days from the onset and shows an extensive lobular hepatitis with intense lymphocytic inflammation of sinusoids and portal spaces, and hepatocellular necrosis.

Plate 27.40 This biopsy (2) was done 1 month later when the total bilirubin was 30 mg/dL. (a,b) Lymphoplasmacytic portal infiltrate which obscures the damaged interlobular bile ducts. (c) Immunostaining with cytokeratin 7 demonstrates that damaged bile ducts are still present. (d) Perivenular (zone 3) cholestasis. Hepatocytes show ballooning, and there is focal dropout associated with a patchy lymphocytic infiltrate. The patient made a full recovery after immunosuppressive treatment for graft-versus-host disease was initiated.

Plate 27.42 Lichen planus-like chronic graft-versus-host disease of the forearm of a 24-year-old African-American male 220 days after bone marrow transplantation of human leukocyte antigen-identical marrow (see also Plates 27.44 and 27.45). Plate 27.41 Hepatic graft-versus-host disease with severe cholangitis lentalike cholangiolar hepatocellular cholestasis. The interlobular bile duct is no longer recognizable. A ductular reaction with large bile-filled cholangioles lies along the periphery of the enlarged irregular portal space. The adjacent hepatocyte cords display marked unrest, disarray, and cytoplasmic ballooning.

Plate 27.43 Periungual erythema and onychodystrophy 100 days after allogeneic bone marrow transplantation in a patient with ocular sicca and oral lesions of chronic graft-versus-host disease.

Plate 27.44 Lichen planus-like chronic graft-versus-host disease before treatment, day 220 (see also Plates 27.42 and 27.45).

Plate 27.45 Same patient shown in Plates 27.42 and 27.44 after treatment, day 770.

Plate 27.46 Poikiloderma in late chronic graft-versus-host disease in a 2year-old girl, day 450. Thinning of the epidermis and dermis is present, along with telangiectasia and reticulated pigmentation.

Plate 27.47 Localized late phase of chronic graft-versus-host disease resembling morphea with atrophic plaque formation, induration, and peripheral hyperpigmentation, day 684.

Plate 27.48 Generalized scleroderma with atrophy, sclerodactyly, and joint contracture, day 540 after human leukocyte antigen-identical bone marrow transplantation.

Plate 27.50 Late chronic graft-versus-host disease (GVHD) of skin, day 959. As this patient’s GVHD progressed, the epidermis became atrophic, with straightening of the dermal-epidermal junction. All dermal appendages were destroyed. The reticular dermal collagen became increasingly sclerotic. A panniculitis seen at the base of the specimen resulted in fibrosis of the subcutaneous fat and large vessels. This histologic picture corresponds to the dense hidebound and sclerodermatous changes of late chronic GVHD.

Plate 27.49 Oral labial biopsy with early chronic graft-versus-host disease (GVHD), day 92. The minor salivary gland exhibits a periductal mononuclear inflammatory infiltrate involving the duct epithelium, in which there is focal epithelial apoptosis. There is inflammation of adjacent acini. This process leads to fibrosis with acinar atrophy in lobules drained by the affected ducts. The squamous surface epithelium (not present here) showed grade II lesions similar to those previously illustrated in skin (see Plate 27.18). The lip biopsy is valuable in the diagnosis and staging of chronic GVHD because it frequently mirrors destructive inflammatory changes in the lacrimal, tracheal, and esophageal glands, as well as bile ducts.

Plate 27.51 Atrophy and fibrosis of the thymus associated with chronic graft-versus-host disease (GVHD) in a 13-year-old, day 350. The patient had not received immunosuppressive drugs, so the destruction was presumably the result of chronic GVHD activity.

Plate 27.52 Poikiloderma, day 719. The patient was treated vigorously for extensive chronic graft-versus-host disease with corticosteroids plus several other immunosuppressive agents. Although he had extensive dyspigmentation, telangiectasia, and atrophy of the skin, there were no contractures or scleroderma. Histologically, the epidermis is atrophic, with loss of rete ridges. The fibrotic papillary dermis contains many telangiectatic vessels, and there is intracellular melanin pigment. The deep dermal sweat glands remain despite earlier inflammatory involvement, and the reticular dermal collagen is normal.

Plate 27.54 Polymyositis in deltoid muscle biopsy associated with chronic graft-versus-host disease, day 1129. There is extensive endomysial chronic inflammation associated with myocyte destruction. The condition responded to immunosuppressive therapy.

Plate 27.55 Coronary arteriopathy associated with chronic graft-versus-host disease in a 15-year-old who died of myocardial infarction preceded by severe anginal symptoms. The lumen and muscular wall of all the major coronary arteries were subtotally to completely obliterated by a cellular infiltrate of lymphoid and monocytoid cells admixed with extracellular matrix. Plate 27.53 Lichenoid early chronic graft-versus-host disease (GVHD) of the skin, day 233. Biopsy of a papulosquamous lesion from the posterior neck demonstrates the characteristic features of the lichen planus-like type of chronic cutaneous GVHD. The thickened epidermis displays hyper- and parakeratosis, hypergranulosis, and acanthosis. The extensive destruction along the dermal-epidermal junction results in sawtooth-like changes of the rete ridges, mimicking lichen planus. The papillary dermis has considerable perivascular inflammation. The lichenoid reaction also involves the hair follicles and eccrine glands seen deep in the reticular dermis. At this stage, treatment can prevent progression to fibrosis (see also Plate 27.50).

Plate 27.56 Two Toxoplasma tachyzoites are present near the ciliated surface of a bronchial epithelial cell. Diagnosis of Toxoplasma pneumonia was established in less than 2 hours after bronchoalveolar lavage. (Wright–Giemsa stain of cytospin preparation)

Plate 27.57 Respiratory syncytial virus pneumonia in a lung biopsy, day 48. The presence of dark purple cytoplasmic inclusions in multinucleated epithelial cells (arrows) suggests the diagnosis but may be found with other viruses such as parainfluenza. During severe community outbreaks, infections may spread to hospitalized bone marrow recipients.

Plate 27.58 Varicella-zoster virus (VZV) hepatitis in a percutaneous needle biopsy, day 79. On the right, a periodic acid–Schiff stain demonstrates a pale necrotic area (*) bordered by cells containing nuclear inclusions (large arrows) visible by hematoxylin and eosin staining on the lower left. Immunofluorescence confirmed the presence of VZV (upper left), and intravenous acyclovir was begun within hours of the biopsy.

Plate 27.60 Fusarium hyphae invade the dermis in pretransplant skin biopsy.

Plate 27.59 Overwhelming adenovirus hepatitis at autopsy 92 days after allogeneic hematopoietic cell transplantation for acute myeloid leukemia. Dark basophilic nuclear inclusions produce “smudge cells” (large, lower arrows) with blurred nuclear margins. Occasional cells with a halo around the inclusion (small, upper arrow) suggestive of cytomegalovirus (CMV) infection are sometimes seen when adenovirus infection is severe. In this case, immunohistology studies for CMV were negative.

Plate 27.61 Pulmonary sinusoidal obstructive syndrome in a lung biopsy from a patient with pulmonary hypertension 48 days post second allogeneic bone marrow transplantation for acute lymphoblastic leukemia. Loose intimal fibrosis partially occludes the interlobular vein. This complication probably results from chemotherapy and may be more frequent than previously suspected. (Verhoeff–Van Gieson elastin stain)

Plate 27.62 Bronchiolitis with evolving subepithelial fibrosis and lymphohistiocytic infiltration. There is marked epithelial damage. In this patient with chronic graft-versus-host disease (GVHD) and obstructive pulmonary function abnormalities, the alterations are consistent with pulmonary GVHD. Similar changes could be seen in respiratory virus infection, which was absent in this biopsy. The ongoing inflammation and subtotal constrictive obliteration of this airway suggest that immunosuppressive therapy may be beneficial.

Plate 27.63 In a patient with a history similar to the preceding case, the bronchiolitis obliterans of chronic pulmonary graft-versus-host disease has progressed. This bronchiole is completely occluded by granulation tissue, and scattered mononuclear inflammatory cells are present. Without successful intervention with immunosuppressive therapy, this airway will finally be replaced by probably permanent scar tissue. The arrows indicate residual smooth muscle. The elastin fibers are black with this Verhoeff–Van Gieson stain. (a)

(c)

(bi)

(bii)

(biii)

(biv)

(d)

Plate 39.1 Proliferative capacity of human cord blood hematopoietic progenitor and stem cells. (a) Colony formation by multipotential (CFU-GEMM) (lower-left) and granulocyte–macrophage (CFUGM) (upper-right) progenitor cells from cord blood cells thawed after 21 years in a frozen cryopreserved state. (b) Colony formation from a high proliferative potential (i and ii) and a smaller proliferative potential (iii and iv) progenitor cell arising from a single high-CD34-expressing single cell sorted into a single microtiter plate (i and iii: magnification × 20; ii and iv: magnification × 80). (Reproduced from [102] with permission.) (c) A secondary umbilical cord blood CFU-GEMM colony derived from replating of a single CFU-GEMM colony onto a secondary plate, a measure of self-renewal of CFUGEMM. See [104] for more information. (d) Immunohistochemical staining of CD34+ cord blood cells that engrafted NOD/SCID mouse bone marrow using human Ki67, a marker of cell proliferation. The conclusion is that cord blood progenitors and stem cells have extensive proliferative capacity. See [238] for more information.

1.0

Probability of survival

0.8

0.6

0.4 Early match, CP (n= 16) Late match, CP (n= 40) Late mismatch, CP (n= 23) Early mismatch, CP (n= 81) Late match, beyond CP (n= 13) Late mismatch, beyond CP (n= 33)

0.2

0.0 0

2

4 6 8 Years after transplant

10

12

Plate 47.1 Impact of disease phase and human leukocyte antigen matching for chronic myeloid leukemia. CP, chronic phase. (Reproduced from [2].)

Plate 62.1 Renal biopsy showing amyloidosis. These images show three views of a renal biopsy: with periodic-acid Schiff (PAS) staining showing amorphous material in glomeruli (top), Congo red staining showing apple-green birefringence in polarized light (middle) and electron microscopy showing nonbranched linear fibrils about 10 nm in length. (See also Chapter 62.)

Plate 70.1 (a, b) Hands of a patient with Sézary syndrome show fissuring, cracking, and nail destruction. (c, d) One year after allogeneic hematopoietic cell transplantation (HCT), there is regression of skin involvement and normalization of the nails. (Reproduced from [94], with permission.)

Plate 79.1 Progression of dysplasia to refractory anemia with excess blasts in a single patient with Fanconi’s anemia over a 9-month period. (a) Marrow aspirate on February 21, 1996 demonstrating pancytopenia and early dysplastic changes in neutrophils. (b) Marrow aspirate on July 3, 1996 demonstrating progressive dysplasia and 3% blasts. (c) Marrow aspirate on September 24, 1996 demonstrating progressive dysplasia and 12% blasts. (d) Marrow biopsy on September 24, 1996 demonstrating severe aplasia. (See Chapter 79.)

Plate 79.2 Above: G-banded karyotypes from an abnormal clone in the marrow of a patient with Fanconi’s anemia. This is a hyperdiploid metaphase cell with 48 chromosomes including two extra derivative chromosomes (designated by arrows); one derivative chromosome is composed of two copies of the long arm of chromosome 1, and the other contains two copies of a small portion of the long arm of chromosome 3. This cell contains a total of four copies of portions of chromosomes 1 and 3. Below: A karyotype of the same abnormal clone as above, as characterized by fluorescent in situ hybridization (FISH) using a multicolor paint probe (Vysis spectral vision M-FISH). MFISH confirmed that the two derivative chromosomes detected by G-banding represented extra copies of portions of chromosomes 1 and 3. (Courtesy of Betsy Hirsch, PhD, Director of the Cytogenetics Laboratory of the University of Minnesota Medical Center, Fairview, Minneapolis, MN.) (See Chapter 79.)

Plate 79.3 Model of the Fanconi’s anemia (FA) pathway. The model shows the sequential assembly of the FA core complex in the cytoplasm and nucleus. DNA replication or DNA damage activates the complex, either directly or indirectly, leading to the activation of FANCD2 by monoubiquination and its targeting to nuclear foci. The association of FANCD2 with BRCA1, FANCD1/BRCA2, FANCN/PALB2, and FANCI, leads to as yet incompletely defined downstream events, such as DNA repair or the activation of DNA synthesis (S) checkpoints. In this model, the core complex is the sensor for DNA damage and participates by activating FANCD2 through ubiquination. FANCD2 and its associates facilitate repair. (Reproduced from [65], with permission.) (See Chapter 79.)

Plate 79.4 Hepatic adenoma. Pathologic specimen obtained at autopsy from a patient with Fanconi’s anemia with multiple adenomata (arrow in upper panel pointing to an adenoma) not revealed by ultrasound prior to hematopoietic cell transplantation. The lower specimen shows area of massive intrahepatic hemorrhage (arrowed) in one previously unidentified adenoma.

Plate 79.5 Before and after blastomere biopsy of the six to eight-cell embryo to diagnose Fanconi’s anemia and assess human leukocyte antigen type. (Electron micrograph provided by Mark Hughes, M.D., Genesis Genetics Institute, Detroit, Michigan.)

Plate 87.1 Poikiloderma, a diagnostic manifestation of chronic graft-versushost disease, which includes both atrophic and pigmentary changes. Note the combination of reticulated pattern, epidermal atrophy (cigarette-paper wrinkling of the skin surface), erythema, and brown pigment forming the borders of skin-colored or depigmented skin.

Plate 87.2 Lichen planus-like eruption involving the palmar surfaces, a diagnostic manifestation of chronic graft-versus-host disease. Note the erythematous/violaceous flat-topped papules or plaques with or without surface reticulations or a silvery or shiny appearance on direct light.

Plate 87.4 Alternative manifestation of deep sclerosis common in areas of excess subcutaneous tissue such as the abdomen, upper arms, and buttocks. Tissue is not hidebound but demonstrates prominent rippling, dimpling or a cellulite-like appearance.

Plate 87.5 Morphea-like superficial sclerotic skin changes, a diagnostic feature of chronic graft-versus-host disease. Note the localized, patchy areas of movable smooth or shiny skin with a leathery-like consistency, often with dyspigmentation. In this picture, there is a fibrotic, hypopigmented area in the center of the plaque with a slightly hyperpigmented border.

Plate 87.3 Diffuse deep sclerotic features involving the lower extremities, a diagnostic feature of chronic graft-versus-host disease. Note the nonmovable/ hidebound subcutaneous tissues, erosions and ulcerations, loss of hair follicles, and toenail dystrophy.

(a)

(b)

Plate 87.6 Lichen sclerosis-like lesion, a diagnostic manifestation of chronic graft-versus-host disease. Note the discrete to coalescent gray to white movable papules or plaques, with a shiny appearance and leathery consistency (a) or cigarette paper-like textural changes (b).

Plate 87.7 Nail dystrophy involving multiple digits is a distinctive feature of chronic graft-versus-host disease (GVHD), although it is insufficient alone to establish the diagnosis of chronic GVHD. Note the longitudinal ridging, splitting and brittle nail features, as well as periungual erythema.

Plate 87.8 New onset of patchy alopecia after recovery from chemoradiotherapy is another distinctive manifestation of chronic graftversus-host disease (but is insufficient alone to establish the diagnosis). Patchy alopecia may involve loss of scalp or body hair.

Plate 87.9 Lichenoid lesions of oral chronic GVHD showing crythema, white striae, plaque, and ulcer formation, day 394. (See also Chapter 23, p. 294; Chapter 50, p. 647; Chapter 67, pp. 920, 922, 923.)

Plate 87.11 Numerous vesicle-like mucoceles (a distinct manifestation of chronic graft-versus-host disease [GVHD]) are seen along the center of the soft palate. Patchy white lichenoid hyperkeratosis (diagnostic of chronic GVHD) and interspersed moderate erythematous changes are also evident across the soft palate. Additionally, note the foamy mucus saliva consistent with GVHD salivary gland dysfunction.

Plate 87.10 Decreased oral range of motion and microstomia in a patient with sclerotic features of chronic graft-versus-host disease. This is a diagnostic manifestation. Note the erythema, edema, and atrophy of the vermillion lip and labial mucosa.

Plate 87.12 Keratoconjunctivitis sicca with blepharitis. The eyelid margins are thickened, edematous, and erythematous. Also note plugging of the meibomian gland orifices (along the eyelid margin) and significant conjunctival hyperemia/injection. A punctal plug can be seen at the lower left margin of the photo.

Plate 87.13 Chronic graft-versus-host disease (GVHD) involving the vulvovaginal tissues. Note lichen planus-like manifestations, which are diagnostic of chronic GVHD. Also seen are erythema and agglutination of the labial tissues.

Plate 87.14 Chronic GVHD of liver, day 166. This biopsy shows a prominent plasmacytic infiltrate in the portal area and a paucity of interlobular bile ducts. The histological dichotomy between acute and chronic GVHD is less clear in liver than in skin. However, increased portal inflammation and loss of bile ducts are more common in chronic GVHD. Despite the frequency of chronic hepatic GVHD, cirrhosis and liver failure are rare and may reflect superinfection with hepatitis C virus. (See also Chapter 23, p. 292.)

Plate 87.15 Bronchiolitis obliterans in a lung biopsy, day 396. The patient had chronic GVHD with severe obstructive pulmonary disease. Similar small airway inflammation with fibrosis is seen in lung allograft rejection. (See also Chapter 23, p. 297; Chapter 50, p. 647.)

(a)

Plate 87.16 Edema in the extremities can be bilateral or, as shown, unilateral. Edema may be an early sign of evolving subcutaneous sclerosis and fasciitis.

(b)

Plate 87.17 (a,b) Manifestations suggestive of fascial involvement by chronic graft-versus-host disease (a diagnostic feature) can include rippling or the groove sign.

Plate 92.1 Modeling varicella zoster virus (VZV) pathogenesis in human tissue xenografts in vivo. Human tissue xenografts are maintained in mice with severe combined immunodeficiency. VZV infection of cells within their tissue microenvironments can be evaluated in mice with skin, T-cell (thymus/liver) or dorsal root ganglia xenografts.

Chronic lymophocytic bronchiolitis

Obstructive bronchiolitis obliterans (OLD)

Acute pneumonitis (IPS)

OLD b

c

Chronic interstitial pneumonitis

Restrictive interstitial fibrosis (RLD)

RLD

a

d Phase one

e Phase two

Phase three

Plate 96.1. A triphasic model of both obstructive lung disease (OLD) and restrictive lung disease (RLD) following allogeneic stem cell transplantation (SCT). Immune-mediated injury to the lung after allogeneic SCT can be conceptualized in three phases. In phase one, acute lung injury develops as a consequence of an allogeneic immune response, and results in the sequential influx of lymphocytes, macrophages, and neutrophils into an inflamed pulmonary parenchyma (a). Persistence of an inflammatory signal in the setting of dysregulated repair mechanisms promotes the transition from acute to chronic injury in phase two (b and d). As chronic inflammation proceeds to phase three, lung fibroblasts increase dramatically in number and contribute to the enhanced deposition of collagen and granulation tissue in and around bronchial structures, ultimately resulting in complete obliteration of the small airways and fixed OLD (c). If, by contrast, the principal target of early damage is the alveolar epithelium, fibroblast proliferation and intraseptal collagen deposition ultimately results in interstitial fibrosis and RLD (e).

(a)

(b)

Plate 103.1 Severe oral mucositis occurring 8 days after hematopoietic cell transplantation following conditioning with VP-16, cyclophosphamide, and total body irradiation (World Health Organization mucositis score of 4). Bleeding ulcerations are noted on the anterior ventral area of the tongue (a) in this patient, with simultaneous pseudomembranous fibrin exudate-covered ulcers evident on the lower labial mucosa (b).

Plate 103.2 Oral chronic graft-versus-host disease in a patient 1 year post hematopoietic cell transplantation, with lichenoid hyperkeratotic changes (stria-, papule-, and plaque-like changes) associated with generalized atrophic and erythematous mucosal changes involving the right buccal mucosa. Small initial pseudomembranous fibrin exudate-covered ulcers are indicated by arrows.

Plate 103.3 Severe oral chronic graft-versus-host disease showing distinct hyperkeratotic lichenoid striae and a pseudomembranous fibrin exudatecovered ulceration with intense surrounding inflammation in a patient approximately 18 months post hematopoietic cell transplantation.

Plate 103.4 A 45-year-old male with a history of multiple myeloma who had been treated with intravenous zoledronate for 2 years prior to having two lower right molars extracted. He presented with three areas of bisphosphonate-associated osteonecrosis of the lower right mandible 6 months following the extractions. Lesions were nonpainful and stable. Arrows indicate areas of bone exposure.

Plate 103.5 Bisphosphonate-associated osteonecrosis of the jaw involving the lower right lingual ridge in a patient with multiple myeloma treated with intravenous zoledronate monthly for 20 months. The patient’s second bicuspid and first molar had been extracted 3 months earlier due to what was thought to be severe peridontal disease; the primary extraction site healed, but an extensive area of osteonecrosis developed along the mandibular lingual torus that was present lingual to the original extraction site.

Section 1 History and Use of Hematopoietic Cell Transplantation

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

1

E. Donnall Thomas

A History of Allogeneic Hematopoietic Cell Transplantation

In the broad sweep of history, it seems strange that our knowledge of the functions of the bone marrow is less than two centuries old, and the knowledge of transplantation of bone marrow occupies little more than half a century. History, as defined in this chapter, will be restricted to those findings which led to human bone marrow transplantation. It will not deal with solid organ transplantation, extensive studies in mice or advances in immunology and immunosuppressive drugs except as related to clinical marrow transplantation. Attempts to employ marrow for therapeutic purposes began in 1939 and 1940 [1,2]. However, infusions of a few milliliters of marrow were not attempts at transplantation, and no useful results were seen. During World War II, some very interesting experiments were carried out by Rekers and colleagues in the then classified laboratories of the Atomic Energy Commission at Rochester, New York. When these studies were published in 1950 [3], they were found to be carefully conducted trials of infusion of bone marrow from normal dogs into dogs exposed to 350 R. The investigators found no significant effect on pancytopenia or on survival. They were not aware that at least twice this exposure is necessary to provide sufficient immunosuppression to achieve engraftment. Thus, they failed in their attempts to achieve marrow recovery by intravenous marrow infusion.

The beginning The seminal studies, first published in 1949, were those of Jacobson and colleagues, who found that a mouse would survive otherwise lethal irradiation if the spleen were exteriorized and protected from the irradiation [4]. They found that an intraperitoneal injection of cells from the spleen (a hematopoietic organ in the mouse) would achieve the same effect. Lorenz et al. [5] extended these observations by demonstrating a similar protective effect by an infusion of bone marrow cells. At first, it was hypothesized that this “irradiation protection” effect was due to some kind of hormone or growth factor contained in the infusion. Most of these early studies involved measuring death at 30 days after lethal irradiation. Infusions of preparations from a donor of different H2 type were effective when survival was measured at 30 days. Such a result favored a humoral mechanism since it would not be expected if cells were involved. However, in 1954, Barnes and Loutit reported that mice protected with syngeneic marrow survived beyond

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

100 days. Mice given allogeneic marrow (A into CBA) survived at 30 days (“academic survivors”) but died thereafter of a “secondary disease” [6]. Another argument in favor of a hormonal effect was that “cell-free” extracts contained the protective effect. The investigators did not yet appreciate the very small number of cells required for transplantation in the syngeneic situation. Barnes and Loutit [7] sounded a warning against the hormonal hypothesis in which they noted that the “cellular hypothesis” had not been excluded as an explanation of the irradiationprotective effect. The demise of the humoral hypothesis became inevitable in 1955 when Main and Prehn published an article in the Journal of the National Cancer Institute [8]. They studied mice given lethal irradiation and marrow from an H2-incompatible (BALB/cAnN into DBA/2JN) strain. Subsequently, the surviving recipient mice given a skin graft from the donor strain accepted the skin graft for the rest of their life. Trentin was able to show that the tolerance of the graft was specific for the marrow donor strain [9]. Survival of the graft could only be explained by the persistence of cells of the donor strain leading to “tolerance.” Proof that these animals protected against lethal irradiation by marrow infusion were true radiation chimeras came from several sources. In 1956, Nowell et al. described the growth and continued function of rat marrow cells in X-irradiated mice [10]. Most convincingly, Ford et al. showed that the marrow of lethally irradiated mice given infusions of syngeneic marrow marked by the T6 chromosome was made up of cells of the cytogenetic type of the donor [11].

Early clinical studies At this point, it became evident that marrow transplantation might be of use not only in irradiation protection, but also in therapeutic application to marrow aplasia, leukemia, and other diseases of the marrow and lymphoid system. In 1956, Barnes et al. described the treatment of mice with leukemia by lethal irradiation and marrow transplantation [12]. In Cooperstown, NY, Thomas and Ferrebee and their colleagues had already begun comparable studies in terminal patients with hematological malignancies. In 1957, they reported six patients treated with irradiation and intravenous infusion of marrow from a normal individual [13]. Only one patient showed a transient marrow graft. These failures were to be duplicated by many investigators, so that in 1970 Bortin was able to compile a list of approximately 200 attempts at allogeneic marrow grafting, all of which had failed [14]. In 1959, Thomas et al. [15] reported an identical twin with terminal leukemia who was given 850 R (748 rad) total body irradiation from

3

4

Chapter 1

Fig. 1.1 A symposium on bone marrow transplantation in Paris in 1971. From left to right: George Mathé, Dirk van Bekkum, George Santos, Don Thomas, Charles Congdon, Delta Uphoff.

opposing cobalt-60 sources and an intravenous infusion of marrow from the healthy twin (a syngeneic transplant). This dose of irradiation would be expected to produce prolonged pancytopenia and death [16]. However, the patient showed prompt recovery and disappearance of the leukemia for 4 months. This study demonstrated that lethal irradiation followed by compatible marrow could have an antileukemic effect even in advanced leukemia. Most importantly, it showed that compatible marrow infused intravenously could restore marrow function in human beings after lethal irradiation. Also in 1959, Mathé et al. reported the infusion of marrow into patients exposed to potentially lethal irradiation in a reactor accident [17]. Subsequent analysis raised doubt about whether there had been a therapeutic effect [18]. Nevertheless, this experience gave a great boost to interest in marrow transplantation and attracted many investigators to the field. One of the driving spirits of the early studies was Charles Congdon of the Oak Ridge National Laboratory, TN. In 1957, he organized an informal series of conferences at Oak Ridge. The early results of these conferences were published in abstract form in small leaflets called Fundamental and Clinical Aspects of Radiation Protection and Recovery. From 1964 to 1970, the abstracts appeared under the title Experimental Hematology. These small booklets may be difficult to find but are well worth the effort. They list the authors and investigations of that early time: there was life before Medline. In 1971, these meetings were organized into the International Society for Experimental Hematology, which publishes the modern journal Experimental Hematology (Fig. 1.1).

Advances from animal studies Thus, it was soon recognized that marrow grafting in man would be very difficult. Many investigators therefore turned to animal studies for definition and resolution of the problems. Much early information came from the work of Brent, Billingham, Medawar, and their colleagues [19]. They studied the induction of tolerance in newborn mice. In the course of these studies, they described the syndrome of “runt disease” in newborn mice given allogeneic cells. The principles they established were to prove the same as for “secondary disease” in irradiated mice and, eventually, for graft-versus-host disease (GVHD) in man. Although there had been great concern about the possibility of runt disease in the early studies of human marrow transplantation [13], it was apparent that this would be a problem only with a successful graft.

In 1959, Billingham and Brent published a landmark study of runt disease in newborn mice [20], and in 1966, Billingham described in detail the biology of graft-versus-host reactions [21]. These authors noted the following: • Syngeneic cells did not result in runt disease. • Persistence of allogeneic cells in the recipient was necessary for the development of runt disease. • The severity and incidence of runt disease were determined by antigenic differences between donor and host. • Tolerance could occur in the absence of runt disease. • The severity of runt disease was enhanced by cells already sensitized by exposure to host strain cells. The principles they elaborated were applicable to all studies of hematopoietic cell transplantation (HCT). The mouse has been used widely in studies of transplantation biology. Numerous studies in the murine system are cited in the book by van Bekkum and de Vries [22]. The availability of inbred strains has permitted extensive studies of genetic factors in transplantation. Uphoff [23] showed that the severity of the secondary disease after irradiation and marrow grafting was controlled by genetic factors. Uphoff [24] and Lochte and colleagues [25] showed that methotrexate (MTX) could prevent or ameliorate the secondary disease now known as GVHD. The dog has been frequently used in transplantation and surgical studies. Dogs are outbred animals comparable to humans. They come in large families, permitting a study of inherited factors. Studies in dogs first demonstrated the utility of autologous marrow grafts [26,27]. Marrow from one group of dogs was collected and set aside. The marrow donor or control animals were then given lethal or supralethal irradiation. The control animals invariably died whereas those treated with their own marrow promptly recovered. Obviously, this type of study could not be done with human beings. Successful cryopreservation of marrow had been accomplished by freezing in glycerol [28] using the technique of Polge and Smith. A careful step-wise removal of the glycerol before infusion of the cells was essential. Cavins et al. showed that canine marrow could be cryopreserved by freezing in dimethylsulfoxide (DMSO), thawed and injected intravenously without removal of the DMSO [29]. This technique was to become standard for marrow transplantation. Goodman and Hodgson had shown that mice could be protected against lethal irradiation by the infusion of blood cells [30]. Cavins et al. showed that autologous canine buffy coat cells from the peripheral blood could be

A History of Allogeneic Hematopoietic Cell Transplantation

collected, cryopreserved in DMSO, and infused after lethal irradiation with subsequent recovery of marrow function [31]. The practical application of peripheral blood cells for transplantation had to await better methods of centrifugation for separation of the buffy coat cells and techniques for mobilizing hematopoietic cells into the blood. The dog was most informative in extensive studies of allogeneic marrow grafting in an outbred species. Ferrebee et al. [32] reported the first allogeneic marrow graft in an irradiated dog. Four dogs were given 400 R on each of 3 days. One control and two dogs given marrow from unrelated dogs died of the acute irradiation syndrome. The male dog given marrow from a female littermate engrafted, and the leukocyte nuclei showed female drumsticks characteristic of the donor. Thomas et al. carried out a series of studies of irradiation and allogeneic marrow grafting in the dog [33,34]. Allogeneic graft recipients showed all of the problems that were soon to be recognized in human patients. The problems were graft failure, graft rejection, GVHD and/or death from opportunistic infections. A few animals did not develop these problems and became long-term survivors with marrow cells of donor type. Presumably these survivors fortuitously had donors of sufficient histocompatibility to permit long-term survival. In 1960, methods for selecting compatible donors were unknown. Early studies of marrow grafting after irradiation were carried out with non-human primates. Autologous marrow grafts were effective in protecting monkeys against lethal irradiation [35]. After allogeneic marrow grafting, they developed early severe secondary disease [35] that was somewhat reduced by treatment with antilymphocyte serum [36]. Rhesus monkeys given blood transfusions before irradiation rarely accepted an allogeneic graft [37]. In the dog, prior transfusions from histocompatible donors also prevented successful engraftment [38]. Unfortunately, marrow-grafting studies in non-human primates were not very useful for application to human patients. The non-human primates were expensive and difficult to work with. Therefore, the number of subjects studied was small. They were also not available in families. In addition, survival studies were difficult because of infection and parasitism in wild-caught animals [39]. In 1960, Medawar and Burnett received the Nobel Prize for their discovery “of acquired immunological tolerance.” When the prize was announced, my wife Dottie and I were attending a transplantation meeting in Switzerland. The joy and celebration knew no bounds. We were not only honoring the winners, but also acknowledging that transplantation biology had become a recognized field of scientific endeavor.

Advances in knowledge of histocompatibility Techniques for defining tissue antigens in humans were crucial to the development of HCT. In 1954, Miescher and Fauconnet recognized antibodies induced by transfusion or pregnancy that reacted with antigens on white blood cells [40]. In 1958, Dausset and van Rood and their colleagues recognized that human leukocyte antigens (HLAs) followed the genetic principles of inheritance [41,42]. There followed numerous studies designed to elucidate the role of these antigens in transplantation. HLA proved, however, to be of minimal value in the prediction of the outcome of skin grafts [43,44]. Epstein et al. [45] developed typing sera for dog leukocyte antigens (DLAs). Storb and colleagues studied dogs given lethal irradiation and a marrow graft from a littermate [46,47]. They showed that those marrow grafts between mismatched littermates always failed. Grafts between DLA-matched donors and recipients showed much improved survival. The administration of a short course of MTX for immunosuppression after grafting improved the long-term survival of matched recipients to 90% or better [48]. Thus, studies in dogs illustrated the importance of

5

DLA matching. At about the same time, Terasaki showed that the outcome of kidney grafts between siblings was highly dependent on HLA matching [49]. Taken together, these studies showed that matching for leukocyte antigens would be essential in human marrow grafting.

Renewed clinical studies After all the disappointments of clinical marrow transplants in the late 1950s and early 1960s, there was general pessimism about the field, and many of the early investigators had moved on to other studies. Nevertheless, improvements in transfusion medicine and the treatment of infections, and especially an improved understanding of the importance of HLA typing, encouraged a renewed attack on the clinical application of marrow grafts. The first good news came from studies of children with an immunological deficiency. Because of their disease, these children could not reject a foreign graft, and therefore immunosuppression before grafting should not be necessary. In November 1968, Gatti et al. performed the first successful allogeneic graft in a patient with severe combined immunodeficiency [50]. Two similar successes were reported immediately thereafter [51,52]. All three patients were alive and well 25 years later [53]. In late 1967, the Seattle marrow transplantation team received a grant to support clinical marrow transplantation. The year 1968 was spent in assembling a team of nurses, technicians, dietitians, etc. who were to be dedicated to patients undergoing intensive therapy and marrow transplantation with a special emphasis on post-transplant clinical care. The first transplant by this team was carried out in March 1969 [54]. The patient was a 46-year-old man with the blastic crisis of chronic myelogenous leukemia. An initial typing showed the patient’s sister to be an HLA match. After an irradiation exposure of a calculated midline dose of 954 rad, marrow was infused intravenously. Engraftment by donor cells was evident in 13 days. The patient developed mild GVHD controlled by MTX. Subsequently, he developed fever and died after 56 days. Autopsy showed cytomegalovirus pneumonia, but there was no evidence of leukemia or GVHD. This patient showed successful engraftment and control of GVHD but illustrated the problem of opportunistic infection. Thus began the Seattle marrow transplant team’s long series of patients given marrow grafts from HLA-matched siblings. In 1972, Thomas et al. reported the first experience with allografting for severe aplastic anemia [55]. The first four patients were referred for marrow grafting after a failure of conventional therapy with steroids and multiple transfusions. Since these patients did not involve the problem of eradication of a malignant disease, they were prepared with immunosuppression by four large doses of cyclophosphamide following the regimen of Santos et al. [56]. All four grafts were initially successful. One patient died of GVHD, and one died of graft rejection. Two patients were long-term survivors. In 1975, the Seattle team published a landmark Medical Progress review in the New England Journal of Medicine that was to become a highly cited article [57]. That article reviewed the rationale and experimental background for marrow transplantation. It emphasized the importance of histocompatibility and the possibility of using unrelated donors. It described the preparation of the patient, the technique of marrow transplantation, and the importance of supportive care. It also described the use of HLA-matched siblings for 37 patients with aplastic anemia and 73 with leukemia who had reached an advanced stage of their disease before transplantation. Death from recurrent disease, opportunistic infection, and GVHD were analysed. Most importantly, the article described a number of survivors. In 1977, the Seattle team reported 100 patients with end-stage acute leukemia treated by chemotherapy, total body irradiation and allogeneic

6

Chapter 1

marrow transplantation [58]. Thirteen of these patients became longterm survivors. Also, by 1977, the time of follow-up of transplanted patients was long enough to show a change in the shape of the survival curve indicating a plateau, which suggested that some patients were cured of their disease [59]. The survival of some patients transplanted in a terminal stage of their disease raised the possibility of transplantation earlier in the course of the disease. Transplantation before the development of drug resistance and before the complications of advanced disease might be expected to improve survival. In 1979, two reports described marrow grafts for patients with acute myeloid leukemia transplanted in first remission [60,61]. As expected, survival was greatly improved, and approximately 50% of the patients became long-term survivors. Mathé had coined the term “adoptive immunotherapy” to indicate the possibility that lymphoid elements in the engrafted marrow might react against malignant cells in the patient to aid in eradication of the remaining malignant cells [62]. Initial attempts to evaluate the possibility that GVHD might include graft-versus-leukemia effects were unrewarding [57]. However, when enough cases had been accumulated, Weiden et al. were able to demonstrate that the more severe the GVHD, the less likely was the recurrence of leukemia [63]. In the 1970s, a major concern was the limitation of allogeneic grafting to HLA-matched sibling pairs. Obviously, only about one-fourth of the patients would have a suitable marrow donor. Because of the complexity of the HLA system, finding a matched unrelated donor seemed a formidable task. In 1979, Hansen et al. performed the first successful marrow graft from an unrelated donor for a patient with leukemia [64].

A young patient with refractory acute leukemia did not have a matched sibling. Quite by chance, it was observed that one of the hematology technicians had an HLA type that matched the patient. The graft was successful without GVHD until the leukemia recurred 2 years later. This patient’s experience stimulated the formation of the National Marrow Donor Program.

History is now The use of donor peripheral blood leukocytes or cord blood cells for HCT has become a routine part of transplantation. The use of donor lymphocytes to cure recurrent leukemia and the use of non-myeloablative preparative regimens are but a few of the advances that are current subjects of research. Molecular biological techniques have greatly improved the accuracy of tissue typing. We are just beginning to appreciate the importance of the microenvironment, the cytokines, and the multitude of genes regulating cell division and senescence. Each advance with new agents, such as imatinib mesylate, forces us to redefine the role of HCT in therapy of disease. Thus, at the end of the 20th century, grafting of hematopoietic cells had become widely employed for research and as a form of therapy for patients with a variety of diseases. Many investigators have been attracted to the field, and many institutions have established HCT programs. Progress in the clinical application of HCT and in new avenues of research is the subject of the remaining chapters of this book and is described by the investigators who carried out much of the work.

References 1. Osgood EE, Riddle MC, Mathews TJ. Aplastic anaemia treated with daily transfusions and intravenous marrow. Ann Intern Med 1939; 13: 357– 67. 2. Morrison M, Samwick AA. Intramedullary (sternal) transfusion of human bone marrow. JAMA 1940; 115: 1708–11. 3. Rekers PE, Coulter MP, Warren S. Effect of transplantation of bone marrow into irradiated animals. Arch Surg 1950; 60: 635–67. 4. Jacobson LO, Marks EK, Robson MJ, Gaston EO, Zirkle RE. Effect of spleen protection on mortality following X-irradiation. J Lab Clin Med 1949; 34: 1538–43. 5. Lorenz E, Uphoff D, Reid TR, Shelton E. Modification of irradiation injury in mice and guinea pigs by bone marrow injections. J Natl Cancer Inst 1951; 12: 197–201. 6. Barnes DWH, Loutit JF. What is the recovery factor in spleen [Letter]? Nucleonics 1954; 12: 68–71. 7. Barnes DWH, Loutit JF. Spleen protection: The cellular hypothesis. In Bacq ZM, editor. Radiobiology symposium. London: Butterworth; 1955. 8. Main JM, Prehn RT. Successful skin homografts after the administration of high dosage X radiation and homologous bone marrow. J Natl Cancer Inst 1955; 15: 1023–9. 9. Trentin JJ. Mortality and skin transplantability in X-irradiated mice receiving isologous or heterologous bone marrow. Proc Soc Exp Biol Med 1956; 92: 688–93. 10. Nowell PC, Cole LJ, Habermeyer JG, Roan PL. Growth and continued function of rat marrow cells in X-radiated mice. Cancer Res 1956; 16: 258– 61.

11. Ford CE, Hamerton JL, Barnes DWH, Loutit JF. Cytological identification of radiation-chimaeras. Nature 1956; 177: 452–4. 12. Barnes DWH, Corp MJ, Loutit JF, Neal FE. Treatment of murine leukaemia with X-rays and homologous bone marrow. Preliminary communication. Br Med J 1956; 2: 626–7. 13. Thomas ED, Lochte HL, Jr., Lu WC, Ferrebee JW. Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. N Engl J Med 1957; 257: 491–6. 14. Bortin MM. A compendium of reported human bone marrow transplants. Transplantation 1970; 9: 571–87. 15. Thomas ED, Lochte HL, Jr., Cannon JH, Sahler OD, Ferrebee JW. Supralethal whole body irradiation and isologous marrow transplantation in man. J Clin Invest 1959; 38: 1709–16. 16. Cronkite EP, Bond VP. Radiation injury in man. Oxford, UK: C.C. Thomas/Blackwell Scientific Publications; 1960. 17. Mathé G, Jammet H, Pendic B et al. Transfusions et greffes de moelle osseuse homologue chez des humains irradiés a haute dose accidentellement. Rev Franc Etudes Clin et Biol 1959; IV: 226–38. 18. Andrews GA. Criticality accidents in Vinca, Yugoslavia, and Oak Ridge Tennessee. Am J Roentgenol Radium Ther Nucl Med 1965; 93: 56–74. 19. Billingham RE, Brent L, Medawar PB. “Actively acquired tolerance” of foreign cells. Nature 1953; 172: 603–6. 20. Billingham RE, Brent L. Quantitative studies on tissue transplantation immunity. IV. Induction of tolerance in newborn mice and studies on the phenomenon of runt disease. Philos Trans R Soc Lond B Biol Sci 1959; 242: 477.

21. Billingham RE. The biology of graft-versus-host reactions. In: The Harvey lectures. New York: Academic Press; 1966. pp. 21–78. 22. van Bekkum DW, de Vries MJ. Radiation chimaeras. London: Logos Press; 1967. 23. Uphoff DE. Genetic factors influencing irradiation protection by bone marrow. I. The F1 hybrid effect. J Natl Cancer Inst 1957; 19: 123–5. 24. Uphoff DE. Alteration of homograft reaction by A-methopterin in lethally irradiated mice treated with homologous marrow. Proc Soc Exp Biol Med 1958; 99: 651–3. 25. Lochte HL, Jr., Levy AS, Guenther D, Thomas ED, Ferrebee JW. Prevention of delayed foreign marrow reaction in lethally irradiated mice by early administration of methotrexate. Nature 1962; 196: 1110–11. 26. Alpen EL, Baum SJ. Modification of X-radiation lethality by autologous marrow infusion in dogs. Blood 1958; 13: 1168–75. 27. Mannick JA, Lochte HL, Jr., Ashley CA, Thomas ED, Ferrebee JW. Autografts of bone marrow in dogs after lethal total-body radiation. Blood 1960; 15: 255–66. 28. Barnes DWH, Loutit JF. The radiation recovery factor: Preservation by the Polge–Smith– Parkes technique. J Natl Cancer Inst 1955; 15: 901–5. 29. Cavins JA, Kasakura S, Thomas ED, Ferrebee JW. Recovery of lethally irradiated dogs following infusion of autologous marrow stored at low temperature in dimethyl-sulphoxide. Blood 1962; 20: 730–4. 30. Goodman JW, Hodgson GS. Evidence for stem cells in the peripheral blood of mice. Blood 1962; 19: 702–14.

A History of Allogeneic Hematopoietic Cell Transplantation

31. Cavins JA, Scheer SC, Thomas ED, Ferrebee JW. The recovery of lethally irradiated dogs given infusions of autologous leukocytes preserved at −80°C. Blood 1964; 23: 38–43. 32. Ferrebee JW, Lochte HL, Jr., Jaretzki A, III, Sahler OD, Thomas ED. Successful marrow homograft in the dog after radiation. Surgery 1958; 43: 516– 20. 33. Thomas ED, Ashley CA, Lochte HL, Jr., Jaretzki A, III, Sahler OD, Ferrebee JW. Homografts of bone marrow in dogs after lethal total-body radiation. Blood 1959; 14: 720–36. 34. Thomas ED, Collins JA, Herman EC, Jr., Ferrebee JW. Marrow transplants in lethally irradiated dogs given methotrexate. Blood 1962; 19: 217–28. 35. Crouch BG, van Putten LM, van Bekkum DW, de Vries MJ. Treatment of total-body X-irradiated monkeys with autologous and homologous bone marrow. J Natl Cancer Inst 1961; 27: 53–65. 36. Merritt CB, Darrow CC, II, Vaal L, Rogentine GN, Jr. Bone marrow transplantation in rhesus monkeys following irradiation. Modification of acute graftversus-host disease with antilymphocyte serum. Transplantation 1972; 14: 9–20. 37. van Putten LM, van Bekkum DW, de Vries MJ, Balner H. The effect of preceding blood transfusions on the fate of homologous bone marrow grafts in lethally irradiated monkeys. Blood 1967; 30: 749–57. 38. Storb R, Epstein RB, Rudolph RH, Thomas ED. The effect of prior transfusion on marrow grafts between histocompatible canine siblings. J Immunol 1970; 105: 627–33. 39. van der Waay D, Zimmerman WMTh. Problems in the sanitation of monkeys for whole-body irradiation experiments. Proceedings of the International Symposium on Bone Marrow Therapy and Chemical Protection in Irradiated Primates 1962. 1962. pp. 231–40. 40. Miescher PP, Fauconnet M. Mise en évidence de différents groupes leucocytaires chez l’homme. Schweiz Med Wochenschr 1954; 84: 597–9. 41. Dausset J. Iso-leuco-anticorps. Acta Haematol 1958; 20: 156–66.

42. van Rood JJ, Eernisse JG, van Leeuwen A. Leukocyte antibodies in sera from pregnant women. Nature 1958; 181: 1735–6. 43. Rapaport FT, Lawrence HS, Thomas L et al. Crossreactions to skin homografts in man. J Clin Invest 1962; 41: 2166–72. 44. Dausset J, Rapaport FT, Legrand L et al. Studies on transplantation antigens (HL-A) by means of skin grafts from 90 children onto their fathers [French]. Nouv Rev Fr Hematol 1969; 9: 215–29. 45. Epstein RB, Storb R, Ragde H, Thomas ED. Cytotoxic typing antisera for marrow grafting in littermate dogs. Transplantation 1968; 6: 45–58. 46. Storb R, Epstein RB, Bryant J, Radge H, Thomas ED. Marrow grafts by combined marrow and leukocyte infusions in unrelated dogs selected by histocompatibility typing. Transplantation 1968; 6: 587–93. 47. Storb R, Rudolph RH, Thomas ED. Marrow grafts between canine siblings matched by serotyping and mixed leukocyte culture. J Clin Invest 1971; 50: 1272–5. 48. Storb R, Epstein RB, Graham TC, Thomas ED. Methotrexate regimens for control of graft-versushost disease in dogs with allogeneic marrow grafts. Transplantation 1970; 9: 240–6. 49. Singal DP, Mickey MR, Terasaki PI. Serotyping for homotransplantation: XXIII analysis of kidney transplants from parental versus sibling donors. Transplantation 1969; 7: 246–58. 50. Gatti RA, Meuwissen HJ, Allen HD, Hong R, Good RA. Immunological reconstitution of sexlinked lymphopenic immunological deficiency. Lancet 1968; 2: 1366–9. 51. Bach FH, Albertini RJ, Joo P, Anderson JL, Bortin MM. Bone-marrow transplantation in a patient with the Wiskott–Aldrich syndrome. Lancet 1968; 2: 1364–6. 52. deKoning J, van Bekkum DW, Dicke KA, Dooren LJ, Rádl J, Van Rood JJ. Transplantation of bonemarrow cells and fetal thymus in an infant with lymphopenic immunological deficiency. Lancet 1969; 1: 1223–7.

7

53. Bortin MM, Bach FH, van Bekkum DW, Good RA, van Rood JJ. 25th anniversary of the first successful allogeneic bone marrow transplants. Bone Marrow Transplant 1994; 14: 211–12. 54. Buckner CD, Epstein RB, Rudolph RH, Clift RA, Storb R, Thomas ED. Allogeneic marrow engraftment following whole body irradiation in a patient with leukemia. Blood 1970; 35: 741–50. 55. Thomas ED, Storb R, Fefer A et al. Aplastic anaemia treated by marrow transplantation. Lancet 1972; 1: 284–9. 56. Santos GW, Sensenbrenner LL, Burke PJ et al. Marrow transplantation in man following cyclophosphamide. Transplant Proc 1971; 3: 400–4. 57. Thomas ED, Storb R, Clift RA et al. Bone-marrow transplantation. I and II. N Engl J Med 1975; 292: 832–43, 895–902. 58. Thomas ED, Buckner CD, Banaji M et al. One hundred patients with acute leukemia treated by chemotherapy, total body irradiation, and allogeneic marrow transplantation. Blood 1977; 49: 511–33. 59. Thomas ED, Flournoy N, Buckner CD et al. Cure of leukemia by marrow transplantation. Leuk Res 1977; 1: 67–70. 60. Thomas ED, Buckner CD, Clift RA et al. Marrow transplantation for acute nonlymphoblastic leukemia in first remission. N Engl J Med 1979; 301: 597–9. 61. Blume KG, Beutler E. Allogeneic bone marrow transplantation for acute leukemia [Letter]. JAMA 1979; 241: 1686. 62. Mathé G, Amiel JL, Schwarzenberg L, Catton A, Schneider M. Adoptive immunotherapy of acute leukemia: Experimental and clinical results. Cancer Res 1965; 25: 1525–31. 63. Weiden PL, Flournoy N, Thomas ED et al. Antileukemic effect of graft-versus-host disease in human recipients of allogeneic-marrow grafts. N Engl J Med 1979; 300: 1068–73. 64. Hansen JA, Clift RA, Thomas ED, Buckner CD, Storb R, Giblett ER. Transplantation of marrow from an unrelated donor to a patient with acute leukemia. N Engl J Med 1980; 303: 565–7.

2

James O. Armitage

The History of Autologous Hematopoietic Cell Transplantation

Introduction The procedure known variously as autologous bone marrow transplantation, autologous peripheral blood cell transplantation, autologous stem cell transplantation, and autologous hematopoietic cell transplantation involves the intravenous infusion of a patient’s own hematopoietic stem cells to rescue the patient from severe bone marrow injury. Usually, the bone marrow injury has been caused by high-dose chemotherapy and/or radiotherapy as part of a treatment for cancer. Usually, but not always, the patient’s hematopoietic stem cells have been cryopreserved and thawed before reinfusion. Autologous hematopoietic cell transplantation tests the hypothesis that the resistance of cancer cells to chemotherapy and/or radiotherapy can be relative rather than absolute, and that the resistance might be overcome by dose escalation. The importance of dose in cancer therapy has been known for some time [1]. While it has been clear that arbitrary dose reductions can reduce the chance of a good outcome with potentially curative cancer chemotherapy, autologous hematopoietic cell transplantation tests the hypothesis that dose escalation to a point that would cause irreversible myelotoxicity (but tolerable because of replacing myeloid function with the infused autologous hematopoietic stem cells) might be able to cure otherwise incurable patients. This hypothesis is illustrated in Fig. 2.1. Both chemotherapeutic agents and total body radiotherapy injure organs other than the bone marrow. Thus, for effective autologous hematopoietic cell transplantation, drugs whose initial life-threatening toxicity is myeloid injury need to be chosen, and radiation doses must be kept below that which would cause lethal pulmonary, gastrointestinal or central nervous system toxicity.

Early attempts at autologous hematopoietic cell transplantation The use of bone marrow to treat cancer has been considered by healthcare providers for a very long time. The first reports of the use of bone marrow as a treatment for cancer appeared in the 1890s [2]. The theoretical basis for the use of hematopoietic stem cells to rescue patients from irreversible myeloid injury was provided by canine studies [3,4] showing that dogs could survive otherwise lethal total body radiotherapy after the infusion of their own autologous hematopoietic cells. A report

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

8

of the first attempts at autologous hematopoietic cell transplantation in humans appeared in 1958 [5] and reported two patients, one suffering from teratocarcinoma and one from renal cell carcinoma, who received wide-field “intensive” radiotherapy followed by the infusion of glycerolpreserved hematopoietic cells derived from the bone marrow. A subsequent report by the same investigators [6] described four patients managed in a similar way who seemed to have hematopoietic recovery after infusion of the autologous bone marrow cells. McFarland et al. in 1959 described five patients with non-Hodgkin’s lymphoma or Hodgkin’s disease who received high doses of mechlorethamine followed by the infusion of noncryopreserved autologous bone marrow cells [7]. Three of the five patients had hematopoietic recovery, but there was only transient improvement in clinical status in a few. Clifford et al., working in Kenya in 1961, reported three patients with lymphoma or anaplastic nasopharyngeal carcinoma who received highdose mechlorethamine followed by an infusion of glycerol-preserved bone marrow cells [8]. All three patients demonstrated tumor regression and clinical improvement in addition to hematopoietic recovery. In 1962, Pegg et al. described 29 patients with cancer who received intensive, large-volume radiotherapy or chemotherapy with one of several alkylating agents followed by autologous bone marrow cell infusion. Most patients showed hematopoietic recovery, and a few had significant tumor regression and clinical improvement [9]. In 1962, Kurnick described 82 patients (presumably including some from his previous reports) who received high-dose chemotherapy and/or radiotherapy for a variety of malignancies including germ-cell tumors, lymphomas, leukemias, carcinomas, and sarcomas. The report expanded his previous observations and described hematopoietic recovery in most patients with, apparently, modest clinical benefit [10]. In many of these early reports, the intensity of chemotherapy would not necessarily have caused extremely prolonged marrow suppression, and it is difficult to be certain of the contribution of the reinfused hematopoietic cells. In the case of cryopreserved cells, it is not certain that viable cells were reinfused. The first patients apparently cured of otherwise incurable malignancy using high-dose therapy and autologous hematopoietic cell transplantation were reported by investigators from the National Cancer Institute in 1978. The authors reported 22 patients with resistant malignant lymphoma who were treated with high-dose chemotherapy utilizing carmustine, cytarabine, cyclophosphamide, and thioguanine [11]. Twelve patients received an infusion of their own cryopreserved autologous bone marrow, and 10 patients served as controls without reinfused cryopreserved marrow. Recovery of peripheral blood counts occurred with a median of 13 days in the patients who received autologous

The History of Autologous Hematopoietic Cell Transplantation

Increasing dose

3

Death secondary to other organ toxicity 2

1

Death secondary to marrow toxicity

Dose level necessary to cure three hypothetical patients

Fig. 2.1 Potential for cure with autologous hematopoietic stem-cell transplantation. The patient represented by column 1 could be cured with doses that would not cause permanent marrow injury. The patient represented by column 2 could be uniquely cured by doses requiring autotransplantation. The patient represented by column 3 would die of nonhematologic toxicity with the doses necessary to cure the cancer.

hematopoietic cells, and at a median of 23 days in those patients who did not receive marrow infusion. In a separate report, the same authors described 14 patients with Burkitt’s lymphoma resistant to conventional therapy who received the same high-dose chemotherapy regimen and autologous hematopoietic cells when these were available. Eight of the 14 patients received autologous hematopoietic cell transplantation; three of those were apparently cured, whereas none of the six patients who did not receive autologous hematopoietic cell transplantation had a long-term, disease-free survival [12]. The success in these patients ushered in the modern era of autologous hematopoietic cell transplantation and was made possible by the reproducible freezing and thawing of autologous hematopoietic cells using dimethylsulfoxide, resulting in viable and transplantable stem cells after thawing. This was initially reported by Cavins et al. [13] in dogs, and the work by Appelbaum et al. at the National Cancer Institute [12] demonstrated that the same approach worked in humans. Although almost all autologous hematopoietic cell transplantations are carried out with cryopreserved cells, some investigators have utilized noncryopreserved hematopoietic progenitor cells for this procedure. For this to be practical, the treatment has to be completed very rapidly, and the rescue product has been reinfused in most studies within 48 hours. Early studies in the United States and Italy showed the feasibility of this approach [14–16]. Carella subsequently reported 10 patients with Hodgkin’s disease and non-Hodgkin’s lymphoma who received highdose therapy with carmustine or vinblastine, cyclophosphamide, and carmustine. Seven of 10 patients with resistant, refractory disease achieved remissions, and hematopoietic recovery occurred in nine of the 10 patients [17]. A subsequent report by the same authors described 13 patients with Hodgkin’s disease and showed similar results, with all but one of the patients having hematopoietic recovery [18]. However, despite occasional other reports, this approach has been rarely used, with almost all patients receiving cryopreserved products to re-establish hematopoiesis.

Autologous hematopoietic cell transplantation utilizing peripheral blood-derived cells The existence of circulating hematopoietic stem cells capable of reestablishing hematopoiesis was established in mice in 1962 [19] and subsequently in other species [20,21]. Initial attempts to utilize circulat-

9

ing autologous hematopoietic stem cells for autologous transplantation in humans failed [22,23], probably related to the infusion of an inadequate number of hematopoietic stem cells. Although an adequate number of hematopoietic stem cells for transplantation can be achieved utilizing multiple apheresis procedures [24], the practical application of this treatment required methods to increase the number of circulating hematopoietic stem cells. This increase in circulating hematopoietic stem cells can be achieved utilizing endotoxin [25] and recovery from chemotherapy [26,27]. However, it required the availability of hematopoietic growth factors, as well as the demonstration that their administration could dramatically increase the number of circulating hematopoietic progenitor cells and decrease the number of apheresis procedures required for an adequate dose [28] to make this approach the standard method for autologous transplantation. The hematopoietic progenitor cells collected after the administration of hematopoietic growth factors altered the kinetics of recovery of hematopoiesis. Both granulocyte colony-stimulating factor (G-CSF) [29] and granulocyte–macrophage colony-stimulating factor (GM-CSF) [30] were found to expand the number of circulating hematopoietic progenitor cells. Sheridan et al. [31] showed that hematopoietic stem cells collected after the administration of G-CSF not only shortened neutrophil recovery time, but also dramatically shortened platelet recovery. This does not appear to be the case using autologous peripheral blood stem cells collected after “priming” with GM-CSF [24,32]. Other hematopoietic growth factors, including erythropoietin [33], for mobilizing circulating progenitor cells have been used, but once again platelet recovery was delayed. However, whatever method of mobilization of circulating hematopoietic progenitor cells is utilized, extensive preceding therapy decreases the ease of collection of an adequate number of hematopoietic progenitor cells and delays recovery of hematopoiesis [34,35]. Peripheral blood-derived hematopoietic progenitor cells and bone marrow-derived hematopoietic progenitor cells have been compared in two randomized trials [36,37]. Both studies showed that autologous peripheral blood hematopoietic progenitor cells were associated with more rapid engraftment and had a similar treatment outcome. The relative ease of collecting peripheral blood-derived hematopoietic progenitor cells has made this the most frequent approach utilized for autologous hematopoietic cell transplantation in the world today.

In vitro treatment of autologous hematopoietic blood or marrow collections The risk of reinfusing cancer cells during autologous hematopoietic cell transplantation has been understood to be a risk for decades. The presence of tumor cells in rescue products has been shown using immunohistochemistry [38], bone marrow culture [39], and the polymerase chain reaction [40,41]. Some studies have found a poorer outcome in patients in whom residual disease in the graft could be identified [41,42], whereas others have questioned the significance of this finding [40]. The description of patients with early pulmonary recurrence after autologous bone marrow infusion in patients with lymphoma suggested that reinfusion of tumor cells may have been the cause [43,44]. Schultz et al., however, used a mathematical model to suggest that, in leukemia, reinfusion of tumor cells was unlikely to be the cause of relapse after autologous hematopoietic cell transplantation [45]. Brenner et al., using gene-marking techniques, showed that in some patients who relapsed after autologous hematopoietic cell transplantation for acute myeloid leukemia, the tumor cells at relapse were derived from the reinfusion product [46]. Attempts at removing tumor cells have used multiple approaches. Nadler et al. first reported the use of antibody treatment of autologous hematopoietic cells in an attempt to eliminate tumor cells [47]. De Fabritiis et al. showed that clonogenic Burkitt’s lymphoma cells could

Chapter 2

be eliminated from human bone marrow using a combination of monoclonal antibodies, compliment, and 4-hydroperoxycyclophosphamide [48]. Hagenbeek and Martens suggested that cryopreservation of the hematopoietic cells was more toxic to leukemic blasts than normal progenitor cells, and that freezing might reduce the risk of reinfusing of clonogenic tumor cells [49]. Attempts to reduce the risk of reinfusion of tumor cells has also been addressed by attempts to concentrate CD34positive hematopoietic stem cells and “leave behind” the tumor cells [50,51]. The use of antibodies directed against some tumor cells administered systemically at the time of peripheral blood cell collection might also reduce the number of tumor cells being collected.

100 Percent surviving disease-free

10

80

60 Sensitive relapse

40

Resistant relapse

20 No remission

0

Diseases treated by autologous hematopoietic cell transplantation Non-Hodgkin’s lymphoma The first malignancy successfully treated by high-dose therapy and autologous hematopoietic cell transplantation was non-Hodgkin’s lymphoma [11] in the late 1970s. In the early 1980s, as techniques of cryopreservation of hematopoietic cells improved, numerous singlecenter publications appeared demonstrating the curative potential of high-dose therapy and autologous hematopoietic cell transplantation in patients who had failed standard chemotherapy for aggressive nonHodgkin’s lymphoma [52–57]. Several of those centers pooled data and reported on the results on autologous hematopoietic cell transplantation in 100 patients with relapsed or refractory aggressive non-Hodgkin’s lymphoma [58]. Observations from that paper have affected the way in which autologous hematopoietic cell transplantation has been applied to patients with lymphoma and other diseases. Patients who had once achieved a complete remission, relapsed, and responded again to salvage chemotherapy before undergoing autologous hematopoietic cell transplantation had a higher complete remission rate following the autotransplant and were more likely to remain in remission than those who had relapsed from complete remission but were no longer chemotherapy sensitive. Both groups had a better result than patients who were primarily refractory to treatment and never achieved a complete remission (Fig. 2.2). These observations were tested in a phase II trial of 50 patients with aggressive non-Hodgkin’s lymphoma who relapsed from complete remission. All patients received dexamethasone, high-dose cytarabine, and cisplatin to test for chemotherapy sensitivity. The actuarial 2-year event-free survival for patients who were chemotherapy sensitive and underwent autotransplantation was 40% [59], confirming the retrospective study and forming the basis for a prospective trial comparing autotransplantation with standard-dose chemotherapy [60]. In that subsequent trial, 215 patients were treated and 109 responded to standard-dose salvage chemotherapy. They were then randomly assigned to continuing standard-dose chemotherapy followed by radiation or autologous hematopoietic cell transplantation. At 5 years, the event-free survival was 46% in the group undergoing transplantation and 12% in the standard therapy group – a difference that was highly significant. In addition, overall survival significantly favored the transplant (53% versus 32%), despite the fact that patients from the standard chemotherapy arm were able to be transplanted at a later time. This study established high-dose therapy and autologous hematopoietic cell transplantation as the standard treatment for patients with relapsed aggressive non-Hodgkin’s lymphoma. The history of the use of high-dose therapy and autologous hematopoietic cell transplantation in other types of non-Hodgkin’s lymphoma has not led to the same consensus as for the common aggressive nonHodgkin’s lymphomas. A number of single centers had reported encour-

1

2

3

4

5

6 Years

7

8

9

10

11

12

Fig. 2.2 Results of a retrospective study of 100 patients with diffuse, aggressive lymphoma showing that patients with chemotherapy-sensitive relapse do better with autologous transplantation than those who are chemotherapy resistant. Both groups have a better outcome than patients never achieving an initial complete remission [58].

aging results with the use of high-dose therapy and autologous hematopoietic cell transplantation in patients with relapsed follicular lymphoma [61–64]. One prospective trial has been completed and showed an advantage in disease-free and overall survival to patients undergoing hematopoietic stem cell transplantation [65]. However, the application of autologous hematopoietic cell transplantation in patients with follicular lymphoma has been much more controversial, and the development of a consensus has been much slower. Patients with Burkitt’s lymphoma were the first to be treated successfully with high-dose therapy and autologous hematopoietic cell transplantation [11]. The advent of high-dose chemotherapy regimens not requiring hematopoietic stem cell support has been so successful in curing patients with Burkitt’s lymphoma that this treatment is rarely utilized today. Several early reports of autologous hematopoietic cell transplantation in the treatment of lymphoblastic lymphoma [66,67] led to an international randomized trial [68]. However, the results did not reach statistical significance, and the use of autotransplantation in lymphoblastic lymphoma has remained controversial. Hodgkin’s disease High-dose therapy and autologous hematopoietic cell transplantation as a successful treatment for patients with relapsed Hodgkin’s disease was first reported in the 1980s [18,69–72]. By the early 1990s. Hodgkin’s disease had become one of the most frequent diagnoses for which autologous transplantation was applied. Subsequently, the British National Lymphoma Investigation carried out a randomized trial of high-dose therapy and autologous hematopoietic cell transplantation for patients with relapsed or refractory Hodgkin’s disease, the control arm being the same drugs used in the high-dose regimen but at lower doses [73]. Another randomized trial carried out by the German Hodgkin’s Lymphoma Study Group compared autologous hematopoietic stem cell transplantation with further conventional therapy in patients with chemotherapy-sensitive relapsed Hodgkin’s disease [74]. Both studies showed an improvement in failure-free survival but no difference in overall survival. Multiple myeloma Although currently one of the diseases most frequently treated using high-dose therapy and autologous transplantation, the fact that visible

The History of Autologous Hematopoietic Cell Transplantation

tumor cells were being reintroduced with the stem cell infusion probably reduced early enthusiasm for this treatment. Multiple myeloma has been treated with alkylating agents for several decades. McElwain and Powles showed in the early 1980s that high doses of melphalan could produce responses in patients refractory to the drug given orally at standard doses [75], but with prolonged and severe myelosuppression. In 1988, Barlogie et al. reproduced the high response rate with high-dose melphalan and showed that the intravenous infusion of autologous hematopoietic stem cells could reduce the duration of myelosuppression [76]. Subsequent studies tested the value of ex vivo treatment of the rescue product to attempt to eliminate tumor cells [77,78], and peripheral blood-derived hematopoietic cells [79,80]. The combination of blood- and marrow-derived hematopoietic cells [81] and the use of CD34-positive enriched hematopoietic stem cells [82] were both tested without any clear advantage being seen to either approach. These studies reflected the increasing interest in autotransplantation for multiple myeloma. However, the widespread acceptance of this treatment followed the publication of the first randomized trial demonstrating the superiority of high-dose therapy and autologous transplantation over standard chemotherapy [83].

Acute leukemia Perhaps surprisingly, autologous hematopoietic cell transplantation has been extensively pursued in the treatment of patients with acute leukemia. This was originally based on the lack of matched, allogeneic donors for most patients, the apparent responsiveness of the leukemia to very high doses of therapy, and the hope that small numbers of reinfused leukemia cells in patients in remission, or the infusion of no or very low numbers of cells after ex vivo treatment of the reinfusion product, might lead to an improved treatment outcome. This treatment was particularly popular in Europe. The first report of autologous hematopoietic stem cell transplantation in acute myeloid leukemia appeared in the late 1970s [84]. Treatment carried out for patients in relapse yielded remission in most, but was usually followed by rapid relapse. The use of ex vivo marrow treatment of the autograft to reduce leukemia cell reinfusion [85,86] and the treatment of patients in remission improved the duration of remission. Similar or somewhat less good results were seen with early attempts of autologous hematopoietic cell transplantation in acute lymphoblastic leukemia [87]. The early prospective trials of autologous hematopoietic cell transplantation in acute myeloid leukemia [88] and acute lymphoid leukemia [89] showed a higher relapse rate with autologous transplantation compared with allogeneic transplantation, but no clear advantage over intensive treatments not involving hematopoietic cell transplantation.

11

remissions but most patients relapsing [94,95]. No prospective randomized trials have been completed.

Carcinomas and sarcomas Probably the carcinoma most frequently treated using high-dose therapy and autologous hematopoietic cell transplantation has been breast cancer. The rationale was a dose response in metastatic breast cancer [96] and the ability to collect autologous cells apparently free of tumor. Early attempts with high-dose therapy and autologous hematopoietic cell transplantation in breast cancer yielded primarily partial responses [97]. There was some evidence of a higher response rate with high-dose alkylating agents as opposed to other cytotoxic agents [98]. A number of high-dose combination chemotherapy regimens were developed in phase II trials, and several randomized trials were subsequently carried out [99]. In the United States, a high level of antipathy developed between physicians using high-dose therapy and autologous hematopoietic cell transplantation, and insurance companies that were reticent to fund this procedure. The borderline or negative results of most randomized trials and the revelation of fraud in one positive trial [100,101] dramatically reduced the frequency of utilization of this treatment approach. High-dose therapy and autologous hematopoietic cell transplantation has been studied in numerous carcinomas and sarcomas, generally with discouraging results. These studies include the treatment of patients with malignant glioma [102,103], malignant melanoma [104–106], neuroblastoma [107], ovarian cancer [108], lung cancer [109–112], and germcell tumors [113]. Of these diseases, only the treatment of germ-cell tumors has continued as a standard therapy [114].

Nonmalignant disorders Allogeneic bone marrow transplantation has been known for some time to have the potential to cure autoimmune disease [115]. It was much less clear that autologous transplantation could be beneficial since cells of the original immune system were being reinfused [116]. Two patients who underwent autologous bone marrow transplantation for lymphoma seemed to show an improvement in their markers for an autoimmune disorder (myasthenia gravis in one case and systemic lupus in another) [117,118]. These results led to attempts at autologous transplantation aimed at treating autoimmune diseases. The first studies focused on rheumatoid arthritis [119–121]. Studies in this and other autoimmune diseases continue.

Conclusion Chronic leukemia Autologous hematopoietic cell transplantation has been utilized to treat patients with chronic myeloid and with chronic lymphocytic leukemia. The rationale for autotransplantation in chronic myeloid leukemia was the existence of surviving normal hematopoietic stem cells [90] and the occasional durable remissions seen in patients who received toxic doses of busulfan [91]. Treatment of patients with chronic myeloid leukemia that had transformed to the acute phase using autologous transplantation of cells stored during the chronic phase of the disease yielded remissions but rapid relapse [92]. Treatment of patients in chronic phase occasionally produced durable remissions [93]. Autologous hematopoietic cell transplantation in chronic lymphocytic leukemia has also been tested. However, early results showed frequent

The history of autologous hematopoietic cell transplantation is a clear example of how clinical advances reflect advances in basic science and come as the result of innovative, but sometimes painstaking, clinical studies. Autologous hematopoietic cell transplantation was developed to test the hypothesis that dose escalation could affect cure in patients who had otherwise incurable malignancies. This turned out to be true for groups of patients with lymphoma, and the treatment continues to be widely utilized today. Studies in most patients with carcinoma and sarcoma showed minimal benefit and, with the exception of germ-cell tumors and certain pediatric malignancies, the treatment is rarely utilized. Although not curative, prolonged survival in patients with multiple myeloma has made the treatment widely utilized, and the results of autologous hematopoietic cell transplantation in nonmalignant disease have been sufficiently interesting to lead to ongoing studies.

12

Chapter 2

References 1. Frei E, 3rd, Canellos GP. Dose: A critical factor in cancer chemotherapy. Am J Med 1980; 69: 585–94. 2. Quine WE. The remedial application of bone marrow. JAMA 1896; 26: 1012–13. 3. Alpen EL, Baum SJ. Modification of X-radiation lethality by autologous marrow infusion in dogs. Blood 1958; 13: 1168–75. 4. Mannick JA, Lochte HL, Jr., Ashley CA, Thomas ED, Ferrebee JW. Autografts of bone marrow in dogs after lethal total-body radiation. Blood 1960; 15: 255–66. 5. Kurnick NB, Montano A, Gerdes JC, Feder BH. Preliminary observations on the treatment of postirradiation hematopoietic depression in man by the infusion of stored autogenous bone marrow. Ann Intern Med 1958; 49: 973–86. 6. Kurnick NB, Feder BH, Montano A, Gerdes JC, Nakamura R. Some observations on the treatment of postirradiation hematopoietic depression in man by the infusion of stored autogenous bone marrow. Ann Intern Med 1959; 51: 1204–19. 7. McFarland W, Granville NB, Dameshek W. Autologous bone marrow infusion as an adjunct in therapy of malignant disease. Blood 1959; 14: 503–21. 8. Clifford P, Clift RA, Duff JK. Nitrogen-mustard therapy combined with autologous marrow infusion. Lancet 1961; 1: 687–90. 9. Pegg DE, Humble JG, Newton KA. The clinical application of bone marrow grafting. Br J Cancer 1962; 16: 417–35. 10. Kurnick NB. Autologous and isologous bone marrow storage and infusion in the treatment of myelo-suppression. Transfusion 1962; 2: 178– 87. 11. Appelbaum FR, Herzig GP, Ziegler JL, Graw RG, Levine AS, Deisseroth AB. Successful engraftment of cryopreserved autologous bone marrow in patients with malignant lymphoma. Blood 1978; 52: 85–95. 12. Appelbaum FR, Deisseroth AB, Graw RG, Jr. et al. Prolonged complete remission following high dose chemotherapy of Burkitt’s lymphoma in relapse. Cancer 1978; 41: 1059–63. 13. Cavins JA, Kasakura S, Thomas ED, Ferrebee JW. Recovery of lethally irradiated dogs following infusion of autologous marrow stored at low temperature in dimethylsulphoxide. Blood 1962; 20: 730–4. 14. Robinson WA, Hartmann DW, Mangalik A, Morton N, Joshi JH. Autologous nonfrozen bone marrow transplantation after intensive chemotherapy: A pilot study. Acta Haematol 1981; 66: 145–53. 15. Carella AM, Giordano D, Santini G et al. High dose BCNU followed by autologous bone marrow infusion in glioblastoma multiforme. Tumori 1981; 67: 473–5. 16. Carella AM, Santini G, Frassoni F et al. Autologous bone marrow transplantation without cryopreservation after megadoses of oncolytic chemotherapy. Pilot study in 18 cases. Haematologica 1983; 68: 620–37. 17. Carella AM, Santini G, Giordano D et al. Highdose chemotherapy and non-frozen autologous bone marrow transplantation in relapsed advanced lymphomas or those resistant to conventional chemotherapy. Cancer 1984; 54: 2836–9.

18. Carella AM, Santini G, Santoro A et al. Massive chemotherapy with non-frozen autologous bone marrow transplantation in 13 cases of refractory Hodgkin’s disease. Eur J Cancer Clin Oncol 1985; 21: 607–13. 19. Goodman JW, Hodgson GS. Evidence for stem cells in the peripheral blood of mice. Blood 1962; 19: 702–14. 20. Storb R, Graham TC, Epstein RB, Sale GE, Thomas ED. Demonstration of hemopoietic stem cells in the peripheral blood of baboons by cross circulation. Blood 1977; 50: 537–42. 21. Appelbaum FR. Hemopoietic reconstitution following autologous bone marrow and peripheral blood mononuclear cell infusions. Exp Hematol 1979; 7 Suppl 5: 7–11. 22. Hershko C, Gale RP, Ho WG, Cline MJ. Cure of aplastic anaemia in paroxysmal nocturnal haemoglobinuria by marrow transfusion from identical twin: Failure of peripheral-leucocyte transfusion to correct marrow aplasia. Lancet 1979; 1: 945–7. 23. Abrams RA, Glaubiger D, Appelbaum FR, Deisseroth AB. Result of attempted hematopoietic reconstitution using isologous, peripheral blood mononuclear cells: A case report. Blood 1980; 56: 516–20. 24. Kessinger A, Armitage JO, Landmark JD, Weisenburger DD. Reconstitution of human hematopoietic function with autologous cryopreserved circulating stem cells. Exp Hematol 1986; 14: 192–6. 25. Cline MJ, Golde DW. Mobilization of hematopoietic stem cells (CFU-C) into the peripheral blood of man by endotoxin. Exp Hematol 1977; 5: 186– 90. 26. Richman CM, Weiner RS, Yankee RA. Increase in circulating stem cells following chemotherapy in man. Blood 1976; 47: 1031–9. 27. Abrams RA, McCormack K, Bowles C, Deisseroth AB. Cyclophosphamide treatment expands the circulating hematopoietic stem cell pool in dogs. J Clin Invest 1981; 67: 1392–9. 28. Gianni AM, Siena S, Bregni M et al. Granulocytemacrophage colony-stimulating factor to harvest circulating haemopoietic stem cells for autotransplantation. Lancet 1989; 2: 580–5. 29. Duhrsen U, Villeval JL, Boyd J, Kannourakis G, Morstyn G, Metcalf D. Effects of recombinant human granulocyte colony-stimulating factor on hematopoietic progenitor cells in cancer patients. Blood 1988; 72: 2074–81. 30. Socinski MA, Cannistra SA, Elias A, Antman KH, Schnipper L, Griffin JD. Granulocytemacrophage colony stimulating factor expands the circulating haemopoietic progenitor cell compartment in man. Lancet 1988; 1: 1194–8. 31. Sheridan WP, Begley CG, Juttner CA et al. Effect of peripheral-blood progenitor cells mobilised by filgrastim (G- CSF) on platelet recovery after high-dose chemotherapy [see comments]. Lancet 1992; 339: 640–4. 32. Haas R, Ho AD, Bredthauer U et al. Successful autologous transplantation of blood stem cells mobilized with recombinant human granulocytemacrophage colony-stimulating factor. Exp Hematol 1990; 18: 94–8. 33. Kessinger A, Bishop MR, Jackson JD et al. Erythropoietin for mobilization of circulating progeni-

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

tor cells in patients with previously treated relapsed malignancies. Exp Hematol 1995; 23: 609–12. Bolwell BJ, Fishleder A, Andresen SW et al. GCSF primed peripheral blood progenitor cells in autologous bone marrow transplantation: Parameters affecting bone marrow engraftment. Bone Marrow Transplant 1993; 12: 609–14. Haas R, Mohle R, Fruhauf S et al. Patient characteristics associated with successful mobilizing and autografting of peripheral blood progenitor cells in malignant lymphoma. Blood 1994; 83: 3787– 94. Kanteti R, Miller K, McCann J et al. Randomized trial of peripheral blood progenitor cell vs bone marrow as hematopoietic support for high-dose chemotherapy in patients with non-Hodgkin’s lymphoma and Hodgkin’s disease: A clinical and molecular analysis. Bone Marrow Transplant 1999; 24: 473–81. Vose JM, Sharp G, Chan WC et al. Autologous transplantation for aggressive non-Hodgkin’s lymphoma: Results of a randomized trial evaluating graft source and minimal residual disease. J Clin Oncol 2002; 20: 2344–52. Shpall EJ, Jones RB, Bearman SI et al. Transplantation of enriched CD34-positive autologous marrow into breast cancer patients following highdose chemotherapy: Influence of CD34-positive peripheral-blood progenitors and growth factors on engraftment. J Clin Oncol 1994; 12: 28–36. Joshi SS, Kessinger A, Mann SL et al. Detection of malignant cells in histologically normal bone marrow using culture techniques. Bone Marrow Transplant 1987; 1: 303–10. Johnson PW, Price CG, Smith T et al. Detection of cells bearing the t(14; 18) translocation following myeloablative treatment and autologous bone marrow transplantation for follicular lymphoma. J Clin Oncol 1994; 12: 798–805. Gribben JG, Freedman AS, Neuberg D et al. Immunologic purging of marrow assessed by PCR before autologous bone marrow transplantation for B-cell lymphoma [see comments]. N Engl J Med 1991; 325: 1525–33. Sharp JG, Joshi SS, Armitage JO et al. Significance of detection of occult non-Hodgkin’s lymphoma in histologically uninvolved bone marrow by a culture technique. Blood 1992; 79: 1074– 80. Glorieux P, Bouffet E, Philip I et al. Metastatic interstitial pneumonitis after autologous bone marrow transplantation. A consequence of reinjection of malignant cells? Cancer 1986; 58: 2136–9. Rossetti F, Deeg HJ, Hackman RC. Early pulmonary recurrence of non-Hodgkin’s lymphoma after autologous marrow transplantation: Evidence for reinfusion of lymphoma cells? Bone Marrow Transplant 1995; 15: 429–32. Schultz FW, Martens AC, Hagenbeek A. The contribution of residual leukemic cells in the graft to leukemia relapse after autologous bone marrow transplantation: mathematical considerations. Leukemia 1989; 3: 530–4. Brenner MK, Rill DR, Moen RC et al. Genemarking to trace origin of relapse after autologous bone-marrow transplantation. Lancet 1993; 341: 85–6.

The History of Autologous Hematopoietic Cell Transplantation 47. Nadler LM, Takvorian T, Botnick L et al. Anti-B1 monoclonal antibody and complement treatment in autologous bone-marrow transplantation for relapsed B-cell non-Hodgkin’s lymphoma. Lancet 1984; 2: 427–31. 48. De Fabritiis P, Bregni M, Lipton J et al. Elimination of clonogenic Burkitt’s lymphoma cells from human bone marrow using 4-hydroperoxycyclophosphamide in combination with monoclonal antibodies and complement. Blood 1985; 65: 1064–70. 49. Hagenbeek A, Martens AC. Cryopreservation of autologous marrow grafts in acute leukemia: Survival of in vivo clonogenic leukemic cells and normal hemopoietic stem cells. Leukemia 1989; 3: 535–7. 50. Gorin NC, Lopez M, Laporte JP et al. Preparation and successful engraftment of purified CD34+ bone marrow progenitor cells in patients with nonHodgkin’s lymphoma. Blood 1995; 85: 1647–54. 51. Berenson RJ, Bensinger WI, Hill RS et al. Engraftment after infusion of CD34+ marrow cells in patients with breast cancer or neuroblastoma. Blood 1991; 77: 1717–22. 52. Philip T, Biron P, Herve P et al. Massive BACT chemotherapy with autologous bone marrow transplantation in 17 cases of non-Hodgkin’s malignant lymphoma with a very bad prognosis. Eur J Cancer Clin Oncol 1983; 19: 1371–9. 53. Gorin NC, Najman A, Douay L et al. Autologous bone marrow transplantation in the treatment of poor prognosis non-Hodgkin’s lymphomas. Eur J Cancer Clin Oncol 1984; 20: 217–25. 54. Appelbaum FR, Sullivan KM, Thomas ED et al. Experimental hematology today. New York: Springer-Verlag; 1985. 55. Verdonck LF, Dekker AW, van Kempen ML et al. Intensive cytotoxic therapy followed by autologous bone marrow transplantation for nonHodgkin’s lymphoma of high-grade malignancy. Blood 1985; 65: 984–9. 56. Armitage JO, Jagannath S, Spitzer G et al. High dose therapy and autologous marrow transplantation as salvage treatment for patients with diffuse large cell lymphoma. Eur J Cancer Clin Oncol 1986; 22: 871–7. 57. Armitage JO, Gingrich RD, Klassen LW et al. Trial of high-dose cytarabine, cyclophosphamide, total-body irradiation, and autologous marrow transplantation for refractory lymphoma. Cancer Treat Rep 1986; 70: 871–5. 58. Philip T, Armitage JO, Spitzer G et al. High-dose therapy and autologous bone marrow transplantation after failure of conventional chemotherapy in adults with intermediate-grade or high-grade nonHodgkin’s lymphoma. N Engl J Med 1987; 316: 1493–8. 59. Philip T, Chauvin F, Armitage J et al. Parma international protocol: Pilot study of DHAP followed by involved-field radiotherapy and BEAC with autologous bone marrow transplantation. Blood 1991; 77: 1587–92. 60. Philip T, Guglielmi C, Hagenbeek A et al. Autologous bone marrow transplantation as compared with salvage chemotherapy in relapses of chemotherapy-sensitive non-Hodgkin’s lymphoma [see comments]. N Engl J Med 1995; 333: 1540–5. 61. Liang R, Chen F, Lee CK et al. Autologous bone marrow transplantation for primary nasal T/NK cell lymphoma. Bone Marrow Transplant 1997; 19: 91–3.

62. Colombat P, Binet C, Linassier C et al. High dose chemotherapy with autologous marrow transplantation in follicular lymphomas. Leuk Lymphoma 1992; 7 Suppl: 3–6. 63. Rohatiner AZ, Johnson PW, Price CG et al. Myeloablative therapy with autologous bone marrow transplantation as consolidation therapy for recurrent follicular lymphoma. J Clin Oncol 1994; 12: 1177–84. 64. Schouten HC, Bierman PJ, Vaughan WP et al. Autologous bone marrow transplantation in follicular non-Hodgkin’s lymphoma before and after histologic transformation. Blood 1989; 74: 2579– 84. 65. Schouten HC, Qian W, Kvaloy S et al. High-dose therapy improves progression-free survival and survival in relapsed follicular non-Hodgkin’s lymphoma: Results from the randomized European CUP trial. J Clin Oncol 2003; 21: 3918– 27. 66. Milpied N, Ifrah N, Kuentz M et al. Bone marrow transplantation for adult poor prognosis lymphoblastic lymphoma in first complete remission. Br J Haematol 1989; 73: 82–7. 67. Santini G, Coser P, Chisesi T et al. Autologous bone marrow transplantation for advanced stage adult lymphoblastic lymphoma in first complete remission. A pilot study of the non-Hodgkin’s Lymphoma Co-operative Study Group (NHLCSG). Bone Marrow Transplant 1989; 4: 399–404. 68. Sweetenham JW, Santini G, Qian W et al. High-dose therapy and autologous stem-cell transplantation versus conventional-dose consolidation/maintenance therapy as postremission therapy for adult patients with lymphoblastic lymphoma: Results of a randomized trial of the European Group for Blood and Marrow Transplantation and the United Kingdom Lymphoma Group. J Clin Oncol 2001; 19: 2927– 36. 69. Jagannath S, Dicke KA, Armitage JO et al. Highdose cyclophosphamide, carmustine, and etoposide and autologous bone marrow transplantation for relapsed Hodgkin’s disease. Ann Intern Med 1986; 104: 163–8. 70. Philip T, Dumont J, Teillet F et al. High dose chemotherapy and autologous bone marrow transplantation in refractory Hodgkin’s disease. Br J Cancer 1986; 53: 737–42. 71. Carella AM, Congiu AM, Gaozza E et al. High-dose chemotherapy with autologous bone marrow transplantation in 50 advanced resistant Hodgkin’s disease patients: An Italian study group report. J Clin Oncol 1988; 6: 1411–6. 72. Phillips GL, Wolff SN, Herzig RH et al. Treatment of progressive Hodgkin’s disease with intensive chemoradiotherapy and autologous bone marrow transplantation. Blood 1989; 73: 2086–92. 73. Linch DC, Winfield D, Goldstone AH et al. Dose intensification with autologous bone-marrow transplantation in relapsed and resistant Hodgkin’s disease: Results of a BNLI randomised trial. Lancet 1993; 341: 1051–4. 74. Schmitz N, Pfistner B, Sextro M et al. Aggressive conventional chemotherapy compared with highdose chemotherapy with autologous haemopoietic stem-cell transplantation for relapsed chemosensitive Hodgkin’s disease: A randomised trial. Lancet 2002; 359: 2065–71.

13

75. McElwain TJ, Powles RL. High-dose intravenous melphalan for plasma-cell leukaemia and myeloma. Lancet 1983; 2: 822–4. 76. Barlogie B, Alexanian R, Smallwood L et al. Prognostic factors with high-dose melphalan for refractory multiple myeloma. Blood 1988; 72: 2015–19. 77. Anderson KC, Andersen J, Soiffer R et al. Monoclonal antibody-purged bone marrow transplantation therapy for multiple myeloma. Blood 1993; 82: 2568–76. 78. Reece DE, Barnett MJ, Connors JM et al. Treatment of multiple myeloma with intensive chemotherapy followed by autologous BMT using marrow purged with 4-hydroperoxycyclophosphamide. Bone Marrow Transplant 1993; 11: 139–46. 79. Dimopoulos MA, Alexanian R, Przepiorka D et al. Thiotepa, busulfan, and cyclophosphamide: A new preparative regimen for autologous marrow or blood stem cell transplantation in high-risk multiple myeloma. Blood 1993; 82: 2324–8. 80. Fermand JP, Chevret S, Ravaud P et al. High-dose chemoradiotherapy and autologous blood stem cell transplantation in multiple myeloma: Results of a phase II trial involving 63 patients. Blood 1993; 82: 2005–9. 81. Jagannath S, Vesole DH, Glenn L, Crowley J, Barlogie B. Low-risk intensive therapy for multiple myeloma with combined autologous bone marrow and blood stem cell support. Blood 1992; 80: 1666–72. 82. Schiller G, Vescio R, Freytes C et al. Transplantation of CD34+ peripheral blood progenitor cells after high-dose chemotherapy for patients with advanced multiple myeloma. Blood 1995; 86: 390–7. 83. Attal M, Harousseau JL, Stoppa AM et al. A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. Intergroupe Francais du Myelome. N Engl J Med 1996; 335: 91–7. 84. Gorin NC, Najman A, Duhamel G. Autologous bone-marrow transplantation in acute myelocytic leukaemia. Lancet 1977; 1: 1050. 85. Gorin NC, Douay L, Laporte JP et al. Autologous bone marrow transplantation using marrow incubated with Asta Z 7557 in adult acute leukemia. Blood 1986; 67: 1367–76. 86. Yeager AM, Kaizer H, Santos GW et al. Autologous bone marrow transplantation in patients with acute nonlymphocytic leukemia, using ex vivo marrow treatment with 4-hydroperoxycyclophosphamide. N Engl J Med 1986; 315: 141– 7. 87. Carella AM, Martinengo M, Santini G et al. Autologous bone marrow transplantation for acute leukemia in remission. The Genoa experience. Haematologica 1988; 73: 119–24. 88. Zittoun RA, Mandelli F, Willemze R et al. Autologous or allogeneic bone marrow transplantation compared with intensive chemotherapy in acute myelogenous leukemia. European Organization for Research and Treatment of Cancer (EORTC) and the Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto (GIMEMA) Leukemia Cooperative Groups. N Engl J Med 1995; 332: 217–23. 89. Kersey JH, Weisdorf D, Nesbit ME et al. Comparison of autologous and allogeneic bone marrow transplantation for treatment of high-risk

14

90.

91.

92.

93.

94.

95.

96.

97.

98.

99.

100.

Chapter 2

refractory acute lymphoblastic leukemia. N Engl J Med 1987; 317: 461–7. Coulombel L, Kalousek DK, Eaves CJ, Gupta CM, Eaves AC. Long-term marrow culture reveals chromosomally normal hematopoietic progenitor cells in patients with Philadelphia chromosome-positive chronic myelogenous leukemia. N Engl J Med 1983; 308: 1493–8. Finney R, McDonald GA, Baikie AG, Douglas AS. Chronic granulocytic leukaemia with Ph 1 negative cells in bone marrow and a ten year remission after busulphan hypoplasia. Br J Haematol 1972; 23: 283–8. Buckner CD, Stewart P, Clift RA et al. Treatment of blastic transformation of chronic granulocytic leukemia by chemotherapy, total body irradiation and infusion of cryopreserved autologous marrow. Exp Hematol 1978; 6: 96–109. Brito-Babapulle F, Apperley JF, Rassool F, Guo AP, Dowding C, Goldman JM. Complete remission after autografting for chronic myeloid leukaemia. Leuk Res 1987; 11: 1115–17. Rabinowe SN, Soiffer RJ, Gribben JG et al. Autologous and allogeneic bone marrow transplantation for poor prognosis patients with B-cell chronic lymphocytic leukemia. Blood 1993; 82: 1366–76. Khouri IF, Keating MJ, Vriesendorp HM et al. Autologous and allogeneic bone marrow transplantation for chronic lymphocytic leukemia: Preliminary results. J Clin Oncol 1994; 12: 748–58. Hryniuk W, Bush H. The importance of dose intensity in chemotherapy of metastatic breast cancer. J Clin Oncol 1984; 2: 1281–8. Tannir N, Spitzer G, Schell F, Legha S, Zander A, Blumenschein G. Phase II study of high-dose amsacrine (AMSA) and autologous bone marrow transplantation in patients with refractory metastatic breast cancer. Cancer Treat Rep 1983; 67: 599–600. Antman KH. Dose-intensive therapy in breast cancer. In: Armitage JO, Antman KH, editors. High-dose cancer therapy – pharmacology, hematopoietins, stem cells. Baltimore, MD: Williams & Wilkins; 1992. pp. 701–718. Rodenhuis S, Richel DJ, van der Wall E et al. Randomised trial of high-dose chemotherapy and haemopoietic progenitor-cell support in operable breast cancer with extensive axillary lymph-node involvement. Lancet 1998; 352: 515–21. Weiss RB, Gill GG, Hudis CA. An on-site audit of the South African trial of high-dose chemo-

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

111.

therapy for metastatic breast cancer and associated publications. J Clin Oncol 2001; 19: 2771–7. Weiss RB, Rifkin RM, Stewart FM et al. Highdose chemotherapy for high-risk primary breast cancer: An on-site review of the Bezwoda study. Lancet 2000; 355: 999–1003. Phillips GL, Wolff SN, Fay JW et al. Intensive 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) monochemotherapy and autologous marrow transplantation for malignant glioma. J Clin Oncol 1986; 4: 639–45. Knight WA, Goodman P, Taylor SA et al. Phase II trial of intravenous melphalan for metastatic colorectal carcinoma. A Southwest Oncology Group Study. Invest New Drugs 1990; 8: S87– S89. Ariel IM, Pack GT. Treatment of disseminated melanoma with phenylalanine mustard (melphalan) and autogenous bone marrow transplants. Surgery 1962; 51: 582. Thomas MR, Robinson WA, Glode LM et al. Treatment of advanced malignant melanoma with high-dose chemotherapy and autologous bone marrow transplantation. Preliminary results – phase I study. Am J Clin Oncol 1982; 5: 611– 22. Cornbleet MA, McElwain TJ, Kumar PJ et al. Treatment of advanced malignant melanoma with high-dose melphalan and autologous bone marrow transplantation. Br J Cancer 1983; 48: 329–34. Pritchard J, McElwain TJ, Graham-Pole J. Highdose melphalan with autologous marrow for treatment of advanced neuroblastoma. Br J Cancer 1982; 45: 86–94. Mulder PO, Willemse PH, Aalders JG et al. Highdose chemotherapy with autologous bone marrow transplantation in patients with refractory ovarian cancer. Eur J Cancer Clin Oncol 1989; 25: 645– 9. Farha P, Spitzer G, Valdivieso M et al. High-dose chemotherapy and autologous bone marrow transplantation for the treatment of small cell lung carcinoma. Cancer 1983; 52: 1351–5. Souhami RL, Harper PG, Linch D et al. Highdose cyclophosphamide with autologous marrow transplantation as initial treatment of small cell carcinoma of the bronchus. Cancer Chemother Pharmacol 1982; 8: 31–4. Glode LM, Robinson WA, Hartmann DW, Klein JJ, Thomas MR, Morton N. Autologous bone marrow transplantation in the therapy of small

112.

113.

114.

115.

116.

117.

118.

119.

120.

121.

cell carcinoma of the lung. Cancer Res 1982; 42: 4270–5. Smith IE, Evans BD, Harland SJ. High-dose cyclophosphamide (7 g/m2) with or without autologous bone marrow rescue after conventional chemotherapy in the treatment of patients with small cell lung cancer. Cancer Treat Rev 1983; 10 Suppl A: 79–81. Nichols CR, Tricot G, Williams SD et al. Doseintensive chemotherapy in refractory germ cell cancer – a phase I/II trial of high-dose carboplatin and etoposide with autologous bone marrow transplantation. J Clin Oncol 1989; 7: 932–9. Einhorn LH, Williams SD, Chamness A, Brames MJ, Perkins SM, Abonour R. High-dose chemotherapy and stem-cell rescue for metastatic germcell tumors. N Engl J Med 2007; 357: 340–8. Ikehara S, Good RA, Nakamura T et al. Rationale for bone marrow transplantation in the treatment of autoimmune diseases. Proc Natl Acad Sci U S A 1985; 82: 2483–7. Marmont AM, Van Bekkum DW. Stem cell transplantation for severe autoimmune diseases: New proposals but still unanswered questions. Bone Marrow Transplant 1995; 16: 497–8. Salzman P, Tami J, Jackson C et al. Clinical remission of myasthenia gravis after high dose chemotherapy and autologous transplantation with CD34+ stem cells [abstract]. Blood 1994; 84 Suppl 1: 206a. Fastenrath S, Dreger P, Schmitz N. Autologous unpurged bone marrow transplantation in a patient with lymphoma and SLE: Short-term recurrence of antinuclear antibodies [abstract]. Arthritis Rheum 1005; 38: S303. Joske DJ, Ma DT, Langlands DR, Owen ET. Autologous bone-marrow transplantation for rheumatoid arthritis. Lancet 1997; 350: 337–8. Burt RK, Georganas C, Schroeder J et al. Autologous hematopoietic stem cell transplantation in refractory rheumatoid arthritis: Sustained response in two of four patients. Arthritis Rheum 1999; 42: 2281–5. Snowden JA, Biggs JC, Milliken ST, Fuller A, Brooks PM. A phase I/II dose escalation study of intensified cyclophosphamide and autologous blood stem cell rescue in severe, active rheumatoid arthritis. Arthritis Rheum 1999; 42: 2286– 92.

3

Mary M. Horowitz

Uses and Growth of Hematopoietic Cell Transplantation

Introduction The first successful transplantations of allogeneic hematopoietic cells were performed in 1968 in three children with congenital immune deficiency diseases [1–4]. In each instance, hematopoietic cells were collected from the bone marrow of sibling donors who were genotypically identical or closely matched to the recipient for human leukocyte antigens (HLAs). Since then, more than 800,000 patients have received hematopoietic cell transplantations (HCTs) to treat life-threatening malignant and nonmalignant diseases. Current estimates of annual number of HCTs are 55,000–60,000, worldwide (Fig. 3.1). Reasons for widespread use include proven and potential efficacy in many diseases, better understanding of the appropriate timing of transplantation and patient selection, greater availability of donors, greater ease of hematopoietic progenitor cell collection, and improved transplantation strategies and supportive care, leading to less transplantation-related morbidity and mortality and an increased ability to perform the procedure in older and sicker patients.

Changing indications for HCT HCT has efficacy in many diseases (Table 3.1). In some, transplantation corrects congenital or acquired defects in blood cell production and/or immune function. In others, it restores hematopoiesis after high-dose (myeloablative) cytotoxic therapy for malignancy and/or provides potent anticancer adoptive immunotherapy. In the 1970s, more than half of the diseases for which HCT was performed were nonmalignant disorders: 40% were for aplastic anemia and 15% for immune deficiencies. Fewer than half were for cancers, and these were mostly for advanced acute leukemia. In the 1970s, Thomas and colleagues showed convincingly that some patients with refractory acute leukemia could achieve longterm leukemia-free survival with high-dose therapy and HLA-identical sibling marrow transplantation (see Chapter 1) [5,6]. Better outcome was subsequently demonstrated in patients transplanted in first or second remission [7–9]. Syngeneic (identical twin) and allogeneic HCTs were shown to produce cytogenetic remissions and long-term leukemia-free survival in chronic myeloid leukemia (CML) in the late 1970s and early 1980s [10–12].

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

Use of allogeneic HCT to treat leukemia increased dramatically in the 1980s. By 1985, about 75% of allogeneic transplantations were for leukemia, with approximately equal numbers for CML, acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL); more than 90% were from HLA-identical sibling donors. Introduction of alternative targeted therapies for CML in the early 2000s led to decreased use of HCT, with current guidelines reserving HCT for patients who fail to respond to nontransplant therapy or who lose their response [13]. However, leukemia treatment still accounts for about 65% of allogeneic HCT procedures (Fig. 3.2) (Center for International Blood and Marrow Transplant Research [CIBMTR] Statistical Center, unpublished data). Experimental and clinical evidence for a dose–response effect of drugs used in lymphoma therapy led to trials of autologous HCT to allow dose intensification in non-Hodgkin’s lymphoma in the middle 1980s (see Chapter 2) [14–16]. Results were promising, and there was rapid acceptance of autologous transplantation as salvage therapy in persons failing conventional chemotherapy for lymphoma. This was followed by increasing use of autologous transplantation as consolidation of primary therapy in patients with high-risk disease. There is now more frequent use of allogeneic transplantation in lymphoma to harness immunemediated graft-versus-lymphoma effects, especially in patients whose disease recurs after autologous transplantation, and in patients with follicular histology who are rarely cured by autologous transplantation [17]. Lymphoma accounted for 12% of allogeneic HCTs in 2006 (CIBMTR Statistical Center, unpublished data). The rationale of dose intensification led to the application of autologous transplants to many other hematologic and nonhematologic cancers over the past 10 years. One striking development was a dramatic increase in their use for breast cancer in the early 1990s. Breast cancer accounted for 16% of autologous transplants done in 1989–90 and 40% in 1994–95 [18]. However, results of randomized clinical trials in early and advanced breast cancer were disappointing (reviewed in [19–21]). In 1999, the use of HCT for breast cancer declined dramatically, and in 2006 breast cancer accounted for fewer than 5% of autologous transplants in North America. Treatment of solid tumors still accounts for about 10% of autologous transplants (Fig. 3.2). In 1996, results of a randomized trial comparing high-dose therapy and autologous HCT with conventional therapy for multiple myeloma were published; this study found a significant survival benefit with autologous transplantation [22]. Autologous HCT for myeloma increased dramatically after this report. Subsequent data suggested improved results with sequential autologous transplantations or autologous transplantation followed by allogeneic transplantation [23,24]. Multiple myeloma is now the single most common indication for HCT, account-

15

16

Chapter 3

ing for 45% of autologous transplants but fewer than 5% of allogeneic transplants (Fig. 3.2). Thirty percent of HCTs for myeloma in 2006 involved a planned sequential autologous–autologous or autologous– allogeneic transplant approach (CIBMTR Statistical Center, unpublished data). The most common indications for allogeneic and autologous HCT in North America in 2006 are shown in Fig. 3.2. About 70% of allogeneic HCTs are for leukemia or preleukemia: 34% for AML, 16% for ALL, 6% CML, 10% for myelodysplastic or myeloproliferative syndromes, and less than 5% for other leukemias. About 15% are for other cancers, including non-Hodgkin’s lymphoma (11%), multiple myeloma (2N DNA content) are less efficient at reconstitution, and are similarly less efficient at homing directly to the bone marrow rather than the spleen or liver [69,70,72]. Within the 2N DNA pool of LT HSCs, the G0 cells transplant much more readily than those in G1 [70]. Following transplantation, a much higher fraction of LT-HSCs remain in cell cycle in the radioprotected host, and this period can extend for at least 4–5 months after transplantation [40,68,69,73]. Given the poor transplantability of cycling HSCs, it is conceivable that protocols that test cells for “stemness” by serial transplantation could be confounded by these lingering effects of mobilization and radiation on cell-cycle status and reconstitution ability [68,69,73]. Therefore, experiments that are designed to measure the life span of expanding and

38

Chapter 5 Kinetics of mobilization of LT-HSC following Cy/G-CSF

Cell-cycle status of LT-HSC in blood 298/307 0 40 80 120 100 200

1,000,000

Total HSC

100,000

10,000 Bone marrow Spleen Blood

1000

100 –1

0

1

2

3

4

5

6

7

8

90 80 70 60 50 40 30 20 10 0

Day of CY/G-CSF treatment

self-renewing HSCs by transplantation should be carried out in ways that remove the induced cell-cycle phenomenon from consideration. Although in the steady state only a small fraction of LT-HSCs is in the cell cycle, this ratio can be dramatically altered by a number of circumstances. For example, upon transplantation into irradiated hosts, probably every HSC that lands in a hematopoietic microenvironment undergoes, at least initially, symmetrical self-renewing cell divisions to expand the HSC pool. In addition, the techniques used to mobilize HSCs into the blood stream usually operate by first causing all HSCs to enter the cell cycle nonrandomly [72,74,75]. As shown in Fig. 5.2, treatment of a mouse with cyclophosphamide (which should kill no more than 4–8% of LT-HSCs, but should have a dramatic effect on both MPPs and oligolineage progenitors [see below] because they are largely in cell cycle), followed by granulocyte-colony-stimulating factor (G-CSF) treatment, results in every HSC entering the cell cycle, which leads to a 12–15-fold increase in the steady state number of LT-HSCs [74]. Even bleeding of mice, up to 25% or 50% of their blood volume two or three times in a week, results in a measurable increase in the fraction of HSCs in the cell cycle [76]. Replacement of RBCs lost due to bleeding suppressed the expansion, whereas replacement of fluid volume or white blood cells did not, indicating that HSCs are selectively responsive to decreases in RBC levels. Thus, positive and negative feedbacks on the regulation of HSC cell division must exist, but currently their nature and mechanisms are unknown. HSCs must go through many self-renewing cell divisions over the life of an animal, and it is reasonable to wonder if they are susceptible to a natural limit on the number of cell divisions they can undergo, often called the “Hayflick limit” [77], and whether HSCs have a special mechanism for maintaining the ends of their chromosomes, called telomeres, to avoid programmed cell senescence resulting from critical telomere shortening [78,79] (see Chapter 6). Both mouse and human HSCs have telomerase activity, but telomere length declines in the progeny of HSCs that have undergone a large number of cell divisions, in the context of normal blood development and in response to radiation and transplant recovery [80]. Therefore, the relatively high telomerase activity in HSCs [81] is not sufficient to completely prevent telomere shortening through several cell division cycles. The role of telomerase is complex. In the absence of a functional telomerase-reverse transcriptase (TERT) component of the telomerase complex, the progeny of dividing HSCs lose telomeres much more rapidly, confirming that it functions to maintain telomere length [27,80]. However, transgenic mouse strains that overexpress the TERT component at high levels in HSCs do not reduce the length of their telomeres, even following successive induced cell cycles by transplantation, and there is also evidence that TERT has a protective effect independent of its enzymatic function [82]. Nevertheless, these HSCs still lose trans-

5.2% S/G2/M

Hoechst 33342

Fig. 5.2 The total number of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow, spleen and blood of mice on successive days of cyclophosphamide/granulocyte colony-stimulating factor (CY/G-CSF) treatment. Total bone marrow hematopoietic stem cells (HSCs) were calculated by assuming that the femurs and tibias (less the epiphyses) contained 15% of all bone marrow in the mouse. Total HSC level in the blood was calculated by assuming that the total blood volume was 1.8 mL. Cell-cycle status of LT-HSCs in the blood was determined by Hoechst DNA staining [72].

plantation activity after the fourth or fifth serial HSC transplant generation [27]. This loss of transplantability with HSCs that retain telomeres points to other phenomena limiting the transplantation of these cells, including perhaps the persistent entry into the cell cycle, programmed cell life spans independent of telomere length, or random factors associated with transplantation and/or cell division [80,83]. Programmed cell death or senescence of HSCs need not occur only as a result of extensive numbers of HSC cell divisions. There are several genetic loci that regulate, in mice, both the frequency of HSCs and the frequency of HSCs that are in the cell cycle [84–86]. One genetic program that likely regulates HSC numbers is that leading to programmed cell death inhibitable by the antiapoptotic protein BCL-2 (and by extension, its antiapoptotic gene relatives such as BCL-x). In mice that have enforced high-level expression of human BCL-2 in HSCs, the steady-state levels of HSCs are increased four- to fivefold, and the competitive repopulation activity of these HSCs, cotransplanted with CD45 congenic HSCs into the same irradiated host, is high [87,88]. Therefore, it is likely that programmed cell death is one regulator of HSC numbers, and it is yet to be determined what stimuli to HSCs enact the programmed death pathway inhibitable by BCL-2. There are several genes whose expression is required to initiate hematopoiesis, hematopoiesis and angiogenesis, or angiogenesis resulting in failed hematopoiesis; these include AML-1/RUNX-1 [89], SCL-TAL [90], FLK1 [91,92]. It is unclear whether these genes act directly within HSCs, within their immediate precursors to generate HSCs or on other cell populations that have a role in the survival or stimulation of HSCs, or whether, as in the case of a developing vasculature, they regulate the movement of HSCs or their precursors from one part of the body to another [93]. Reverse transcription polymerase chain reaction (RT-PCR) analysis of highly purified HSCs, or the use of highly purified HSC messenger RNA probes to interrogate extensive mouse complementary DNA libraries has revealed that at least some of these genes are expressed intrinsically in HSCs [55,94–98]. It is important to point out that many factors could be playing a part in the movement of pre-HSCs or HSCs during embryonic and fetal development. Factors affecting the release of cells from a particular microenvironment, their movement through tissues or the blood stream, the recognition of endothelium in tissues, and their homing to stem cell niches could easily be read out as genes affecting HSCs without directly affecting either their development or their self-renewal capacity (see below). Genetic pathways for self-renewal of HSCs Self-renewal is the single distinguishing characteristic between LTHSCs and the rest of the multipotent and oligopotent progenitors in the hematopoietic system (see Chapter 6). While LT-HSCs can be expanded

Biology of Hematopoietic Stem and Progenitor Cells

in a variety of situations in vivo, the attempt to expand rigorously purified mouse or human HSCs in vitro with conventional cytokines such as steel factor (SLF), thrombopoietin (TPO), interleukin-11 (IL-11), IL-6, IL-3, and Flt3L, alone or in combination in serum-containing medium, has never resulted in more than a minor expansion in HSCs by phenotype, and little or no expansion by function [99–105]. Many investigators were led astray by the expansion of cells expressing CD34, but a broad variety of non-HSCs are also CD34+, and these cells were the result of proliferation of HSCs and progenitors coupled with cell differentiation. The introduction of the antiapoptosis protein BCL-2 into HSCs in mice allowed HSCs to survive serum-free culture conditions, so the response of LT-HSCs to factors in the absence of differentiating factors present in serum, such as monocyte/macrophage-CSF (M-CSF), G-CSF, granulocyte–macrophage CSF (GM-CSF), and others, could be studied [87,88]. Any combination of these cytokines and others when added to these BCL-2-containing HSCs led to a massive burst of proliferation and progenitor expansion by most HSCs, but no detectable HSC expansion [87,88]. These HSCs could respond to three factors as single factors in vitro: IL3, SLF, and TPO [88]. HSCs responding to IL-3 give large bursts of proliferation with concomitant maturation along the myeloid and mast cell lineages, without self-renewal of HSCs. One might have hoped that the HSC response to SLF alone might have been self-renewing divisions, as mutations in the SLF gene or its receptor, c-kit, lead to fetal or early neonatal lethality, usually because of profound anemia [106–111] (reviewed in [112]). However, the response of single highly purified Bcl-2-expressing LT-HSCs to SLF in serum-free medium was proliferation without detectable self-renewal, but with maturation through both CLP and CMP pathways [88]. Stimulation with TPO led to mild proliferation, and dedicated differentiation along the megakaryocytic lineage. Interestingly, RT-PCR analysis of messenger RNA from HSCs and highly purified myeloid progenitors revealed that HSCs might have the highest expression of the TPO receptor c-mpl of any of these cell populations [26,94,113]. Recent reports support a complex role for TPO in HSC biology. Neither TPO nor c-mpl is necessary for fetal HSC development, but HSCs in adult TPO- or c-mpl-deficient mice are cycling more and have impaired reconstitution ability compared with wild-type HSCs [114]. Further, TPO is produced by osteoblasts in the bone marrow, and blocking tpo antibody induces release from the niche and increased proliferation [115]. Taken together, these data suggest that TPO is important for HSC proliferation and self-renewal after transplantation, but also promotes quiescence by promoting retention in the niche with increased integrin expression and preventing cycling and subsequent HSC exhaustion. Some early experiments indicated that other factors might be involved in HSC expansion. For example, activation of the Notch-1 receptor, mimicked by retroviral transduction with the intracellular activated form of Notch-1, occasionally led to the generation of mouse cell lines with many of the characteristics of HSCs, although this response was neither general nor robust [116,117]. Provision of HoxB4 as a retroviral insert to semipurified mouse hematopoietic progenitors led to vigorous proliferation and at least some retention of hematopoietic multilineage and long-term reconstituting activity in vitro and in vivo [118–120], although a rigorous demonstration that this outcome was caused by expansion of HSCs alone, rather than provision of other signals that in addition enable their engraftment has not yet been established [119–121]. The Wnt/Fzd/Dsh/GSK-3β/β-catenin pathway is also implicated in HSC self-renewal (reviewed in [122,123]). Wnt, a highly hydrophobic protein, binds to surface frizzled (Fzd), a 7TM protein, in combination with a low-density lipoprotein-associated receptor, usually LRP5. Binding of Wnt to Fzd leads to activation of disheveled (Dsh). The action of Dsh prevents GSK-3 (glycogen synthase kinase) β-phosphory-

39

lation of β-catenin, and also allows β-catenin to dissociate from the complex and translocate to the nucleus, where it becomes available for binding to Lef/TCF factors and activation of transcription [122,123]. The addition of partially or fully purified Wnt3A to highly purified LT-HSCs in serum-free medium, with or without SLF, results in clonal proliferation of HSCs. In vitro, these HSCs go through a significant expansion, and up to 50% of the expanded progeny are cells of the LTHSC phenotype [124,125]. Transfection of nondegradable β-catenin lacking the N-terminal phosphorylation sites was reported to drive LTHSCs into massive and prolonged expanding cell divisions, with expansion of LT-HSCs both by phenotype and by function upon transplantation [124]. Inhibitors of Wnt signaling even blocked SLF and other cytokine-mediated proliferation of LT-HSCs [124]. When LTHSCs are transfected with a reporter of LEF/TCF + β-catenin activity in the nucleus and then transplanted into lethally irradiated hosts, LTHSCs reading out the reporter are retained in vivo long term, whereas myeloid progenitors derived from these LT-HSCs do not express the reporter [124]. Taken together, these results indicated that the Wnt/Fzd/ β-catenin pathway is likely to be an important pathway utilized by LTHSCs in self-renewing cell division. When activated by Wnt 3A, LTHSCs upregulate expression of both Notch-1 and HoxB4, genes also implicated in the self-renewal of HSCs. However, the regulated deletion of the β-catenin gene in transplanted bone marrow failed to affect the engraftment of hematopoiesis, or the retransplantation capacity of this marrow [126]. Contradictory results were recently reported with a different gene promoter encoding the cre recombinase required for βcatenin deletion [127]. The bmi-1 gene is a member of the polycomb family, which has a role in silencing gene expression by site-specific deacetylation of histones associated with those genes [128]. BMI-1 is expressed in high levels in mouse and human LT-HSCs, and mouse-made mutants for the BMI-1 gene have a profound defect in the transplantation and selfrenewal of LT-HSCs [97,128]. The two transcripts of the p16 locus, P16INK4A and P19ARF, are overexpressed selectively in the hematopoietic tissues in the BMI-mutant mice, thus implicating a role of BMI1 in the downregulation of genes whose expression is contrary to self-renewal of HSCs [129]. These could include decreased viability, enforced differentiation, or direct regulation or inhibition of genes involved in the self-renewing pathways such as those of the Wnt/Fzd/βcatenin and Notch pathways [116,117,122,124]. BMI-1 mutants do not show differences in expression in either HoxA9 or HoxB4 (Clarke et al., unpublished data), genes that also have some role in HSC functions [121,130]. JunB negatively regulates mouse LT-HSCs, and in the absence of JunB the HSC pool expands; in the expanded pool, p16ink4a and p19arf are expressed at lower levels than wild-type HSC, and Bcl-2 and Bcl-xl are increased in expression in JunB knockout compared with wild-type HSCs [131]. A number of genes have recently been reported to play a role in HSC self-renewal including junb, sox17, cited2, Mcl1, Tel/Etv6, Gfi1, Pten, and Stat5. Chapter 6 covers self-renewal in more detail, but taken together these results indicate that the control of self-renewal is a complex, multipathway process, much of which is probably controlled by the HSC niche. Migration of HSCs Hematopoietic capacity is known to be present in the adult mouse at highest levels in bone marrow, at about one-tenth that level in spleen, and at one hundredth or less of the level of bone marrow in blood [132]. In those early experiments, the ability of marrow or tissue transplants to retain erythropoiesis could not be assigned directly to actions of HSCs. There was no clear explanation as to why hematopoietic activity might

40

Chapter 5

Rapid clearance of mobilized Lin−Sca-1+c-kit+ progenitor cells from the blood of mobilized mice 100

10

Bone marrow (7%)

90 80 Clearance (%)

Clearance

1

0.1

0.01

70 50 40 30

Liver (39%)

20

Thymus (0%) PP (0.5%) ILN (2%) MLN (0%)

10 0.001

0 0

2

4

6

8

Spleen (51%)

60

10

Time (min) +

GFP progenitors PKH-26+ RBC Predicted progenitor frequency Predicted RBC frequency

be present in organs other than the bone marrow, and certainly not in the blood. It was only when clinical HCT groups described the phenomenon of hematopoietic activity increasing in the blood of cytotoxic drug-treated individuals that the possibility of HSC mobilization into the blood was considered [133,134]. Empirical protocols using cyclophosphamide plus cytokines such as G-CSF, GM-CSF, SLF alone or in various combinations with G-CSF, IL-1, and IL-3, led to efficient mechanisms to mobilize hematopoietic cells into the blood stream [135–139]. While at the early stages, this activity was measured in terms of in vitro tests for hematopoietic CFCs, but CFC assays alone are not specific for HSC versus oligolineage progenitor activities [37,58]. However, there could be no doubt that HSCs were involved when MPB transplants were used in hosts conditioned with high-dose chemotherapy, resulting in both early and sustained hematopoiesis. When cyclophosphamide and G-CSF are used in the mouse model, wherein one can analyze directly the daily changes in marrow, spleen, and blood, it is clear that nearly every HSC and MPP enters the cell cycle rapidly [74]. These reach a peak in the marrow, at which time the cells appear rapidly in blood and spleen (Fig. 5.2). At all times, the HSCs appearing in the blood have a 2N amount of DNA, indicating that they are not proliferating in the blood; in fact, the fold increase in their number would have ruled out a blood-only expansion of the HSC pool [74]. Bromodeoxyuridine incorporation into their chromosomal DNA during the expansion phase of HSC in the “mobilized” bone marrow results in the labeling of virtually every HSC, and, interestingly, just 1 or 2 days later the 2N HSCs found in the blood are all labeled with bromodeoxyuridine [74]. Thus, mobilization appears to result as a consequence of marrow HSC proliferation, and accumulation of emigrant HSCs in extramedullary sites such as the spleen, liver, and blood. The proliferation that precedes mobilization has been described above, and it is notable that the cytokines (e.g. G-CSF) that show high efficiency in the mobilization process do not have receptors on native HSCs [26,113,140]. In the early phases of mobilization, there is a rise in cells of the myelomonocytic series, and it has been shown that the elaboration locally by myeloid cells of matrix metalloproteinases such as MMP-9 can serve to cleave cell–cell interaction molecules known to be active between HSCs and marrow stroma [141]. Similarly, elaboration of the chemokine IL-8 occurs, and infusion of IL-8 alone can cause a significant and rapid mobilization of HSCs [142,143]. The actual nature of the molecules involved in HSC release is not clear, although it is known that HSCs express integrin α4β1, which allows them to attach to hematopoietic stromal cell vascular cell adhe-

Fig. 5.3 Rapid clearance of mobilized hematopoietic progenitor cells from the blood stream. Clearance from the blood of eGFP+ progenitors and PKH-26+ red blood cells (RBCs), and predicted progenitor and RBC frequencies, respectively. In vivo homing to different organs of mobilized and consecutively transplanted hematopoietic progenitor and stem cells 3 hours after injection. (From [72] with permission.)

sion molecule [144–148]. Also, HSCs express the c-kit receptor that allows binding to, at least, cell surface SLF on stroma. Although HSCs have a number of other members of the adhesion and integrin family members, the role of each of these in cell proliferation, detachment from stroma, local emigration into sinusoids, movement through the blood stream, margination on distant blood vessels, transendothelial migration at these vessel sites, and localization to microniches that support LTHSCs are still unclear. In the adult mouse, homing of infused HSCs appears to involve both cell surface integrin α4β1 and cell surface CXCR4, a receptor for the chemokine stromal cell-derived factor-1 (SDF-1) [149–151]. Of all known chemokines, adult mouse HSCs migrate in vitro only in response to SDF-1, and this is chemotactic movement [151]. However released, co-infusion of labeled HSCs and labeled RBCs from mobilized blood into the blood of same-stage mobilized animals results in the rapid emigration of HSCs out of the blood, with retention of RBCs in the vessels [72]. In fact, most HSCs are gone by 1–5 minutes, and they do not reappear in the blood stream within the next several hours (Fig. 5.3). These HSCs nonrandomly home to hematopoietic sites in mobilized animals: bone marrow, spleen, and liver, all tissues with sinusoids lined by macrophages. This observation fits with previous studies on the rapid egress of infused lymphocytes out of the blood stream into tissues via recognition of particular vascular addressins for which they have cognate homing receptors [152,153], as well as the rapid egress from the blood stream of other nucleated cells such as monocytes [154]. There are similar HSC, MPP, and myeloid progenitor fluxes in normal mice. To maintain the approximate 100 HSCs found in the blood of mice, given a residence time in the blood of 5 minutes or less, would require fluxes of well over tens of thousands of HSCs and MPPs per day. Recent data suggest that HSCs and progenitors circulate through the blood and reside in the tissues for up to 36 hours before returning to the blood via the lymphatics [155]. It is not yet clear whether these cells are passing through the blood in one pass or whether they form a specialized pool of HSCs and MPPs yet have a higher probability of re-entering the blood than cells resident in deep marrow niches. If HSCs found in the blood are also derived from dividing HSCs, nearly one out of every two daughter cells from a self-renewing HSC division would have to enter the blood stream. Consistent with this emigration rate, infusion of purified LT-HSCs into untreated immunodeficient mice (to avoid immune rejection of cells with antigenic markers) led to a dose–response engraftment of marrow sites of between 0 and 250 HSCs, while up to 5000 HSCs engrafted no

41

% granulocyte chimerism

Biology of Hematopoietic Stem and Progenitor Cells

(a)

1st transplant (GFP = CD45.1 +) 2.0

2nd transplant (GFP = CD45.1 +)

1.5

re

p

e lac

me

n

o tm

de

l

1.0

0.5

0.0 0

200

400

600

800

1000

#HSCs 1st transplant: (b) 10

5

10

5

10

5

10

4

10

4

10

4

10

5

10

4

10

10

2

10

0

3

10

CD45.1

3

CD45.1

10

SlamF1

c-kit

43% 1.1%

3

10

57%

3

2

0

0

0 0

10

2

3

10

Sca-1

10

4

10

5

0

2

10

3

10

4

10

10

5

CD34

0

3

10

4

10

5

10

CD45.2

0

10

2

3

10

4

10

5

10

GFP

Fig. 5.4 Hematopoietic stem cell (HSC) niches can be transiently saturated in unconditioned animals [157]. (a) RAG2−/−γc−/− (CD45.2) mice were transplanted with 10, 50, 250, 500 or 1000 HSCs from green fluorescent protein (GFP) transgenic mice (shaded bars). Eight weeks after transplantation, mice were given a second transplant of 4000 non-GFP HSCs from CD45.1 donors (open bars). Peripheral blood granulocyte chimerism was analyzed at 16 weeks after the first transplantation. The dashed line indicates theoretical chimerism if endogenous HSCs were displaced from bone marrow niches. (b) Additive donor HSC engraftment following repetitive transplantation. Unconditioned CD45.2 RAG2−/−γc−/− mice were transplanted with saturating doses of GFP+ CD45.1 HSCs (250–1000) and retransplanted with 4000 GFP− CD45.1 HSCs 8 weeks after the first transplantation. Bone marrow was harvested and donor HSCs were quantified as Lin−c-kit+Sca-1+Slamf1+CD34−CD45.1+ cells that are either GFP+ (first transplant) or GFP− (second transplant).

more niches than 50 cells. This plateau was about 0.5% of marrow sites. However, 50 cells on day 1 and another 50 cells on day 2 gave about 0.5% of each population (Fig. 5.4) [156]. Thus, about 0.5% of the marrow niches for HSC appear to be available at any moment, and these appear to be occupied by the next interval. Preclearing of HSC with a cytoreductive anti-c-kit antibody alone in immunodeficient mice leads to up to 80% engraftment with purified HSCs given in large numbers at the point that the monoclonal antibody reaches its nadir concentration in the blood [157]. The migration of HSCs into hematopoietic tissues involves a step of crossing macrophage-lined sinusoids. We have recently found that, during the mobilization process, HSCs and progenitors upregulate the expression of the integrin-associated protein CD47 [158]. One function of CD47 is as a ligand for the signal regulatory protein-α macrophage receptor [159]. In the absence of CD47, macrophage-bound cells are phagocytosed, whereas cells expressing high levels of CD47 signal the macrophage through the receptor to block phagocytosis. We have found that leukemias transfected with mouse CD47 at levels approximating those found on mobilized HSCs are protected from phagocytosis in vitro and engraft in vivo, whereas the same leukemias lacking these levels of CD47 are phagocytosed in vitro and fail to engraft in vivo [158]. Further, HSCs from CD47 knockout mice fail to engraft wild-type congenic mice [158]. Taken together, these experiments provide evidence that, during

migration, itinerant HSCs express high levels of CD47 to gain entry to hematopoietic sites. This interpretation of the fate of recently self-renewed and also resting HSCs calls for a deeper examination of hematopoietic niches in the marrow; niches that commit recently entering HSCs into the myeloid or lymphoid pathways need not be adjacent to the HSC niche if the daughter cells of HSCs are constantly migrating. In addition, the existence of a large flux of recirculating HSCs likely provides for the continuous replacement of empty HSC niches with functional HSCs; in the absence of such a pool of migrating HSCs, the death of an HSC could mean the permanent loss of that functional niche. This finding also has relevance when one considers claims of plasticity of stem cells in one or more tissues [5]. If tens of thousands of HSCs are fluxing through tissues every day, finding HSC activity in those tissues might only result from these itinerant HSCs, rather than from transdifferentiation of local tissue-specific stem cells into hematopoietic fates [5]. Ontogeny and aging of HSCs It is tacitly assumed that HSCs derive from the embryonic mesoderm. In mammals, the first site of definable blood cells appears to be the yolk sac blood islands, in mice at about 7.5 dpc [160,161]. Although the terminology used for development from the time of conception is E

42

Chapter 5

Yolk sac

Liver

AGM

Spleen

Bone marrow

Blood

8

10

12

14

16

18

birth

dpc

Fig. 5.5 The ontogeny of hematopoietic progenitors. The sites of and duration of hematopoiesis in the fetus and neonate are shown. (Adapted from [178], with permission.)

(embryonic day) 1–21 (for mouse), in fact the transition from the embryo to fetus, at organogenesis, occurs at about 8–9 days post conception or coitus (dpc), so we will hereafter use the “dpc” abbreviation. At dpc 7–8, mouse yolk sacs contain cells capable of responding in vitro to produce hematopoietic CFCs [160,161]. The yolk sac connects to the developing embryo/fetus [162] at about dpc 8.5 later via the development of the vitelline vessels, which feed into the developing fetal liver. At very nearly that dpc 8–8.5 time interval, hematopoietic cells appear in and around the dorsal aorta of the developing fetus (reviewed in [163]). Transplantation of dpc 8–9 yolk sac blood island cells into the yolk sac cavity of dpc 8 allogeneic hosts results in mice that have a lifelong presence of donor-derived day 10 CFU-spleen (CFU-S) in marrow as well as thymocytes [93]. At about dpc 10–11, hematopoiesis begins in the fetal liver in the mouse, and continues there throughout fetal life (Fig. 5.5). Where do the first HSCs develop [164]? At dpc 3.5, the blastocyst implants into the uterus, and its inner cell mass appears to be a population of pluripotent cells without defined commitments [162]. Between dpc 3.5 and dpc 5.5, gastrulation occurs with the formation of the three germ layers [162]. It has been a mystery whether the developing postgastrulation mesoderm cells emigrate separately to yolk sac and embryo to give rise to independent origins of hematopoiesis, or whether only one of these sites is the initial site of commitment to hematopoiesis, the later appearance of hematopoiesis at the other site resulting from migration [93,163,165,166]. The idea of separate origins for extraembryonic (yolk sac) hematopoiesis and intraembryonic hematopoiesis was supported by the fact that yolk sac erythropoiesis resulted in the production of embryonic and fetal hemoglobins, while lifelong hematopoiesis resulted in the production of mainly adult hemoglobin [165,167]. These were called, respectively, primitive and definitive hematopoiesis [167]. Until recently, no experiment directly assessed whether these two kinds of hematopoiesis have distinct or common origins at the cellular level, To address this question, Nishikawa’s group used an inducible Runx1/ aml1 reporter system to label extraembryonic HSCs [168]. When embryos were induced at dpc 7.5, the time that HSCs first appear in the yolk sac blood islands [93,169], labeled hematopoietic cells were observed in the fetus and adult. As circulation was not established at this time point, these data suggest that the labeled HSCs that persist and

give rise to ongoing hematopoiesis must have derived from yolk sac HSCs and migrated into the embryo once circulation was established [168]. Because the allantois at dpc 9 has some runx1+ cells, it is another possible site of HSC generation in these experiments if the tamoxifen inducer has a half-life in the embryo of greater than a day. There is a close association of hematopoiesis and angiogenesis in both the developing blood islands and the developing dorsal aorta, and there is good evidence that mutant mice lacking the cell surface receptors for angiogenic peptides fail to develop either hematopoietic or angiogenic cells [91,170]. While these experiments suggest that a bipotent precursor called the hemangioblast exists that can give rise to both angiogenic stem cells and HSCs, only a small number of hemangioblast cells capable of producing both outcomes in vitro can be prospectively isolated from the pre-yolk sac embryo [171]. Rather than there being a hemangioblast precursor, it is possible that mutant mice that cannot make blood vessels properly cannot make blood because primitive stem cells travel via the blood, or that blood vessels make factors important for hematopoiesis. A recent set of experiments supports the hypothesis that the majority of embryo blood cells derive from distinct progenitors for hematopoiesis and endothelium [172]. Tetrachimeric mice were generated by the injection of four distinctly colored single cells into developing blastocysts. Analysis of the color combinations observed in the yolk sac of the resulting embryos revealed endothelial and hematopoietic cells of different colors in all individual blood islands, indicating that they developed from more than one cell (Plate 5.1). If both hematopoietic and endothelial cells derived from a common hemangioblast progenitor, each blood island would be monochromatic in these mice. However, single-color blood islands were not observed. Instead, the majority of blood islands contained blood and endothelial cells of multiple but not necessarily the same colors, indicating that multiple progenitors of each type came together to form each blood island [172]. About 15–20% of the blood islands examined could have derived from hemangioblasts; when we lineage-marked cells with the Flk1.cre/floxed Lacz-GFP (green fluorescent protein) constructs in the same ES cells, about 20% of blood cells were GFP+, showing an activated flk1 locus, but 80% were lacz+GFP−. Taken together, these data support about 20% of early blood cells deriving from a possible hemangioblast progenitor, and about 80% being derived from a hematopoietic progenitor that has not gone through a hemagioblast pathway. Similar conclusions were drawn by Shalaby et al. [91]. There is an interesting point coming out of ES cell biology that might be relevant to these issues. ES cells are derived from the inner cell mass of blastocysts, and likely represent pluripotent stem cells in the mouse at approximately dpc 3.5 age. They are maintained as pluripotent cells by the addition of leukemia inhibitory factor to their cultures, at least in mouse ES lines [173]. Removal of leukemia inhibitory factor from these pluripotent ES lines results in the onset of hematopoiesis, and during this development, at about 4 days, cells capable of in vitro hematopoiesis emerge, which express some markers shared with HSCs (Yamane, Domen, and Weissman, unpublished data). This interval, 3.5–4.0 days, correlates in developmental time to about dpc 7.5, when HSCs first appear in yolk sac blood islands [93,169]. Thus, these two systems appear in parallel to produce embryonic HSCs. The major site of the next (fetal) stage of hematopoiesis appears to be the fetal liver [161]. The numbers of HSCs in the fetal liver increase logarithmically from dpc 11–15. After dpc 15, fetal liver HSC numbers level off, but hematopoiesis continues to expand in the liver [53,174,175]. At dpc 16 hematopoiesis appears in the fetal spleen in mice, and at dpc 18 in the fetal bone marrow. The initiation of fetal liver hematopoiesis is blocked in mice with mutations in integrin α4β1 (very late antigen-4 [VLA-4]) or integrin α5β1

Biology of Hematopoietic Stem and Progenitor Cells

(VLA-5) [176,177]. Because integrins are involved in cell migration, it is reasonable to propose that immigration of these cells into or beyond the fetal liver might require steps that include the expression of α4β1, or induce the expression of integrin α5β1 at this stage of the liver. Integrin α4β1 is used by trafficking HSCs throughout life to enter sites of hematopoiesis [148], so the endowment of expression of integrin α4β1 permits fetal liver HSCs to be assayed by transplantation in fetuses or adults. It is important to note that neither yolk sac blood island HSCs nor ESderived HSCs in their native state can be transplanted into adult irradiated mice. It has been reported that the transition of yolk sac HSCs and ES-derived HSCs from that embryonic stage to the fetal stage can be facilitated by enforced expression by HoxB4, or by HoxB4 and cdx4 in these cells [118]. Therefore, there are important clues to how embryonic HSCs, presumably capable of primitive hematopoiesis, can develop into fetal HSCs capable of definitive hematopoiesis. During the various stages of fetal HSC function, it seemed possible that waves of HSCs emerge from tissues such as the fetal liver at specific time points to be accepted by and to engraft in secondary sites such as fetal spleen, fetal bone marrow, and lymphoid sites such as the thymus [93]. However, HSCs are found constitutively in the blood throughout fetal life, and it must be the preparedness of the developing organs such as the spleen, marrow, and thymus that results in the movement of hematopoiesis to those sites [178]. A detailed examination of HSCs in the developing fetal liver with respect to T-cell developmental potentials has revealed that changes are occurring at the level of HSCs which show that HSCs develop in a quantal but dynamic process [179]. Developing T cells gain their specificity and function in a coordinated series of molecular events, including selection of which class of T-cell receptor (TCR) genes are rearranged and selected, and to which distant sites the T cells will migrate. Within the thymus during fetal development, the first T cells to emerge use products of the TCR Vγ3 and TCR δ gene families, to be followed by use of the TCR Vγ4, then Vγ2 and Vγ5 and TCR αβ gene families. In mice, the TCR V genes at the γ locus that must rearrange to be expressed are, extending from the most proximal site of potential rearrangements, Vγ3, Vγ4, Vγ2, and then Vγ5. The first T cells to emerge in the thymus are Vγ3 cells, which colonize the skin only during fetal life [180]. While Vγ3 T-cell development is shutting down in the thymus, Vγ4 T-cell development arises, and these T cells will migrate during their fetal life to the presumptive female genital tract epithelia and the tongue epithelium of both sexes [179,180]. The development of Vγ4 T cells decreases at about day 16 or 17 of fetal life, to be overtaken by the almost simultaneous onset of that will inhabit the entire gastrointestinal tract, and αβ T cells that are largely restricted to lymphoid tissues. The kinetics of T-cell development can be studied with the use of fetal thymic organ cultures, although it should be noted that these are not physiologic cultures. When single clonogenic dpc 12 fetal liver HSCs are added to fetal thymic organ cultures, those thymuses produce Vγ3, then Vγ2, then Vγ5, and TCR αβ T cells [179]. When dpc 14 fetal liver HSCs are placed at clonal levels into the same stage fetal thymic organ culture, Vγ3 T cells do not emerge, but the rest of the repertoire is developed. Finally, dpc 12 fetal liver HSCs added directly into the adult thymus cannot produce the fetal program of Vγ3 T cells, but can produce the mature program of Vγ2, Vγ5, and αβ TCR cells [179]. Taken together, these studies indicate that, probably with each cell division, the fetal liver HSCs are changing (usually reducing) their developmental fates that would only be read out when their progeny entered the fetal thymus, and the change in these fates and the readout of these fates is dependent upon factors within HSCs and within the thymus microenvironment. After birth, and at least in young adult mice, the numbers of LT-HSCs, ST-HSCs, and MPPs are regulated at a relatively constant level.

43

HSCs in the mouse go through cell autonomous changes in number and developmental potential during aging. We first reported that there is a gradual loss of cells of the MPP phenotype, and an increase in both the number of LT-HSCs and the fraction of LT-HSCs that are in the cell cycle [83], most dramatically seen in the marrow of geriatric mice. With a refinement of markers for LT-HSC versus all other MPPs, we found that the number of LT-HSCs increased as reported, but that they retained a low level of cell-cycle activity; the increase was found primarily in an MPP fraction [96]. In old mice, myelopoiesis is retained or even enhanced, while lymphopoiesis in both T and B lineages falls. These fates are cell intrinsic; co-transplantation of 2-month-old and 2-year-old LT-HSCs into irradiated young mice led to increased output of LT-HSCs from the old versus young stem cells, and decreased output of CLP and T and B cells from the old HSCs compared with the young stem cells [96]. These same trends were seen in the small number of aged hosts transplanted with young and old HSCs. The cell intrinsic biologic properties of old versus young HSCs were mirrored by a microarray analysis of these highly purified cells: young HSCs had both myeloid and lymphoid transcripts, but old HSCs overexpressed myeloid and underexpressed lymphoid transcripts [96]. Of the top 32 myeloid transcripts overexpressed in old HSCs, 13 have been identified as proto-oncogene or translocation partners of myelogenous leukemia proto-oncogenes in humans. As myelogenous leukemias increase in incidence with age, it is reasonable to propose that initiating events may occur in HSCs, and if so, it is possible that genomic events such as translocation, inversion, etc, occur preferentially at highly transcribed loci. Does hematopoiesis only derive from HSCs, and do HSCs only give rise to blood? Several years ago, there were reports that bone marrow hematopoietic cells could give rise to brain cells, that brain stem cell populations could give rise to blood, that fat cells could give rise to neurons and mesenchymal fates, that muscle stem cells could give rise to hematopoietic and myogenic outcomes, and a host of others (reviewed in [181]). What was lacking in most of these experiments was the demonstration that the transplanted population was in fact a stem cell purified to homogeneity that could give rise at the clonal level to two different tissue types, and that the tissue types that had been generated were robustly characterized as functional mature cells of that tissue without cell fusion. Attempts to repeat the experiments showing transdifferentiation of CNS stem cells to hematopoietic tissues have thus far failed. A reexamination of the cells in muscle that give rise to blood show them to be CD45+ committed HSCs, separable from muscle progenitors that, on their own, give rise to muscle [182,183]. In some circumstances, regenerating muscle following an injury can incorporate cells whose markers are expressed in the regenerated muscle tissue, and the most common transplantable source of these cells is bone marrow [184,185]. While some claims have been made that these are derived from HSCs, in fact single purified HSCs, even over long intervals, cannot contribute to muscle or any other tissue, except a rare set of liver cells in irradiated hosts [5]. In another example, liver regeneration of hepatocytes bearing markers from injected purified HSCs, following multiple rounds of selection through liver toxicity, can occur [186], but recent evidence [187,188] indicates that these are mainly a result of rare cell fusion events between myelomonoctic progeny of the HSCs and some precursor of liver cells. Furthermore, the marrow or HSC or circulating precursor cells that enter undamaged or kainite-damaged brains are limited to microglia and CD45+ cells, as well as rare binucleate Purkinje cells derived by cell fusion [189]. We had previously proposed that cases of transdifferentiation of one tissue-specific stem cell to another tissuespecific stem cell might not really be transdifferentiation but differen-

44

Chapter 5

tiation from a more pluripotent precursor [1]. That possibility is still viable. The many claims that transdifferentiation could occur reflect a problem in stem cell biology as a young field. Most new investigators to the field did not appreciate the importance of purifying the populations to homogeneity, populations of cells of apparent phenotypic and functional homogeneity, and did not mark cells retrovirally or with other genetic markers to follow the fate of clonogenic precursors. The transition from discovery to accepted scientific fact It is important to note that, in order for a reported discovery to become accepted as scientific fact, the following several criteria need to be met: 1. The initial discovery must be published in fully peer-reviewed journals. 2. The experiment as published must be repeatable in many independent laboratories. 3. The phenomenon described should be so robust that other experimental methods must reveal it. 4. In the case of transplantation, the tissue regeneration must be therapeutic in quantity and quality. Based on these criteria, we do not believe there is sufficient evidence for any of the transplant claims of transdifferentiation. In addition, bone marrow is not the same as HSCs, but is a population of mature or maturing cells, and certainly includes at least two or three stem and progenitor populations: HSCs, mesenchymal stem cells (MSCs), and endothelial precursors, which might really be MSCs [18]. Thus, it is possible that the transdifferentiation ability or “plasticity” ascribed to HSCs to interpret formerly unexpected outcomes from cell transplantations, if not the result of cell fusion, is more likely derived from a class of previously unappreciated adult pluripotent cells, or perhaps the normal developmental fates of MSCs are not yet appreciated. It is conceivable that some of these very rare cell fusions could be part of a regenerative process, but there is no evidence today that such is the case. Thus, it is inappropriate for such initial claims to be considered true enough to be the basis of clinical trials or care protocols or public funding and policy decisions.

Lineage-committed hematopoietic progenitor cells Considerations for the definition/isolation of hematopoietic progenitors The developmental process from HSCs to mature cells must involve intermediates that have lost stem cell potentials but are not yet terminally differentiated. An important question and active area of research is the sequence in which lineage commitment occurs. Several findings have suggested the existence of oligopotent progenitors. First, the lack of lymphocyte in patients with severe combined immunodeficiency syndromes was taken as evidence for the existence of oligopotent, lymphoid-committed progenitor cells or lymphoid stem cells. However, the loss of lymphocytes caused by adenosine deaminase deficiency [190,191] or by mutations of the common cytokine receptor γ-chain (γc) [192,193] does not necessarily imply a common progenitor. Rather, several lymphoid cell types or their progenitors might be most susceptible to the induced alterations [194] or might be dependent on the same signal transduction mechanisms. This same caution needs to be applied to the interpretation of hematopoietic phenotypes of genetically altered mice, as mutants of Ikaros [195] or Notch-1 [196] that could either be essential factors for a common or multiple different progenitors [197]. Second, leukemia cells can be viewed as cells that are arrested at early developmental stages but have gained self-renewal capacity. The occur-

rence of leukemias that either coexpress antigens normally associated with myeloid or lymphoid cell types (mixed-lineage leukemias) or that harbor two leukemia populations of clonal origin (bilineal leukemias) could be taken as evidence for the existence of bipotent B-myeloid or T-myeloid progenitors [198–203] (reviewed in [204–206]). However, leukemia clones could be derived from HSCs, from MPPs or from restricted progenitors that, because of their altered gene expression profile, display cell surface gene products which are normally not present at these developmental stages. Third, the fact that single cells in bone marrow give rise to colonies that contain all myeloid but no lymphoid progeny in vitro (colonyforming unit-granulocyte/erythrocyte/macrophage/megakaryocyte [CFUGEMM]) or in vivo (mixed CFU-S) is suggestive for the existence of oligopotent myeloid progenitors. However, cells that give myeloid readout could also be multipotent but did not find the conditions to read out all possible progeny [48,207,208]. In light of these problems, several prerequisites need to be met to define restricted progenitors in a developmental hierarchy. First, in order to prospectively identify candidate populations, they need to be isolated to highest possible homogeneity/purity. Second, if a population shows oligopotent differentiation activity, it must be demonstrated that at least some single cells within this population give oligopotent readout, i.e. that this population is not a mixture of different monopotent progenitors. Third, it further needs to be shown that single oligopotent progenitors are not HSCs or MPPs. These prerequisites are difficult to achieve. At any given time point, progenitor cells might undergo intrinsic “stochastic” random commitment events, might not find suitable microenvironments to read out in all their possibilities or might even not read out at all [209,210]. However, by applying the above criteria, we and others were able to identify oligopotent developmental intermediates in mice and humans. These data strongly support the hypothesis that multipotent hematopoietic progenitors first lose their self-renewal ability and consecutively commit to either the lymphoid or the myeloid developmental pathway. Although not formally proven, based on frequency, cycling status, and in vitro and in vivo expansion potential of bone marrow lymphoid and myeloid committed progenitors, it seems possible that all hematopoiesis develops through either a common lymphoid or common myeloid developmental stage. CLP cells and lymphoid development Lymphoid cell development from HSCs is dependent on externally provided differentiation, growth, and survival factors. Of those, IL-7 might be most important. Its cognate high-affinity receptor is a complex composed of the IL-7 receptor-α (IL-7Rα) chain [211] and the γc chain [193,212]. Neutralizing antibodies to IL-7 or genetic ablation of either IL-7 or IL-7Rα inhibits both T- and B-cell development in vivo [213,214]. Targeted deletion of the γc gene leads to the loss of T and B cells and the additional loss of NK cells [215–217], presumably because of the impaired formation not only of the IL-7R, but also the IL-2R and IL-15R that might be nonredundant cytokines for NK cell development [218– 220]. Further, mice that lack Jak3, a signal transduction molecule associated with γc [221,222], display a phenotype similar to the γc-deficient mice [223–225]. Also, patients with genetic defects in Jak3 show similar defects in lymphoid development to patients with γc gene disruptions (X-linked severe combined immunodeficiency syndrome) [226,227]. Based on these data, IL-7 is recognized as a nonredundant cytokine for both T- and B-cell development that could act as a proliferation factor or a survival factor and/or could initiate lineage-specific developmental programs. IL-7R is expressed in both developing T and B cells, and in mature T cells [228]. In genetically modified mice that are either IL-7Rα−/−,

Biology of Hematopoietic Stem and Progenitor Cells

45

HSC

Population

MPP

HSC

Lin–KSCD150+ Flk2–Thylo(CD105lo CD34–FcγRlo)

MPP

Lin–KSCD150–Flk2+ Thylo/–(CD105lo, CD34–/+ FcγRlo)

GMLP GMLP

MEP

GMP ProT

Lin–KitintScaintIL7Rα+Flk2+

MCP

Lin–Kit+Sca–Ly6c–FcεRIα–CD27–T1/ST2+β7+

CMP

Lin–Kit+ScaloCD150+CD34+FcγR+

GMP

Lin–Kit+Sca–CD150+ Flk2–/+FcγRlo

MEP

Lin–Kit+Sca–CD150+ Flk2–FcγRlo

MkP

Lin–Kit+Sca–CD150+ Flk2–CD41+CD9+

BLP

MCP

MkP

Lin–Kit+ScaloFlk2++

CLP CLP

CMP

Phenotype

EP T cells

Mast cells

B cells

EP

Lin–Kit+Sca–CD150+ Flk2–CD105+

Dendritic cells Platelets

RBC

Myelomonocytic cells

Fig. 5.6 Schematic of hematopoietic development. Composite of current data from mice indicating intermediates in the hierarchy of hematopoietic differentiation. Hematopoietic cells (HSC), long-term reconstituting, self-renewing; MPP multipotent progenitors with limited or no self-renewal leading to transient but multilineage reconstitution; GMLP; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; BLP, B-lymphocyte progenitor; ProT, T-cell progenitor; GMP, granulocyte–macrophage progenitor; MEP, megakaryocyte–erythroid progenitor; MCP, mast cell progenitor; MkP, megakaryocyte progenitor; EP, erythroid progenitor.

γc−/− or IL-7−/−, T-cell progenitors express low levels of Bcl-2, an antiapoptotic protein [229]. However, incubation of T cells from IL-7−/− mice with recombinant IL-7 results in the upregulation of Bcl-2 [230], and enforced expression of Bcl-2 in either IL-7Rα−/− or γc−/− mice leads to significant rescue of αβ T-cell development [25,230,231]. Therefore, a critical role of IL-7 in developing and mature αβ T cells is the promotion of survival via expression of Bcl-2 or other antiapoptotic proteins as possibly Bcl-Xl [232]. However, enforced expression of Bcl-2 was not sufficient to rescue B-cell development and αβ T-cell development [25,87,230]. Here, IL-7R-mediated signals are necessary for the rearrangement of immunoglobulin heavy chain V segments via Pax-5 gene activation [233,234] and for V–J recombination of αβ TCR genes via Stat5 [235,236]. Therefore IL-7R signaling can either lead to the transmission of “trophic” survival signals in the T-cell lineage or “mechanistic” differentiation and proliferation signals in the B and αβ T-cell lineages, respectively. Thus, it was reasonable to search for oligopotent CLPs within the Lin−IL-7Rα+ fraction in adult mouse bone marrow. We identified a population of Lin−IL-7Rα+ Thy-1−Sca-1loc-kit lo cells that also express γc, indicating that this population possesses functional IL-7R (Fig. 5.6) [25]. In contrast, both LT-HSC and ST-HSC populations are IL-7Rα−. These CLPs possess rapid and potent T-, B-, and NK-cell-restricted differentiation activity in reconstitution assays. In both in vitro and in vivo assays, Lin−IL-7Rα+ Thy-1−Sca-1loc-kit lo cells completely lack myeloid differentiation activity [25]. Also no day 8 and day 12 CFU-S activity could be detected [25,37]. Injection of 1000 CLPs could generate 0.6– 1.1 × 107 CD3+ spleen T cells by 4–6 weeks, and 1.4 × 107 B220+ spleen B cells by 2 weeks in vivo. CLP-derived T- and B-cell generation peaks

7–10 days earlier than similar numbers of T and B cells derived from the same number of HSCs [25]. However, in contrast to HSC-derived cells, numbers of CLP-derived T and B cells begin to decline after 4–6 weeks, indicating that this population has no or limited self-renewal activity. Using a two-step assay, we demonstrated that CLPs contain clonogenic progenitors for both T and B cells. Single cells were cultured in methylcellulose in the presence of SLF, IL-7, and Flt3L to expand cell numbers, and a portion of the day 3 colonies were picked up and injected directly into the thymus. Those injected into the thymus differentiated into all stages of T-cell development and, in some cases, differentiated into both T- and B-lineage cells. The further cultured cells formed Blineage colonies composed of pro-B and pre-B cells [25]. Therefore, the defined CLPs match the criteria for the definition of oligopotent progenitor cells. We have subsequently shown that the purity of CLPs is further refined based on their expression of Flk2, whereas the Flk2 fraction of the previously defined CLPs contains B-committed cells [237]. Accordingly, CLPs exist downstream of Flk2+ ST-HSCs and MPPs in normal hematopoiesis [51,52,238]. Based on their frequency in bone marrow, their cycling status and their in vivo T-, NK-, and B-cell generation potential, CLPs theoretically could be a developmental intermediate for most if not all mature lymphoid cells. The commitment of CLPs to either the T-, B- or dendritic cell lineage may be determined simply by the microenvironment and signals that CLPs encounter. However, it is still unclear whether CLPs are an indispensable stage of T- and B-cell development. Coffman and Weissman identified the B220 marker that is present on all B-lineage cells [239], and early B-cell progenitors were found to be

46

Chapter 5

B220+, CD43+, CD24-BP1/6C3− [240,241]. Hardy and his colleagues further subdivided B-cell progenitors into fractions: pre-pro-B cells (fraction A0: AA4.1+CD4loB220−HSA−; A1: AA4.1+CD4+B220+HSA−; A2: AA4.1+CD4−B220+HSA−) and pro-B cells (fraction B: B220+CD43+HSA+6C3/BP-1−; C: B220+CD43+HSA+6C3/BP-1+) [242– 244] (reviewed in [245]). Some fraction A0 cells express c-kit and Sca1 and can give rise not only to B cell, but also to myeloid and T-cell progeny. Therefore, they might contain MPPs. The majority of fraction A1 cells are c-kit − and lack myeloid potential, but minor T-cell potential can be detected after intrathymic injection. Pro-B cells are B-lineage committed, rearrange DH–JH genes (fraction B) and undergo V–DJ recombination (fraction C). In view of their T- and B-cell readout capacity, the A0 or A1 fraction could contain CLPs, but because different markers were used for their isolation, a direct comparison of data from these populations is not possible. However, a majority of A0 and A1 fraction cells do not express IL-7Ra, while IL-7Ra is expressed on fraction A2 cells. B-lineage development has subsequently been studied in detail by a variety of groups using different combinations of markers that prevented direct comparison of the data [242,244,246–252]. Recently, we performed analyses to clarify these data by incorporating the majority of these described markers and others in our experiments. These studies allowed us to identify a committed B-lineage progenitor downstream of the CLP with the surface phenotype B220+CD43+AA4.1+Flk2+CD27+IL 7Ra+c-kitloCD24−CD19−CD11c−Ly6c−CD4− [253]. This population is distinguished from CLPs by the expression of B220 and comprises only approximately 5% of fraction A/pre-pro B cells, while the remaining cells are dendritic cell (DC) progenitors. Using the same markers described above, we also identified heterogeneity within the fraction B cells, which are thought to lie immediately downstream of the fraction A cells. One subset, which we term fraction B1, is highly proliferative, while the second subset, fraction B2, is largely quiescent and smaller in size. In contrast to the refined pre-pro B cells, fraction B1 cells do not respond strongly to IL-1, suggesting that the sensitivity to inflammatory signals within B-committed progenitors is restricted to pre-pro B cells and is lost immediately upon differentiation to their immediate downstream progeny. The earliest thymic progenitors (double-negative-1 cells) are CD4loCD8−CD44+CD25−c-kit+ cells [254–257] and therefore closely resemble the phenotype of HSCs. Most of these early thymic progenitors are T-cell committed (pro-T cells); however, some cells within this population are capable of differentiating into B, NK, and dendritic cells and, at a low frequency, into myeloid cells [257], but their clonal origin has not been established [256–258]. Whether the earliest thymic progenitor population contains a small number of recently homed CLPs that, within this microenvironment, preferentially differentiate into T cells, or whether CLPs commit to the T-cell lineage while still in bone marrow and successively home to the thymus [259], has been controversial. Allman et al. [260] suggest that thymopoiesis is maintained through early thymic progenitors that might develop from bone marrow progenitors more closely related to HSCs and not via a CLP-dependent pathway. This is based on findings that some cells (early thymic progenitors) within the double-negative-1 fraction do not express IL-7Rα at high levels, have some myeloid potential, give rise to T and B cells with kinetics that resemble those of ST-HSCs [52] more closely than those of CLPs, and were present at near-normal frequencies in Ikaros−/− mice while CLPs were not detectable by phenotype. While these data add important information on possible alternative T-cell developmental pathways, they do not assess directly how many early thymic progenitors or mature T cells are CLP-derived or whether most thymopoiesis is independent of CLPs. Both HSCs and CLPs might home to the thymus,

where T-cell commitment might immediately be initiated and thymus homing receptors might be lost. The issue of progenitor commitment to the T-cell lineage in bone marrow versus thymus is further complicated by the observation that, at least in experimental settings, extrathymic T-cell development can occur [261–263]. Recent studies comparing (Flk2+CD27+) MPPs and CLPs for their ability to seed the thymus and generate T cells indicate that CLPs are indeed the major source of normal thymic development (Serwold, Erhlich, and Weissman, submitted for publication). Both MPPs and CLPs injected intravenously give rise to thymocytes, but the MPPs do so with delayed kinetics reflective of their homing to the bone marrow and giving rise to CLPs that then go on to seed the thymus directly. Additionally, thymic myeloid cells were observed to derive from transplanted MPPs and not CLPs, suggesting that these cells are the progeny of myeloid progenitors that also enters the thymus. Further, when Flk2+ CLPs were plated in conditions capable of supporting differentiation into multiple lymphoid and myeloid lineages, single CLPs gave rise to T, B, NK, and DC lineages [264]. Approximately 50% of single CLP clones gave rise to both B- and T-lineage cells in this assay, and none gave rise to any macrophages or neutrophils. Taken together, these data confirm the role of CLPs as potent progenitors of both B and T lymphocytes in vivo and in vitro. Also of note, CLPs co-transplanted in addition to HSCs in autologous or allogeneic transplantation models can rescue mice from otherwise lethal cytomegalovirus (murine CMV) infections [265], providing additional evidence for the robust and functional T- and/or NKcell reconstitution capacity of CLPs in vivo. In humans, evidence for a T/NK bipotential precursor population in fetal thymus [266] and a terminal deoxynucleotidyl transferase (TdT)positive candidate lymphoid precursor population in CD34+ bone marrow cells have been reported [267]. However, clonal analysis was not carried out. A subpopulation of the CD34+TdT+ cells expresses the neutral endopeptidase CD10 [268]. Later, it was shown that fetal and adult bone marrow Lin−CD34+Thy-1−CD38+CD10+ cells contain clonal progenitors of B, NK, and dendritic cells [269]. As a population, these cells give rise to T cells in the SCID-hu thymus assay but could not generate myeloid progeny. In another report, it was shown that cord blood CD34+CD38−CD7+ cells contain clonal B, NK, and dendritic cell precursors; however, T-cell readout was not evaluated [242]. Therefore, both studies suggest but do not formally demonstrate the existence of CLPs in humans. IL-7 signaling might not be essential for normal B-cell development in humans because B cells can be generated without IL-7 in vitro [270], and disruption of the IL-7R in vivo causes T-cell but not consistently B-cell deficiencies [271,272] (reviewed in [268]). Human early lymphoid progenitors, in analogy to mouse CLPs, express IL-7Rα. Indeed, Lin−CD34+CD38+CD10+ cells can be subdivided into a CD10+IL-7Rα− and a CD10loIL-7Rα+ fraction, with the latter being highly enriched in clonal B-cell progenitors [273]. It has also been shown that pro-B cells proliferate and differentiate in culture with IL7 and bone marrow stromal cells, whereas pre-B cells are no longer responsive to IL-7 [274]. It is important to determine whether CD10loIL-7Rα+ cells or CD10+IL-7Rα− cells contain both T- and B-cell progenitors and whether thymus seeding cells share some of these phenotypes (for a review of human early thymocyte development, see [275]). CMP cells and myeloid development The identification of CLPs in the IL-7Rα-expressing fraction of mouse bone marrow which holds no myeloid differentiation activity [25] suggested that complementary progenitors common to all myeloid cells might exist in the IL-7Rα-negative cell fraction. In addition, we had noted that the Sca-1− subset of Thy-1loLin− cells contained myeloery-

Biology of Hematopoietic Stem and Progenitor Cells

throid progenitors [40]. Therefore, to exclude HSCs and CLPs, we searched for myeloid progenitors within the Lin−IL-7Rα−Sca-1−c-kit + bone marrow fraction. We identified three myeloid progenitor populations [26]: CMPs, FcgRloCD34+ (now known to be Sca-1lo not Sca-1− [276]), and their lineal descendants; megakaryocyte/erythrocyte progenitor cells (MEPs; FcgRloCD34−); and granulocyte/macrophage progenitor cells (GMPs; FcgRhiCD34+). These three myeloid progenitor subsets likely represent the major pathways for myeloid cell differentiation because they contain the vast majority of myeloid progenitor activity in steady-state bone marrow. CMPs give rise to all myeloid colonies in vitro, including CFU-mixed; GMPs give rise to CFU-granulocytes (CFU-G) and CFU-macrophage (CFU-M) as well as CFU-granulocyte–macrophage (CFU-GM); and MEPs give rise to burst-forming unit-erythroid (BFU-E), CFU-megakaryocyte (CFU-Meg) as well as CFU-megakaryocyte/erythroid (CFUMegE), each with high cloning efficiency. We demonstrated that CMPs as a population differentiate in vitro into cells with MEP and GMP phenotype and function [26]. Further, the majority of single CMPs generate both GM- and MegE-related progeny. Upon in vivo transfer, the three cell populations give short-term but not long-term readout corresponding to their in vitro activities, indicating that they have only limited if any self-renewal activity [37]. MEPs provide radioprotective cells for lethally irradiated mice housed under infection control conditions [37]. Lethally irradiated mice injected with MEPs, CMPs, ST-HSCs or MPPs show transient donor-derived hematopoiesis sufficient to sustain survival while reconstitution with residual host HSCs occurs [37,38,87]. In addition, the majority of day 8 CFU-S activity resides within the MEP population but is absent from GMPs [37], supporting previous findings that day 8–9 CFU-S are largely erythroid [40,277]. Our data also confirm previous findings that the majority of day 12 CFU-S activity resides not within lineage-committed progenitors but within the more primitive HSC populations with some activity in CMPs and MEPs [35,38,40]. Neither B- nor T-cell differentiation activity was detectable in either MEPs or GMPs. CMPs could not generate T cells; however, a small number of B-cell progeny were detectable in vitro and in vivo [26]. We also isolated a pure population of megakaryocyte progenitors in mice. These Lin−c-kit+Sca-1− CD34+CD9+CD41+ cells are highly efficient at producing megakaryocyte CFCs in vitro, micromegakaryocyte loci in vivo, and donor-derived platelet production in vivo, while they are devoid of erythrogenic activity [278]. The subsequent identification of additional markers has enabled higher-resolution delineation of myeloid developmental pathways. First, we and others found that the CMP population, like the CLP, was heterogenous for Flk2 expression [237,279] and the Flk2+ fraction contained the CMP activity and could give rise to GMPs, MEPs, and all myeloerythroid progeny including DCs. More recently, the addition of CD150 (Slamf1), CD105 (endoglin), and CD41 to the analysis revealed further heterogeneity within the myeloid progenitor populations [280]. These markers revealed that the previously defined CMPs could be further subdivided into CD150+ and CD150− fractions, and that MEPs can be divided into three subpopulations based on endoglin and CD150 expression. Detailed analyses with these markers led to the identification of isolatable pre-GM, pre-MegE, and pre-CFU-erythrocyte populations that give appropriate lineage restricted readouts in vitro and in vivo [280]. These data are consistent with another study that used a Gata1-GFP reporter to identify a population that refines the CMP phenotype or precedes it in the developmental scheme [276] (Fig. 5.6). Specifically, this population is Lin−Kit+ScaintFlk2−Gata1lo and gives rise in vivo to all myeloid lineages but very low lymphoid readout. Using a PU.1 reporter, this study also described a granulocyte–lymphocyte–macrophage progenitor that could give rise to lymphocytic and myeloid lineage cells but

47

not platelets, similar to results from Adolfsson et al. which indicated that those MPPs that express the highest levels of Flk2 have lost platelet-forming ability [281]. This is also consistent with data suggesting that PU.1 represses MegE development [282]. Alternative developmental pathways There are several recent in vivo studies as well as in vitro data suggesting that myeloid versus lymphoid differentiation may not always be the first decision made during hematopoietic development, and either bipotent B-cell/myeloid or T-cell/myeloid progenitors, or both, might exist. In support of a B/macrophage progenitor, it was demonstrated that several B-cell lines can be modified to produce cells with features of macrophages by in vitro manipulations [283–285]. Importantly, transfection of v-raf into B-cell lines established from Eμ-myc transgenic mice could convert them into macrophages that maintained the identical rearrangement of an immunoglobulin H (IgH) gene [284]. This observation suggests that latent developmental potentials that are not accessible under normal conditions might be regained through genetic alteration. Indeed, we have shown that CLPs, which under normal conditions never generate myeloid readout, produced granulocytes and macrophages after they were genetically engineered to express the human IL-2 receptor β chain (IL-2Rβ) or the granulocyte/macrophage colony-CSF receptor and were stimulated with the cognate ligands [286]. This was blocked by enforced expression of Pax-5 [287]. Also in vitro, single fetal liver cells could be demonstrated to give rise to both B/myeloid and T/myeloid progeny [207,288,289]. In these experiments using a fetal thymic organ culture system in the presence of 60% oxygen for single cell readout, Kawamoto et al. [289] detected B/T/myeloid, B/myeloid, T/myeloid, but never T/B-bipotent progenitors from fetal liver cell populations. However, in these high oxygen concentration cultures, only approximately 5% of single Lin−Sca-1+c-kit + HSCs gave rise to multilineage (T/B/myeloid) outcomes, suggesting that this system is not reproducible enough to demonstrate all differentiation capacities of HSCs. Also, differentiation into T/myeloid or B/myeloid lineages might simply be because of the “random” commitment of HSCs in these culture conditions. Alternatively, T/B-committed bipotent progenitors might not be contained in the Lin−Sca-1+c-kit + population but only in the Lin−Sca-1loc-kit lo CLP fraction [25,290]. Recently, bipotent B/macrophage progenitors were also described at a very low frequency (~0.02%) in adult hematopoiesis in vitro [291], supporting the hypothesis that a B/macrophage developmental line might be conserved beyond fetal hematopoiesis. As described above, recent data also suggest that HSCs and progenitors circulate through the blood and reside in the tissues before returning to the blood via the lymphatics [155]. Further, these cells were shown to differentiate in the tissue directly into dendritic, myeloid, and B220+ cells. This result, taken together with studies that HSCs respond to reduced hematocrit [76], in vitro data providing evidence for direct differentiation to megakaryocyte development [88], and studies suggesting oligo- and bipotent progenitors, suggest that HSCs percolate through the tissues as a means of directly assessing the specific hematopoietic and immune demands. It is reasonable to hypothesize that these cells reflect the responsiveness of the hematopoietic system and indicate that steadystate hematopoiesis when demand for each lineage is constant is distinct from hematopoieis that occurs during infection or blood loss, when there is a specific need for an increase in certain lineages. Being able to shut down production of lineages not in demand would also be a means of conserving resources such that white blood cells are not generated at the expense of RBCs during anemia and, conversely, RBCs and platelets are not generated at the expense of innate or specific immune cells when they are in demand.

48

Chapter 5

Human myeloid progenitors As in the case of CLPs, multiple studies suggested the existence of human oligopotent myeloid progenitors [292–296]. Recently, we were able to identify human bone marrow and cord blood cell populations that were counterparts of mouse CMPs, GMPs, and MEPs [273]. They are negative for multiple mature lineage markers (including early lymphoid markers that might define human CLPs as CD7, CD10 or IL-7Rα), and all are CD34+CD38+. They are distinguished by the expression of CD45RA, an isoform of the CD45 cell surface tyrosine phosphatase that can negatively regulate at least some classes of cytokine receptor signaling [297], and IL-3Rα, a receptor that upon activation supports the proliferation and differentiation of primitive progenitors [88,298,299]. CD45RA−IL-3Ralo (CMPs), CD45RA+IL-3Rαlo (GMPs), and CD45RAIL-3Rα− (MEPs) cells show high myeloid cloning efficacy but low long-term (stromal hematopoietic) culture-initiating cell capacity, indicating that they do not self-renew. Although FcγRII–III (CD16/CD32) expression distinguishes mouse CMPs and GMPs, it was not detectable on any of the human myeloid progenitor populations, and all human clonal myeloid progenitors express CD34 while mouse MEPs are CD34− [26]. Interestingly, in mouse myeloid progenitors, IL-3Rα as well as CD45RA expression were both negative to low in MEPs, intermediate in CMPs, and positive in GMPs, without providing sufficient discrimination to clearly set a cut-off level between the different cellular fractions. As with the mouse myeloid progenitors, CMPs give rise to MEPs and GMPs in vitro, and a significant proportion of CMPs have clonal granulocyte/macrophage and megakaryocyte/erythrocyte readout potential. We did not test T-cell differentiation ability; however, no in vitro B- or NK-cell readout could be detected. However, some B cells might develop from large numbers of transplanted CMPs. As in the case of mouse CMPs, it is important to clarify whether the CMP population contains oligopotent B/myeloid progenitors, or monopotent B-cell progenitors occurred because of small numbers of contaminating B-cell progenitors in the large number of transplanted cells (see below).

Lineage commitment in fetal hematopoiesis While fetal liver HSCs show major similarities to adult HSCs, some phenotypic and functional differences exist [175,300–302]. Remarkably, fetal liver HSCs possess some cell fate potentials such as the generation of Vγ3+ and Vγ4+ T cells [179] and B-1a lymphocytes [262,303] that are not detectable in adult HSCs. Therefore, it was important to determine the phenotype and developmental capacities of fetal liver CLPs and CMPs. We recently identified fetal liver CLPs (Lin−IL-7Rα+ B220− /lo Sca-1loc-kit lo) [290], and the myeloid progenitors fetal liver CMPs (Lin−IL-7Rα−Sca-1−c-kit+AA4.1−FcgRloCD34+), fetal liver GMPs (Lin− IL-7Rα−Sca-1−c-kit +AA4.1−FcgRhiCD34+), and fetal liver MEPs (Lin−IL7Rα−Sca-1−c-kit+AA4.1−FcgRloCD34−) [304] that show high similarities to their adult counterparts. However, some significant differences in proliferative potential colony-forming activity, and lineage differentiation capacities exist. Of special note, fetal liver CLPs generate CD45+CD4+ CD3−LTb+ cells that are candidate initiators of lymph node and Peyer’s patch formation [305–308]. About 5% of these IL-7R+ fetal liver CLPs differentiated into macrophages as well as into B cells, at least in vitro. Interestingly, the fetal liver CLPs fail to express Pax-5, a suppressor of macrophage development [306] while adult marrow CLPs that lack myeloid potential are Pax-5+ [290]. Conversely, although fetal liver CMP and downstream fetal liver MEPs or fetal liver GMPs do not give rise to T cells, fetal liver CMPs possess some in vitro and in vivo B-cell potential. A close relation between B-cell and macrophage development for adult and fetal hematopoiesis was suggested before (see below). It can therefore be speculated that, during commitment to the

lymphoid or myeloid lineage in fetal liver hematopoiesis, the developmental capacity for macrophages and B cells might be the last to be lost in commitment to either the lymphoid or myeloid lineage. DC development DCs were initially defined as bone marrow-derived nonmacrophage leukocyte populations with high antigen presentation capacities and the ability to prime naïve T cells [309–311]. To date, with their numbers still growing, multiple DC subpopulations that differ in tissue distribution, surface marker expression, and functional capacities have been described in humans and mice (reviewed in [312,313] and Chapter 19). From an evolutionary point of view, DCs might belong to the myeloid as well as to the lymphoid lineage because they link the innate and adaptive immune responses. Based on current data, two opposing models of DC ontogeny can be suggested: either the mature DC subpopulation is determined at the level of an early hematopoietic progenitor or, alternatively, immature DCs retain the capacity to mature to distinct DC subpopulations in response to different environmental instructions. In mice, two major DC subpopulations are distinguishable by their expression of the CD8α chain [314]. It was reported that early intrathymic progenitors (CD4loCD44+CD25−c-kit +) and thymic pro-T cells (CD44+CD25+c-kit +) contain precursors capable to differentiate into CD8α+ DCs in vivo [258,315]. Therefore, and because the majority of thymic DCs and only a minority of secondary lymphoid organ DCs express CD8α+, the CD8α+ DCs had been considered to be related to the T-lymphoid lineage and were therefore termed “lymphoid DCs” in contrast to the CD8α− “myeloid DCs.” This model seemed to be supported, but not directly demonstrated, in mice deficient for the transcription factors RelB [316], PU.1 [317], and Ikaros [318] that lack only CD8α− DCs. However, other mutants, as in c-kit −/− , γc−/− [319], and Notch1−/− [320] mice, showed a developmental dissociation of CD8α+ DC and T cells. We and others therefore tested DC developmental potentials in adult and fetal lymphoid and myeloid committed progenitors. Surprisingly, CD8α+ and CD8α− DCs were produced both by highly purified CLPs and CMPs with similar efficacy on a per cell basis, therefore disproving the concept of CD8α+ “lymphoid”- and CD8α− “myeloid”-derived DCs in mice [290,320–322]. Further, beyond CLPs and CMPs, DC developmental potential is conserved in the lymphoid lineage in pro-T cells and in the myeloid lineage in GMPs, but it is lost once B-cell or megakaryocyte/erythrocyte commitment occurs [320,321]. In contrast to mice wherein DCs or their precursors are usually isolated from the lymphoid organs or bone marrow, human DCs and their precursors have usually been isolated from peripheral blood and only in rare cases from blood-forming organs, making direct comparisons difficult (reviewed in [313]). However, DCs could be generated from total CD34+ progenitor populations, lymphoid-restricted progenitor populations, and peripheral blood monocytes, suggesting that, in analogy to mice, DC developmental potential is conserved in myeloid- and lymphoid-restricted lineages [269,323–328]. Recently, an immediate DC precursor population that upon stimulation can produce high amounts of interferon-α, displays a set of distinct pattern recognition receptors, and can mature into DCs in vitro, was identified in humans [329–332] and later in mice [333–335]. This DC subpopulation has alternatively been referred to as plasmacytoid DCs, DC2 cells, and interferon-α-producing cells [312]. These cells can derive from both myeloid and lymphoid progenitors (reviewed in [336]) but are predominantly of myeloid origin [337]. Taken together, DCs are developing along a lymphoid and myeloid pathway in vitro and in vivo. By using simple assays as immunophenotyping, or functional readout as mixed lymphocyte reaction and cytokine

Biology of Hematopoietic Stem and Progenitor Cells

production, we did not detect differences in lymphoid precursor- and myeloid precursor-derived CD8α+ DC fraction DCs [321]. This is an unexpected finding, because it suggests developmental redundancy for DCs. Of note, DC developmental capacities in CLPs and pro-T cells correlate with their latent myeloid developmental potential that can be “rescued” by artificial introduction and activation of the IL-2β or GMCSF receptor [286,338]. It would therefore be important to characterize the signals that are involved to retain DC developmental capacities in the otherwise exclusively lymphoid- or myeloid-committed progenitor cells. Activation of the Flt3 tyrosine kinase could be one of them [237].

Gene expression profiles of HSCs Several powerful technologies have allowed the characterization of transcripts expressed in defined cells, populations of cells, tissues, and organs. These technical advances allow for reductionist approaches to understand the complexity of RNA transcripts found in these cells or tissues, and how the transcriptional profile changes upon differentiation, self-renewal or other functional events. One of the most powerful technologies is microarray analysis, wherein the expression profile of transcripts for a high fraction of all known genes in the species can be ascertained for purified cell populations. Several groups have performed microarray expression profiling of hematopoietic stem and progenitor cells from fetal, adult, and aged mice [55,56,94,97,98,238,280,339,340]. These studies have identified several genes that have been subsequently validated as important for HSC function, such as Bmi1 [97], or useful for HSC purification, such as Slamf1 [55,56]. Gene subtractions or other differential expression techniques have revealed a subset of genes that appear to be expressed in HSCs but not broadly elsewhere. In fact, some genes have been found that are expressed in HSCs and neurosphere cultures (highly enriched for CNS stem cells) [341], or HSCs and ES cells, or all three [140,342]. These gene expression arrays cannot detect important events that are essentially post-translational, for example the phosphorylation and dephosphorylation of proteins in signal transduction pathways, or the changes in subcellular location of proteins such as β-catenin under conditions when they are held within the degradation complex, or attached to the cytoplasmic face of cadherins, or allowed to be free in the cytoplasm and appear in the nucleus [122]. Most of the oligolineage progenitors downstream of mouse HSCs [25,26] and human HSCs [63,269,273,323] have been isolated to homogeneity, and in some instances a comparison of their gene expression profiles reveals important clues as to genes responsible for particular commitments to differentiated fates [140,342] (see below). It is reasonable to expect that the reductionist approach to cataloging genes in this way will reveal many new clues about stem cell behaviors, but it will require a similar cataloging of cellular proteins (proteomics) to begin to reveal cell protein modifications that lead one to identify important pathways in stem cell behavior.

Gene expression profile of stem and progenitor cells An important question is how the progeny of multipotent cells adopt one fate from a choice of several. Lineage commitment and subsequent differentiation of multipotent cells should involve the selective activation and silencing of particular gene expression programs. There are single or pairs of transcription factors that have “master” roles in hematopoiesis in altering the phenotype of hematopoietic cells in defined circumstances. Combinatorial control is operated at the level of two or more lineage-specific regulators. This transcriptional control might be more complicated, considering protein–protein interactions mounted by the

49

formation of multiprotein hematopoietic-specific protein complexes [343], and changes in the expression levels of transcription factors [344]. Furthermore, as in the case of cytokine signals, roles in transcription factors are also sometimes redundant. Finally, single MPPs appear to coexpress transcription factors and cytokine receptors related to multiple lineages [345]. Promiscuous expression of multiple myeloid or lymphoid genes in hematopoietic branchpoints Prospectively purified LT-HSCs, oligopotent progenitors, and lineagecommitted progenitors have been used for a definitive sampling of the transcriptional profiles of cells at these particular stages of physiologic hematopoiesis. The expression patterns of lineage-affiliated genes in each stage of hematopoiesis are largely consistent with their known functions. Using semiquantitative RT-PCR assay targeted for each purified population, SCL (stem cell leukemia) [346,347] is expressed in HSCs and all myeloid progenitors. GATA-2 [348] and c-mpl are expressed in HSCs, CMPs, and MEPs, but not in GMPs [26,94,113]. Other MegErelated genes such as NF-E2 [347], GATA-1 [349,350], and the erythropoietin receptor are expressed in CMPs and MEPs but not in GMPs, and their expression levels are highest in MEPs [113,340]. Granulocyteaffiliated transcription factor C/EBPα [351] is expressed in HSCs, CMPs, and GMPs but not in MEPs, and its expression level is highest in GMPs [113,340]. All of these genes are expressed in CMPs, but not in CLPs or other late lymphoid progenitors, suggesting potential roles for each in myeloid-specific cell fate decisions. In contrast, the lymphoid-related transcription factor Aiolos [352] is not expressed in any myeloid progenitors, nor in HSCs. Ikaros, an Aiolos partner nuclear factor, thought initially to be a lymphoid-specific transcription factor [195,353], is expressed in HSCs and all stages of hematopoiesis [354]. Aiolos first appears at the CLP stage and is highest in T- and B-cell progenitors [26,113]. Lymphoid-related GATA-3 [355] and Pax-5 [356] are expressed at a low level in adult marrow CLPs but not in myeloid progenitors and fetal liver CLPs [26,290]. These data provide a clearer view of the regulation of gene expression in hematopoietic development. First, the expression of many myeloid and lymphoid genes is mutually exclusive in each pathway. Second, CMPs express both MegE- and GM-affiliated genes, and CLPs express both T- and B-lymphoid genes, although CMPs and CLPs have not committed to myeloid and lymphoid sublineages, respectively [26,113]. These data are consistent both with the priming hypothesis [357] and a model wherein chromatin is open prior to commitment, but the transcripts from that open chromatin are not critical for maturation [113]. The view of myeloid or lymphoid promiscuity has been significantly extended by using an oligonucleotide microarray analysis targeted for purified CMPs and CLPs. Genome-wide profiling using microarray methods revealed that CMPs and CLPs coexpress a vast majority of GM- and MegE-affiliated genes, and T-, B-, and NK-lymphoid genes, respectively [94,340]. Furthermore, coexpression of both GM- and MegE-related genes in CMPs and of both T- and B-lymphoid genes in CLPs have been formally demonstrated at the level of single cells [94]. By using RT-PCR analysis targeted for single cells, CMPs coexpress MegE-related β-globin, erythropoietin receptor, NF-E2 and GM-related myeloperoxidase, G-CSF receptor and PU.1, while CLPs coexpress Bcell-related l5 and/or Pax-5 and T-cell-related CD3d and/or GATA-3 [113]. These results indicate that the expression of lineage-related genes can precede commitment, and suggest that the “promiscuous” expression of multiple lineage-related genes may allow a flexibility of commitment at these oligopotent stages: for differentiation towards T- and B-cell lineages in CLPs, and towards GM and MegE lineages in CMPs.

50

Chapter 5

It is still unclear whether the primary mechanism of hematopoietic commitment could be operated first by opening chromatin at programmed sites, or whether the “priming” seen in promiscuous transcription patterns results from the presence of open chromatin and transcription factors that can form “sterile” transcripts. Nonetheless, in priming stages, multiple differentiation programs might collaborate or compete with each other until one becomes dominant. These programs should include cross-talk among transcription factors or transcriptional complexes, because transcription factor activity can be potentiated [358] or suppressed [359,360] by interaction with other transcription factors. Changes in levels of key transcription factors [344,361] may also be critical for the lineage decision. Cross-talk between different cytokine signals may also be important, as single progenitors coexpress multiple cytokine receptors. Further, fluctuations of gene expression of multiple differentiation programs in progenitors may lead to different outcomes depending on the microenvironment the cell inhabits. In this context, both intrinsic and extrinsic factors could play a part in lineage determination. However, data comparing HSCs from young and old mice show an intrinsic change in their differentiation potential with decreasing lymphoid potential with age, even when transplanted into a young mouse [96]. These data suggest that at least some portion of lineage determination is independent of the environment or that aging HSCs irreversibly lose responsiveness to signals provided by it. One way to view the hematopoietic system is to assume that, at or around the stage of yolk sac hematopoiesis, a pool of HSCs are created that are the only source of hematopoiesis for life. Each early HSC then is a clonogenic precursor of other HSCs, which self-renew, and their progeny, which do not. Over time and perhaps with a cell divisioncounting developmental clock, the clones begin to diversify in terms of responsive potential; these intrinsic epigenetic, and rarely genetic, changes prime the clones for their responsiveness to normal cues, such as diffusible or membrane-bound factors, or to pathologic cues, such as bacterial endotoxins. At any point, the clones that are present represent clonal competitions up to that point. In this way, intrinsic programs can select certain clones over others, but within an inbred colony of pretty clean mice, the clones chosen tend to be consistent, mouse-to-mouse. We later discuss changes that not only enable clonal selection, but are also proto-oncogenic in nature, so that the long process of preleukemic clonal progression is initiated and spread through the HSC clones.

Downregulation of genes irrelevant to committed lineages as a critical mechanism of lineage restriction According to the “priming” model, commitment could be dependent on two independent molecular events: upregulation of differentiation programs related to selected lineages, and the transcriptional abrogation of master control genes in the unselected lineages. A striking example is Pax-5, a key molecule for restriction to B-cell lineage [356]. In normal hematopoiesis, Pax-5 expression initiates at the CLP stage, and is upregulated in pro-B cells, but undetectable in pro-T cells or myeloid cells. Expression of Pax-5 in pro-B cells is important not to differentiate into other lineage cells, because pro-B cells from Pax-5-deficient mice (Pax5−/− pro-B cells) can differentiate into T cells, NK cells, granulocytes, macrophages, and osteoclasts as well as B cells [356]. Pax-5−/− pro-B cells rearrange the D–J locus of IgH genes and express other pro-B related genes. Thus, even after a set of B lineage-associated genes begins to be transcribed, differentiation programs for other lineages are still accessible. Conversely, enforced Pax-5 expression in myeloid progenitors suppresses myeloid development, presumably blocking signals from myelomonocytic cytokine receptors such as G-CSF receptor and GMCSF receptor [362].

Similarly, enforced expression of PU.1 in pro-T cells can block T-cell differentiation [363]. Ectopic GATA-1 expression in GMPs also inhibits differentiation into granulocyte/monocyte lineages, but induces transdifferentiation into megakaryocytes and erythrocytes [364]. A similar phenomenon is observed in ectopic cytokine receptors. Ectopic expression of IL-2Rβ or GM-CSF receptor in CLPs can deliver signals that redirect cells to myelomonocytic differentiation [286]. Further characterization of this latent myeloid potential revealed that CCAAT/enhancerbinding protein (CEBPα) is upregulated in response to the ectopic IL2 signal and leads to GM-CSF receptor expression and further myeloid differentiation [365]. This study also revealed that enforced expression of CEBPα in CLPs is itself sufficient to induce the myeloid outcome and, conversely, enforced Pax-5 expression blocks the myeloid differentiation induced from IL2Rβ. Similarly, Laiosa et al. showed that committed T cell progenitors could be induced to develop into macrophages or DCs by enforced expression of CEBPα or PU.1, respectively. These data suggest that committed T-cell progenitors are also susceptible to the lineage-instructive effects of myeloid transcription factors, but that T-lineage development occurs by the normal role of Notch signaling downregulating CEBPα and PU.1 in multilineage precursors. These data indicate that termination of lineage-related gene expression is equally important for completion of lineage commitment. First, in committed progenitors, abrogated differentiation programs for unselected lineages are not totally erased immediately after commitment, but could be reactivated by “instructive” signals mediated by ectopic cytokine receptors and transcription factors. For example, GMPs might be restricted to GM lineages simply because GATA-1 is downregulated, and CLPs are likely lymphoid restricted because they downregulate myeloid cytokine receptors. Second, programs for differentiation into committed lineages can be operated on condition that transcription factors for unselected lineages are downregulated, as Pax-5 and PU.1 inhibit myeloid and T-cell differentiation programs, respectively. Thus, a hierarchy of transcription factors and cytokine receptors organized at the epigenetic level is critical to maintain hematopoietic homeostasis. Changes in chromatin structure, allowing access for RNA polymerase to initiate transcription, are essential for genetic programs to be transcribed [366,367]. The activation of chromatin structure can occur prior to significant expression of genes [368]. It has been hypothesized that a wide-open chromatin structure is maintained in early hematopoietic progenitors, enabling multilineage-affiliated programs to be accessible [369]. This phenomenon of open chromatin in early hematopoietic progenitors could be the mechanism by which lineage priming occurs in stem or progenitor cells prior to their lineage determination [140,358,370,371]. Standard methods for studying chromatin structure require large numbers of cells, which has made their application to stem cell biology difficult, but we and others have made recent technical advances that have allowed chromatin immunoprecipitation to be performed on rare cells [372–374] so that we have been able to determine whether the epigenetic marks in control regions of lineage-directing genes modulate during hematopoiesis. Specifically, we analyzed histone modifications and DNA methylation patterns within the regulatory regions of erythroid (Gata1 and β-globin), myeloid (c-fms), and lymphoid lineage-affiliated genes (Gata3 and Ptcrα) in 50,000 prospectively purified stem and progenitor cells. By performing bisulfite sequencing analysis, we found that methylated H3K4 and AcH3 and unmethylated CpG dinucleotides localized at defined regulatory regions of these lineage-affiliated genes in HSC. These histone modifications are epigenetic markers of active transcription and either accumulated or were replaced by increased DNA methylation and H3K27 trimethylation in committed progenitors consistent with the pattern of gene expression. We also observed bivalent

Biology of Hematopoietic Stem and Progenitor Cells

domains at lymphoid-affiliated genes in HSC and downstream transitamplifying progenitors. Bivalent domains described by Bernstein et al. contain both activating and silencing histone modifications [375], and are associated with genes such as transcription factors that are expressed at low levels and affect developmental decisions. These data support the role of epigenetic modifications as being reflective of HSC multipotency and suggest that the remodeling of chromatin is an important mechanism of driving gene expression changes associated with developmental decisions.

Transplantation of HSCs in mouse and humans It is fair to say that the field of bone marrow transplantation, now more accurately called HCT, has provided one of the most striking medical advances in our lifetime and, similarly, the desire to understand cells involved in such transplants has opened the field of stem cell biology, first to HSC but then to other tissue-specific stem cells. It is important at the outset, however, to make sure that the scientific and clinical community uses the appropriate nomenclature for the type of transplant used as misconceptions as to the cell type transplanted lead to misconceptions about the scientific or clinical results obtained [14,15]. Thus, the transplantation of complex mixtures of hematopoietic cells in bone marrow or MPB is not the same as transplanting purified HSCs. Clinical HCTs have been used for a variety of purposes, but their primary use is the ability to regenerate the hematopoietic tissues of a cancer patient who has received otherwise lethal doses of a high-dose regimen of chemoradiotherapy [376,377]. Other uses have been the correction of inherited or acquired hematopoietic cell defects and dysplasias, and in animals the induction of transplantation tolerance to other cells, tissues or organs co-transplanted from the HSC donor, the hematopoietic cell donor, and also the reversal of autoimmune disease [19] (see Chapter 20). It was not clear whether purified HSCs could replace HCT, as it was not clear whether a rapid and sustained regeneration of a particular hematopoietic outcome could be accomplished with the primitive HSCs alone, without having oligolineage progenitors in the transplant. Several experiments have demonstrated in mice and in humans that HSCs are the principal cells that are functional in the transplant setting, and that

51

by varying cell dose one could achieve both rapid and sustained regeneration of hematopoietic tissues [378]. Prospective isolation of HSCs with multiple markers allows the possibility that grafts would consist solely of HSCs and of no other contaminating cells. There are two important advantages, at least theoretically, to the transplantation of HSCs rather than HCT removal of cancer cells and removal of T cells. Autologous HCT from cancer patients can be accompanied by transplantation of the patient’s cancer cells; in some diseases, such as stage 4 breast cancer and many lymphomas, the HCT populations to be transplanted that are contaminated with cancer cells vary from 20% to 40% of all transplants [379–381]. In patients with multiple myeloma, contamination of the graft is the rule and not the exception [382]. Therefore, it was important to test whether positive selection of HSCs would effectively eliminate or purge cancer cells from the transplant. Deliberate spiking of MPB with cell lines representing human breast cancer, human non-Hodgkin’s lymphoma and human myeloma was performed, and four methods of HCT/HSC isolation were carried out to determine the full reduction of contaminating cancer cells. Two independent methods to identify breast cancer cells were used: microscopy to detect cells carrying breast cell cytokeratins, and a flow-based method. Cells representative of myeloma and B-cell lymphoma were detected by PCR specific for the immunoglobin heavy chain rearrangements in these B-lineage cells. All three techniques have a sensitivity of between 10−5 and 10−6. Over 105-fold reductions of contaminating cancer cells were accomplished with the multiparameter cell sorting of CD34+Thy+ cells from MPB, whereas techniques based on CD34 alone were at least two to three orders of magnitude less efficient (Fig. 5.7). The time to reach engraftment with purified HSCs (measured by absolute neutrophil count >500/mL and platelets >20,000/mL of blood [379,380,383]) (Fig. 5.8) is very similar to the time obtained when unmanipulated MPB is used as the HCT transplant, and similar to the dose–response observed for mouse HSCs in syngeneic transplants (Fig. 5.9) [378]. Even in acute myeloid leukemia (AML), HSCs can be separated from leukemic stem cells (LSCs) by surface markers [384]. Therefore, the stage is set to test whether the transplantation of cancer-depleted HSCs will lead to clinical benefits when compared with the transplantation of cancer-containing unmanipulated HCT grafts.

Comparison of fold depletion of cancer cells from mobilized peripheral blood CD34+ Thy1.1+ (flow) CD34+ (flow) CD34+ (paramagnetic beads) CD34+ (magnetic beads)

Myeloma (CDRIII)

Non-Hodgkin’s lymphoma (BCL-2)

Breast cancer (IFM)

0

50,000

100,000

150,000

200,000

250,000

300,000

Fig. 5.7 Comparison between CD34 only and CD34+Thy-1 isolation of human stem and progenitor cells in terms of fold depletion of human cancer cells. MPB (1.6 × 1010) was spiked with 1.2 × 108 cancer cells (T47D breast cancer, SU DHL 6 non-Hodgkin’s lymphoma cells, or RPMI-LAV myeloma cells) prior to cell sorting. The CD34+Thy-1 cells were flow sorted with a modified high-flow cytometer, as were the first CD34 flow sort test. The other devices were a commercial paramagnetic bead-coupled monoclonal passed through magnetized steel mesh, and the last a large magnetic bead device. Breast cancer cells were analyzed by immunofluorescence microscopy for cytokeratin, the non-Hodgkin’s lymphoma by Igh-BCL-2 real-time polymerase chain reaction (PCR), and the myeloma by CDR III real-time PCR. The data are represented as fold reduction of cancer cells from the sorted CD34 or CD34+Thy-1 cells. (Hanania, unpublished data.)

52

Chapter 5

50 Platelets >20,0000/μL ANC >500/μL

40

Days

30

20

10

0

0

20

40

60

80

100

150

Fig. 5.8 Transplantation of highly purified human hematopoietic stem cells in patients with metastatic breast cancer. Shown are the times to engraftment of neutrophils (absolute neutrophil count [ANC] > 500/mL] ) following transplantation with purified CD34+Thy1-1+ hematopoietic stem cells. (Reproduced from [379].)

200

CD34+Thy1+ × 105/kg

Transplantation of high doses of purified stem cells results in early hematopoietic recovery 5000

WBC/μl (log)

1000

100

5000 & 10,000

1000

500 WBC/μl 100

Platelets × 103/μl (log)

10,000

7

14

21

100

200,000 plts/μl 100

10 5

10 0

1000

5000 & 10,000

1000

28

0

Days post transplantation

7

14

21

28

Days post transplantation

Fig. 5.9 Hematopoietic recovery in lethally irradiated C57BL/Ka mice syngeneically transplanted with different doses of purified hematopoietic stem cells (HSCs). Irradiated mice were injected with 100 (solid circles), 1000 (triangles), 5000 (solid triangles) or 10,000 (squares) HSCs. Shown are the recovery kinetics for white blood cell (WBC), and platelet (plts) counts. (From [378] with permission.)

Days to reach

KTLS cell dose 10,000 5000 1000 100

500 WBC/μl (10% of normal)

200,000 plts/μl (20% of normal)

11 11 15 22

11 12 14 23

KTLS . . . c-kit+ Thy-1.1lo Lin−/lo Sca-1+ cells

HSC cell dose effects in “autologous” versus allogeneic transplants in mouse Allogeneic barrier = H-2 differences at both major and minor loci WBC engraftment kinetics: Delayed (radioprotective dose)

Intermediate

10,000 Syn 100

WBC /μl (log)

Allo 1000

Allo 5000

Rapid

Syn 1000

Syn 5000

Allo 10,000

1000 500 WBC/μl 100 10 0

7

14

21 22

28

0

7

14 15

21

28

0

7 11 14

Days post transplantation

Allogeneic HCT transplants have been plagued from the beginning by the appearance of graft-versus-host disease as a result of T cells contained within the graft [385]. Mice receiving allogeneic HSCs have few or no contaminating T cells, and never develop graft-versus-host disease [378,386]. Dose–response comparisons of purified HSC transplants in syngeneic, in matched unrelated donor allogeneic, and in fully allogeneic circumstances have been carried out, and, for the most part, doses of HSCs sufficient to allow rapid and sustained engraftment can be achieved without contaminating T cells (Fig. 5.10) (see Chapter 20) [386]. However, the number of HSCs required for engraftment in the allogeneic setting can be 10 times higher than that required for syngeneic

21

28

Fig. 5.10 Engraftment kinetics of allogeneic versus congeneic purified hematopoietic stem cells (HSCs). Comparison of stem cell dose required to achieve early hematopoietic recovery in syngeneic versus allogeneic irradiated mice. Syngeneic or allogeneic mice were transplanted with purified Thy-1loSca-1+ Lin−/loc-kit + (KTLS) cells and bled on sequential days post transplant. The kinetics of white blood cell (WBC) recovery were categorized into delayed, intermediate, and rapid recovery. (Adapted from [378] with permission.)

HSC engraftment (Fig. 5.10) [378]. One can achieve nearly equal levels of engraftment at the same HSC dose with syngeneic and allogeneic models if the allogeneic hosts are preconditioned with high-dose conditioning regimens that include either facilitator cells [387–389] from the donor, or monoclonal antibodies that eliminate NK- and T-cell populations [386,390]. High-dose conditioning alone results in donor B-celland myeloid-lineage conversion [386–390], but persistent donor and host T-cell chimerism requires elimination of the host residual T and NK cells with facilitator cells or antibodies. There are two kinds of facilitator cell found in mouse bone marrow and lymphoid tissues: classic CD8α+TCR+CD3+ T cells and CD8α+ TCR−

Biology of Hematopoietic Stem and Progenitor Cells

CD3− facilitator cells [387–389]. The immunocompetent CD8α+ TCR+ T cells have some capacity for graft-versus-host disease, so there is great interest in understanding the nature and function of CD8α+ TCR−CD3− facilitator cells [387]. There are two other kinds of cell that share this phenotype: CD8α+ immature DCs, and veto cells [387,391,392]. We have proposed that all three cell types are in fact a single cell class, and that it is only the assays that makes one believe that they are different lymphoid cell subsets [387]. This view is strengthened by the recent findings of Steinman and Nussenzweig [393] that CD8α+ DCs that do not have active or appropriate co-stimulation can in fact lead to the inactivation or disappearance of T cells (and, we propose, NK cells) that have receptors which recognize them. Thus, a cell that facilitates engraftment of allogeneic HSCs might be a cell that presents major histocompatibility complex/peptide antigens to reactive T and NK cells without delivering a co-stimulatory signal, or induces the cells to undergo activation-induced cell death. These same cells might also be responsible for the veto of active CD4 and CD8 T cells that recognize and respond to them. It has been known since the pioneering work of Main and Prehn that bone marrow chimeras are usually transplantation tolerant to cells, tissues or organs from the hematopoietic cell donor [359]. This method of inducing transplantation tolerance has been substantiated with pure HSC transplants [387,390], in either the high-dose or reduced-intensity setting, and whether the transplant is concurrent with the HSC transplant or occurs months later [387,390]. Hematopoietic chimeras in humans are also usually specifically transplant tolerant of donor tissues, and new trials for inducing tolerance with bone marrow cells in a reducedintensity but lymphoablative setting along with organ allografts from the bone marrow donor are underway [394]. Partial or complete replacement of an autoimmune diabetes-prone hematopoietic system in mice with purified HSCs from diabetes-resistant strains of mice in the prediabetic period completely precludes progression to diabetes (see Chapter 20), whereas the same treatment of already-diabetic hosts along with donor strain islet cell transplants cures the small numbers of mice undergoing this protocol [395]. This too has been accomplished in both high-dose and reduced-intensity conditioning settings. Additionally, in a mouse model of systemic lupus erythematosus, Smith-Berdan was able to achieve greatly increased overall survival by treating mice with established disease with reduced-intensity conditioning and transplants of major histocompatibility complex haplomismatched allogeneic HSCs [396]. Mice that received transplants exhibited stabilization or reversal of their lupus symptoms, including proteinuria, and elevated circulating immune complexes or autoantibodies compared with control mice. These results indicate that induction of durable mixed chimerism by transplantation of purified allogeneic HSCs after reducedintensity conditioning has the potential to reverse symptoms of established autoimmune disease.

Co-transplantation of HSCs and hematopoietic progenitors to protect against opportunistic infections The bane of clinical HCT in terms of infectious disease is the often lethal infections with Aspergillus fumigatus, CMV, and Pseudomonas [397]. Small numbers of each of these infectious organisms given to HSC transplanted mice can result in the morbidity and mortality that occurs in patients [397]. With the isolation of clonal CLPs, CMPs, and GMPs, cellular therapies for each of these disorders have been investigated. Co-transplantation of HSCs and CLPs can completely preclude the lethality of murine CMV infection in both syngeneic and allogeneic protocols [265]. Co-transplantation of HSCs and CMPs and GMPs can prevent the lethal consequences of Aspergillus infections if the Aspergil-

53

lus is given on day 9, 11 or 14 after HSC transplantation, and addition of G-CSF to these HSC/CMP/GMP transplant protocols precludes Aspergillus infections given just 3 days after transplantation [397]. Similar CMP plus GMP protocols are sufficient to eliminate Pseudomonas infections, and in both cases these treatments work in both the syngeneic and allogeneic settings.

Origin of leukemia: LSCs Poorly regulated or unregulated self-renewal is a property that all cancers have, and we call those cells within a cancer that have self-renewal capacity “cancer stem cells” [122]. Therefore, it is important for the reader to keep in mind that the major distinguishing property of normal and neoplastic stem cells is their capability of self-renewal, and thus it becomes important to understand whether the pathways that lead to self-renewal are novel in normal HSCs, or might be shared between different classes of tissue-specific multipotent stem cells, and might be acquired by cancer cell progenitors as they emerge as cancer stem cells [122]. Pten, a tumor suppressor that negatively regulates proliferation, is required to sustain long-term hematopoiesis and self-renewing LSCs [398]. To understand the mechanisms of leukemic transformation, it is important to understand the life history of leukemic progression (Plate 5.2). If one views leukemia development as a series of steps in malignant progression, malignant self-renewal may be obtained through several transformation steps that include a block in differentiation, the retention or acquisition of self-renewal, and the avoidance of programmed cell death by several independent cell-intrinsic pathways and by intrinsic and adaptive immune system immunosurveillance. A set of early stem/progenitor cells with extensive proliferation, or with reduced programmed cell death, that are not yet malignant may form “preleukemic” clones, and these clones may receive additional oncogenic events that induce differentiation blocks at a progenitor stage to become leukemia. Accumulation of mutations as a precondition for malignant transformation in human acute leukemias Molecular markers for leukemia, including oncogenic fusion proteins resulting from leukemia-specific chromosomal translocations, have disclosed the multistep nature of acute leukemia development. A typical case is chronic myelogenous leukemia (CML). CML-specific t(9;22) Philadelphia chromosome and its resultant Bcr–Abl fusion gene has been shown to be present in all myeloid cells, B cells, and rare T cells. Additional mutation(s) and/or epigenetic changes in patterns of gene expression are required for blastic transformation into acute leukemia. There are many other examples of human adult and childhood leukemias that support the early acquisition of predisposing mutations occurring in HSCs that require additional events to result in leukemic transformation. AML1/ETO chimeric protein that is the product of the t(8;21) translocated genomic region is continuously detectable in patients with t(8;21) AML who maintain long-term remission for more than 10 years. Although these patients are clinically “cured,” AML1/ETO remains detectable in HSCs (Thy-1+CD34+Lin−CD38), B cells, and erythroid and megakaryocyte CFCs [384]. In all of these cells, only HSCs self-renew, setting up clones that can acquire additional proto-oncogenic events. In childhood t(8;21) AML, AML1–ETO fusions are retrospectively detected in neonatal Guthrie spots [399], further suggesting that AML1–ETO fusion is at the level of HSCs (in some cases in utero). Acquisition of chromosomal translocations in utero is also demonstrated in infant t(4;11) and childhood t(12;21) acute leukemias. Clonotypic MLL–AF4 and TEL–AML1 genomic fusion sequences were demonstrated to be present in neonatal blood spots from patients who were

54

Chapter 5

diagnosed with acute lymphoblastic leukemia (ALL) [399–401] and were also detected in identical twins, whose genomic fusion sequence was identical [369]. In these cases, t(12;21) ALL developed at approximately 10 years of age, indicating that cells with TEL–AML1 persisted more than 10 years before leukemic transformation. Further, TEL– AML1+ ALL cells in twins sometimes possess an identical IgH rearrangement, implicating in utero transfer of preleukemic cells. In childhood ALL, preleukemic B- or T-cell progenitors appear to persist in the long term; “leukemic clones” possessing clonal TCR or IgH rearrangements are reported to persist for more than 3 years after achieving complete remission [402]; these might involve the only other blood cells that self-renew – memory T and B cells. These data strongly suggest that leukemic transformation commonly occurs by multistep processes. In mice and in humans, the only cell in the myeloid lineage that self-renews over long periods of time is the LT-HSC [1]. The preleukemic clone should exist in the LT-HSC to sustain multiple mutational events over time. Therefore, it appears that the accumulation of mutations occur as a precondition for leukemia development at the level of HSCs, or in progenitors that have gained the capacity of self-renewal.

Final leukemic transformation can occur at the level of myeloid progenitors Several lines of evidence show that final myeloid leukemic transformation can only occur at the level of progenitors that have acquired the property of self-renewal. In contrast, lymphoid leukemias are made up of neoplastic cells with clonally rearranged TCR or immunoglobulin genes. Whereas myeloid progenitors and myelomonocytic cells are not self-renewing, lymphocytes that contribute to the memory pool are selfrenewing. Thus, the final transformation to lymphatic neoplasms can occur in cells that already have regulated self-renewal capacity. The search for LSCs involves the one property shared by all malignancies – the ability to initiate and spread the disease. Transfer of human AML to immunodeficient mice was first demonstrated to be limited to CD34+CD38− or CD34+CD71−HLA-DR− cells, and was attributed to early progenitors such as HSCs [403]. We [396] and others [404] have shown that, in at least some AMLs, the LSCs lack CD90 expression. In our study of AML1/ETO AML, LSCs were in the MPP phenotype (CD34+CD38lo90− Lin−). Surprisingly, in the same patients, 1–40% of HSCs were positive for the t(8;21) translocation, but these HSCs in vitro gave rise to normal myeloerythroid colonies, not leukemic blasts [396]. As shown in Plate 5.2, these represent preleukemic clones; thus, AML1/ETO may be necessary but not sufficient for full leukemic transformation. A mouse model that expresses AML1/ETO driven by multidrug resistance protein-8 (MRP8) (GMP, granulocyte, and monocyte specific) promoter did not develop leukemia. After the injection of a DNA alkylating mutagen, N-ethyl-N-nitrosourea, however, almost 55% of mice develop AML-M2 [405]. Similarly, promyelocytic leukemia/retinoic receptor-α (PML/RARα) is a product of the t(15;17) translocation, and MRP8–PML/RARα transgenic mice develop acute promyelocytic leukemia-like disorders [406]. The frequency and rapidity of PML-RARα leukemias increases when they are crossed to MPR8–Bcl-2 mice [407]. In these mice, the proto-oncogenes were present in all cells of the GMP lineage, allowing rare subsequent events to be fixed. Inhibition of apoptotic pathways alone can allow leukemia development at progenitor stages. MRP8–Bcl-2 mice display increased numbers of mature monocytes, resembling chronic myelomonocytic leukemia, but do not develop acute leukemia. However, an additional introduction of Fas (lpr/lpr) mutations into these mice results in transformation into AML in about 15% of progeny, but only if leukemia emerges by age 8 weeks [408]. The transplantable GMP-stage leukemias are tert++, over-

express CD47, and have acquired the property of self-renewal, implicating a multistep process. Even mpr8–bcr–abl mice have increased frequencies of conversions to acute blastic leukemias if crossed to mpr8– bcl-2 strains. The general rules so far are that mouse AML LSCs induced as described are at the stage of the GMP, as is human CML myeloid blast crisis [409–412]. Most human AMLs are at the level of the MPP. No HSC-stage LSC has been found. LSC markers The identification and characterization of LSCs is crucial to the development of more effective treatment strategies. Ideal therapies will be those that target the LSCs while sparing normal HSCs, so identifying LSCspecific markers is an important strategy. To date we have identified several candidate LSC-specific markers, including CD96 and CD47 [158,413]. Fluorescence-activated cell sorter analysis indicates that, in many cases, CD96 is expressed on the majority of CD34+CD38− AML cells, whereas few, if any, normal HSCs (Lin−CD34+CD38−CD90+) expressed CD96 [413]. Further, in four of five samples, only CD96+ cells were able to propagate the leukemia in recipient immunocompromised mice, supporting its expression on LSCs and its usefulness as a potential therapeutic target. We identified CD47 in the mouse AML microarray screen, and have verified it as specifically upregulated in human AML. Subsequently, we have found it to be expressed in all mouse models of myeloid leukemia we have examined, and in human AML and blast crisis CML (manuscript submitted). Recent data suggests that expression of mouse CD47, which inhibits phagocytosis, is sufficient to allow the human myeloid leukemia cell line (MOLM-13) to engraft hematopoietic organs efficiently and develop into large tumors, whereas control MOLM-13 cells fail to engraft in immunocompromised Rag2−/−γc−/− cells [158]. Thus, CD47 does seem to play a role in the spread and/or life span of AML LSCs. These data also suggest that macrophagemediated killing may be a significant player in leukemia surveillance. There are increasing descriptions of mutations that occur at the posttranslational and epigenetic levels, and would not be detected by gene expression profiling. A study of 100 CML blood and marrow samples has demonstrated that GMPs from CML blast crisis have been shown to have increased levels of the active β-catenin in the nucleus compared with normal GMPs and to exhibit increased self-renewal in in vitro replating assays [412]. Dysregulation of microRNAs, small RNAs that bind to specific transcripts and either suppress their translation or target them for deletion, is another potential leukemogenic mechanism [414–417]. The important point here, as outlined in Plate 5.2, is that the elucidation of the phenotypes of normal HSCs and hematopoietic progenitors allows the classification of the various types of LSC, and the comparison of genes and gene transcripts between the normal and the neoplastic counterparts has revealed a plethora of new, and perhaps useful diagnostic and therapeutic, targets undetected when whole blood or marrow populations are analyzed.

Conclusion With the development of a general method to prospectively isolate hematopoietic stem and progenitor cells [1,40,45], it has become possible to construct increasingly detailed lineage pathways from LT-HSCs to mature blood cells. With the exception of memory T and B lymphocytes in the entire hematopoietic lineage, only LT-HSCs self-renew, and this self-renewal capacity is tightly regulated [1]. The genetic regulation of HSC self-renewal includes regulation of programmed cell death, regulation of cell-division frequency, and regulation of transition to

Biology of Hematopoietic Stem and Progenitor Cells

multipotent and to oligopotent progenitors. Gene expression analyses of prospectively isolated HSCs and progenitors are revealing insights into the mechanisms of self-renewal and those in lineage commitment. These same isolated HSCs and progenitors are increasingly useful in clinical HCT, whether to regenerate the myeloablated hematopoietic system in cancer patients, to condition allogeneic hosts for donor tissue or organ grafts, or even to replace an autoimmune-prone hematolymphoid system

55

with a nonautoreactive donor population. Finally, the concept of selfrenewal as the critical regulated event limited to HSCs in normal hematopoiesis has now been extended to the hypothesis that within leukemias and cancers are relatively infrequent populations of LSCs that undergo poorly regulated self-renewal. Prospective isolation of LSCs should allow more direct approaches for immune and drug therapies for target molecules restricted to these malignant stem cells.

References 1. Weissman IL. Stem cells: units of development, units of regeneration, and units in evolution. Cell, 2000; 100: 157–68. 2. Becker AJ, Mc CE, Till JE. Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature 1963; 197: 452–4. 3. Metcalf D, Moore MAS. Hematopoietic Cells. Amsterdam: North Holland; 1971. 4. Smith LG, Weissman IL, Heimfeld S. Clonal analysis of hematopoietic stem-cell differentiation in vivo. Proc Natl Acad Sci U S A 1991; 88: 2788–92. 5. Wagers AJ, Sherwood RI, Christensen JL, Weissman IL. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 2002; 297: 2256–9. 6. Spangrude GJ, Aihara Y, Weissman IL, Klein J. The stem cell antigens Sca-1 and Sca-2 subdivide thymic and peripheral T lymphocytes into unique subsets. J Immunol 1988; 141: 3697–707. 7. Uchida N. Characterization of mouse hematopoietic stem cells. In Pathology. PhD thesis, Stanford University School of Medicine, Stanford, CA; 1992. 8. Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34- low/negative hematopoietic stem cell. Science 1996; 273: 242–5. 9. Lemischka IR, Raulet DH, Mulligan RC. Developmental potential and dynamic behavior of hematopoietic stem cells. Cell 1986; 45: 917–27. 10. Abramson S, Miller RG, Phillips RA. The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems. J Exp Med 1977; 145: 1567–79. 11. McCulloch EA, Siminovitch L, Till JE. Spleencolony formation in anemic mice of genotype Ww. Science 1964; 144: 844–6. 12. Till JE, McCulloch EA. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 1961; 14: 213–22. 13. Siminovitch L, McCulloch EA, Till JE. The distribution of colony-forming cells among spleen colonies. J Cell Physiol 1963; 62: 327–36. 14. Weissman IL, Anderson DJ, Gage F. Stem and progenitor cells: origins, phenotypes, lineage commitments, and transdifferentiations. Annu Rev Cell Dev Biol 2001; 17: 387–403. 15. Anderson DJ, Gage FH, Weissman IL. Can stem cells cross lineage boundaries? Nat Med 2001; 7: 393–5. 16. Lagasse E, Shizuru JA, Uchida N, Tsukamoto A, Weissman IL. Toward regenerative medicine. Immunity 2001; 14: 425–36. 17. Weissman IL. Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science 2000; 287: 1442–6. 18. Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

from adult marrow. Nature 2002; 418: 41– 9. Morrison SJ, White PM, Zock C, Anderson DJ. Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells. Cell 1999; 96: 737–49. Palmer TD, Takahashi J, Gage FH. The adult rat hippocampus contains primordial neural stem cells. Mol Cell Neurosci 1997; 8: 389–404. Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992; 255: 1707–10. Stemple DL, Anderson DJ. Isolation of a stem cell for neurons and glia from the mammalian neural crest. Cell 1992; 71: 973–85. Suhonen JO, Peterson DA, Ray J, Gage FH. Differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo. Nature 1996; 383: 624–7. Taylor G, Lehrer MS, Jensen PJ, Sun TT, Lavker RM. Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 2000; 102: 451–61. Kondo M, Weissman IL, Akashi K. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 1997; 91: 661– 72. Akashi K, Traver D, Miyamoto T, Weissman IL. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 2000; 404: 193–7. Allsopp RC, Morin GB, Horner JW, DePinho R, Harley CB, Weissman IL. Effect of TERT overexpression on the long-term transplantation capacity of hematopoietic stem cells. Nat Med 2003; 9: 369–71. Lorenz E, Uphoff D, Reid TR, Shelton E. Modification of irradiation injury in mice and guinea pigs by bone marrow injections. J Natl Cancer Inst 1951; 12: 197–201. Jacobson LO, Simmons EL, Marks EK, Eldredge JH. Recovery from radiation injury. Science 1951; 113: 510–11. Jacobson LO, Simmons EL, Marks EK, Robson MJ, Bethard WF, Gaston EO. The role of the spleen in radiation injury and recovery. J Lab Clin Med 1950; 35: 746–70. Gengozian N, Urso IS, Congdon CC, Conger AD, Makinodan T. Thymus specificity in lethally irradiated mice treated with rat bone marrow. Proc Soc Exp Biol Med 1957; 96: 714–20. Ford CE, Hamerton JL, Barnes DW, Loutit JF. Cytological identification of radiation-chimaeras. Nature 1956; 177: 452–4. Nowell PC, Cole LJ, Habermeyer JG, Roan PL. Growth and continued function of rat marrow cells in x-radiated mice. Cancer Res 1956; 16: 258–61.

34. Wu AM, Till JE, Siminovitch L, McCulloch EA. Cytological evidence for a relationship between normal hematopoietic colony-forming cells and cells of the lymphoid system. J Exp Med 1968; 127: 455–64. 35. Magli MC, Iscove NN, Odartchenko N. Transient nature of early haematopoietic spleen colonies. Nature 1982; 295: 527–9. 36. Visser JW, Van Bekkum DW. Purification of pluripotent hemopoietic stem cells: past and present. Exp Hematol 1990; 18: 248–56. 37. Na Nakorn T, Traver D, Weissman IL, Akashi K. Myeloerythroid-restricted progenitors are sufficient to confer radioprotection and provide the majority of day 8 CFU-S. J Clin Invest 2002; 109: 1579–85. 38. Morrison SJ, Weissman IL. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1994; 1: 661–73. 39. Jones RJ, Wagner JE, Celano P, Zicha MS, Sharkis SJ. Separation of pluripotent haematopoietic stem cells from spleen colony-forming cells. Nature 1990; 347: 188–9. 40. Morrison SJ, Wandycz AM, Hemmati HD, Wright DE, Weissman IL. Identification of a lineage of multipotent hematopoietic progenitors. Development 1997; 124: 1929–39. 41. Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science 1988; 241: 58–62. 42. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256: 495–7. 43. Hulett HR, Bonner WA, Barrett J, Herzenberg LA. Cell sorting: automated separation of mammalian cells as a function of intracellular fluorescence. Science 1969; 166: 747–9. 44. Ezine S, Weissman IL, Rouse RV. Bone marrow cells give rise to distinct cell clones within the thymus. Nature 1984; 309: 629–31. 45. Muller-Sieburg CE, Whitlock CA, Weissman IL. Isolation of two early B lymphocyte progenitors from mouse marrow: a committed pre-pre-B cell and a clonogenic Thy-1-lo hematopoietic stem cell. Cell 1986; 44: 653–62. 46. Wu AM, Till JE, Siminovitch L, McCulloch EA. A cytological study of the capacity for differentiation of normal hemopoietic colony-forming cells. J Cell Physiol 1967; 69: 177–84. 47. Dexter TM, Allen TD, Lajtha LG. Conditions controlling the proliferation of haemopoietic stem cells in vitro. J Cell Physiol 1977; 91: 335–44. 48. Weilbaecher K, Weissman I, Blume K, Heimfeld S. Culture of phenotypically defined hematopoietic stem cells and other progenitors at limiting dilution on Dexter monolayers. Blood 1991; 78: 945–52. 49. Eaves CJ, Sutherland HJ, Cashman JD et al. Regulation of primitive human hematopoietic

56

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

Chapter 5

cells in long-term marrow culture. Semin Hematol 1991; 28: 126–31. Visser JW, Bauman JG, Mulder AH, Eliason JF, de Leeuw AM. Isolation of murine pluripotent hemopoietic stem cells. J Exp Med 1984; 159: 1576–90. Adolfsson J, Borge OJ, Bryder D et al. Upregulation of Flt3 expression within the bone marrow Lin(−)Sca1(+)c-kit(+) stem cell compartment is accompanied by loss of self-renewal capacity. Immunity 2001; 15: 659–69. Christensen JL, Weissman IL. Flk-2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long-term stem cells. Proc Natl Acad Sci U S A 2001; 98: 14541– 6. Ikuta K, Weissman IL. Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation. Proc Natl Acad Sci U S A 1992; 89: 1502–6. Uchida N, Weissman IL. Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin− Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow. J Exp Med 1992; 175: 175–84. Forsberg EC, Prohaska SS, Katzman S, Heffner GC, Stuart JM, Weissman IL. Differential expression of novel potential regulators in hematopoietic stem cells. PLoS Genet 2005; 1: e28. Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, Terhorst C, Morrison SJ. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 2005; 121: 1109–21. Civin CI, Strauss LC, Brovall C, Fackler MJ, Schwartz JF, Shaper JH. Antigenic analysis of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells. J Immunol 1984; 133: 157–65. Baum CM, Weissman IL, Tsukamoto AS, Buckle AM, Peault B. Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci U S A 1992; 89: 2804–8. Uchida N, Sutton RE, Friera AM et al. HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/ G1 human hematopoietic stem cells. Proc Natl Acad Sci U S A 1998; 95: 11939–44. Petzer AL, Zandstra PW, Piret JM, Eaves CJ. Differential cytokine effects on primitive (CD34+CD38−) human hematopoietic cells: novel responses to Flt3-ligand and thrombopoietin. J Exp Med 1996; 183: 2551–8. Larochelle A, Vormoor J, Hanenberg H et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat Med 1996; 2: 1329–37. Bhatia M, Wang JC, Kapp U, Bonnet D, Dick JE. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci U S A 1997; 94: 5320– 5. Majeti R, Park CS, Weissman IL. Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood. Cell Stem Cell 2007; 1: 635–45. DiGiusto D, Chen S, Combs J et al. Human fetal bone marrow early progenitors for T, B, and myeloid cells are found exclusively in the popula-

65.

66.

67.

68.

69.

70.

71. 72.

73.

74.

75.

76.

77.

78.

79. 80.

tion expressing high levels of CD34. Blood 1994; 84: 421–32. Bhatia M, Bonnet D, Murdoch B, Gan OI, Dick JE. A newly discovered class of human hematopoietic cells with SCID-repopulating activity. Nat Med 1998; 4: 1038–45. Goodell MA, Rosenzweig M, Kim H et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 1997; 3: 1337–45. Zanjani ED, Almeida-Porada G, Livingston AG, Flake AW, Ogawa M. Human bone marrow CD34− cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells. Exp Hematol 1998; 26: 353–60. Allsopp RC, Cheshier S, Weissman IL. Telomere shortening accompanies increased cell cycle activity during serial transplantation of hematopoietic stem cells. J Exp Med 2001; 193: 917– 24. Cheshier SH, Morrison SJ, Liao X, Weissman IL. In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells. Proc Natl Acad Sci U S A 1999; 96: 3120–5. Passegue E, Wagers AJ, Giuriato S, Anderson WC, Weissman IL. Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates. J Exp Med 2005; 202: 1599–611. Ooi L, Bhattacharya D, Rossi D, Weissman IL. Unpublished observations, 2008. Wright DE, Cheshier SH, Wagers AJ, Randall TD, Christensen JL, Weissman IL. Cyclophosphamide/granulocyte colony-stimulating factor causes selective mobilization of bone marrow hematopoietic stem cells into the blood after M phase of the cell cycle. Blood 2001; 97: 2278– 85. Fleming WH, Alpern EJ, Uchida N, Ikuta K, Spangrude GJ, Weissman IL. Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells. J Cell Biol 1993; 122: 897–902. Morrison SJ, Wright DE, Weissman IL. Cyclophosphamide/granulocyte colony-stimulating factor induces hematopoietic stem cells to proliferate prior to mobilization. Proc Natl Acad Sci U S A 1997; 94: 1908–13. Wright DE, Wagers AJ, Gulati AP, Johnson FL, Weissman IL. Physiological migration of hematopoietic stem and progenitor cells. Science 2001; 294: 1933–6. Cheshier SH, Prohaska SS, Weissman IL. The effect of bleeding on hematopoietic stem cell cycling and self-renewal. Stem Cell Dev 2007; 16: 707–17. Hayflick L. The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 1965; 37: 614– 36. Blackburn EH, Greider CW, Henderson E, Lee MS, Shampay J, Shippen-Lentz D. Recognition and elongation of telomeres by telomerase. Genome 1989; 31: 553–60. Cohn M, Blackburn EH. Telomerase in yeast. Science 1995; 269: 396–400. Allsopp RC, Cheshier S, Weissman IL. Telomerase activation and rejuvenation of telomere length in stimulated T cells derived from serially transplanted hematopoietic stem cells. J Exp Med 2002; 196: 1427–33.

81. Morrison SJ, Prowse KR, Ho P, Weissman IL. Telomerase activity in hematopoietic cells is associated with self-renewal potential. Immunity 1996; 5: 207–16. 82. Zimmermann S, Martens UM. Telomeres and telomerase as targets for cancer therapy. Cell Mol Life Sci 2007; 64: 906–21. 83. Morrison SJ, Wandycz AM, Akashi K, Globerson A, Weissman IL. The aging of hematopoietic stem cells. Nat Med 1996; 2: 1011–16. 84. Morrison SJ, Qian D, Jerabek L et al. A genetic determinant that specifically regulates the frequency of hematopoietic stem cells. J Immunol 2002; 168: 635–42. 85. Muller-Sieburg CE, Riblet R. Genetic control of the frequency of hematopoietic stem cells in mice: mapping of a candidate locus to chromosome 1. J Exp Med 1996; 183: 1141–50. 86. de Haan G. Latexin is a newly discovered regulator of hematopoietic stem cells. Nat Genet 2007; 39: 141–2. 87. Domen J, Gandy KL, Weissman IL. Systemic overexpression of BCL-2 in the hematopoietic system protects transgenic mice from the consequences of lethal irradiation. Blood 1998; 91: 2272–82. 88. Domen J, Weissman IL. Hematopoietic stem cells need two signals to prevent apoptosis; BCL-2 can provide one of these, Kitl/c-Kit signaling the other. J Exp Med 2000; 192: 1707–18. 89. Lacaud G, Gore L, Kennedy M et al. Runx1 is essential for hematopoietic commitment at the hemangioblast stage of development in vitro. Blood 2002; 100: 458–66. 90. Endoh M, Ogawa M, Orkin S, Nishikawa S. SCL/ tal-1-dependent process determines a competence to select the definitive hematopoietic lineage prior to endothelial differentiation. EMBO J 2002; 21: 6700–8. 91. Shalaby F, Rossant J, Yamaguchi TP et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 1995; 376: 62–6. 92. Shalaby F, Ho J, Stanford WL et al. A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell 1997; 89: 981–90. 93. Weissman I, Papaioannou VE, Gardner RL. Fetal hematopoietic origins of the adult hematolymphoid system. In: Clarkson B, Marks PA, Till JE, editors. Cold Spring Harbor Meeting on Differentiation of Normal and Neoplastic Hematopoietic Cells. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1978. pp. 577– 89. 94. Terskikh AV, Miyamoto T, Chang C, Diatchenko L, Weissman IL. Gene expression analysis of purified hematopoietic stem cells and committed progenitors. Blood 2003; 102: 94–101. 95. Venezia TA, Merchant AA, Ramos CA et al. Molecular signatures of proliferation and quiescence in hematopoietic stem cells. PLoS Biol 2004; 2: e301. 96. Rossi DJ, Bryder D, Zahn JM et al. Cell intrinsic alterations underlie hematopoietic stem cell aging. Proc Natl Acad Sci U S A 2005; 102: 9194–9. 97. Park IK, He Y, Lin F et al. Differential gene expression profiling of adult murine hematopoietic stem cells. Blood 2002; 99: 488–98. 98. Phillips RL, Ernst RE, Brunk B et al. The genetic program of hematopoietic stem cells. Science 2000; 288: 1635–40.

Biology of Hematopoietic Stem and Progenitor Cells 99. Bryder D, Jacobsen SE. Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro. Blood 2000; 96: 1748–55. 100. Conneally E, Cashman J, Petzer A, Eaves C. Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice. Proc Natl Acad Sci U S A 1997; 94: 9836–41. 101. Ema H, Takano H, Sudo K, Nakauchi H. In vitro self-renewal division of hematopoietic stem cells. J Exp Med 2000; 192: 1281–8. 102. Fraser CC, Eaves CJ, Szilvassy SJ, Humphries RK. Expansion in vitro of retrovirally marked totipotent hematopoietic stem cells. Blood 1990; 76: 1071–6. 103. Glimm H, Tang P, Clark-Lewis I, von Kalle C, Eaves C. Ex vivo treatment of proliferating human cord blood stem cells with stroma-derived factor1 enhances their ability to engraft NOD/SCID mice. Blood 2002; 99: 3454–7. 104. Miller CL, Eaves CJ. Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability. Proc Natl Acad Sci U S A 1997; 94: 13648–53. 105. Yagi M, Ritchie KA, Sitnicka E, Storey C, Roth GJ, Bartelmez S. Sustained ex vivo expansion of hematopoietic stem cells mediated by thrombopoietin. Proc Natl Acad Sci U S A 1999; 96: 8126–31. 106. McCulloch EA, Siminovitch L, Till JE, Russell ES, Bernstein SE. The cellular basis of the genetically determined hemopoietic defect in anemic mice of genotype Sl-Sld. Blood 1965; 26: 399– 410. 107. Huang E, Nocka K, Beier DR et al. The hematopoietic growth factor KL is encoded by the Sl locus and is the ligand of the c-kit receptor, the gene product of the W locus. Cell 1990; 63: 225– 33. 108. Sarvella PA, Russel LB. Steel, a new dominant gene in the house mouse. J Hered 1956; 47: 390. 109. Williams DE, Eisenman J, Baird A et al. Identification of a ligand for the c-kit proto-oncogene. Cell 1990; 63: 167–74. 110. Geissler EN, Ryan MA, Housman DE. The dominant-white spotting (W) locus of the mouse encodes the c-kit proto-oncogene. Cell 1988; 55: 185–92. 111. Chabot B, Stephenson DA, Chapman VM, Besmer P, Bernstein A. The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus. Nature 1988; 335: 88–9. 112. Lev S, Blechman JM, Givol D, Yarden Y. Steel factor and c-kit protooncogene: genetic lessons in signal transduction. Crit Rev Oncog 1994; 5: 141–68. 113. Miyamoto T, Iwasaki H, Reizis B et al. Myeloid or lymphoid promiscuity as a critical step in hematopoietic lineage commitment. Dev Cell 2002; 3: 137–47. 114. Qian H, Buza-Vidas N, Hyland CD et al. Critical role of thrombopoietin in maintaining adult quiescent hematopoietic stem cells. Cell Stem Cell 2007; 1: 671–84. 115. Yoshihara H, Arai F, Hosokawa K et al. Thrombopoietin/MPL signaling regulates hematopoietic stem cell quiescence and interaction with the

116.

117.

118.

119.

120.

121.

122.

123.

124.

125.

126.

127.

128.

129.

130.

131.

132.

133.

134.

osteoblastic niche. Cell Stem Cell 2007; 1: 685– 97. Karanu FN, Murdoch B, Gallacher L et al. The notch ligand jagged-1 represents a novel growth factor of human hematopoietic stem cells. J Exp Med 2000; 192: 1365–72. Varnum-Finney B, Xu L, Brashem-Stein C et al. Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nat Med 2000; 6: 1278–81. Kyba M, Perlingeiro RC, Daley GQ. HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell 2002; 109: 29– 37. Antonchuk J, Sauvageau G, Humphries RK. HOXB4 overexpression mediates very rapid stem cell regeneration and competitive hematopoietic repopulation. Exp Hematol 2001; 29: 1125–34. Antonchuk J, Sauvageau G, Humphries RK. HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell 2002; 109: 39–45. Sauvageau G, Thorsteinsdottir U, Eaves CJ et al. Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes Dev 1995; 9: 1753–65. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature 2001; 414: 105–11. Katoh M. Networking of WNT, FGF, Notch, BMP, and Hedgehog signaling pathways during carcinogenesis. Stem Cell Rev 2007; 3: 30–8. Reya T, Duncan AW, Ailles L et al. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 2003; 423: 409–14. Willert K, Brown JD, Danenberg E et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 2003; 423: 448–52. Cobas M, Wilson A, Ernst B et al. Beta-catenin is dispensable for hematopoiesis and lymphopoiesis. J Exp Med 2004; 199: 221–9. Zhao C, Blum J, Chen A et al. Loss of betacatenin impairs the renewal of normal and CML stem cells in vivo. Cancer Cell 2007; 12: 528– 41. Park IK, Qian D, Kiel M et al. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature 2003; 423: 302–5. Jacobs JJ, Kieboom K, Marino S, DePinho RA, van Lohuizen M. The oncogene and Polycombgroup gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature 1999; 397: 164–8. Sauvageau G, Lansdorp PM, Eaves CJ et al. Differential expression of homeobox genes in functionally distinct CD34+ subpopulations of human bone marrow cells. Proc Natl Acad Sci U S A 1994; 91: 12223–7. Passegue E, Wagner EF, Weissman IL. JunB deficiency leads to a myeloproliferative disorder arising from hematopoietic stem cells. Cell 2004; 119: 431–43. Goodman JW, Hodgson GS. Evidence for stem cells in the peripheral blood of mice. Blood 1962; 19: 702–14. Richman CM, Weiner RS, Yankee RA. Increase in circulating stem cells following chemotherapy in man. Blood 1976; 47: 1031–9. Juttner CA, To LB, Haylock DN, Branford A, Kimber RJ. Circulating autologous stem cells col-

135.

136.

137.

138.

139.

140.

141.

142.

143.

144.

145.

146.

147.

148.

57

lected in very early remission from acute nonlymphoblastic leukaemia produce prompt but incomplete haemopoietic reconstitution after high dose melphalan or supralethal chemoradiotherapy. Br J Haematol 1985; 61: 739–45. Brugger W, Bross K, Frisch J et al. Mobilization of peripheral blood progenitor cells by sequential administration of interleukin-3 and granulocytemacrophage colony-stimulating factor following polychemotherapy with etoposide, ifosfamide, and cisplatin. Blood 1992; 79: 1193–200. Duhrsen U, Villeval JL, Boyd J, Kannourakis G, Morstyn G, Metcalf D. Effects of recombinant human granulocyte colony-stimulating factor on hematopoietic progenitor cells in cancer patients. Blood 1988; 72: 2074–81. McNiece IK, Langley KE, Zsebo KM. Recombinant human stem cell factor synergises with GMCSF, G-CSF, IL-3 and epo to stimulate human progenitor cells of the myeloid and erythroid lineages. Exp Hematol 1991; 19: 226–31. Siena S, Bregni M, Brando B, Ravagnani F, Bonadonna G, Gianni AM. Circulation of CD34+ hematopoietic stem cells in the peripheral blood of high-dose cyclophosphamide-treated patients: enhancement by intravenous recombinant human granulocyte-macrophage colony-stimulating factor. Blood 1989; 74: 1905–14. Socinski MA, Cannistra SA, Elias A, Antman KH, Schnipper L, Griffin JD. Granulocyte-macrophage colony stimulating factor expands the circulating haemopoietic progenitor cell compartment in man. Lancet 1988; 1: 1194–8. Terskikh AV, Easterday MC, Li L et al. From hematopoiesis to neuropoiesis: evidence of overlapping genetic programs. Proc Natl Acad Sci U S A 2001; 98: 7934–9. Fibbe WE, Pruijt JF, Velders GA et al. Biology of IL-8-induced stem cell mobilization. Ann N Y Acad Sci 1999; 872: 71–82. Heissig B, Hattori K, Dias S et al. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kitligand. Cell 2002; 109: 625–37. Laterveer L, Lindley IJ, Hamilton MS, Willemze R, Fibbe WE. Interleukin-8 induces rapid mobilization of hematopoietic stem cells with radioprotective capacity and long-term myelolymphoid repopulating ability. Blood 1995; 85: 2269–75. Miyake K, Weissman IL, Greenberger JS, Kincade PW. Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis. J Exp Med 1991; 173: 599– 607. Kina T, Majumdar AS, Heimfeld S et al. Identification of a 107-kD glycoprotein that mediates adhesion between stromal cells and hematolymphoid cells. J Exp Med 1991; 173: 373–81. Elices MJ, Osborn L, Takada Y et al. VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site. Cell 1990; 60: 577–84. Schweitzer KM, Drager AM, van der Valk P et al. Constitutive expression of E-selectin and vascular cell adhesion molecule-1 on endothelial cells of hematopoietic tissues. Am J Pathol 1996; 148: 165–75. Wagers AJ, Allsopp RC, Weissman IL. Changes in integrin expression are associated with altered homing properties of Lin(−/lo)Thy1.1(lo)Sca1(+)c-kit(+) hematopoietic stem cells following

58

149.

150.

151.

152.

153.

154.

155.

156.

157.

158.

159.

160.

161.

162.

163.

164.

165.

Chapter 5

mobilization by cyclophosphamide/granulocyte colony-stimulating factor. Exp Hematol 2002; 30: 176–85. Mohle R, Bautz F, Rafii S, Moore MA, Brugger W, Kanz L. The chemokine receptor CXCR-4 is expressed on CD34+ hematopoietic progenitors and leukemic cells and mediates transendothelial migration induced by stromal cell-derived factor1. Blood 1998; 91: 4523–30. Peled A, Petit I, Kollet O et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 1999; 283: 845–8. Wright DE, Bowman EP, Wagers AJ, Butcher EC, Weissman IL. Hematopoietic stem cells are uniquely selective in their migratory response to chemokines. J Exp Med 2002; 195: 1145–54. Gutman GA, Weissman IL. Homing properties of thymus-independent follicular lymphocytes. Transplantation 1973; 16: 621–9. Gallatin M, St John TP, Siegelman M, Reichert R, Butcher EC, Weissman IL. Lymphocyte homing receptors. Cell 1986; 44: 673–80. Lagasse E, Weissman IL. Enforced expression of Bcl-2 in monocytes rescues macrophages and partially reverses osteopetrosis in op/op mice. Cell 1997; 89: 1021–31. Massberg S, Schaerli P, Knezevic-Maramica I et al. Immunosurveillance by hematopoietic progenitor cells trafficking through blood, lymph, and peripheral tissues. Cell 2007; 131: 994– 1008. Bhattacharya D, Rossi DJ, Bryder D, Weissman IL. Purified hematopoietic stem cell engraftment of rare niches corrects severe lymphoid deficiencies without host conditioning. J Exp Med 2006; 203: 73–85. Czechowicz A, Kraft D, Weissman IL, Bhattacharya D. Efficient transplantation via antibodybased clearance of hematopoietic stem cell niches. Science 2007; 318: 1296–9. Jaiswal S, Jamieson C, Park CY, Traver D, Weissman IL. Human and mouse leukaemias upregulate CD47 to evade macrophage killing (submitted for publication). Oldenborg PA, Zheleznyak A, Fang YF, Lagenaur CF, Gresham HD, Lindberg FP. Role of CD47 as a marker of self on red blood cells. Science 2000; 288: 2051–4. Moore MA, Metcalf D. Ontogeny of the haemopoietic system: yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo. Br J Haematol 1970; 18: 279– 96. Johnson GR, Moore MA. Role of stem cell migration in initiation of mouse foetal liver haemopoiesis. Nature 1975; 258: 726–8. Gardner RL. The initial phase of embryonic patterning in mammals. Int Rev Cytol 2001; 203: 233–90. Cumano A, Ferraz JC, Klaine M, Di Santo JP, Godin I. Intraembryonic, but not yolk sac hematopoietic precursors, isolated before circulation, provide long-term multilineage reconstitution. Immunity 2001; 15: 477–85. Weissman I, O’Neill WW, Juni JE. Evaluation of chest pain in the emergency department. Ann Intern Med 1995; 123: 314; author reply 317–18. Keller G, Lacaud G, Robertson S. Development of the hematopoietic system in the mouse. Exp Hematol 1999; 27: 777–87.

166. Yoder MC. Introduction: spatial origin of murine hematopoietic stem cells. Blood 2001; 98: 3–5. 167. Palis J, Robertson S, Kennedy M, Wall C, Keller G. Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse. Development 1999; 126: 5073–84. 168. Samokhvalov IM, Samokhvalova NI, Nishikawa S. Cell tracing shows the contribution of the yolk sac to adult haematopoiesis. Nature 2007; 446: 1056–61. 169. Haar JL, Ackerman GA. A phase and electron microscopic study of vasculogenesis and erythropoiesis in the yolk sac of the mouse. Anat Rec 1971; 170: 199–223. 170. Carmeliet P, Ferreira V, Breier G et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996; 380: 435–9. 171. Huber TL, Kouskoff V, Fehling HJ, Palis J, Keller G. Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature 2004; 432: 625–30. 172. Ueno H, Weissman IL. Clonal analysis of mouse development reveals a polyclonal origin for yolk sac blood islands. Dev Cell 2006; 11: 519–33. 173. Williams RL, Hilton DJ, Pease S et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 1988; 336: 684–7. 174. Rebel VI, Miller CL, Eaves CJ, Lansdorp PM. The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their liver adult bone marrow counterparts. Blood 1996; 87: 3500–7. 175. Morrison SJ, Hemmati HD, Wandycz AM, Weissman IL. The purification and characterization of fetal liver hematopoietic stem cells. Proc Natl Acad Sci U S A 1995; 92: 10302–6. 176. Hirsch E, Iglesias A, Potocnik AJ, Hartmann U, Fassler R. Impaired migration but not differentiation of haematopoietic stem cells in the absence of beta1 integrins. Nature 1996; 380: 171–5. 177. Potocnik AJ, Brakebusch C, Fassler R. Fetal and adult hematopoietic stem cells require beta1 integrin function for colonizing fetal liver, spleen, and bone marrow. Immunity 2000; 12: 653–63. 178. Christensen JL, Wright DE, Wagers AJ, Weissman IL. Circulation and chemotaxis of fetal hematopoietic stem cells. PLoS Biol 2004; 2: E75. 179. Ikuta K, Kina T, MacNeil I et al. A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells. Cell 1990; 62: 863–74. 180. Havran WL, Allison JP. Developmentally ordered appearance of thymocytes expressing different Tcell antigen receptors. Nature 1988; 335: 443–5. 181. Wagers AJ, Weissman IL. Plasticity of adult stem cells. Cell 2004; 116: 639–48. 182. Kawada H, Ogawa M. Bone marrow origin of hematopoietic progenitors and stem cells in murine muscle. Blood 2001; 98: 2008–13. 183. McKinney-Freeman SL, Jackson KA, Camargo FD, Ferrari G, Mavilio F, Goodell MA. Musclederived hematopoietic stem cells are hematopoietic in origin. Proc Natl Acad Sci U S A 2002; 99: 1341–6. 184. Ferrari G, Cusella-De Angelis G, Coletta M et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998; 279: 1528– 30.

185. Gussoni E, Soneoka Y, Strickland CD et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 1999; 401: 390– 4. 186. Lagasse E, Connors H, Al-Dhalimy M et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000; 6: 1229– 34. 187. Wang X, Willenbring H, Akkari Y et al. Cell fusion is the principal source of bone-marrowderived hepatocytes. Nature 2003; 422: 897– 901. 188. Willenbring H, Grompe M. Delineating the hepatocyte’s hematopoietic fusion partner. Cell Cycle 2004; 3: 1489–91. 189. Massengale M, Wagers AJ, Vogel H, Weissman IL. Hematopoietic cells maintain hematopoietic fates upon entering the brain. J Exp Med 2005; 201: 1579–89. 190. Giblett ER, Anderson JE, Cohen F, Pollara B, Meuwissen HJ. Adenosine-deaminase deficiency in two patients with severely impaired cellular immunity. Lancet 1972; 2: 1067–9. 191. Ochs HD, Yount JE, Giblett ER, Chen SH, Scott CR, Wedgwood RJ. Adenosine-deaminase deficiency and severe combined immunodeficiency syndrome. Lancet 1973; 1: 1393–4. 192. Kondo M, Takeshita T, Higuchi M et al. Functional participation of the IL-2 receptor gamma chain in IL-7 receptor complexes. Science 1994; 263: 1453–4. 193. Noguchi M, Nakamura Y, Russell SM et al. Interleukin-2 receptor gamma chain: a functional component of the interleukin-7 receptor. Science 1993; 262: 1877–80. 194. Hirschhorn R, Yang DR, Insel RA, Ballow M. Severe combined immunodeficiency of reduced severity due to homozygosity for an adenosine deaminase missense mutation (Arg253Pro). Cell Immunol 1993; 152: 383–93. 195. Georgopoulos K, Moore DD, Derfler B. Ikaros, an early lymphoid-specific transcription factor and a putative mediator for T cell commitment. Science 1992; 258: 808–12. 196. Radtke F, Wilson A, Stark G et al. Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 1999; 10: 547– 58. 197. Weissman IL. Stem cells, clonal progenitors, and commitment to the three lymphocyte lineages: T, B, and NK cells. Immunity 1994; 1: 529–31. 198. Akashi K, Shibuya T, Harada M et al. Acute ‘bilineal-biphenotypic’ leukaemia. Br J Haematol 1990; 74: 402–7. 199. Akashi K, Harada M, Shibuya T et al. Simultaneous occurrence of myelomonocytic leukemia and multiple myeloma: involvement of common leukemic progenitors and their developmental abnormality of “lineage infidelity”. J Cell Physiol 1991; 148: 446–56. 200. Akashi K, Mizuno S, Harada M et al. T lymphoid/ myeloid bilineal crisis in chronic myelogenous leukemia. Exp Hematol 1993; 21: 743–8. 201. Akashi K, Taniguchi S, Nagafuji K et al. B-lymphoid/myeloid stem cell origin in Ph-positive acute leukemia with myeloid markers. Leuk Res 1993; 17: 549–55. 202. Barlogie B, Epstein J, Selvanayagam P, Alexanian R. Plasma cell myeloma – new biological insights and advances in therapy. Blood 1989; 73: 865–79.

Biology of Hematopoietic Stem and Progenitor Cells 203. Gale RP, Ben Bassat I. Hybrid acute leukaemia. Br J Haematol 1987; 65: 261–4. 204. Schmidt CA, Przybylski GK. What can we learn from leukemia as for the process of lineage commitment in hematopoiesis? Int Rev Immunol 2001; 20: 107–15. 205. Altman AJ. Clinical features and biological implications of acute mixed lineage (hybrid) leukemias. Am J Pediatr Hematol Oncol 1990; 12: 123–33. 206. Lefterova P, Schmidt-Wolf IG. Coexpression of lymphoid and myeloid markers on cell surfaces. Leuk Lymphoma 1997; 26: 27–33. 207. Lacaud G, Carlsson L, Keller G. Identification of a fetal hematopoietic precursor with B cell, T cell, and macrophage potential. Immunity 1998; 9: 827–38. 208. Hirayama F, Ogawa M. Cytokine regulation of early lymphohematopoietic development. Stem Cells 1996; 14: 369–75. 209. Ogawa M. Stochastic model revisited. Int J Hematol 1999; 69: 2–5. 210. Buza-Vidas N, Luc S, Jacobsen SE. Delineation of the earliest lineage commitment steps of haematopoietic stem cells: new developments, controversies and major challenges. Curr Opin Hematol 2007; 14: 315–21. 211. Goodwin RG, Friend D, Ziegler SF et al. Cloning of the human and murine interleukin-7 receptors: demonstration of a soluble form and homology to a new receptor superfamily. Cell 1990; 60: 941–51. 212. Kondo M, Takeshita T, Ishii N et al. Sharing of the interleukin-2 (IL-2) receptor gamma chain between receptors for IL-2 and IL-4. Science 1993; 262: 1874–7. 213. Peschon JJ, Morrissey PJ, Grabstein KH et al. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J Exp Med 1994; 180: 1955–60. 214. von Freeden-Jeffry U, Vieira P, Lucian LA, McNeil T, Burdach SE, Murray R. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J Exp Med 1995; 181: 1519–26. 215. Cao X, Shores EW, Hu-Li J et al. Defective lymphoid development in mice lacking expression of the common cytokine receptor gamma chain. Immunity 1995; 2: 223–38. 216. DiSanto JP, Dautry-Varsat A, Certain S, Fischer A, de Saint Basile G. Interleukin-2 (IL-2) receptor gamma chain mutations in X-linked severe combined immunodeficiency disease result in the loss of high-affinity IL-2 receptor binding. Eur J Immunol 1994; 24: 475–9. 217. Ohbo K, Suda T, Hashiyama M et al. Modulation of hematopoiesis in mice with a truncated mutant of the interleukin-2 receptor gamma chain. Blood 1996; 87: 956–67. 218. Lodolce JP, Boone DL, Chai S et al. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity 1998; 9: 669–76. 219. Ogasawara K, Hida S, Azimi N et al. Requirement for IRF-1 in the microenvironment supporting development of natural killer cells. Nature 1998; 391: 700–3. 220. Suzuki H, Duncan GS, Takimoto H, Mak TW. Abnormal development of intestinal intraepithelial lymphocytes and peripheral natural killer cells in mice lacking the IL-2 receptor beta chain. J Exp Med 1997; 185: 499–505.

221. Miyazaki T, Kawahara A, Fujii H et al. Functional activation of Jak1 and Jak3 by selective association with IL-2 receptor subunits. Science 1994; 266: 1045–7. 222. Russell SM, Johnston JA, Noguchi M et al. Interaction of IL-2R beta and gamma c chains with Jak1 and Jak3: implications for XSCID and XCID. Science 1994; 266: 1042–5. 223. Park SY, Saijo K, Takahashi T et al. Developmental defects of lymphoid cells in Jak3 kinasedeficient mice. Immunity 1995; 3: 771–82. 224. Nosaka T, van Deursen JM, Tripp RA et al. Defective lymphoid development in mice lacking Jak3. Science 1995; 270: 800–2. 225. Thomis DC, Gurniak CB, Tivol E, Sharpe AH, Berg LJ. Defects in B lymphocyte maturation and T lymphocyte activation in mice lacking Jak3. Science 1995; 270: 794–7. 226. Macchi P, Villa A, Giliani S et al. Mutations of Jak-3 gene in patients with autosomal severe combined immune deficiency (SCID). Nature 1995; 377: 65–8. 227. Russell SM, Tayebi N, Nakajima H et al. Mutation of Jak3 in a patient with SCID: essential role of Jak3 in lymphoid development. Science 1995; 270: 797–800. 228. Akashi K, Kondo M, Weissman IL. Role of interleukin-7 in T-cell development from hematopoietic stem cells. Immunol Rev 1998; 165: 13–28. 229. Vaux DL, Cory S, Adams JM. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 1988; 335: 440–2. 230. Akashi K, Kondo M, von Freeden-Jeffry U, Murray R, Weissman IL. Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor-deficient mice. Cell 1997; 89: 1033–41. 231. Maraskovsky E, O’Reilly LA, Teepe M, Corcoran LM, Peschon JJ, Strasser A. Bcl-2 can rescue T lymphocyte development in interleukin-7 receptor-deficient mice but not in mutant rag-1−/− mice. Cell 1997; 89: 1011–19. 232. Pestano GA, Zhou Y, Trimble LA, Daley J, Weber GF, Cantor H. Inactivation of misselected CD8 T cells by CD8 gene methylation and cell death. Science 1999; 284: 1187–91. 233. Corcoran AE, Smart FM, Cowling RJ, Crompton T, Owen MJ, Venkitaraman AR. The interleukin7 receptor alpha chain transmits distinct signals for proliferation and differentiation during B lymphopoiesis. Embo J 1996; 15: 1924–32. 234. Corcoran AE, Riddell A, Krooshoop D, Venkitaraman AR. Impaired immunoglobulin gene rearrangement in mice lacking the IL-7 receptor. Nature 1998; 391: 904–7. 235. Ye SK, Maki K, Kitamura T et al. Induction of germline transcription in the TCRgamma locus by Stat5: implications for accessibility control by the IL-7 receptor. Immunity 1999; 11: 213–23. 236. Ye SK, Agata Y, Lee HC et al. The IL-7 receptor controls the accessibility of the TCRgamma locus by Stat5 and histone acetylation. Immunity 2001; 15: 813–23. 237. Karsunky H, Merad M, Cozzio A, Weissman IL, Manz MG. Flt3 ligand regulates dendritic cell development from Flt3+ lymphoid and myeloidcommitted progenitors to Flt3+ dendritic cells in vivo. J Exp Med 2003; 198: 305–13. 238. Forsberg EC, Serwold T, Kogan S, Weissman IL, Passegue E. New evidence supporting megakaryocyte-erythrocyte potential of flk2/flt3+ mul-

239.

240.

241.

242.

243.

244.

245.

246.

247.

248.

249.

250.

251.

252.

253.

254.

59

tipotent hematopoietic progenitors. Cell 2006; 126: 415–26. Coffman RL, Weissman IL. B220: a B cell-specific member of the T200 glycoprotein family. Nature 1981; 289: 681–3. Wu Q, Tidmarsh GF, Welch PA, Pierce JH, Weissman IL, Cooper MD. The early B lineage antigen BP-1 and the transformation-associated antigen 6C3 are on the same molecule. J Immunol 1989; 143: 3303–8. Sherwood PJ, Weissman IL. The growth factor IL-7 induces expression of a transformation-associated antigen in normal pre-B cells. Int Immunol 1990; 2: 399–406. Allman D, Li J, Hardy RR. Commitment to the B lymphoid lineage occurs before DH-JH recombination. J Exp Med 1999; 189: 735–40. Hardy RR, Carmack CE, Shinton SA, Kemp JD, Hayakawa K. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J Exp Med 1991; 173: 1213– 25. Li YS, Wasserman R, Hayakawa K, Hardy RR. Identification of the earliest B lineage stage in mouse bone marrow. Immunity 1996; 5: 527–35. Hardy RR, Wei CJ, Hayakawa K. Selection during development of VH11+ B cells: a model for natural autoantibody-producing CD5+ B cells. Immunol Rev 2004; 197: 60–74. Mojica MP, Perry SS, Searles AE et al. Phenotypic distinction and functional characterization of pro-B cells in adult mouse bone marrow. J Immunol 2001; 166: 3042–51. Nikolic T, Dingjan GM, Leenen PJ, Hendriks RW. A subfraction of B220(+) cells in murine bone marrow and spleen does not belong to the B cell lineage but has dendritic cell characteristics. Eur J Immunol 2002; 32: 686–92. Ogawa M, ten Boekel E, Melchers F. Identification of CD19(−)B220(+)c-Kit(+)Flt3/Flk-2(+)cells as early B lymphoid precursors before pre-B-I cells in juvenile mouse bone marrow. Int Immunol 2000; 12: 313–24. Rolink A, Grawunder U, Winkler TH, Karasuyama H, Melchers F. IL-2 receptor alpha chain (CD25, TAC) expression defines a crucial stage in pre-B cell development. Int Immunol 1994; 6: 1257–64. Rumfelt LL, Zhou Y, Rowley BM, Shinton SA, Hardy RR. Lineage specification and plasticity in CD19- early B cell precursors. J Exp Med 2006; 203: 675–87. Tung JW, Mrazek MD, Yang Y, Herzenberg LA. Phenotypically distinct B cell development pathways map to the three B cell lineages in the mouse. Proc Natl Acad Sci U S A 2006; 103: 6293–8. Tudor KS, Payne KJ, Yamashita Y, Kincade PW. Functional assessment of precursors from murine bone marrow suggests a sequence of early B lineage differentiation events. Immunity 2000; 12: 335–45. Bhattacharya D, Inlay M, Karsunky H, Serwold T, Rich BE, Weissman IL. High resolution analysis of the earliest stages of B lineage commitment reveals an inhibitory role for IL-1 signaling (submitted for publication). Adkins B, Mueller C, Okada CY, Reichert RA, Weissman IL, Spangrude GJ. Early events in Tcell maturation. Annu Rev Immunol 1987; 5: 325–65.

60

Chapter 5

255. Godfrey DI, Kennedy J, Suda T, Zlotnik A. A developmental pathway involving four phenotypically and functionally distinct subsets of CD3−CD4−CD8− triple-negative adult mouse thymocytes defined by CD44 and CD25 expression. J Immunol 1993; 150: 4244–52. 256. Matsuzaki Y, Gyotoku J, Ogawa M et al. Characterization of c-kit positive intrathymic stem cells that are restricted to lymphoid differentiation. J Exp Med 1993; 178: 1283–92. 257. Wu L, Scollay R, Egerton M, Pearse M, Spangrude GJ, Shortman K. CD4 expressed on earliest T-lineage precursor cells in the adult murine thymus. Nature 1991; 349: 71–4. 258. Ardavin C, Wu L, Li CL, Shortman K. Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population. Nature 1993; 362: 761–3. 259. Antica M, Wu L, Shortman K, Scollay R. Thymic stem cells in mouse bone marrow. Blood 1994; 84: 111–17. 260. Allman D, Sambandam A, Kim S et al. Thymopoiesis independent of common lymphoid progenitors. Nat Immunol 2003; 4: 168–74. 261. Blais ME, Louis I, Perreault C. T-cell development: an extrathymic perspective. Immunol Rev 2006; 209: 103–14. 262. Dejbakhsh-Jones S, Garcia-Ojeda ME, Chatterjea-Matthes D, Zeng D, Strober S. Clonable progenitors committed to the T lymphocyte lineage in the mouse bone marrow; use of an extrathymic pathway. Proc Natl Acad Sci U S A 2001; 98: 7455–60. 263. Garcia-Ojeda ME, Dejbakhsh-Jones S, Weissman IL, Strober S. An alternate pathway for T cell development supported by the bone marrow microenvironment: recapitulation of thymic maturation. J Exp Med 1998; 187: 1813–23. 264. Karsunky H, Inlay MA, Serwold T, Bhattacharya D, Weissman IL. Flk2+ common lymphoid progenitors possess equivalent differentiation potential for the B and T lineages. Blood. Epub 2008 April 18. doi: 10.1182/blood-2007-11126219. 265. Arber C, BitMansour A, Sparer TE et al. Common lymphoid progenitors rapidly engraft and protect against lethal murine cytomegalovirus infection after hematopoietic stem cell transplantation. Blood 2003; 102: 421–8. 266. Sanchez MJ, Muench MO, Roncarolo MG, Lanier LL, Phillips JH. Identification of a common T/ natural killer cell progenitor in human fetal thymus. J Exp Med 1994; 180: 569–76. 267. Gore SD, Kastan MB, Civin CI. Normal human bone marrow precursors that express terminal deoxynucleotidyl transferase include T-cell precursors and possible lymphoid stem cells. Blood 1991; 77: 1681–90. 268. LeBien TW. Fates of human B-cell precursors. Blood 2000; 96: 9–23. 269. Galy A, Travis M, Cen D, Chen B. Human T, B, natural killer, and dendritic cells arise from a common bone marrow progenitor cell subset. Immunity 1995; 3: 459–73. 270. Prieyl JA, LeBien TW. Interleukin 7 independent development of human B cells. Proc Natl Acad Sci U S A 1996; 93: 10348–53. 271. Puel A, Ziegler SF, Buckley RH, Leonard WJ. Defective IL7R expression in T(−)B(+)NK(+) severe combined immunodeficiency. Nat Genet 1998; 20: 394–7.

272. Roifman CM, Zhang J, Chitayat D, Sharfe N. A partial deficiency of interleukin-7R alpha is sufficient to abrogate T-cell development and cause severe combined immunodeficiency. Blood 2000; 96: 2803–7. 273. Manz MG, Miyamoto T, Akashi K, Weissman IL. Prospective isolation of human clonogenic common myeloid progenitors. Proc Natl Acad Sci U S A 2002; 99: 11872–7. 274. Dittel BN, LeBien TW. The growth response to IL-7 during normal human B cell ontogeny is restricted to B-lineage cells expressing CD34. J Immunol 1995; 154: 58–67. 275. Spits H, Blom B, Jaleco AC et al. Early stages in the development of human T, natural killer and thymic dendritic cells. Immunol Rev 1998; 165: 75–86. 276. Arinobu Y, Mizuno SI, Chong Y et al. Reciprocal activation of GATA-1 and PU.1 marks initial specification of hematopoietic stem cells into myeloerythroid and myelolymphoid lineages. Cell Stem Cell 2007; 1: 416–27. 277. Humphries RK, Jacky PB, Dill FJ, Eaves AC, Eaves CJ. CFU-S in individual erythroid colonies derived in vitro from adult mouse marrow. Nature 1979; 279: 718–20. 278. Nakorn TN, Miyamoto T, Weissman IL. Characterization of mouse clonogenic megakaryocyte progenitors. Proc Natl Acad Sci U S A 2003; 100: 205–10. 279. Nutt SL, Metcalf D, D’Amico A, Polli M, Wu L. Dynamic regulation of PU.1 expression in multipotent hematopoietic progenitors. J Exp Med 2005; 201: 221–31. 280. Pronk CJH, Rossi D, Mansson R et al. Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy. Cell Stem Cell 2007; 1: 428– 42. 281. Adolfsson J, Mansson R, Buza-Vidas N et al. Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential: a revised road map for adult blood lineage commitment. Cell 2005; 121: 295–306. 282. DeKoter RP, Kamath MB, Houston IB. Analysis of concentration-dependent functions of PU.1 in hematopoiesis using mouse models. Blood Cells Mol Dis 2007; 39: 316–20. 283. Katoh S, Tominaga A, Migita M, Kudo A, Takatsu K. Conversion of normal Ly-1-positive B-lineage cells into Ly-1-positive macrophages in long-term bone marrow cultures. Dev Immunol 1990; 1: 113–25. 284. Klinken SP, Alexander WS, Adams JM. Hemopoietic lineage switch: v-raf oncogene converts Emu-myc transgenic B cells into macrophages. Cell 1988; 53: 857–67. 285. Martin M, Strasser A, Baumgarth N et al. A novel cellular model (SPGM 1) of switching between the pre-B cell and myelomonocytic lineages. J Immunol 1993; 150: 4395–406. 286. Kondo M, Scherer DC, Miyamoto T et al. Cellfate conversion of lymphoid-committed progenitors by instructive actions of cytokines. Nature 2000; 407: 383–6. 287. Hsu C-L, King-Fleischman AG, Lai AY et al. Antagonistic effect of CCAAT enhance-binding protein-alpha and Pax5 in myeloid or lymphoid lineage choice in common lymphoid progenitors. Proc Natl Acad Sci U S A 2006; 103: 672– 7.

288. Cumano A, Paige CJ, Iscove NN, Brady G. Bipotential precursors of B cells and macrophages in murine fetal liver. Nature 1992; 356: 612–15. 289. Kawamoto H, Ohmura K, Katsura Y. Direct evidence for the commitment of hematopoietic stem cells to T, B and myeloid lineages in murine fetal liver. Int Immunol 1997; 9: 1011–19. 290. Mebius RE, Miyamoto T, Christensen J et al. The fetal liver counterpart of adult common lymphoid progenitors gives rise to all lymphoid lineages, CD45+CD4+CD3− cells, as well as macrophages. J Immunol 2001; 166: 6593–601. 291. Montecino-Rodriguez E, Leathers H, Dorshkind K. Bipotential B-macrophage progenitors are present in adult bone marrow. Nat Immunol 2001; 2: 83–8. 292. Fritsch G, Buchinger, Printz D et al. Rapid discrimination of early CD34+ myeloid progenitors using CD45-RA analysis. Blood 1993; 81: 2301– 9. 293. Huang S, Chen Z, Yu JF et al. Correlation between IL-3 receptor expression and growth potential of human CD34+ hematopoietic cells from different tissues. Stem Cells 1999; 17: 265–72. 294. Lansdorp PM, Sutherland HJ, Eaves CJ. Selective expression of CD45 isoforms on functional subpopulations of CD34+ hemopoietic cells from human bone marrow. J Exp Med 1990; 172: 363– 6. 295. de Wynter EA, Heyworth CM, Mukaida N et al. CCR1 chemokine receptor expression isolates erythroid from granulocyte-macrophage progenitors. J Leukoc Biol 2001; 70: 455–60. 296. Rappold I, Ziegler BL, Kohler I et al. Functional and phenotypic characterization of cord blood and bone marrow subsets expressing FLT3 (CD135) receptor tyrosine kinase. Blood 1997; 90: 111– 25. 297. Irie-Sasaki J, Sasaki T, Matsumoto W et al. CD45 is a JAK phosphatase and negatively regulates cytokine receptor signalling. Nature 2001; 409: 349–54. 298. Ogawa M. Differentiation and proliferation of hematopoietic stem cells. Blood 1993; 81: 2844– 53. 299. Kimura T, Sakabe H, Tanimukai S et al. Simultaneous activation of signals through gp130, c-kit, and interleukin-3 receptor promotes a trilineage blood cell production in the absence of terminally acting lineage-specific factors. Blood 1997; 90: 4767–78. 300. Holyoake TL, Nicolini FE, Eaves CJ. Functional differences between transplantable human hematopoietic stem cells from fetal liver, cord blood, and adult marrow. Exp Hematol 1999; 27: 1418– 27. 301. Jordan CT, Astle CM, Zawadzki J, Mackarehtschian K, Lemischka IR, Harrison DE. Long-term repopulating abilities of enriched fetal liver stem cells measured by competitive repopulation. Exp Hematol 1995; 23: 1011–15. 302. Pawliuk R, Eaves C, Humphries RK. Evidence of both ontogeny and transplant dose-regulated expansion of hematopoietic stem cells in vivo. Blood 1996; 88: 2852–8. 303. Hayakawa K, Hardy RR. Development and function of B-1 cells. Curr Opin Immunol 2000; 12: 346–53. 304. Traver D, Miyamoto T, Christensen J, IwasakiArai J, Akashi K, Weissman IL. Fetal liver myelopoiesis occurs through distinct, prospectively

Biology of Hematopoietic Stem and Progenitor Cells

305.

306.

307.

308.

309.

310.

311.

312.

313.

314.

315.

316.

317.

318.

319.

320.

321.

isolatable progenitor subsets. Blood 2001; 98: 627–35. Adachi S, Yoshida H, Kataoka H, Nishikawa S. Three distinctive steps in Peyer’s patch formation of murine embryo. Int Immunol 1997; 9: 507–14. Nutt SL, Urbanek P, Rolink A, Busslinger M. Essential functions of Pax5 (BSAP) in pro-B cell development: difference between fetal and adult B lymphopoiesis and reduced V-to-DJ recombination at the IgH locus. Genes Dev 1997; 11: 476–91. Mebius RE, Streeter PR, Michie S, Butcher EC, Weissman IL. A developmental switch in lymphocyte homing receptor and endothelial vascular addressin expression regulates lymphocyte homing and permits CD4+ CD3− cells to colonize lymph nodes. Proc Natl Acad Sci U S A 1996; 93: 11019–24. Mebius RE, Rennert P, Weissman IL. Developing lymph nodes collect CD4+CD3− LTbeta+ cells that can differentiate to APC, NK cells, and follicular cells but not T or B cells. Immunity 1997; 7: 493–504. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998; 392: 245– 52. Hart DN. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 1997; 90: 3245–87. Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 1991; 9: 271–96. Liu YJ, Kanzler H, Soumelis V, Gilliet M. Dendritic cell lineage, plasticity and cross-regulation. Nat Immunol 2001; 2: 585–9. Shortman K, Liu YJ. Mouse and human dendritic cell subtypes. Nat Rev Immunol 2002; 2: 151– 61. Vremec D, Zorbas M, Scollay R et al. The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells. J Exp Med 1992; 176: 47–58. Wu L, Li CL, Shortman K. Thymic dendritic cell precursors: relationship to the T lymphocyte lineage and phenotype of the dendritic cell progeny. J Exp Med 1996; 184: 903–11. Wu L, D’Amico A, Winkel KD, Suter M, Lo D, Shortman K. RelB is essential for the development of myeloid-related CD8alpha− dendritic cells but not of lymphoid-related CD8alpha+ dendritic cells. Immunity 1998; 9: 839–47. Guerriero A, Langmuir PB, Spain LM, Scott EW. PU.1 is required for myeloid-derived but not lymphoid-derived dendritic cells. Blood 2000; 95: 879–85. Wu L, Nichogiannopoulou A, Shortman K, Georgopoulos K. Cell-autonomous defects in dendritic cell populations of Ikaros mutant mice point to a developmental relationship with the lymphoid lineage. Immunity 1997; 7: 483–92. Radtke F, Ferrero I, Wilson A, Lees R, Aguet M, MacDonald HR. Notch1 deficiency dissociates the intrathymic development of dendritic cells and T cells. J Exp Med 2000; 191: 1085–94. Manz MG, Traver D, Miyamoto T, Weissman IL, Akashi K. Dendritic cell potentials of early lymphoid and myeloid progenitors. Blood 2001; 97: 3333–41. Traver D, Akashi K, Manz M et al. Development of CD8alpha-positive dendritic cells from a

322.

323.

324.

325.

326.

327.

328.

329.

330.

331.

332.

333.

334.

335.

336.

common myeloid progenitor. Science 2000; 290: 2152–4. Wu L, D’Amico A, Hochrein H, O’Keeffe M, Shortman K, Lucas K. Development of thymic and splenic dendritic cell populations from different hemopoietic precursors. Blood 2001; 98: 3376–82. Hao QL, Zhu J, Price MA, Payne KJ, Barsky LW, Crooks GM. Identification of a novel, human multilymphoid progenitor in cord blood. Blood 2001; 97: 3683–90. Caux C, Vanbervliet B, Massacrier C et al. CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GMCSF+TNF alpha. J Exp Med 1996; 184: 695– 706. Young JW, Szabolcs P, Moore MA. Identification of dendritic cell colony-forming units among normal human CD34+ bone marrow progenitors that are expanded by c-kit-ligand and yield pure dendritic cell colonies in the presence of granulocyte/macrophage colony-stimulating factor and tumor necrosis factor alpha. J Exp Med 1995; 182: 1111–19. Randolph GJ, Beaulieu S, Lebecque S, Steinman RM, Muller WA. Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking. Science 1998; 282: 480–3. Romani N, Gruner S, Brang D et al. Proliferating dendritic cell progenitors in human blood. J Exp Med 1994; 180: 83–93. Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 1994; 179: 1109–18. Cella M, Jarrossay D, Facchetti F et al. Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon. Nat Med 1999; 5: 919–23. Grouard G, Rissoan MC, Filgueira L, Durand I, Banchereau J, Liu YJ. The enigmatic plasmacytoid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand. J Exp Med 1997; 185: 1101–11. Jarrossay D, Napolitani G, Colonna M, Sallusto F, Lanzavecchia A. Specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells. Eur J Immunol 2001; 31: 3388–93. Kadowaki N, Antonenko S, Lau JY, Liu YJ. Natural interferon alpha/beta-producing cells link innate and adaptive immunity. J Exp Med 2000; 192: 219–26. Kadowaki N, Ho S, Antonenko S et al. Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. J Exp Med 2001; 194: 863–9. Asselin-Paturel C, Boonstra A, Dalod M et al. Mouse type I IFN-producing cells are immature APCs with plasmacytoid morphology. Nat Immunol 2001; 2: 1144–50. Bjorck P. Isolation and characterization of plasmacytoid dendritic cells from Flt3 ligand and granulocyte-macrophage colony-stimulating factor-treated mice. Blood 2001; 98: 3520– 6. Wu L, Dakic A. Development of dendritic cell system. Cell Mol Immunol 2004; 1: 112–18.

61

337. Karsunky H, Merad M, Mende I, Manz MG, Engleman EG, Weissman IL. Developmental origin of interferon-alpha-producing dendritic cells from hematopoietic precursors. Exp Hematol 2005; 33: 173–81. 338. King AG, Kondo M, Scherer DC, Weissman IL. Lineage infidelity in myeloid cells with TCR gene rearrangement: a latent developmental potential of proT cells revealed by ectopic cytokine receptor signaling. Proc Natl Acad Sci U S A 2002; 99: 4508–13. 339. Rossi DJ, Bryder D, Seita J, Nussenzweig A, Hoeijmakers J, Weissman IL. Deficiencies in DNA damage repair limit the function of haematopoietic stem cells with age. Nature 2007; 447: 725–9. 340. Prohaska SS, Weissman I. Gene expression profiling of highly purified myeloid progenitor populations (submitted for publication). 341. Geschwind DH, Ou J, Easterday MC et al. A genetic analysis of neural progenitor differentiation. Neuron 2001; 29: 325–39. 342. Ivanova NB, Dimos JT, Schaniel C, Hackney JA, Moore KA, Lemischka IR. A stem cell molecular signature. Science 2002; 298: 601–4. 343. Sieweke MH, Graf T. A transcription factor party during blood cell differentiation. Curr Opin Genet Dev 1998; 8: 545–51. 344. DeKoter RP, Singh H. Regulation of B lymphocyte and macrophage development by graded expression of PU.1. Science 2000; 288: 1439– 41. 345. Hu M, Krause D, Greaves M et al. Multilineage gene expression precedes commitment in the hemopoietic system. Genes Dev 1997; 11: 774– 85. 346. Porcher C, Swat W, Rockwell K, Fujiwara Y, Alt FW, Orkin SH. The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages. Cell 1996; 86: 47–57. 347. Shivdasani RA, Mayer EL, Orkin SH. Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL. Nature 1995; 373: 432–4. 348. Tsai FY, Keller G, Kuo FC et al. An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature 1994; 371: 221–6. 349. Fujiwara Y, Browne CP, Cunniff K, Goff SC, Orkin SH. Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1. Proc Natl Acad Sci U S A 1996; 93: 12355–8. 350. Shivdasani RA, Fujiwara Y, McDevitt MA, Orkin SH. A lineage-selective knockout establishes the critical role of transcription factor GATA-1 in megakaryocyte growth and platelet development. EMBO J 1997; 16: 3965–73. 351. Zhang DE, Zhang P, Wang ND, Hetherington CJ, Darlington GJ, Tenen DG. Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice. Proc Natl Acad Sci U S A 1997; 94: 569–74. 352. Morgan B, Sun L, Avitahl N et al. Aiolos, a lymphoid restricted transcription factor that interacts with Ikaros to regulate lymphocyte differentiation. EMBO J 1997; 16: 2004–13. 353. Klug CA, Morrison SJ, Masek M, Hahm K, Smale ST, Weissman IL. Hematopoietic stem cells and lymphoid progenitors express different Ikaros isoforms, and Ikaros is localized to heterochromatin

62

354.

355.

356.

357. 358.

359.

360.

361.

362.

363.

364.

365.

366. 367. 368.

369.

370.

371.

Chapter 5

in immature lymphocytes. Proc Natl Acad Sci U S A 1998; 95: 657–62. Georgopoulos K, Bigby M, Wang JH et al. The Ikaros gene is required for the development of all lymphoid lineages. Cell 1994; 79: 143– 56. Ting CN, Olson MC, Barton KP, Leiden JM. Transcription factor GATA-3 is required for development of the T-cell lineage. Nature 1996; 384: 474–8. Nutt SL, Heavey B, Rolink AG, Busslinger M. Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature 1999; 401: 556–62. Enver T, Greaves M. Loops, lineage, and leukemia. Cell 1998; 94: 9–12. Marks PA, Richon VM, Rifkind RA. Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells. J Natl Cancer Inst 2000; 92: 1210–16. Sieweke MH, Tekotte H, Frampton J, Graf T. MafB is an interaction partner and repressor of Ets-1 that inhibits erythroid differentiation. Cell 1996; 85: 49–60. Zhang P, Zhang X, Iwama A et al. PU.1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding. Blood 2000; 96: 2641–8. Kulessa H, Frampton J, Graf T. GATA-1 reprograms avian myelomonocytic cell lines into eosinophils, thromboblasts, and erythroblasts. Genes Dev 1995; 9: 1250–62. Chiang MY, Monroe JG. BSAP/Pax5A expression blocks survival and expansion of early myeloid cells implicating its involvement in maintaining commitment to the B-lymphocyte lineage. Blood 1999; 94: 3621–32. Anderson MK, Weiss AH, Hernandez-Hoyos G, Dionne CJ, Rothenberg EV. Constitutive expression of PU.1 in fetal hematopoietic progenitors blocks T cell development at the pro-T cell stage. Immunity 2002; 16: 285–96. Iwasaki H, Mizuno S, Wells RA, Cantor AB, Watanabe S, Akashi K. GATA-1 converts lymphoid and myelomonocytic progenitors into the megakaryocyte/erythrocyte lineages. Immunity 2003; 19: 451–62. Hsu CL, King-Fleischman AG, Lai AY, Matsumoto Y, Weissman IL, Kondo M. Antagonistic effect of CCAAT enhancer-binding protein-alpha and Pax5 in myeloid or lymphoid lineage choice in common lymphoid progenitors. Proc Natl Acad Sci U S A 2006; 103: 672–7. Berger SL, Felsenfeld G. Chromatin goes global. Mol Cell 2001; 8: 263–8. Felsenfeld G. Chromatin unfolds. Cell 1996; 86: 13–19. Weintraub H. High-resolution mapping of S1- and DNase I-hypersensitive sites in chromatin. Mol Cell Biol 1985; 5: 1538–9. Cross MA, Enver T. The lineage commitment of haemopoietic progenitor cells. Curr Opin Genet Dev 1997; 7: 609–13. Jung M. Inhibitors of histone deacetylase as new anticancer agents. Curr Med Chem 2001; 8: 1505–11. Skov S, Rieneck K, Bovin LF et al. Histone deacetylase inhibitors: a new class of immunosuppressors targeting a novel signal pathway essential for CD154 expression. Blood 2003; 101: 1430–8.

372. Attema JL, Papathanasiou P, Forsberg EC, Xu J, Smale ST, Weissman IL. Epigenetic characterization of hematopoietic stem cell differentiation using miniChIP and bisulfite sequencing analysis. Proc Natl Acad Sci U S A 2007; 104: 12371– 6. 373. Dahl JA, Collas P. Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay unravels epigenetic dynamics of developmentally regulated genes in human carcinoma cells. Stem Cells 2007; 25: 1037–46. 374. O’Neill LP, VerMilyea MD, Turner BM. Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations. Nat Genet 2006; 38: 835–41. 375. Bernstein BE, Mikkelsen TS, Xie X et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 2006; 125: 315–26. 376. Thomas ED. Bone marrow transplantation from bench to bedside. Ann N Y Acad Sci 1995; 770: 34–41. 377. Thomas ED. A history of haemopoietic cell transplantation. Br J Haematol 1999; 105: 330–9. 378. Uchida N, Tsukamoto A, He D, Friera AM, Scollay R, Weissman IL. High doses of purified stem cells cause early hematopoietic recovery in syngeneic and allogeneic hosts. J Clin Invest 1998; 101: 961–6. 379. Negrin RS, Atkinson K, Leemhuis T et al. Transplantation of highly purified CD34+Thy-1+ hematopoietic stem cells in patients with metastatic breast cancer. Biol Blood Marrow Transplant 2000; 6: 262–71. 380. Vose JM, Bierman PJ, Lynch JC et al. Transplantation of highly purified CD34+Thy-1+ hematopoietic stem cells in patients with recurrent indolent non-Hodgkin’s lymphoma. Biol Blood Marrow Transplant 2001; 7: 680–7. 381. Stadtmauer EA, O’Neill A, Goldstein LJ et al. Conventional-dose chemotherapy compared with high-dose chemotherapy plus autologous hematopoietic stem-cell transplantation for metastatic breast cancer. Philadelphia Bone Marrow Transplant Group. N Engl J Med 2000; 342: 1069–76. 382. Gazitt Y, Tian E, Barlogie B et al. Differential mobilization of myeloma cells and normal hematopoietic stem cells in multiple myeloma after treatment with cyclophosphamide and granulocyte-macrophage colony-stimulating factor. Blood 1996; 87: 805–11. 383. Michallet M, Philip T, Philip I et al. Transplantation with selected autologous peripheral blood CD34+Thy1+ hematopoietic stem cells (HSCs) in multiple myeloma: impact of HSC dose on engraftment, safety, and immune reconstitution. Exp Hematol 2000; 28: 858–70. 384. Miyamoto T, Weissman IL, Akashi K. AML1/ ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation. Proc Natl Acad Sci U S A 2000; 97: 7521–6. 385. Ferrara JL, Levy R, Chao NJ. Pathophysiologic mechanisms of acute graft-vs.-host disease. Biol Blood Marrow Transplant 1999; 5: 347–56. 386. Shizuru JA, Jerabek L, Edwards CT, Weissman IL. Transplantation of purified hematopoietic stem cells: requirements for overcoming the barriers of allogeneic engraftment. Biol Blood Marrow Transplant 1996; 2: 3–14.

387. Gandy KL, Domen J, Aguila H, Weissman IL. CD8+TCR+ and CD8+TCR− cells in whole bone marrow facilitate the engraftment of hematopoietic stem cells across allogeneic barriers. Immunity 1999; 11: 579–90. 388. Kaufman CL, Colson YL, Wren SM, Watkins S, Simmons RL, Ildstad ST. Phenotypic characterization of a novel bone marrow-derived cell that facilitates engraftment of allogeneic bone marrow stem cells. Blood 1994; 84: 2436–46. 389. Martin PJ. Donor CD8 cells prevent allogeneic marrow graft rejection in mice: potential implications for marrow transplantation in humans. J Exp Med 1993; 178: 703–12. 390. Shizuru JA, Weissman IL, Kernoff R, Masek M, Scheffold YC. Purified hematopoietic stem cell grafts induce tolerance to alloantigens and can mediate positive and negative T cell selection. Proc Natl Acad Sci U S A 2000; 97: 9555–60. 391. Heeg K, Wagner H. Induction of peripheral tolerance to class I major histocompatibility complex (MHC) alloantigens in adult mice: transfused class I MHC-incompatible splenocytes veto clonal responses of antigen-reactive Lyt-2+ T cells. J Exp Med 1990; 172: 719–28. 392. Thomas JM, Verbanac KM, Carver FM et al. Veto cells in transplantation tolerance. Clin Transplant 1994; 8: 195–203. 393. Steinman RM, Nussenzweig MC. Avoiding horror autotoxicus: the importance of dendritic cells in peripheral T cell tolerance. Proc Natl Acad Sci U S A 2002; 99: 351–8. 394. Millan MT, Shizuru JA, Hoffmann P et al. Mixed chimerism and immunosuppressive drug withdrawal after HLA-mismatched kidney and hematopoietic progenitor transplantation. Transplantation 2002; 73: 1386–91. 395. Beilhack GF, Scheffold YC, Weissman IL et al. Purified allogeneic hematopoietic stem cell transplantation blocks diabetes pathogenesis in NOD mice. Diabetes 2003; 52: 59–68. 396. Smith-Berdan S, Gille D, Weissman IL, Christensen JL. Reversal of autoimmune disease in lupus-prone New Zealand black/New Zealand white mice by nonmyeloablative transplantation of purified allogeneic hematopoietic stem cells. Blood 2007; 110: 1370–8. 397. BitMansour A, Burns SM, Traver D et al. Myeloid progenitors protect against invasive aspergillosis and Pseudomonas aeruginosa infection following hematopoietic stem cell transplantation. Blood 2002; 100: 4660–7. 398. Yilmaz OH, Valdez R, Theisen BK et al. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature 2006; 441: 418–19. 399. Wiemels JL, Xiao Z, Buffler PA et al. In utero origin of t(8;21) AML1-ETO translocations in childhood acute myeloid leukemia. Blood 2002; 99: 3801–5. 400. Gale KB, Ford AM, Repp R et al. Backtracking leukemia to birth: identification of clonotypic gene fusion sequences in neonatal blood spots. Proc Natl Acad Sci U S A 1997; 94: 13950–4. 401. Hjalgrim LL, Madsen HO, Melbye M et al. Presence of clone-specific markers at birth in children with acute lymphoblastic leukaemia. Br J Cancer 2002; 87: 994–9. 402. Cave H, van der Werff ten Bosch J, Suciu S et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. Euro-

Biology of Hematopoietic Stem and Progenitor Cells

403.

404.

405.

406.

407.

pean Organization for Research and Treatment of Cancer – Childhood Leukemia Cooperative Group. N Engl J Med 1998; 339: 591–8. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997; 3: 730–7. Blair A, Hogge De, Ailles LE, Lansdorp PM, Sutherland HJ. Lack of expression of Thy-a (CD90) on acute myeloid leukemia cells with long-term proliferative ability in vitro and in vivo. Blood 1997; 89: 3104–12. Yuan Y, Zhou L, Miyamoto T et al. AML1-ETO expression is directly involved in the development of acute myeloid leukemia in the presence of additional mutations. Proc Natl Acad Sci U S A 2001; 98: 10398–403. Brown D, Kogan S, Lagasse E et al. A PMLRARalpha transgene initiates murine acute promyelocytic leukemia. Proc Natl Acad Sci U S A 1997; 94: 2551–6. Kogan SC, Brown DE, Shultz DB et al. BCL-2 cooperates with promyelocytic leukemia retinoic

408.

409.

410.

411.

412.

acid receptor alpha chimeric protein (PMLRARalpha) to block neutrophil differentiation and initiate acute leukemia. J Exp Med 2001; 193: 531–43. Traver D, Akashi K, Weissman IL, Lagasse E. Mice defective in two apoptosis pathways in the myeloid lineage develop acute myeloblastic leukemia. Immunity 1998; 9: 47–57. Passegue E, Jamieson CH, Ailles LE, Weissman IL. Normal and leukemic hematopoiesis: are leukemias a stem cell disorder or a reacquisition of stem cell characteristics? Proc Natl Acad Sci U S A 2003; 100(Suppl 1): 11842–9. Passegue E, Jochum W, Schorpp-Kistner M, Mohle-Steinlein U, Wagner EF. Chronic myeloid leukemia with increased granulocyte progenitors in mice lacking junB expression in the myeloid lineage. Cell 2001; 104: 21–32. Jamieson CH, Weissman IL, Passegue E. Chronic versus acute myelogenous leukemia: a question of self-renewal. Cancer Cell 2004; 6: 531–3. Jamieson CH, Ailles LE, Dylla SJ et al. Granulocyte-macrophage progenitors as candidate leuke-

413.

414.

415.

416.

417.

63

mic stem cells in blast-crisis CML. N Engl J Med 2004; 351: 657–67. Hosen N, Park CY, Tatsumi N et al. CD96 is a leukemic stem cell-specific marker in human acute myeloid leukemia. Proc Natl Acad Sci U S A 2007; 104: 11008–13. Isken O, Maquat LE. Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function. Genes Dev 2007; 21: 1833– 56. Mi S, Lu J, Sun M et al. MicroRNA expression signatures accurately discriminate acute lymphoblastic leukemia from acute myeloid leukemia. Proc Natl Acad Sci U S A 2007; 104: 19971– 6. Nicoloson MS, Kipps TJ, Croce CM, Calin GA. MicroRNAs in the pathogeny of chronic lymphocytic leukaemia. Br J Haematol 2007; 139: 709– 16. Zanette DL, Rivadavia F, Molfetta GA et al. miRNA expression profiles in chronic lymphocytic and acute lymphocytic leukemia. Braz J Med Biol Res 2007; 40: 1435–40.

6

Peter M. Lansdorp

Molecular Biology of Stem Cell Renewal

Several factors have led to intensive investigations of the hematopoietic system over the past few decades. The hematopoietic system represents the prototype of a self-renewing biologic system in that large numbers of blood cells have to be produced daily in order to compensate for the loss of relatively short-lived mature blood cells. The relative ease with which blood and marrow samples can be obtained and single-cell suspensions prepared has greatly facilitated in vitro and in vivo studies of hematopoietic progenitor and stem cells. Techniques to purify increasingly rare cells on the basis of cell surface antigens or functional properties have been refined and are available to most researchers. A large number of purified recombinant proteins and other molecules with activity on various hematopoietic cells have been discovered and have become available as well. In addition, a variety of in vitro and in vivo assays to measure functional properties of hematopoietic cells have been developed over the years, and some of these are now available as standardized tests. Together, these advances have allowed various studies with purified cells and molecules, some of which have revealed unexpected characteristics of stem cell “candidates.” From such studies, it has become clear that hematopoietic stem cells (HSCs) cannot be considered a homogeneous population of cells. Instead, HSCs represent a heterogeneous population of cells with functional properties that vary with replicative history, developmental stage, and age. This is illustrated by the pronounced developmental changes in the functional properties of purified “candidate” stem cell populations, the marked change in stem cell turnover in the first few years of life, and the age-related loss of telomeric DNA in HSCs. The age-related decline in stem cell renewal and replicative potential is clinically relevant as limited engraftment and failure of stem cell transplants could result from properties of HSCs that are predictable and, increasingly, measurable. In this chapter, some aspects related to the “self-renewal” of HSCs are reviewed. No attempt is made to cover the extensive literature in this general area. Specifically, the large number of studies aimed at dissecting the role of various transcription factors in stem cell properties and function are only briefly mentioned here. Instead, the focus is on the role of telomerase and the loss of telomere repeats in stem cells relative to their “self-renewal” in vitro and in vivo.

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

64

The stem cell hierarchy A recurrent theme in experimental hematology over the past 40 years has been the redefinition of what HSCs are, based on changes and improvements in assays. The trend has invariably been towards the identification of cells with a lower frequency than was previously reported or proposed [1]. The inevitable conclusion of this trend will no doubt be the identification of the fertilized egg as the “mother of all stem cells.” But what could be the explanation for the seemingly endless debate about HSC definition, HSC purification, and the apparent inability of an extensive field to agree on basic principles? The fact that the behavior of HSCs upon transplantation depends on many experimental variables, including the number and type of transplanted cells as well as host factors, has certainly contributed to the confusion. In animal models, only a limited number of blood- or bone marrow-derived HSCs are required to completely regenerate hematopoiesis in suitably conditioned recipients. Although the minimum number of HSCs required for sustained engraftment in human patients is not known, it has been shown that a single purified HSC in the mouse can reconstitute hematopoiesis [2]. This remarkable fact has supported the older definition of HSCs as “pluripotent cells with self-renewal potential” [3]. Implicit in the definition of “self-renewal” is that the two daughter cells, derived from the division of a single parental HSC, are identical to each other and indistinguishable from the parental cell. However, this is most likely an oversimplification as the behavior of HSCs is known to vary markedly with the developmental stage of the donor [4]. Thus, the source of HSCs is an important variable in transplant studies. Indeed, stem cell “candidates” purified from human [5] or murine [6] fetal liver versus adult bone marrow show remarkable functional differences that correlate with measurable molecular changes occurring with age [7] and upon transplantation [8]. Loss of telomere repeats in stem cells, developmental changes in HSC function, and the facultative, cell dosedependent symmetric versus asymmetric (self-renewal versus maintenance) cell divisions of HSCs [9] greatly complicate generalizations about the nature and function of HSCs. Nevertheless, the central dogma and current paradigm in experimental hematology has remained that perhaps the frequency of HSCs, but not HSCs themselves, are subject to significant change. In a way, it is understandable that experimental hematologists hate to part with simple notions about stem cells as “units” and “self-renewal” as a concept. After all, it is these notions that allow, in theory, an external control of HSC properties and the net “expansion” of HSCs in vitro. Although the inclusion of “relative” in the definition of “self-renewal”

Molecular Biology of Stem Cell Renewal

may seem annoying, the fact remains that hematopoietic tissues typically contain a spectrum of “stem” cells, each endowed with different replicative potential and functional properties. The relative frequency of various HSCs within the hierarchy is likely to vary with the age of the donor. The optimal clinical use of HSCs in transplantation settings will take not only the number and viability of HSCs in the transplant into account, but also their developmental stage and their estimated replicative potential.

Hematopoiesis: how many stem cell divisions? The realization that stem cells are subject to pronounced developmental changes has focused attention on the question, “How many times can stem cells divide?” Unfortunately, no techniques to address this important question are currently available, and possible answers range from fewer than 100 times [1] to over 5000 times [10,11]. The total number of blood cells required for the lifelong maintenance of normal levels of blood cells can be calculated to be in the order of 4.1016 cells (∼1012 cells per day × 365 days × 100 years). In theory, only 55 divisions of a single cell would satisfy this need (255 = 4.1016). Because many hundreds of different HSCs are contributing to hematopoiesis in a normal individual at any given time, an absolute replicative potential of fewer than 100 cell divisions per HSC could presumably easily accommodate a lifetime of blood cell production and allow for the known apoptosis of cytokinedeprived committed progenitor cells and the remarkable regenerative capacity of HSCs displayed in response to marrow injury and upon (serial) transplantation. An important related question is how many HSC clones are actually present in normal adults. Because genetic markers such as point mutations can occur at any given cell division, the presence of such a clonal marker does not a priori allow the conclusive identification of a single HSC clone. For example, in patients with paroxysmal nocturnal hemoglobinuria, the presence of a mutation in the PIG-A gene is often used to identify the abnormal HSC “clone” [12]. However, such a mutation could have occurred relatively early in development, for example before the major expansion of HSC numbers in early childhood [13]. As a result, many hundreds of adult HSC clones would be expected to show the mutation and “clonal” marker. Conversely, when a mutation takes place very late in the stem cell hierarchy, only committed progenitors could be affected, resulting in the transient appearance of a clonal marker in the peripheral blood. The transient presence of BCR–ABL fusion genes in the circulation of normal individuals could be an example of such a late occurrence [14]. Presumably the likelihood of PIG-A, BCR–ABL or other mutations will be directly related to the number of cells at risk: relatively few in early development and many in adult tissues. The notion that HSCs have a limited replicative potential is further supported by several observations. Both the in vivo regenerative capacity [15,16] and the in vitro expansion potential [4,5] of HSCs appear to be under developmental control (see also below). Most HSCs in adult tissue are quiescent cells in line with expectations for cells that divide very infrequently and have only a limited replicative potential that lack “self-renewal” properties in an absolute sense. The loss of telomere repeats in adult hematopoietic cells (including purified “candidate” HSCs) relative to fetal hematopoietic cells [7] also fits a model that postulates a finite and limited replicative potential of HSCs [1,17]. Recent longitudinal studies of telomere length in baboons provide further support for the idea that each cell division in HSC is accompanied by a measurable loss of telomere repeats [18]. In this study, it was found that the rate of telomere loss was greatly reduced after the first year of life in line with the rapid expansion of the HSC compartment in the first few years deduced from cross-sectional telomere length measurements in human leukocytes [13].

65

Development and organization of hematopoietic tissue The hematopoietic system has been subdivided into a hierarchy of three distinct populations [19,20]. In this model, the most mature cells are morphologically identifiable as belonging to a particular lineage and have very limited proliferative potential. The cells in this most mature compartment are derived from committed progenitor cells with a higher but finite proliferative potential. Committed progenitor cells are in turn produced by a population of multipotential HSCs with variable “selfrenewal” potential. Hematopoiesis in embryos is first observed in “blood islands” in the yolk sac [15]. Blood island precursor cells are mesenchymal cells derived from the primitive streak region of the early blastoderm. In the mouse, the yolk sac does not contain precursors of adult hematopoiesis, which can first be found in the aorta–gonad–mesonephros (AGM) region at day 10 of gestation [21]. From the AGM region, hematopoiesis appears to move to the fetal liver before reaching its final destination in the marrow. The concept of subsequent waves of progenitor cells seeding different tissues is compatible with various observations. However, the origin and number of cells seeding a particular hematopoietic niche still needs to be defined. Interestingly, some cells from yolk sac, fetal liver, and marrow are all capable of producing spleen colonies in irradiated adult recipients [15]. This observation, together with the questionable use of spleen colony assays to measure HSCs [22], is probably responsible for what appears to be an incomplete appreciation of the developmental changes in the biologic properties of primitive hematopoietic cells. Results showing that cells from early stages of development can function, to a large extent, in adult hosts and vice versa [23] illustrate a remarkable functional adaptive capacity or “plasticity” of hematopoietic cells at various stages of development. However, in the mouse, cells giving rise to spleen colonies upon transplantation into lethally irradiate recipients (colony-forming unit-spleen or CFU-S) from different tissues were found to be qualitatively different in that the number of CFU-S per individual spleen colony decreased during ontogeny [15]. In these experiments, yolk sac CFU-S could be transferred in vivo seven times, fetal liver CFU-S four to six times, and marrow CFU-S from young and adult mice up to three times [15]. These findings support a functional decline in stem cell self-renewal properties with each subsequent cell division and/or a decreasing and limited replicative potential of HSCs (see below). Several more recent observations provide further evidence of ontogeny-related differences between populations of primitive hematopoietic cells at various stages of development (reviewed in [4,24]). In general, fetal cells have a higher turnover rate and a higher replating potential than their functional counterparts from adult marrow. This observation is illustrated in Fig. 6.1 [5,25], which shows the production of CD34+ cells in culture from “candidate” HSCs with a CD34+CD45RAloCD71lo phenotype purified from, respectively, human bone marrow, cord blood, and fetal liver. Using identical growth factors and culture conditions, no significant production of CD34+ cells was observed in cultures initiated with adult bone marrow HSCs, whereas the number of CD34+ cells in cultures of fetal liver HSCs increased several thousand-fold. As the same number of cells was cultured under identical conditions, these observations point to intrinsic differences between the candidate HSCs themselves. Developmental switches have also been described in the immune system (reviewed in [26]). The switches from embryonic to fetal to adult hemoglobin in red cells reflect epigenetic changes in HSCs that could also affect their biologic properties upon transplantation. Because such epigenetic differences between HSCs at different developmental stages

66

Chapter 6 1011

Bone marrow

Cord blood

Fetal liver

1010

Cell number

109 108 107 106 105 104 103 0

10

20

30

40 0

10

20

30

40 0

10

20

30

40

Days

Fig. 6.1 Ontogeny-related differences in production of CD34+ cells in culture. Human candidate hematopoietic stem cells (HSCs) with a CD34+CD45RAloCD71lo phenotype were purified from bone marrow, umbilical cord blood and fetal liver, and cultured in serum-free culture medium supplemented with interleukin-6 (IL-6), IL-3, stem cell factor, and erythropoietin (Epo) as described in [25]. Cultures (1 mL each) were initiated with 104 sorted cells. At the indicated time interval, the total number of nucleated (䊐) and CD34+ (䊏) cells present in the cultures was calculated from the cell counts and the percentage of viable CD34+ cells measured by flow cytometry. All CD34+ cells from bone marrow cultures and fractions of the CD34+ cells from cord blood and fetal liver cultures were sorted and used for continuation of the cultures. Reproduced with permission from [5]. Copyright (1993), The Rockefeller University Press.

are likely, many assumptions about HSCs in adults need experimental verification. For example, it is currently not clear that adult HSCs have a similar potential to their fetal counterparts to produce lymphoid progenitors. In the mouse, the ability of HSCs to produce specific lymphocyte precursors is under developmental control [27], and the number of HSCs with full lymphoid potential in adults is expected to be limited. Similarly, the cells of the microenvironment are expected to show changes as a function of normal development and as a function of age. Uncertainties regarding the composition of the stem cell microenvironment and its function over time, combined with similar questions about the cells in the stem cell compartment, represent ongoing challenges in the basic science of HSCs and HSC transplantation.

Molecular control of stem cell fate Of fundamental interest to experimental hematologists and developmental biologists are the mechanisms that control the fate of HSCs. At any point in time, a “stem” cell has a choice to contribute to either the immediate future (by differentiating and producing committed progenitor cells) or the more distant requirements for mature cells (by undergoing a functional self-renewal division). It has been proposed that such decisions at the level of pluripotent HSCs can be depicted as stochastic processes that are intrinsic to the cells [20,28]. If this concept is correct, the distinction between HSCs with limited and extensive in vivo repopulation potential will likely remain elusive as the critical distinction between the cells, the number of actual self-renewal divisions executed in vivo, would be governed by chance. This possibility is certainly in agreement with the difficulties encountered by many investigators in purifying repopulating HSCs to homogeneity. However, as was outlined above, the situation is more complex as the self-renewal properties of HSCs are also subject to developmental control. Furthermore, it seems likely that the two daughter cells resulting from cell division of a single HSC may each differ in their functional properties. Such asymmetric cell divisions are key in animal development [29] and need to be taken into account in models of HSC biology and hematopoiesis [9,30]. The ability of a limited number of HSCs to reconstitute hematopoiesis in an irradiated recipient strongly suggests

that factors in the environment are also crucial in directing stem cell fate. Most likely, HSCs switch upon transplantation from symmetric “self-renewal” divisions (which increase the absolute number of stem cells, albeit at the expense of telomere loss) to asymmetric “maintenance” cell divisions as the bone marrow and its HSC “niches” become occupied with cells. Although the molecular details that govern this switch remain to be elucidated, it seems likely that the orientation of the mitotic spindle relative to the basal layer is involved [9,31]. One interesting possibility is that, following DNA replication but prior to mitosis, the two sister chromatids in the stem cells carry distinct epigenetic marks at the centromeric DNA as well as at the regulatory regions of specific genes [32]. The nonrandom segregation of sister chromatids could support asymmetric cell divisions and maintenance of stem cells, whereas the random segregation of sister chromatids could favor self-renewal and increased stem cell numbers (Plate 6.1).

Transcriptional control of stem cell function Despite intensive studies, the role of soluble factors involved in stem cell fate decisions is not clear. The dramatic clinical effect of cytokines such as erythropoietin, granulocyte colony-stimulating factor, stem cell factor, thrombopoietin, and others have underscored the important and critical role that cytokines play in the proliferation and differentiation of committed hematopoietic progenitor cells. However, attempts to modulate cell fate decisions in early hematopoietic cells using cytokines have invariably been unsuccessful [28,30,33,34]. Perhaps the primary role of cytokines and the microenvironment in HSC biology is to provide the essential but primarily permissive environment required for survival, proliferation, and differentiation. But if growth factors or factors in the microenvironment do not dictate stem cell fate decisions, what factors do? Important clues to this crucial question have been obtained by studies of the expression of homeobox genes in human hematopoietic cells [35]. It was found that expression of Hox genes is restricted to the most primitive hematopoietic progenitors and that the overexpression of HoxB4 in murine HSCs results in a remarkable expansion of cells with long-term lymphomyeloid repopulat-

Molecular Biology of Stem Cell Renewal

ing potential [36,37]. The involvement of Hox genes in stem cell biology is particularly striking because Hox genes are better known as key regulators of patterning along the body axis of developing embryos [38]. It is possible that Hox genes are part of the network that controls stem cell expansion during development. However, the precise mechanism by which overexpression of Hox (fusion) genes increases self-renewal is currently not understood. Another signaling pathway that is clearly very important for stem cell self-renewal is the WNT signaling pathway [39–41]. This evolutionary conserved signaling pathway spans all the way from the cell membrane to regulate transcription at several stages of B- and T-cell development, as well as HSC self-renewal. Unlike the Hox genes, for which the connection with extracellular regulation is not clear, WNT signaling provides a degree of extracellular control over the self-renewal of HSCs. How the WNT pathway regulates stem cell fate in relation to other regulatory factors, including HOX proteins and other transcription factors, remains to be established [42,43]. None of the current models of how stem cell fate is controlled has led to procedures that allow a manipulation of HSCs ex vivo in a way that is clinically meaningful. Given the known complexity of transcriptional regulation and the difficulty of assaying stem cell function, mentioned above, a detailed understanding of the factors that control HSC self-renewal is likely to remain elusive for the foreseeable future.

Telomere structure and function Without extremely reliable mechanisms to duplicate and segregate complete copies of genomes into daughter cells, life in any form could not exist. Although much progress has been made in deciphering the many factors and pathways involved in securing the fidelity of DNA replication and chromosome segregation [44], many questions in this general area remain. The ends of chromosomes are crucially important to maintain genome stability, and telomeres are of particular interest in relation to DNA damage responses and DNA repair pathways. The DNA component of telomeres in all vertebrates consists of (TTAGGG)n. Telomeres distinguish intact from broken chromosomes, stabilize chromosome ends, and provide protection against nuclear degradation and end-to-end fusion events [45]. Telomeres are furthermore involved in the positioning of chromosomes within the nucleus and are also important for chromosome segregation [46]. Almost 50 years ago, Hayflick suggested that most normal human cells are unable to divide indefinitely but are programmed for a given number of cell divisions [47]. In 1990, several papers described a loss of telomeres with replication and with age, and suggested that progressive telomere shortening could explain Hayflick’s original observation [48–50]. This model was confirmed by subsequent studies showing that transfer of the telomerase reverse transcriptase gene could prevent telomere erosion and resulted in immortalizing of the cells that Hayflick studied in most detail: normal diploid human fibroblasts [51,52]. Since then, many papers have appeared that are compatible with the notion that telomere shortening limits the number of times most normal diploid cells can divide (reviewed in [53]). Telomeric DNA is lost in human cells via several mechanisms that are related to DNA replication, remodeling, and repair. Causes of telomere loss include the “end replication problem” [54,55], the nucleolytic processing of 5′ template strands following DNA replication to create a 3′ single-strand overhang [56,57], and the failed repair of oxidative DNA damage to telomeric DNA [58–60]. The relative contribution of the different mechanisms of telomere shortening that have been proposed (reviewed in [61]) to the overall decline in telomere length with age is not known and most likely varies between cell types and individuals, and as a function of age.

67

To compensate for the inevitable loss of telomere repeats with each cell division, most cells express telomerase. Telomerase is a ribonucleoprotein containing the reverse transcriptase telomerase protein (hTERT), the telomerase RNA template component (hTERC), and the dyskerin protein as essential elements [62]. In addition, a number of proteins have been described that are important for telomerase assembly (sub) nuclear localization [63] and stability (reviewed in [64]). Telomerase is capable of extending the 3′ ends of telomeres. Telomerase levels are typically high in immortal cells that maintain a constant telomere length, such as the stem cells of the germline in the testis and embryonic stem cells. For reasons that remain to be precisely defined, the telomerase activity that is readily detected in human candidate HSCs [65] (reviewed in [66]) appears to be incapable of maintaining telomere length in these cells. Nevertheless, existing telomerase levels in HSCs are functionally important, as is highlighted in patients with the disorder dyskeratosis congenita (DKC). One possibility is that only very short telomeres provide a suitable substrate for telomerase in somatic cells. Patients with the autosomal dominant form of DKC have one normal and one mutated copy of the telomerase RNA template gene [67]. As expected, such patients show a modest reduction in telomerase levels, yet they eventually typically suffer from marrow failure, immune deficiencies, pulmonary fibrosis or cancer and rarely live past the age of 50 [68–70]. These findings are in stark contrast to those in the mouse, where a complete lack of telomerase activity is tolerated for up to six generations [71]. An emerging consensus is that telomere shortening in long-lived mammals acts as a tumor suppressor mechanism that prevents an unlimited and life-threatening proliferation of organ-specific stem cells and lymphocytes [72]. Current data are compatible with a model in which telomerase levels in human HSCs are tightly regulated and sufficient to elongate only a limiting number of short telomeres. As a result, the telomere length in human HSCs declines with cell proliferation and with age, eventually triggering replicative senescence or apoptosis. In patients with inherited telomerase deficiencies, limitations in the proliferation of various stem cells imposed by telomere loss occur earlier in life than normal.

Telomeres in the hematopoietic system Since the important original observation that telomeres in adult blood leukocytes are significantly shorter compared with germline material (sperm) from the same donor [73], the decline in somatic telomeres has been documented in three ways. The original observation was confirmed [48,74], it was shown that telomeres in various tissues were shorter in older donors [49,50], and telomere shortening was documented during in vitro culture of human cells [49,75]. In 1994, it was reported that cells produced in culture by highly purified human “candidate” HSCs from fetal liver, cord blood, and adult bone marrow could be distinguished by reproducible differences in telomere length, and that these cells showed a measurable decline in telomere length upon culture in vitro (Fig. 6.2) [7]. In the years that followed these initial reports, a large number of papers have appeared that have greatly refined our understanding of telomere shortening in human nucleated blood cells (reviewed in [17]). Studies in this general area have been facilitated by the development of quantitative fluorescence in situ hybridization (FISH) techniques to measure the telomere length in suspension cells using flow cytometry (“flow FISH” [76, 77]). With this technique, it has been shown that the age-related decline in telomere length in lymphocytes is much more pronounced than in granulocytes, and that rapid telomere shortening early in life is followed by a much more gradual decline thereafter (Fig. 6.3) [13].

68

Mean TRF length (kbp)

Chapter 6 15 14 13 12 11 10 9 8 7 6 5

Fetal liver

5

0

Cord blood

10

15

20

25

0

5

10

Bone marrow

15

20

25

0

5

10

15

20

25

Mean population doublings

Fig. 6.2 Loss of telomeric DNA in cells from cultures of purified human candidate hematopoietic stem cells (HSCs) with a CD34+CD45RA10 CD710 phenotype from fetal liver, cord blood, and bone marrow. The purified cells were cultured in serum-free medium supplemented with a mixture of cytokines to stimulate their proliferation and obtain sufficient numbers of cells for telomere restriction fragment (TRF) length measurements by Southern analysis. The mean ± SE of TRF measurements for two different donors of each tissue is shown for the cells produced in culture (䊐) as well as for the total nucleated cells from each donor before culture and cell purification ( ). Reproduced with permission from [7]. Copyright (1994), National Academy of Sciences, USA.

30

30

25 25

20 15

20 0

1

2

3

4

5

15

Telomere fluorescence (MESF × 10−3)

10

5

Lymphocyte gate: −59 bp/year

0 0

10

20

30

40

50

60

70

80

90

100

30

30

25 25

20 15

20 0

1

2

3

4

5

15

10

5

Granulocyte gate: −39 bp/year

0 0

10

20

30

40

50

60

70

80

90

100

Age (years)

If one assumes that the number of cell divisions between HSCs and granulocytes is relatively constant throughout life and that telomere shortening in HSCs is (i) primarily resulting from replication, and (ii) relatively constant with each cell division, the telomere length in granulocytes can be used as a surrogate marker for the average telomere length

Fig. 6.3 Age-related loss of telomere length in lymphocytes and granulocytes from peripheral blood of normal individuals measured by flow fluorescence in situ hybridization (FISH). The specific telomere fluorescence of lymphocytes and granulocytes was analyzed after gating on these cells based on light scatter properties and DNA fluorescence. Note the heterogeneity in telomere fluorescence values, the overall decline in telomere fluorescence with age in both cell types, and the higher rate of telomere attrition in lymphocytes. In this study, it was found that a bi-segmented line resulted in a significantly better fit with the data than the linear fit shown on the left. Inserts show the results of bi-segmented fit analysis for lymphocytes (top) and granulocytes (bottom). Arrows indicate the optimal intersection of calculated regression lines which for both cell types was before 2 years of age. Reproduced with permission from [13]. Copyright (1999), the Rockefeller University Press.

in the “average” HSC clone. While the telomere length in leukocytes shows an overall decline with age [13,78], the telomere length at any given age in humans is highly heterogeneous, as is illustrated in Fig. 6.3 below. This variation appears to be primarily genetically determined [13,79]. For example, monozygous twins of over 70 years of age were

Molecular Biology of Stem Cell Renewal

shown to have a very similar telomere length in both granulocytes and lymphocytes, whereas such values in dizygotic twins differed more but not as much as between unrelated individuals [13]. Using further refinements in the flow FISH method ([76]), it was recently shown that the rapid decline early in life is followed by a slow decline until the age of 50–60 years, after which the decline again accelerates [80]. The decline in both granulocytes and lymphocytes is nonlinear and fits a cubic curve. The pronounced decline in telomere length in specific T and natural killer cells could trigger apoptosis in these cells during a normal lifetime and compromise immune responses in the elderly. In contrast, granulocytes show a much more modest decline in telomere length with age, and critical telomere shortening in HSCs during normal hematopoiesis does not seem very plausible. More likely, the total production of blood cells from a single HSC is primarily determined by differentiation into committed progenitor cells and not by replicative senescence. Furthermore, the occasional loss of individual stem cells via telomere shortening is not expected to impact on overall hematopoiesis (or on the overall telomere length in granulocytes) as long as an excess of HSCs exists in the marrow. Indeed, studies in mice suggest that HSCs accumulate with age, be it that their functional properties in terms of replicative potential and production of B lymphocytes is diminished [81,82]. Nevertheless, the age-related loss of telomeres in granulocytes, the (modest) loss of telomeres following allogeneic transplantation [8,83], and the aplastic anemia that follows partial telomerase deficiency [84–86] all indicate that telomerase levels in normal HSCs are limiting and that the proliferation of HSCs is ultimately also limited by progressive telomere shortening. The number of mature “end” cells, such as granulocytes, that is produced by an individual HSC is expected to be highly variable as a result of the poorly understood regulation of symmetric versus asymmetric cell divisions. Even a very limited number of additional self-renewal divisions in a HSC will greatly increase cell output, and if endowed with a modest proliferative advantage, individual HSCs can produce a staggering number of cells. This phenomenon is illustrated in clonal proliferative disorders such as paroxysmal nocturnal hemoglobinuria and chronic myeloid leukemia. However, even in chronic myeloid leukemia, clonally expanded Philadelphia-positive stem cells eventually appear to encounter a telomere crisis [87]. Unfortunately, with a large number of cells to select from, the genetic instability triggered by the loss of functional telomeres appears to facilitate the emergence of subclones in blast crisis that typically show additional genetic abnormalities, more malignant properties, and higher levels of telomerase.

The telomere checkpoint Most human somatic cells, including HSCs, express limiting levels of telomerase, and as a result, telomere shortening effectively limits their proliferative potential. Most likely, telomere shortening evolved as a checkpoint function to suppress tumor growth in long-lived species. The function of this “telomere checkpoint” may help explain poorly understood aspects of stem cell biology, including stem cell “exhaustion” in aplastic anemia and other disorders. Cells may bypass the telomere checkpoint by expressing high levels of telomerase or by inactivating downstream signaling events, for example by loss of p53 function. Some cells, including subsets of B cells [88], appear to avoid the telomere checkpoints altogether, and this process could make these cells more vulnerable to tumor development. Loss of p53 function also inactivates the telomere checkpoint. This is expected to be a rare event as both copies of the normal p53 allele in a cell must typically be lost or mutated in order to continue proliferation in the presence of many short and dysfunctional telomeres [89]. Loss of p53 function allows survival of cells with dysfunctional telomeres. The resulting chromosome fusions result in chromosome breakage and, as a result,

69

in the loss and amplification of genes [90]. This leaves us with a paradox: telomere shortening can act both as a tumor suppressor mechanism (by imposing limits to the replication of somatic cells) and as a tumor promoter mechanism (by favoring genetic instability once cells have reached their telomere-imposed limit in replicative potential). Whether tumor growth is suppressed or promoted by progressive telomere shortening may depend on the number of cells that encounter such a telomere crisis and the genetic changes acquired by such cells prior to this encounter.

Conclusion In this chapter, some molecular aspects related to stem cell renewal were discussed. Developmental changes in the functional properties of HSCs and loss of telomeric DNA in HSCs are important considerations in both laboratory studies and the clinical use of HSCs. It is clear that, despite much progress over the last several decades, we are still left with many questions. Some of the most urgent are: How many times can HSCs divide, and what is the actual replicative potential of HSCs at various stages of development? To what extent are factors in the microenvironment (i.e., cytokines) capable of modulating the self-renewal properties of HSCs? How does the microenvironment change during development and with age? What are the mechanisms controlling the turnover time of HSCs in steady state hematopoiesis and during hematopoietic regeneration? Are differences in the replicative history of HSCs reflected in their anatomic location, cytokine response, and mobilization properties? Hopefully, answers to these important questions will become available in the near future. Studies with cultured hematopoietic cells have shown that the formation of hematopoietic colonies, as well as the proliferation and survival of various hematopoietic cells, typically requires the presence of “hematopoietic growth factors,” many of which have been cloned over the last decade and produced for clinical trials (reviewed in Chapter 41). The availability of recombinant growth factors has led to frantic research efforts over the past two decades to achieve clinically useful manipulation of blood cell production in vitro. Two sets of observations that were discussed in this chapter cast doubt about the feasibility of some of the perceived applications of ex vivo “expansion” and should probably be taken into account when laboratory or clinical experiments involving limited numbers of HSCs are con-templated. First, there are the observations indicating that currently ill-defined, intrinsic (genetic) mechanisms with a developmental component control the fate of HSCs. Second, hematopoietic cells, including purified HSCs, appear to lose telomeric DNA with each cell division [7]. Recent studies indicate that inherited telomerase deficiencies can give rise to a large number of disorders including idiopathic pulmonary fibrosis [91], aplastic anemia [86,92], and dyskeratosis [93]. The expectation is that inherited defects in telomere maintenance pathways will prove to be involved in many more diseases. The studies of telomeres have focused attention on possible genetic limitations in the replicative potential of somatic cells, including HSCs, and such restrictions are compatible with most experimental evidence produced in clinical and laboratory studies. In view of these considerations, it seems prudent to use reasonably large numbers of HSCs for transplantation, especially from adult donors [83]. Although the number of HSCs in fetal liver or cord blood is small, such cells are likely to be superior in terms of replicative potential. This genetic superiority must be balanced against practical disadvantages including the small number of cells available for transplantation. The small size of fetal or neonatal stem cell grafts is expected to compromise clinical results, especially in the presence of histocompatibility differences between donor and recipient [94,95]. Possibly, this limitation could be addressed by more effective immunosuppression and conditioning regimens. In addition, the outcome of fetal liver and cord blood transplants could possibly be improved by increasing the number of cells in the transplant, for example by culture ex vivo.

70

Chapter 6

References 1. Lansdorp PM. Self-renewal of stem cells. Biol Blood Marrow Transplant 1997; 3: 171–8. 2. Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 1996; 27: 242–5. 3. Siminovitch L, McCulloch EA, Till JE. The distribution of colony-forming cells among spleen colonies. J Cell Physiol 1963; 62: 327–36. 4. Lansdorp PM. Developmental changes in the function of hematopoietic stem cells. Exp Hematol 1995; 23: 187–91. 5. Lansdorp PM, Dragowska W, Mayani H. Ontogeny-related changes in proliferative potential of human hematopoietic cells. J Exp Med 1993; 178: 787–91. 6. Rebel VI, Miller CL, Thornbury GR, Dragowska WH, Eaves CJ, Lansdorp PM. A comparison of long-term repopulating hematopoietic stem cells in fetal liver and adult bone marrow from the mouse. Exp Hematol 1996; 24: 638–48. 7. Vaziri H, Dragowska W, Allsopp RC, Thomas TE, Harley CB, Lansdorp PM. Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age. Proc Natl Acad Sci U S A 1994; 91: 9857–60. 8. Rufer N, Brümmendorf TH, Chapuis B, Helg C, Lansdorp PM, Roosnek E. Accelerated telomere shortening in hematological lineages is limited to the first year following stem cell transplantation. Blood 2001; 97: 575–7. 9. Morrison SJ, Kimble J. Asymmetric and symmetric stem-cell divisions in development and cancer. Nature 2006; 441: 1068–74. 10. Potten CS, Owen G, Booth D. Intestinal stem cells protect their genome by selective segregation of template DNA strands. J Cell Sci 2002; 115(Pt 11): 2381–8. 11. Cairns J. Somatic stem cells and the kinetics of mutagenesis and carcinogenesis. Proc Natl Acad Sci U S A 2002; 99: 10567–70. 12. Karadimitris A, Luzzatto L. The cellular pathogenesis of paroxysmal nocturnal haemoglobinuria. Leukemia 2001; 15: 1148–52. 13. Rufer N, Brümmendorf TH, Kolvraa S et al. Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood. J Exp Med 1999; 190: 157–67. 14. Bose S, Deininger M, Gora-Tybor J, Goldman JM, Melo JV. The presence of typical and atypical BCR–ABL fusion genes in leukocytes of normal individuals: biologic significance and implications for the assessment of minimal residual disease. Blood 1998; 92: 3362–7. 15. Moore MA, Metcalf D. Ontogeny of the haemopoietic system: yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo. Br J Haematol 1970; 18: 279–96. 16. Pawliuk R, Eaves C, Humphries RK. Evidence of both ontogeny and transplant dose-regulated expansion of hematopoietic stem cells in vivo. Blood 1996; 88: 2852–8. 17. Greenwood MJ, Lansdorp PM. Telomeres, telomerase, and hematopoietic stem cell biology. Arch Med Res 2003; 34: 489–95. 18. Baerlocher GM, Rice K, Vulto I, Lansdorp PM. Longitudinal data on telomere length in leukocytes from newborn baboons support a marked drop in

19. 20.

21.

22.

23.

24. 25.

26.

27.

28.

29.

30.

31.

32. 33.

34.

35.

stem cell turnover around 1 year of age. Aging Cell 2006; 6: 121–3. Metcalf D. The Hemopoietic Colony Stimulating Factors. Amsterdam: Elsevier; 1984. Till JE, McCulloch EA, Siminovitch L. A stochastic model of stem cell proliferation, based on the growth of spleen colony-forming cells. Proc Natl Acad Sci U S A 1964; 51: 29–36. de Bruijn MF, Ma X, Robin C, Ottersbach K, Sanchez MJ, Dzierzak E. Hematopoietic stem cells localize to the endothelial cell layer in the midgestation mouse aorta. Immunity 2002; 16: 673–83. Ploemacher RE, Brons RH. Separation of CFU-S from primitive cells responsible for reconstitution of the bone marrow hemopoietic stem cell compartment following irradiation: evidence for a pre-CFU-S cell. Exp Hematol 1989; 17: 263– 6. Fleischman RA, Mintz B. Development of adult bone marrow stem cells in H-2-compatible and – incompatible mouse fetuses. J Exp Med 1984; 159: 731–45. Zon LI. Developmental biology of hematopoiesis. Blood 1995; 86: 2876–91. Lansdorp PM, Dragowska W. Long-term erythropoiesis from constant numbers of CD34+ cells in serum-free cultures initiated with highly purified progenitor cells from human bone marrow. J Exp Med 1992; 175: 1501–9. Jane SM, Cunningham JM. Molecular mechanisms of hemoglobin switching. Int J Biochem Cell Biol 1996; 28: 1197–209. Ikuta K, Uchida N, Friedman J, Weissman IL. Lymphocyte development from stem cells. Annu Rev Immunol 1992; 10: 759–83. Ogawa M. Differentiation and proliferation of hematopoietic stem cells. Blood 1993; 81: 2844– 53. Knoblich JA. Asymmetric cell division during animal development. Nat Rev Mol Cell Biol 2001; 2: 11–20. Brummendorf TH, Dragowska W, Zijlmans JMJM, Thornbury G, Lansdorp PM. Asymmetric cell divisions sustain long-term hematopoiesis from singlesorted human fetal liver cells. J Exp Med 1998; 188: 1117–24. Lee CY, Andersen RO, Cabernard C et al. Drosophila Aurora-A kinase inhibits neuroblast selfrenewal by regulating aPKC/Numb cortical polarity and spindle orientation. Genes Dev 2006; 20: 3464–74. Lansdorp PM. Immortal strands? Give me a break. Cell 2007; 129: 1244–7. Fairbairn LJ, Cowling GJ, Reipert BM, Dexter TM. Suppression of apoptosis allows differentiation and development of a multipotent hemopoietic cell line in the absence of added growth factors. Cell 1993; 74: 823–32. Mayani H, Dragowska W, Lansdorp PM. Lineage commitment in human hemopoiesis involves asymmetric cell division of multipotent progenitors and does not appear to be influenced by cytokines. J Cell Physiol 1993; 157: 579–86. Sauvageau G, Lansdorp PM, Eaves CJ et al. Differential expression of homeobox genes in functionally distinct CD34+ subpopulations of human bone marrow cells. Proc Natl Acad Sci U S A 1994; 91: 12223–7.

36. Antonchuk J, Sauvageau G, Humphries RK. HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell 2002; 109: 39–45. 37. Sauvageau G, Thorsteinsdottir U, Eaves CJ et al. Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes Dev 1995; 9: 1753–65. 38. Krumlauf R. Hox genes in vertebrate development. Cell 1994; 78: 191–201. 39. Reya T, Duncan AW, Ailles L et al. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 2003; 423: 409–14. 40. Staal FJ, Clevers HC. WNT signalling and haematopoiesis: a WNT–WNT situation. Nat Rev Immunol 2005; 5: 21–30. 41. Willert K, Brown JD, Danenberg E et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 2003; 423: 448–52. 42. Georgopoulos K. Haematopoietic cell-fate decisions, chromatin regulation and ikaros. Nat Rev Immunol 2002; 2: 162–74. 43. Mikkola HK, Orkin SH. The journey of developing hematopoietic stem cells. Development 2006; 133: 3733–44. 44. Hoeijmakers JH. Genome maintenance mechanisms for preventing cancer. Nature 2001; 411: 366–74. 45. Sandell LL, Zakian VA. Loss of a yeast telomere: arrest, recovery, and chromosome loss. Cell 1993; 75: 729–39. 46. Dernburg AF, Sedat JW, Cande WZ et al. Cytology of telomeres. In: Blackburn EH, Greider CW, editors. Telomeres. Plainview, NY: Cold Spring Harbor Laboratory Press; 1995. pp. 295–338. 47. Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp Cell Res 1961; 25: 585–621. 48. de Lange T, Shiue L, Myers RM et al. Structure and variability of human chromosome ends. Mol Cell Biol 1990; 10: 518–27. 49. Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature 1990; 345: 458–60. 50. Hastie ND, Dempster M, Dunlop MG, Thompson AM, Green DK, Allshire RC. Telomere reduction in human colorectal carcinoma and with ageing. Nature 1990; 346: 866–8. 51. Bodnar AG, Ouellette M, Frolkis M et al. Extension of life-span by introduction of telomerase into normal human cells. Science 1998; 279: 349–52. 52. Vaziri H, Benchimol S. Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span. Curr Biol 1998; 8: 279–82. 53. Shin JS, Hong A, Solomon MJ, Lee CS. The role of telomeres and telomerase in the pathology of human cancer and aging. Pathology 2006; 38: 103–13. 54. Olovnikov AM. A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon. J Theor Biol 1973; 41: 181–90. 55. Watson JD. Origin of concatemeric T7 DNA. Nat New Biol 1972; 239: 197–201. 56. Makarov VL, Hirose Y, Langmore J. Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening. Cell 1997; 88: 657–66.

Molecular Biology of Stem Cell Renewal 57. Wellinger RJ, Ethier K, Labrecque P, Zakian VA. Evidence for a new step in telomere maintenance. Cell 1996; 85: 423–33. 58. Kruk PA, Rampino NJ, Bohr VA. DNA damage and repair in telomeres: relation to aging. Proc Natl Acad Sci U S A 1995; 92: 258–62. 59. Lansdorp PM. Repair of telomeric DNA prior to replicative senescence. Mech Ageing Dev 2000; 118(1–2): 23–34. 60. Petersen S, Saretzki G, von Zglinicki T. Preferential accumulation of single-stranded regions in telomeres of human fibroblasts. Exp Cell Res 1998; 239: 152–60. 61. Lansdorp PM. Major cutbacks at chromosome ends. Trends Biochem Sci 2005; 30: 388–95. 62. Cohen SB, Graham ME, Lovrecz GO, Bache N, Robinson PJ, Reddel RR. Protein composition of catalytically active human telomerase from immortal cells. Science 2007; 315: 1850–3. 63. Wong JM, Kusdra L, Collins K. Subnuclear shuttling of human telomerase induced by transformation and DNA damage. Nat Cell Biol 2002; 4: 731–6. 64. Collins K, Mitchell JR. Telomerase in the human organism. Oncogene 2002; 21: 564–79. 65. Chiu CP, Dragowska W, Kim NW et al. Differential expression of telomerase activity in hematopoietic progenitors from adult human bone marrow. Stem Cells 1996; 14: 239–48. 66. Ohyashiki JH, Sashida G, Tauchi T, Ohyashiki K. Telomeres and telomerase in hematologic neoplasia. Oncogene 2002; 21: 680–7. 67. Vulliamy T, Marrone A, Goldman F et al. The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita. Nature 2001; 413: 432–5. 68. Collins K. The biogenesis and regulation of telomerase holoenzymes. Nat Rev Mol Cell Biol 2006; 7: 484–94. 69. Drachtman RA, Alter B. Dyskeratosis congenita. Dermatol Clin 1995; 13: 33–9. 70. Vulliamy T, Marrone A, Szydlo R, Walne A, Mason PJ, Dokal I. Disease anticipation is associated with progressive telomere shortening in families with dyskeratosis congenita due to mutations in TERC. Nat Genet 2004; 36: 447–9. 71. Blasco MA, Lee HW, Hande MP et al. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 1997; 91: 25–34.

72. Maser RS, DePinho RA. Connecting chromosomes, crisis, and cancer. Science 2002; 297: 565– 9. 73. Cooke HJ, Smith BA. Variability at the telomeres of the human X/Y pseudoautosomal region. Cold Spring Harb Symp Quant Biol 1986; 51(Pt 1): 213–19. 74. Allshire RC, Gosden JR, Cross SH et al. Telomeric repeat from T. thermophila cross hybridizes with human telomeres. Nature 1988; 332: 656– 9. 75. Counter CM, Avilion AA, LeFeuvre CE et al. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. Embo J 1992; 11: 1921–9. 76. Baerlocher GM, Vulto I, de Jong G, Lansdorp PM. Flow cytometry and FISH to measure the average length of telomeres (flow FISH). Nat Protoc 2006; 1: 2365–76. 77. Rufer N, Dragowska W, Thornbury G, Roosnek E, Lansdorp PM. Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat Biotechnol 1998; 16: 743– 7. 78. Frenck RW, Jr., Blackburn EH, Shannon KM. The rate of telomere sequence loss in human leukocytes varies with age. Proc Natl Acad Sci U S A 1998; 95: 5607–10. 79. Slagboom PE, Droog S, Boomsma DI. Genetic determination of telomere size in humans: a twin study of three age groups. Am J Hum Genet 1994; 55: 876–82. 80. Baerlocher GM, Rice K, Vulto I, Lansdorp PM. Longitudinal data on telomere length in leukocytes from newborn baboons support a marked drop in stem cell turnover around 1 year of age. Aging Cell 2007; 6: 121–3. 81. Bryder D, Rossi DJ, Weissman IL. Hematopoietic stem cells: the paradigmatic tissue-specific stem cell. Am J Pathol 2006; 169: 338–46. 82. Linton PJ, Dorshkind K. Age-related changes in lymphocyte development and function. Nat Immunol 2004; 5: 133–9. 83. Awaya N, Baerlocher GM, Manley TJ et al. Telomere shortening in hematopoietic stem cell transplantation: a potential mechanism for late graft failure? Biol Blood Marrow Transplant 2002; 8: 597–600.

71

84. Harrington L. Making the most of a little: dosage effects in eukaryotic telomere length maintenance. Chromosome Res 2005; 13: 493–504. 85. Vulliamy T, Dokal I. Dyskeratosis congenita. Semin Hematol 2006; 43: 157–66. 86. Yamaguchi H, Calado RT, Ly H et al. Mutations in TERT, the gene for telomerase reverse transcriptase, in aplastic anemia. N Engl J Med 2005; 352: 1413–24. 87. Brummendorf TH, Holyoake TL, Rufer N et al. Prognostic implications of differences in telomere length between normal and malignant cells from patients with chronic myeloid leukemia measured by flow cytometry. Blood 2000; 95: 1883–90. 88. Martens UM, Brass V, Sedlacek L et al. Telomere maintenance in human B lymphocytes. Br J Haematol 2002; 119: 810–18. 89. Martens UM, Zijlmans JM, Poon SS et al. Short telomeres on human chromosome 17p. Nat Genet 1998; 18: 76–80. 90. de Lange T. Telomere dynamics and genome instability in human cancer. In: Blackburn EH, Greider CW, editors. Telomeres. Plainview, NY: Cold Spring Harbor Laboratory Press; 1995. pp. 265–93. 91. Armanios MY, Chen JJ, Cogan JD et al. Telomerase mutations in families with idiopathic pulmonary fibrosis. N Engl J Med 2007; 356: 1370–2. 92. Fogarty PF, Yamaguchi H, Wiestner A et al. Late presentation of dyskeratosis congenita as apparently acquired aplastic anaemia due to mutations in telomerase RNA. Lancet 2003; 362: 1628–30. 93. Vulliamy TJ, Marrone A, Knight SW, Walne A, Mason PJ, Dokal I. Mutations in dyskeratosis congenita: their impact on telomere length and the diversity of clinical presentation. Blood 2006; 107: 2680–5. 94. Petersdorf EW, Malkki M. Genetics of risk factors for graft-versus-host disease. Semin Hematol 2006; 43: 11–23. 95. Sierra J, Storer B, Hansen JA et al. Transplantation of marrow cells from unrelated donors for treatment of high-risk acute leukemia: the effect of leukemic burden, donor HLA-matching, and marrow cell dose. Blood 1997; 89: 4226–35. 96. Yamashita YM, Mahowald AP, Perlin JR, Fuller MT. Asymmetric inheritance of mother versus daughter centrosome in stem cell division. Science 2007; 315: 518–21.

7

Catherine M. Flynn & Catherine M. Verfaillie

Cellular Biology of Hematopoiesis

Introduction

Stem cells

Hematopoiesis

Stem cells can be defined based on their obligatory characteristics of (1) self-renewal and (2) differentiation, as well as (3) functional repopulation of a tissue in vivo. Self-renewal is defined as the ability of a cell to divide without maturation and produce progeny identical to the parent cell. This process can be symmetric, wherein a single stem cell gives rise to two daughter stem cells, or asymmetric, generating a single new stem cell and a second daughter cell that has differentiated features. What governs asymmetric versus symmetric cell divisions is not yet known. All stem cells can differentiate, in response to the correct intrinsic and environmental cues, towards progeny of one or more lineages. Depending on the number of cell types that stem cells can generate, they are termed totipotent, pluripotent, and multipotent (Table 7.1). Totipotent stem cells, or the fertilized oocytes, can form a fully functional organism. Pluripotent stem cells differentiate into all cells of the embryo, i.e. the three germ layers – endoderm, ectoderm, and mesoderm – and the germline. Multipotent stem cells can be isolated from various tissues in fetal and adult animals. Such multipotent stem cells, sometimes referred to as adult stem cells or tissue-specific stem cells, differentiate into a limited range of cell types that are specific to the tissue from where the stem cell was obtained [1]. Stem cells are the key ingredient of hematopoietic cell grafts and have been used clinically since 1968 when the first successful BM transplant was performed at the University of Minnesota [2]. Since then, hematopoietic cell transplantation (HCT) has become a well-established treatment modality for malignant and nonmalignant diseases [3]. HSCs, the best-described stem cells, are categorized as multipotent stem cells because they self-renew, differentiate into all the cells of the blood lineage, and reconstitute the hematolymphoid system for the life of an individual.

“Hematopoiesis” is derived from the two words, “(h)aima” for blood and “poiein” meaning to make. Hematopoiesis involves generating an entire hematopoietic and immune system for the life of an individual. The cellular biology of hematopoiesis encompasses three components: the hematopoietic stem cell and its progeny, the environment in which they reside, and the signals and cytokines that regulate their fate. In adults, hematopoiesis occurs in the bone marrow (BM), which supports the lifelong maintenance of stem cells and their regulated trilineage differentiation. The earliest hematopoietic cells of the developing mammalian embryo are detectable in the blood islands in the extraembryonic yolk sac at 2–3 weeks. Subsequently, hematopoietic cells are found within the embryo proper in the gonad–mesonephros region, from where hematopoietic stem cells (HSCs) migrate to the fetal liver and later to the BM. The regulation of HSC fate is complex, and the exact mechanisms that govern HSC self-renewal are not yet fully understood. This process involves factors intrinsic to the HSC and cues from the surrounding microenvironment, which plays a critical role as a supporting structure for HSCs. The BM microenvironment is composed of, aside from HSC progeny, such as macrophages, cells of mesenchymal origin like osteoblasts and adipocytes, and endothelial cells. The mesenchymal lineage cells are purportedly derived from a stem cell different from the HSC, namely the mesenchymal stem cell (MSC), also termed the marrow stromal cell. These three cell types create the HSC niche wherein the HSCs reside, and from which HSCs are released or whereto they home in manipulations aimed at the mobilization and transplantation, respectively, of HSCs. Both HSCs and MSCs continue to be a subject of intense interest and excitement, particularly with the wealth of recent information outlining mechanisms of HSC homing and mobilization, as well as the concept of stem cell plasticity. The focus of this chapter is to discuss HSC biology, HSC regulation, and a critical assessment of the presumed greater potentiality or plasticity of BM stem cells, whether HSCs or MSCs.

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

72

Stem cell plasticity A number of studies during the last decade have suggested that the therapeutic potential of BM cells may be more extensive than originally thought, because investigators have noted that HSCs and other cells residing in the BM may have differentiation ability beyond their tissuespecific stem cell fate. While conventional dogma dictates that multipotent adult stem cells are irreversibly committed to the organ in which they reside, the cloning of mammals from the nucleus of an adult somatic cell refuted the notion of irreversible commitment, and observations from a number of additional experimental models have challenged this paradigm [4]. Likely more than 1000 reports have suggested that

Cellular Biology of Hematopoiesis

73

Table 7.1 Stem cell hierarchy Stem cell

Example

Explanation

Totipotent

Fertilized oocyte

Pluripotent

Embryonic stem cells Embryonic germ cells Inner cell mass of the blastocyst Primordial germ cells of the early embryo Hematopoietic stem cells Skin stem cells Neural stem cells

Cells with the capacity to form mesoderm, endoderm, and ectoderm, including germ cells and extraembryonic tissue Cells with the capacity to form mesoderm, endoderm, ectoderm, and germ cells. Cannot give rise to trophoblast

Multipotent

Cells within various adult organs that have the capacity to self-renew and differentiate into all the organ-specific cell types

stem cells residing in adult tissues can generate, under certain environmental conditions, specialized cell types other than those of the tissue from where the stem cells were obtained, and this even irrespective of the particular germ layer designation (mesoderm, ectoderm, and endoderm). This concept has been termed “stem cell plasticity” and has led to considerable excitement as well as skepticism. If correct, this would defy the developmental biologic principles that lineage-specific stem cells generate only differentiated cells from the tissue of origin, and would signify that adult stem cells may hold much broader therapeutic potential. BM-derived cells have been central to this paradigm shift, as they have been reported to contribute to a number of seemingly unrelated tissues and organs, including the central nervous system [5], skeletal muscle [6], liver [7,8], and cardiac muscle [9], particularly in the context of tissue injury. While evaluating reported plasticity of BM-derived cells, it is important to recognize that fresh adult BM contains multiple stem cell populations that include HSCs, MSCs, and endothelial progenitor cells [10]. In addition, cells with molecular features of hepatic progenitors and other cell types have been described [11]. It is thus possible that somatic stem cells may circulate from their tissue of origin and reside or pass through the BM. Plasticity would then only represent appropriate differentiation of a passenger progenitor/stem cell population. As will be discussed further in the chapter, there is also mounting evidence that, in particular, macrophages derived from HSCs can fuse with a differentiated host cell, creating the false impression that HSCs transdifferentiate in a cell-autonomous fashion to cell lineages other than the hematopoietic lineage [12]. It will be important to further decipher what possible mechanisms underlie BM cell plasticity, whether these be fusion or true transdifferentiation, to determine the clinical implications of the apparent plasticity.

Stem cells in BM HSCs During ontogeny, HSCs can be found in different locations, including the blood islands in the placenta, aorta–gonad–mesonephos region, fetal liver, and BM. In postnatal life, HSCs are found in the BM at a low frequency (1 : 105 cells of mouse BM [13]). At birth, HSCs are also found in the placenta [14,15] and in the peripheral blood of the infant, and hence umbilical cord blood (UCB) [16]. Postnatally, HSCs can also be found at very low frequency in peripheral blood [17], and they circulate at higher frequency following administration of cytokines such as granulocyte colony-stimulating factor [18] or of chemotherapeutic agents like

cyclophosphamide [19]. The observation that HSCs can leave the BM environment and be found in the circulation is of importance when considering possible plasticity of stem cells from tissues other than BM. As HSCs can only be defined based on their functional attributes, i.e. repopulation of the hematopoietic system following transplantation, murine HSCs remain the best characterized at present, chiefly because very accurate functional assays for murine HSCs exist that do not exist for human HSCs. The most accurate enumeration of murine HSCs is done via competitive repopulation assays [20], wherein a population of test cells are co-transplanted with a fixed number of BM cells and repopulation by the test cells is evaluated at 4–6 months following transplantation. To assess the self-renewal ability of the test cells, the BM from the initial recipient is grafted into secondary animals, where again repopulation by the test cells is evaluated. Using such in vivo studies, murine HSCs were found to be highly enriched (1 : 10 cells) within the c-Kit receptor (c-Kit)-positive, T-cell antigen (Thy-1.1)-low, lineage (Lin)-negative, stem cell antigen (Sca-1)-positive (KTLS) fraction of murine BM [21]. Subsequent studies have further enriched murine HSCs by evaluating additional stem cell markers, including CD50 and Flk1 among others [22]. Additional tools to identify murine HSCs include evaluation of the efflux of a number of dyes such as rhodamine (via the multidrug resistance pump) [23] or the DNA-binding dye Hoechst-33342 (via the breast cancer resistance protein) used to define the side population cells, or Sp cells [24]). Isolation of human HSCs has been more challenging owing to the lack of accurate assays such as the reconstitution of lethally irradiated animals. Surrogate in vitro and in vivo assays have been used to screen for biologic activity, including a number of long-term stromal cultures (long-term culture-initiating cells [25], cobblestone area-forming cells [26], myeloid–lymphoid initiating cells [27]) or transplantation into immunodeficient hosts such as the nonobese diabetic severe combined immunodeficiency (NOD-SCID) mouse (SCID-repopulating cell) [28] or the preimmune fetal sheep [29]. However, due to the xenogeneic nature of such transplantation models, accurate assessment of HSC quantity and quality is not possible. As in the mouse, investigators have used fluorescence-activated cell sorter (FACS) separation to enrich for candidate human HSCs. A number of different sorting strategies have allowed enrichment of human HSCs to 1 : 100–500, including selecting for CD34+Thy-1+Lin− [30], CD34+Lin−CD38− [31], CD34+Lin−CD38−ckit+Rho− [32] or CD34+CD38−Lin−Sp [33] cells from BM or UCB [28,34]. Despite the best selection procedures for human HSCs based on cell-surface markers or their ability to extrude dyes, the resultant population remains heterogeneous and contains less than 1% SCID repopulating cells.

74

Chapter 7

MSCs The BM stroma plays an important role in sustaining the survival, proliferation, and differentiation of mature blood cells from HSCs. Cells within the BM microenvironment that contribute to this support are, in addition to HSC-derived macrophages, endothelial cells and mesenchymal cells, including osteoblasts and adipocytes. The latter cells are progeny from a stem/progenitor cell, the MSCs. Like HSCs, MSCs are rare [35], comprising 0.01–0.001% of BM cells [36]. MSCs were first described in 1976 [37] as plastic adherent cells from murine BM that, at the clonal level, gave rise to colonies of fibroblast-like cells which could be induced to differentiate to adipocytes, osteoblasts, and chrondrocykes. Owing to their fibroblastic appearance, Friedenstein named these cells “colony-forming fibroblasts” or “CFU-Fs.” Since the initial identification of MSCs in BM, MSCs have also been isolated from a number of other tissues, including UCB, placental tissue, fetal blood and liver, adipose tissue, exfoliated deciduous teeth, amniotic fluid, and adult lung and pancreas [38–43]. MSCs are self-renewing clonal precursors of nonhematopoietic stromal tissues. They are isolated and expanded in culture based on their ability to adhere to plastic in response to factors present in fetal calf serum. However, cultures of MSC are highly heterogeneous with only rare multipotent, uncommitted cells between a sea of more differentiated precursors. MSCs differentiate into cells found in limb bud mesoderm, including osteoblasts, chondroblasts, adipocytes, fibroblasts, smooth muscle-like cells or myofibroblasts, and skeletal muscle cells. However, this differentiation potential is quickly lost from ex vivo expanded MSCs, likely due to the fact that – as is true for HSCs – self-renewing cell divisions of MSCs in vitro occur rarely [44]. Morphologically, cultured MSCs are spindle-like cells with a fibroblast-like appearance. The phenotype of cultured MSCs has been established by a number of investigators as positive for CD44, CD29, CD90, CD105, CD73 but negative for hematopoietic markers CD34, CD45, and CD14, and endothelial markers CD34, CD31, and von Willebrand factor. The phenotype of freshly isolated MSCs is less clear, although enrichment can be obtained by sorting for cells positive for CD105 (SH2) and CD73 (SH3), or for Stro-1brightVCAMbright cells [45–47]. Whether one should even consider MSCs to be “stem cells” depends on how one defines stem cells. If the definition includes the notion of in vivo repopulation of a given tissue, few if any studies have demonstrated that MSCs are true stem cells. What the role is of MSCs within BM is not clear, although they likely are responsible for replacing osteoblasts and adipocytes. MSCs express a wide variety of cell adhesion proteins, interact with endothelial cells, and possess the ability to respond to a wide variety of cytokines and chemokines. Their differentiated progeny secrete a multitude of cytokines and growth factors, which play a role in supporting hematopoiesis. The ability of MSCs to enhance HSC engraftment has been tested and proven in murine models [48,49]. Specifically, co-transplantation of murine MSCs with limiting doses of murine HSCs results in significantly more mice in which hematopoietic reconstitution is seen. Similar results have been observed when human MSCs and HSCs are co-transplanted into immunodeficient mice [50]. This has led to ongoing clinical trials wherein MSCs are co-transplanted with – for instance – haploidentical CD34+ cells or UCB grafts to enhance engraftment [51]. Although MSCs are present in BM harvests, it is unclear whether host mesenchymal cells are replaced by donor-derived cells. Horwitz et al. found that 1–2% of osteoblasts from a patient after HCT were of donor origin [52], and Cilloni et al. [53] showed that 7 of 41 patients who underwent a sex-mismatched leukodepleted transplant displayed donor stromal engraftment. By contrast, a significantly larger number of studies concluded that MSCs, adipocytes, osteoblasts or stromal cells were not

donor derived following HCT. The reason for these discrepancies is not known. It is also not known why MSCs do not engraft. One possible explanation is that, for at least culture-expanded MSCs, over 95% remain within the lung vascular bed and rarely move beyond this capillary bed [54]. As MSCs secrete many cytokines that support hematopoiesis, investigators have examined the possibility that defects in MSCs may contribute to the pathogenesis of hematologic malignancies, including leukemia and myelodysplastic syndrome (MDS). One question is whether the stromal compartment is clonally abnormal. Stromal cultures from patients with MDS, chronic myeloid leukemia (CML), and acute myeloid leukemia have been examined [55–58]. Some have found that stromal cells are devoid of any chromosomal abnormality and are functionally normal, while others have found chromosomal abnormalities in stromal cells. Contaminating HSC-derived macrophages may account for some of the aberrations seen, as, for instance, macrophages (CD14+) in stromal cultures established from CML BM were enriched for Philadelphia chromosome (Ph)-positive cells whereas mesenchymal (CD14−) cells in these same cultures were Ph− [58]. Some reports suggest that endothelial cells in CML patients may be clonally related to the Ph+ HSC clone [59]. However, as has become clear in recent studies, some apparent endothelial progenitors are monocytic in origin [60]. Two recent studies examined the karyotype of hematopoietic cells and stromal cells from patients with MDS [56,57]. In both studies, karyotypic abnormalities were detected in stromal cells and hematopoietic cells, but there was a striking lack of overlap between the karyotypic abnormalities of the hematopoietic and stromal cells. It is not known whether the stromal chromosomal abnormality predates the leukemic process or whether the stromal abnormalities are caused by the same event that caused the initial genetic damage to the HSCs. Whether the stromal cells are initiators, supporters or simply bystanders in the clonal process is not clear. One also needs to be cognizant of the fact that chromosomal alterations may be acquired in culture after several passages, which result in the development of sarcomas when such MSCs are transplanted [61]. The ability of MSCs to secrete cytokines and growth factors may also be exploited for tissue repair. A large number of studies has been published wherein the trophic effects of MSCs on damaged tissues, ranging from radiation-induced damage to the gastrointestinal epithelium, to ischemic renal, cardiac or central nervous system lesions among others, have been described [62–66]. This has led to the contemplation of clinical use of MSCs for therapy of such disorders. A word of caution, however, comes from recent reports that transplantation of MSCs in ischemic tissues may not only have beneficial effects, but also cause aberrant tissue formation. For instance, two recent reports demonstrated that encapsulated structures were found in the infarcted areas of 51% of hearts after injecting MSCs and to a lesser extent unfractionated BM cells [67–69]. An interesting observation is that cultured MSCs can modulate the immune system. Specifically, MSCs are poorly recognized by allogeneic T lymphocytes [70] or by natural killer cells [71], while at the same time inhibiting T-cell [72] and B-cell [73–75] responses. The mechanism underlying this immunomodulatory effect, specifically the inhibition of mixed lymphocyte reactions, is not totally clear, although cell–cell interactions as well as the secretion of soluble T-cell inhibiting factors play a role. This has led to several provoking clinical reports wherein dramatic graft-versus-host disease (GVHD) modulation has been described following MSC administration [51,76,77]. A number of prospective studies are now ongoing testing the efficacy of MSC-based prevention or treatment of GVHD. In addition, the possible usefulness of MSCbased therapies for autoimmune disorders such as inflammatory bowel disease is being evaluated.

Cellular Biology of Hematopoiesis

A number of reports have suggested that MSCs may generate differentiated progeny other than mesenchymal progeny, including endothelial cells, cardiomyocytes, hepatic cells, and neural cells. These studies will be further assessed below.

Plasticity of BM-derived stem cells That BM-derived cells reconstitute the hematopoietic system is well established. Since the late 1990s, numerous reports have appeared concluding that BM-derived cells may also contribute to other tissues, a phenomenon termed plasticity. Some of these surprising results are thought to be due to plasticity of hematopoietic cells, whereas others have been ascribed to plasticity of MSCs. These studies will be discussed in the following paragraphs. While evaluating these studies, we will critically determine to what extent real transdifferentiation has been demonstrated. For that reason, we will here first define criteria that should be fulfilled to demonstrate true plasticity or transdifferentiation. Full proof of plasticity requires the following: 1 A single cell that ordinarily is destined to differentiate towards hematopoietic or mesenchymal progeny can generate the expected progeny as well as cells of another lineage. 2 Differentiated cell types are defined based on morphologic and phenotypic criteria as well as expression of the expected transcripts and proteins, and the acquisition of functional attributes of the unexpected progeny. 3 Finally, the differentiated cell should also integrate into a tissue in vivo and perform the function of the unexpected lineage cell in vivo. When most studies are evaluated based on this set of criteria, relatively few studies have proven that transdifferentiation is occurring. A seminal paper demonstrating the plasticity of BM-derived HSCs was published by Krause et al. in 2001 [78]. In this study, murine, male donor BM cells were HSC enriched by fractionation and lineage depletion, labeled with the membrane intercalating dye PKH26 and injected into irradiated female recipient mice. After 2 days, BM was harvested, bright PKH26 cells were reisolated by FACS, single PKH26 cells were injected into 30 irradiated female hosts, and donor reconstitution was assessed at 5 and 11 months. The HSC-enrichment process, by homing to the BM within 48 hours, identified a population of HSCs capable of durable reconstitution. At 11 months, donor cells contributed 12–86% of the peripheral blood in five mice, and BM from 4 of 5 of these animals could be serially transplanted. Among the five mice who survived to 11 months, HSC-derived progeny could also be found in nonhematopoietic tissues, as proven by presence of the Y chromosome in epithelial cells in the lung, gastrointestinal tract, epidermis, and dermis. Differentiation to the unexpected phenotype was defined based on morphology and expression of cell-type specific antigens, such as cytokeratin 8, 18, and 19. As Y chromosome-positive pulmonary epithelial cells also expressed mRNA for surfactant, it was suggested that these cells had functional properties of alveolar type II pneumocytes. A number of reports subsequently have suggested that the plasticity seen by Krause et al. may not be as extensive. For instance, Wagers et al. [79] evaluated mice grafted with single HSCs (defined as KTLS cells) and found vanishingly rare cells in the brain, liver, and heart that were donor in origin. In this study, donor origin was defined based on the expression of green fluorescent protein (GFP) in the donor HSCs. In addition, they evaluated tissues of parabiotic animals, where wild-type (WT) and enhanced GFP (eGFP) transgenic mice were surgically joined such that they rapidly develop a common, anastomosed circulatory system. As HSCs spontaneously migrate in and out the BM and can be found circulating, one would expect to find unexpected lineage differentiation from HSCs in this model. Even though extensive hematopoietic chimerism was seen, analysis of multiple tissues failed to demonstrate

75

significant engraftment of nonhematopoietic tissues by circulating GFPpositive cells. One possible explanation for the discrepancies between the studies was the differing method of identifying donor cells (in situ hybridization of the Y chromosome versus a transgenic marker gene). A subsequent study by Krause et al. reconfirmed the ability of HSCs to transdifferentiate into lung epithelial cells, now using transgenic gene markers [80]. Hence the debate remains whether HSCs in this setting can transdifferentiate into epithelial cells. Since this initial seminal report (and prior to that) a multitude of papers has been published wherein authors describe the possibility that BM-derived stem cells can transdifferentiate. Before the studies are reviewed, we will, however, address possible mechanisms that could explain the perceived and unexpected lineage switch, as well as the many methods that can or have been employed to address plasticity, each with their own caveats.

Possible explanations of plasticity (Fig. 7.1) If plasticity is real, the long-held view that, during development at gastrulation, cells are definitively committed to a single germ layer

(a) Multiple stem cells

=

+

(b) cell fusion

+

=

(c) Trans de-/re-differentiation

(d) Pluripotency

TRENDS in Cell Biology

Fig. 7.1. Possible explanations for the perceived plasticity. (a) Stem cells for a given tissue might exist in an unrelated organ. (b) Perceived plasticity might be caused by the transplanted cells fusing with a host cell of a different lineage, leading to transfer of the genetic information of the transplanted cell to the host-derived cell. (c) Plasticity might occur via de- and redifferentiation, as is seen in cloning or in limb regeneration in amphibians. (d) Cells with pluripotent characteristics might persist even after the initial steps of embryological development. Reproduced with permission from Verfaillie [192]. Copyright (2002), Elsevier.

76

Chapter 7

(mesoderm, endoderm, ectoderm) would not be totally correct as, in plasticity, tissue-specific stem cells would overcome their definitive commitment to a single germ layer when they were placed in a different and suitable environment. That this might be possible follows from studies as far back as the 1970s in which it was demonstrated that, by transferring the nucleus of Xenopus cells into enucleated, unfertilized Xenopus egg cytoplasm, frogs could be generated with the genetic information of the transferred nucleus. These observations were extended in the 1990s with the cloning of sheep and subsequently other mammals from the nucleus of an adult somatic cell [4]. In addition, dedifferentiation and redifferentiation is a phenomenon known in lower animal species such as newts [81]. How cell fate is altered is not fully understood, although recent studies have shown that overexpression of four transcription factors can reprogram fibroblasts to cells with embryonic stem cell (ESC)-like properties [82,83]. Hence, it is not inconceivable that, in certain circumstances, such reprogramming may occur. What role tissue injury plays in its initiation is controversial. Cells may be summoned to sites of injury and, on arrival in the “damaged” environment, cytokines and external factors may induce changes in gene expression patterns and result in an altered phenotype, and possibly function. Alternative explanations for the perceived plasticity include the fact that tissue-specific stem cells other than HSCs also circulate and home to tissues other than the tissue of origin. In this scenario, neural or liver progenitor/stem cells may be “hiding out’ among the heterogeneous populations of cells existing in the BM. Migration of HSCs from the fetal liver to the BM during development is mediated by a CXCR4– SDF1 gradient, and homing of HSCs to the BM following transplantation is mediated by the same CXCR4–SDF1 gradient [84]. As many stem cells from other tissues also express CXCR4, it is not inconceivable that, during embryogenesis or in postnatal life, stem/progenitor cells other than HSCs may home to the BM [11]. That such cells may act as reserves for tissue regeneration or repair in adult life is tempting to speculate; however, there is no proof for such a hypothesis. A third explanation for plasticity arose in 2002, namely that donor cells can fuse with host cells whether in intact tissue or damaged tissue. Such fusion leads to the generation of 4N cells, which express cell surface and cytoplasmic markers derived from both the donor cell and the cell it fuses with. Terada et al. [85] and Ying et al. [86] found that in co-culture experiments with BM cells or neurospheres and ESCs, tetraploid hybrids could be created that adopted ESC characteristics. Similar findings were subsequently documented in vivo. In an elegant study, Alvarez-Dolado et al. [87] used a Cre–Lox recombination system to detect cell fusion events. Following transplantation of BM cells into lethally irradiated mice, they found that BM-derived cells fused with Purkinje cells, hepatocytes, and cardiomyocytes. Camargo et al. [88] also used a single HSC transplant model to demonstrate that HSCderived hepatocytes occur as a result of cell fusion. Which cell fuses with target cells in vivo has not been addressed in all studies. Nevertheless, two independent groups have demonstrated that HSCs themselves do not fuse with hepatocytes, rather that monocytes generated from HSCs are responsible [12,88]. It was furthermore shown that, even though part of the genetic program of the host cell is preserved, reprogramming by both activating and silencing of genes occurs, so that the hybrid cell resembles the host cell with which the donor cell fused [89]. A final explanation may be that rare, more pluripotent precursors of postnatal stem cells persist. Although pluripotent stem cells are present throughout the body of planaria, such cells were not thought to exist in higher mammalian species. However, as will be discussed below, there is mounting evidence that cells expressing the ESC transcription factors Oct4, Sox2, and Nanog can be isolated from human and murine BM or UCB [90–93]. The frequency of such cells decreases very fast with age.

The natural role of such cells in postnatal tissues is not known. However, the possibility exists that when such cells are placed in a different environment, such as the brain or liver, they have the inherent ability, without need for significant reprogramming, to respond to cues from these new environments and differentiate in a tissue-appropriate manner.

Techniques to assess plasticity One of the most important technical considerations in demonstrating plasticity is to identify without any ambiguity the donor cell in the target organ. A robust, unambiguous molecular marker for the donor cell allows accurate assessment of the clonal relationship of donor cells and facilitates identification of the donor cells following transplantation. A variety of methods has been used to mark the donor cells. For all detection methods, proper fixation, particularly fixative concentration, and staining is essential to ensure maximal signal strength. Donor markers used include the Y chromosome (in sex-mismatched transplantations), membrane or cytoplasmic dyes, labels that incorporate in the DNA, and genetic labels such as β-galactosidase (β-gal) and GFP transgenes. More recently, the advent of conditional genetic marking using the Cre–Lox technology has provided another method of detection. Each of these approaches has advantages and limitations. A recent paper by Brazelton and Blau provides a comprehensive review of stem cell tracking methodology [94]. Equally important when trying to identify a cell whose fate has changed is to definitively prove acquisition of a new cell fate. It is essential to demonstrate that the donor marker and the lineage-specific marker are expressed in the same cell. Cellular morphology can be complex, and it may be difficult to discriminate between two closely associated cells. If tissue sections are examined in two dimensions with standard microscopy, a donor cell may overlay a host cell with the typical host-tissue specific marker, and this may be mistaken for a transdifferentiated donor cell. These events are rare, but the reported frequency of adult stem cells contributing to nonhematopoietic tissues is often equally rare. Methods that allow visualization of thin optical sections and three-dimensional reconstructions of cells in tissue sections are therefore required. The important aspect of the detection of any specific signal in a tissue is the use of appropriate negative controls to evaluate potential confounding factors, including autofluorescence. Careful experimental design and appropriate controls can avoid many of the common pitfalls.

Fluorescent in situ hybridization for the Y chromosome In sex-mismatched transplantation in which male donor cells are transplanted into female recipients, a Y chromosome is present in all intact donor cells. Typically, it is detected using fluorescent in situ hybridization (FISH) with fluorescent probes for specific regions of the Y chromosome. However, the Y chromosome cannot be visualized in all male cells if thin tissue sections that only partially sample the nuclei are assessed, and it is technically difficult to use Y chromosome FISH on thicker sections that contain the full thickness of most cells (greater than 10 μm). Nevertheless, the specificity and sensitivity of Y-chromosome FISH are very high. False-positive results can be caused by cell overlay and from nonspecific binding of the probe. Experience and evaluation of good negative controls in parallel can help to avoid these problems. As discussed earlier, it is critical to demonstrate that the Y chromosome is in the cell of interest, which can be accomplished by threedimensional reconstruction of double-labeled thin sections obtained by confocal microscopy.

Cellular Biology of Hematopoiesis

DNA or cell membrane labels 3

Bromo-deoxyuridine (BrdU) or H-thymidine labels dividing cells by incorporating into DNA during the S phase of cell division and has been extensively used to identify cells transplanted into the brain. However, two recent studies have shown that BrdU is present in the DNA of grafted cells, which can be taken up by actively dividing cells in the injected/injured area if the grafted cells die [95,96]. This then creates the incorrect impression that transplanted cells have adopted a neuronal fate. Membrane dyes such as PKH26 and CM-Dil (3,3′-dioactadecyl-5,5′di(4-sulfophenyl)-oxacarbocyanine, sodium salt, SP-DIOC18) have also been used to mark donor cells [78,97]. Both these dyes are nontoxic fluorescent lipophilic dyes that incorporate into the lipid bilayer of cytoplasmic membranes; hence, the amount of dye per cell diminishes as the cell undergoes cell division, which makes monitoring of proliferating cells difficult. Another pitfall of this marker is that fusion or phagocytosis of a labeled cell with an unlabeled cell results in transfer of the dye to the host cell, and many studies using membrane dyes have not controlled for this possibility [98]. Similarly to other markers, tissue preparation for evaluation is critical as the embedding procedure can influence the detection of fluorescence; for example, PKH26 does not withstand embedding in poly(methyl methacrylate). Another method, used in combination with magnetic resonance imaging, is passive labeling of donor cells with small particles of iron oxide [99]. The label can also be visualized by Prussian blue staining on histologic examination. As with membrane dyes, the label can be diluted so that it becomes no longer visible with cell division, and the possibility exists for false-positive results from microglia that uptake iron particles released from dead cells. Another downside of iron oxide contrast agents is that once cells home to a lesioned area, it becomes difficult to distinguish contrast originating from the stem cells or from bleeding in the damaged area.

Transgenic markers One of the earliest tracking markers was bacterial β-gal. The ROSA26 strain of mice constitutively expresses β-gal in most cells and can be used as a marker in transplantation studies. However, two problems arise with its use: the expression of β-gal as a marker is relatively weak, and it can be difficult to distinguish it from endogenous mammalian β-gal activity [100]. Therefore, when rare events are observed using ROSA cells, it is necessary to exclude unanticipated upregulation of endogenous β-gal in response to the injury itself. Several mammalian cell types, such as Purkinje cells, have substantially higher endogenous β-gal activity than others, and these normal variations can be misleading. Mammalian β-gal can be distinguished by its intracellular location and activity at optimal pH, and techniques on how to optimize β-gal-based tracking in the central nervous system have been recently reviewed [101]. eGFP is a modified protein originating from a naturally occurring fluorophore, and several lines of transgenic mice have been generated that ubiquitously express eGFP [102,103]. However, transgene expression varies among mice, may not be completely ubiquitous, and may weaken with time, age, and tissue differentiation [104]. One can use antibodies to GFP to detect lower levels of GFP expression; however, many anti-GFP antibodies are not totally specific, and several may need to be tested to ensure specificity for the tissue of interest. Other limitations of GFP include its rapid diffusion out of unfixed cells and apparent GFP positivity due to autofluorescence of (injured) tissues. However, careful evaluation of the emission spectrum of GFP compared with autofluorescence may circumvent the latter problem [105]. Some have

77

suggested that the presence of GFP in itself may promote differentiation, although abundant evidence suggests that the fluorophore is biologically inert [106]. Others have suggested that decreased GFP expression is a function of cellular differentiation and that expression in “transdifferentiated cells” is decreased, making detection of transdifferentiation difficult [6]. If this were true, it might account for many publications in the literature stating that no donor cells were detected when using GFP as a reporter gene to label BM.

Cre–Lox technology Cre–Lox technology has been a useful addition to the armamentarium to understand cellular plasticity and has been used to address the likelihood of cell fusion as a contributing mechanism. Cre–Lox recombination is a technique used to conditionally turn gene expression on and off in specific cell types and tissues. Cells with Cre recombinase under the control of a ubiquitous or cell-specific promoter are grafted in conditional Cre reporter animals, in which a loxP-flanked stop cassette is located in front of a LacZ or eGFP reporter gene. Only when Cre recombinase excises the floxed stop cassette is the LacZ/eGFP gene expressed. Alvarez-Dolado et al. used this method to show that BM-derived cells could fuse with hepatocytes, cardiomyocytes, and Purkinje cells [87]. Oh et al. showed that intravenously infused progenitor cells from the adult heart could fuse with cardiomyocytes in the setting of myocardial infarction [107], and Reinecke et al. extended this by adding skeletal myoblasts as a clinically relevant cell type that can fuse with cardiac myocytes in vitro and in vivo [108]. This method has failed to detect cell fusion in the pancreas [109]. Keeping these possible explanations and the many methodological pitfalls in mind, the following sections will review papers suggesting differentiation of BM cells into mesodermal, endodermal, and ectodermal cells. We will first discuss studies wherein investigators describe transdifferentiation of HSCs or MSCs into unexpected lineages, and then focus briefly on studies that have suggested that more pluripotent adult stem cells may be responsible for the observed apparent transdifferentiation.

Transdifferentiation of HSCs or MSCs BM-derived cells – mesoderm BM-derived skeletal muscle Already in the late 1990s, some studies suggested that BM-derived cells could contribute to skeletal muscle [110–112]. Ferrari et al. noted that, following muscle damage, more myogenic precursors than expected from resident muscle stem cells, also termed satellite cells, could be found in muscle [110]. To elucidate the source of these additional cells, they chemically injured the tibialis anterior muscle and injected into the limb either whole BM cells or satellite cells containing a LacZ reporter driven by a myogenic promoter. β-gal+ cells were found in the BM cells injected into muscle, even though more βgal+ cells were detected in the satellite cell-injected muscle. The myogenic potential of BM cells appeared to be derived from adherent cells. They also injured hematopoietically reconstituted mice grafted with BM cells from C57/MlacZline (H-2b) mice and found, in 5 of 6 animals, β-gal+ cells in the muscle. Together, these studies suggest that cells present in BM cells can contribute to muscle, and this contribution is enhanced when the muscle is injured. Gussoni et al. [6,113] isolated murine HSCs from WT male mice based on the SP property and transplantated these into female mice with a defect in the dystrophin gene (mdx mice). As expected, hematopoietic reconstitution was seen. In addition, in the recipient muscle,

78

Chapter 7

Y chromosome-positive cells expressing dystrophin were detected. When Sp cells were isolated from muscle of WT male mice and grafted into mdx mice, radioprotection was seen with a contribution of donor cells to muscle. As studies were not done using single cells, it is not clear whether the same cell that gave rise to hematopoietic reconstitution also contributed to muscle. By contrast, Corbel et al. used single HSC transplantation to address whether HSCs or their progeny contributed to muscle. Single GFP+SPckit+Lin−Sca-1+ (SPKLS) cells from CD45.1 mice were injected into CD45.2 recipients [114]. After 4 weeks, over 30% of blood cells were donor in origin. In these animals, muscle, without and with toxin injury, was evaluated at after 16 weeks for the presence of donor contribution. Donor-derived muscle cells were defined as cells with muscle morphology, encased in laminin displaying the characteristic sarcomeric pattern following antimyosin staining that were GFP+. In the mice reconstituted from a single SPKLS cell, GFP+ cells were detected in the uninjured and injured tibialis anterior muscle of three mice. Damage promoted integration of HSC-derived cells into the regenerating muscle. When BM from the primary transplanted mice was grafted in secondary mice, hematopoietic reconstitution confirmed the self-renewal capability of the original single cell. Moreover, in 1 of the 3 secondary recipients, GFP+ myofibers were also seen, strongly suggesting that the progeny of a single HSC can contribute to the hematopoietic system as well as muscle. The mechanism of contribution, i.e. direct transdifferentiation versus fusion of HSC-derived progeny with myofibers, was not addressed. That human BM contains cells with myogenic potential was shown by identifying rare dystrophin-positive cells in muscle of a 12year-old boy who underwent a BM transplant for immunodeficiency [115]. BM-derived cardiomyocytes Several studies have suggested that cardiomyocytes can be generated from BM, although controversy exists as to the degree and mechanism via which BM cells contribute to improved cardiac function. The cardiomyogenic potential of ESCs from a variety of species including human is well established [116,117]. Whether any other stem cell, except perhaps stem/progenitor cells found in the heart itself [107], can differentiate into bona fide functional cardiomyocytes is still not clear. Fukuda et al. demonstrated that treatment of MSCs with 5-azacytidine gives rise to spontaneously contractile cells with some features consistent with cardiomyocytes [113]. Such MSC-derived cardiomyocyte-like cells formed myotubes with branching fibers and at 4 weeks stained positive for antimyosin, antiactinin and antidesmin. The cells had a cardiomyocyte-like ultrastructure and, by reverse transcription-polymerase chain reaction had cardiomyocyte-specific gene expression. More importantly, electrophysiological studies demonstrated the presence of action potentials consistent with sinus node ventricular action potentials. While the phenotype of these cells derived in vitro at a transcript, protein, and functional level is convincing, no in vivo studies were performed. Toma et al. injected human LacZ-labeled MSCs into the uninjured left ventricle of an adult SCID/beige mouse [118]. At 2 weeks, rare (0.4%) β-gal-, desmin-, cardiac troponin T-, and α-actinin-positive cardiomyocyte-like cells were found. Sarcomeric organization of the contractile proteins in the β-gal+ cells was indistinguishable from that of neighboring recipient cardiomyocytes. Dzau and colleagues performed a number of studies wherein MSCs were grafted into the borderzone surrounding the infarct in mice, and demonstrated functional improvement. The functional improvement was higher when MSCs were genetically modified to express Akt, which was associated with prolonged and more abundant persistence of the grafted cells [119]. However, in subsequent studies, they demonstrated that a

similar effect could be obtained when media conditioned by Akt-transduced MSCs was injected, suggesting strongly that the effect was trophic, i.e. secretion by the MSCs of factor(s), such as secreted frizzled2, chiefly protecting at-risk cardiomyocytes in the ischemic borderzone, and perhaps also inducing recruitment of endogenous progenitors or endothelial cells [120]. Others evaluated whether hematopoietic cells could differentiate into cardiomyocytes. Bittner et al. described that, following male BM cell transplantation into irradiated normal and mdx female mice, not only skeletal, but also cardiac myofibers appeared to be derived from donor BM cells 10 weeks following transplantation [111]. The transdifferentiated phenotype was determined to be skeletal and cardiac muscle based on coexpression of skeletal and cardiac muscle-specific proteins in Y chromosome-positive cells. Jackson et al. transplanted 2000 SP cells from male C57BL/6-Rosa-Ly-5.2 mice into lethally irradiated female C57BL/6-Ly-5.1 mice and after 10 weeks induced a myocardial infarct [121]. Evaluation 2–4 weeks later indicated that 0.02% of the cardiomyocytes were β-gal+α-actinin+CD45− and 3.3% of the endothelial cells were β-gal+Flt-1+CD45− and hence apparently donor derived. Neither study determined the mechanism underlying the apparent transdifferentiation. Orlic and colleagues injected murine GFP+Lin−ckithi BM cells into the borderzone myocardium of female mice several hours after coronary artery ligation [9]. Evaluation 9–14 days afterwards suggested that 68% of the cells in the infarct were GFP+Y chromosome+ cells that coexpressed the early cardiomyocyte transcription factors myocyte enhancer factor-2 and gata-4. In addition, some GFP+Y chromosome+ endothelial cells and smooth muscle cells were also detected. Significant functional improvement was also seen. When Lin−c-kitlo cells were injected, no functional improvement was detected. However, two studies performed subsequently by independent groups demonstrated significantly less contribution of murine BM Lin−ckithi cells to the injured cardiac tissue, and significantly less significant improvement in cardiac function than in the original Orlic et al. report [9]. Murry et al. failed to demonstrate generation of cardiomyocytes from Lin−ckithi cells grafted into the borderzone myocardium, even though some functional improvement was seen [122]. Similarly, following grafting of Lin−ckithi cells from myosin heavy chain (MHC)–LacZ transgenic animals into infarcted hearts of WT mice, no β-gal+ cells could be detected in the heart. Unfractionated BM cells from β-actin– eGFP transgenic mice were also transplanted into lethally irradiated, nontransgenic recipients. Two months after hematopoietic engraftment, a myocardial infarct was induced. Although some eGFP+αMHC+ cells were detected in the borderzone, the apparent transdifferentiated cells were infrequent. Balsam and colleagues transplanted both GFP+Lin− ckithi and GFP+KTLS cells into the infarcted heart of WT mice and could not find cells of donor origin with cardiac characteristics [123]. They also infarcted the heart of WT mice parabiosed with β-actin–EGFP transgenic mice and did not find donor-derived cardiomyocytes in the infarct zone [124]. The latter study did not assess the impact on the cardiac function. Both authors cautioned therefore that the data initially obtained by Orlic et al. [9] might not be sufficient to warrant clinical studies. Nevertheless, a now large number of clinical trials in humans have ensued in which different types of BM cell have been injected following balloon angioplasty into patients who have suffered from a cardiac infarct. Despite initial encouraging results in nonrandomized studies, four studies have been published in 2006 wherein patients were treated in a randomized blind fashion with BM cells or vehicle control, demonstrating little functional benefit for the majority of patients treated with cells [125–128]. Of note, a clinical study in which the hearts of patients were examined at autopsy after sex-mismatched BM transplantation showed that Y

Cellular Biology of Hematopoiesis

chromosome-positive cardiomyocytes contributed 0.23 ± 0.06% of the myocardium, and no Y chromosome-positive cells were seen in gendermatched controls. These data are consistent with the murine studies indicating that very rare human BM cells have the potential to transdifferentiate into cardiomyocytes. Although this has been rarely assessed, the study by Alvarez-Dolado et al. has definitively demonstrated that rare apparent transdifferentiation may be due to cell fusion between donor cells and cardiomyocytes [87]. BM-derived cells – endoderm A number of reports have suggested that BM cells have the potential to generate endodermal tissue. Even though, during embryonic development, the mesoderm and endoderm lie anatomically close to each other and there is considerable crosstalk between these two tissues that enables each to develop, the transdifferentiation between two germ layers would perhaps be even more astounding than that between mesodermal cell types themselves. Petersen et al. were the first to suggest that BM cells contribute to liver regeneration [123]. Hepatic oval cells are regarded as facultative hepatic stem cells that can generate both hepatocytes and cholangiocytes. Their precise origin is not clear, but it is likely that they are derived from canals of Herring or reside as blast-like cells next to the bile ducts. Hepatic oval cells express CD34, Thy-1 and c-kit, markers also present on HSCs. Previous studies had shown that oval cells proliferate when hepatocyte proliferation is suppressed following liver injury. To test whether BM cells could contribute to liver regeneration, female rats were transplanted with BM from syngeneic male rats, and liver injury was induced using a protocol to stimulate oval cell proliferation (2-acetylaminofluorene [2-AAF] and carbon tetrachloride [CCL4]). Thirteen days following transplantation, Y chromosomepositive hepatocytes were detected by in situ hybridization. In a second experiment, irradiated dipeptidyl peptidase-IV (DPPIV)-negative female rats received BM from DPPIV-positive male rats. Liver injury was generated using the 2-AAF–CCL4 protocol. DPPIV expression was observed in oval cells and hepatocytes in the recipient liver, that coexpressed proteins found in mature hepatocytes. Finally, a liver transplant was performed from L21.6 antigen-negative donor rats into allogeneic L21.6-positive rats and hepatic ductular structures containing L21.6+ cells were present in the transplanted liver, suggesting that oval cells were derived from extrahepatic cells. The authors concluded that BM cells can under certain conditions become hepatic oval cells. Similar findings were seen in a murine model [8], and together these results support the concept of a BM or extrahepatic stem cell generating hepatic oval cells. Neither study established the exact phenotype of the “plastic” BM cells or their use in the therapy of liver diseases. Lagasse et al. [7] used an animal model of fatal hereditary tyrosinemia type 1 (fuarylacetoacetate hydrolase [FAH]-deficient mice) in which mutant mice develop progressive liver failure and renal tubular damage unless treated with 2-(2-nitro-4-trifluro-methylebenzyol)-1,3-cyclohexanedione (NTBC). Unfractionated BM or 50 KTLS cells from male FAH–WT β-gal−transgenic mice were injected into lethally irradiated female FAH−/− mice treated with NTBC. Three weeks after transplantation, NTBC was discontinued intermittently to allow selection of the liver repopulating cells. Four out of nine mice grafted with unfractionated BM and 9 of 20 mice grafted with KTLS cells survived and had donor-derived β-gal+FAH+Y chromosome+ cells in the liver with an improvement in liver function. These donor-derived cells coexpressed albumin with β-gal when cultured on a mouse embryonic feeder layer. However, later studies [89,129] demonstrated that the differentiation of HSCs into hepatocytes did not occur in a cell-autonomous manner but following fusion of HSC-derived monocytes with hepatocytes.

79

A number of studies have also addressed whether apparent hepatic transdifferentiation occurs in humans. Theise et al. [129] demonstrated that liver tissue examined from male recipients of female livers and female recipients of male BM contained marrow-derived hepatocytes and cholangiocytes using in situ hybridization for the Y chromosome with colabeling with the monoclonal antibody CAM5.2, which identifies hepatocytes and cholangiocytes but not Kuppfer cells, stromal cells or circulating blood cells. The frequency of Y chromosomepositive hepatocytes and cholangiocytes varied but appeared higher in patients with GVHD or liver rejection. Engrafted human cells were most often scattered throughout the parenchyma as isolated cells [130]. Microchimerism for the Y chromosome can occur in the human female liver as a result of transplacental passage of male fetal blood cells during pregnancy; this was excluded as a contributing factor in a similar study by Alison et al. [131]. Both studies conclude that human BM cells can differentiate into mature hepatocytes and cholangiocytes in the absence of severe injury. Korbling et al. published that, following mobilized peripheral blood transplantation, up to 7% of epithelial cells of the liver, gastrointestinal tract, and skin appear donor in origin [132]. The frequency was low, and GVHD did not affect apparent donor cell engraftment in epithelia. Similarly, human hematopoietic progenitor cells transplanted into fetal sheep have been reported to differentiate into hematopoietic cells and hepatocytes [133]. While many studies as outlined have suggested that BM-derived cells may, at low level, contribute to liver, gut or lung tissue, and that this contribution increases in the setting of organ failure, the cell responsible is not known, and whether these phenomena occur outside the setting of cell fusion is not clear. One exception perhaps is the study by Krause et al. [80] demonstrating that contribution to lung epithelium may happen, at least in part, outside the setting of cell fusion [134]. A final tissue in which possible transdifferentiation has been suggested is the pancreas. To address the question of whether a cell within the BM can contribute to pancreatic β cells, Ianus et al. used mice transgenic for the Cre recombinase expression under the mouse Insulin2 promoter crossed into mice ubiquitously expressing the Rosa–Flox–GFP (RosaGFP) transgene (Ins2cre–RosaGFP) [109]. β cells derived from this donor mouse would hence express GFP. Irradiated WT mice were transplanted with Ins2cre–RosaGFP BM and analyzed 4–6 weeks post transplant for pancreatic engraftment. Approximately 1.7–3% of all cells within the pancreas were GFP positive, expressed characteristic β-cell markers (including Hnf3β, Pdx1, Glut2, Insulin1, and Insulin2), and secreted insulin in a glucose-responsive manner at levels similar to that of mature β cells. To exclude fusion as a mechanism, they transplanted Ins2cre BM into irradiated mice with the RosaGFP reporter. As no GFP-expressing β cells were seen, Ianus et al. concluded that the presence of GFP expressing β-cells in the first transplant model was not caused by fusion. In support of these data, Wang et al. reported that neonatal immunocompromised mice transplanted with GFP+ BM cells acquired significant engraftment in pancreatic epithelium (up to 40% with a mean of 4.6%) as well as rare GFP+Ins+ cells (0.03%) [135]. Other studies have questioned these results. Hess et al. demonstrated that BM cells transplanted into a diabetic model could reverse hyperglycemia, but that the mechanism was not direct differentiation to β cells [136]. Within the transplanted BM, the ability to reduce hyperglycemia was accounted for by a c-Kit+ subpopulation [136]. A similar study utilizing the same diabetic model revealed that BM contribution and support during regeneration was likely due to BM-derived endothelial progenitor cells (EPCs) [134]. The donor-derived endothelial cells may have facilitated the proliferation and ultimate recovery of remaining endogenous β cells.

80

Chapter 7

BM-derived cells – ectoderm Ectoderm is generated from the outer layer of the embryo and produces a surface layer of skin, the epidermis, and the central nervous system. One of the most difficult lineage boundaries to cross is the bridge between mesoderm and ectoderm, yet there are studies suggesting that this too might be possible. Eglitis and Mezey in 1997 were the first to report that BM-derived cells could contribute to, as expected, microglia (F4/80 antigen) but also (unexpected) astroglial (glial fibrillary acidic protein [GFAP]-positive) cells in the adult murine brain [5]. Sublethally irradiated female mice were transplanted with genetically marked male donor cells (enriched for HSCs using 5-flurouracil), the mice were sacrificed at intervals post transplantation, and the brain was examined using a combination of in situ hybridization histochemistry and immunohistochemistry. Doublelabeling analyses revealed that some BM-derived cells acquired glial markers. BM-derived cells in several mice were localized to the subependymal zone, an area known to be neurogenic during development, which would provide cues for glial differentiation. The type of BM cell contributing to astroglial cells was not determined. Mezey et al. transplanted WT BM cells intraperitoneally into PU.1−/− mice to rescue the animal from the typical early postnatal mortality [137]. Brains were examined at 1 and 4 months following transplantation using a combination of in situ hybridization to detect the Y chromosome and immunohistochemistry to visualize the neuronal nuclear marker, NeuN. BM-derived cells were found in the brain of all the animals examined, and the Y chromosome was present in 0.3–2.3% of the NeuN-immunoreactive nuclei. Mezey et al. concluded that BM cells can enter the brain and differentiate into cells that express neuronal markers, supporting the idea that mesodermally derived cells can adopt a neuronal (ectodermal) cell fate. In the same issue of Science, Brazelton et al. documented that when GFP-transgenic BM was grafted in lethally irradiated syngeneic mice, both NF200+ neurons and GFAP+ astrocytes could be found that were GFP+, concluding that cells from BM could generate neuroectodermal cells in vivo [138]. However, as indicated earlier, Wagers et al. were unable to find significant contribution of HSC to cells within the brain, except for rare Purkinje cells [79]. Likewise, Castro et al. could not find the same degree of transdifferentiation from BM to neuronal cells [139]. In a second series of studies, the ability of MSC to differentiate into neurons in vitro or in vivo was evaluated. Agents used to induce neural differentiation in vitro included nonspecific inducers, whereas others used cytokines and growth factors known to induce differentiation of ESCs to neuronal cells or induce differentiation of neural stem cells [140–143]. In most studies, “transdifferentiation” was shown only by the acquisition of neural morphology and the expression of a few neuronor glia-specific proteins or transcripts, which can already be detected at low level in undifferentiated MSCs [144]. A recent study has also suggested that dimethylsulfoxide induces rapid disruption of the actin cytoskeleton, leading to an apparent change in morphology of fibroblasts to cells that appear to extend neurites [145]. A few studies have assessed whether MSC-derived neuron-like cells have the functional attributes of neurons. Kohyama et al. showed that single-cell-derived immortalized BM stromal cells acquired the morphological and surface phenotype of neurons when treated with 5-azacytidine and the BMP2/4 inhibitor Noggin [146]. The resting potential of such cells of −5.0 mV and acquisition of potassium voltage-gated channels suggested the acquisition of some functional features of neurons. Wislet-Gendebien et al. co-cultured MSC-derived nestin-positive cells with cerebellar granule neurons, which led to the expression of astrocyte- and neuron-specific proteins [147]. More importantly, the resting membrane potential increased to −57 mV, at which time the cells also

acquired functional sodium voltage-gated channels, a phenotype similar to that of cells at an intermediate phase of neural development. The fact that MSCs co-cultured with paraformaldehyde-fixed cerebellar granule neurons yielded similar results suggests directed differentiation and not cell fusion. Dezawa et al. expressed the intracellular domain of Notch in MSC [148]. This led to the expression of early neural transcripts, and upon addition of forskolin and basic fibroblast growth factor, more than 95% of MSCs expressed markers for postmitotic neurons, and 50% of the cells acquired outward rectified potassium ion currents, but no voltagegated fast sodium currents. Following exposure to ciliary neurotrophic factor, 40% of the cells expressed genes found in dopaminergic neurons and secreted dopamine following depolarization. When grafted in 6hydroxydopamine treated animals, rotational behavior improved, as is seen with fetal midbrain transplantations. These studies suggest therefore that, under certain conditions, MSCs may acquire not only phenotypic, but also functional characteristics of neuroectodermal cells. Several groups have grafted rodent or human MSCs in vivo in intact or injured brain [149,150]. Although some authors have stated that MSCs transdifferentiate into neuronal cells, these conclusions have in general been based solely on the identification of neuron- (nestin) or glia- (GFAP) specific markers in rare donor cells. Moreover, in many studies, donor origin was determined by evaluation of BrdU or 3Hthymidine incorporated in the donor cells prior to grafting. As discussed earlier, recent studies have shown that such labels, previously thought to be superior over, for instance, membrane dyes, can be transferred from dying grafted cells to endogenously proliferating cells such as astroglia or neural stem/progenitor cells [95]. In addition, no study has excluded conclusively that the presumed neural differentiation is not caused by fusion between MSCs and host brain cells. Finally, no study has demonstrated that presumed MSC-derived neurons are functional. Nevertheless, as is true for grafting of BM cells in the heart, grafting MSCs in the brain may have beneficial effects by secreting trophic factors that enhance vascularization, recruit endogenous progenitors, and/or induce mature neural cell plasticity [151–154]. Endothelial cells in the BM Another cell type present in BM is the endothelial cell. HSCs proliferate and differentiate, and their progeny migrate into the blood circulation lined by endothelium. The endothelium forms the vascular niche within the BM, and at least some HSCs lie in close proximity to it [155]. The vascular niche is thought to promote HSC proliferation and differentiation, whereas the osteoblastic niche may favor quiescence [156]. Explanted endothelial cells can maintain HSCs in culture [157]. Its location at the interface between the BM and the circulation allows the endothelium to regulate transendothelial migration, homing, and mobilization of HSCs [84,158]. It has also been hypothesized that endothelial cells outside the BM medullary cavity may provide a back-up niche when the BM is stressed [159]. The boundaries separating the hematopoietic and vascular developmental programs are somewhat indistinct. HSCs are an accepted entity with a defined hierarchy based on proliferative potential. There remains controversy over the definition of an endothelial progenitor, and a similar organization for endothelial precursors, while postulated, is not proven. There is, at least during development and using in vitro embryonic stem cultures, evidence of a common endothelial and hematopoietic progenitor called a “hemangioblast.” This concept was proposed over 80 years ago when Florence Sabin observed the formation of blood and blood vessels from primitive mesoderm in chick blastoderm [160]. The term “hemangioblast” was coined by P.D.F. Murray in 1932, who noted that cells from the primitive streak of chick embryos formed simultane-

Cellular Biology of Hematopoiesis

ously both endothelial cells and blood islands [161]. Hematopoietic and endothelial cells develop in close proximity within the mammalian yolk sac [162,163], and in the embryo proper, current information suggests the presence of “hemogenic” endothelium in the dorsal aorta as a source of definitive HSCs [164–169]. Investigations in humans have shown similar results [170]. Using murine ESCs, several investigators have isolated hemangioblasts in vitro, which has allowed more extensive evaluation than cells from the embryo proper. ESCs generate embryoid bodies that can be induced to differentiate into hematopoietic lineage [171]. They generate a transient precursor with genes in common with both the hematopoietic and endothelial cell lineages. These transient precursors, referred to as the blast colony-forming cell population, may be an in vitro equivalent of a hemangioblast [168]. Whether circulating endothelial cells originate as outgrowths from vascular endothelium or reside in the circulation or BM is not clear [169]. Whatever the origin (reviewed more extensively in [172]), the endothelium has an intimate relationship with hematopoiesis throughout development. It has been difficult to study this association as EPCs share many phenotypic and functional characteristics with HSCs: they exist in the same fluid microenvironment and respond to the same stimuli. EPCs express KDR (VEGFR1), CD31, and Tie-2 (angiopoietin-1 receptor), ingest acetylated low-density lipoprotein, and bind lectins (a feature of most macrophages). That EPCs are present in BM comes from murine

81

and human BM transplantation studies demonstrating that at least some endothelial cells are of donor origin [173]. Whether such endothelial cells could be derived from HSCs rather than EPCs has not been fully addressed. Grant et al. showed that, following transplantation of a single long-term repopulating HSC, perfusable endothelial cells in the retina of a mouse developed following vascular injury [174]. However, recent studies have demonstrated that, aside from EPCs that generate selfrenewing colonies of endothelial cells in vitro, HSC-derived macrophages can also generate cells with features of endothelial cells, such as expression of CD31, von Willebrand factor, and ability to take up acetylated low-density lipoprotein [175]. The difference between the two endothelial cell-like cell progenitors is the time after which they start generating colonies in in vitro cultures, the ability to replate the colonies, and the ability to generate perfused vessels in vivo. The fact that the progeny of HSCs can acquire features of endothelial cells makes evaluation of the possible lineage relationship of HSCs and EPCs in in vivo studies as well as in vitro studies more difficult.

Persistence of more pluripotent adult stem cells? Adult pluripotent stem cells (Table 7.2) There are an increasing number of studies suggesting that cells with greater potency than classical tissue-specific stem cells can be isolated

Table 7.2 Adult pluripotent stem cells isolated from different murine and human sources, with their phenotype and “plasticity” towards alternative dermal lineages Name

Source

Phenotype

Differentiation capacity

MAPC [90]

Murine BM

Endothelium, hepatocytes, neuron-like cells

MIAMI cell [186]

Human BM

hBMSC [97]

Human BM

MASC [93]

Human BM, liver, heart

SSEA-1 cell [91]

Murine BM

VSEL cell [92]

Murine BM

USSCs [187]

Human umbilical cord blood

AFS [188]

Human amniotic fluid

maGSCs [189]

Murine testis

Oct4+, Rex1+ Thy-1.1lo, Sca-1+lo, Flk-1+lo, SSEA-1+, CD13+ CD34−, CD44−, CD45−, CD117−, MHC-I−MHC-II− Oct3a+ Rex-1+ Thy-1+, CD10+, CD29+, CD44+, CD49e+, CD103+ CD34−, CD36−, CD45−, CD54−, CD56−, CD117−, HLA-II− Oct3/4− Thy-1lo, CD105lo, Cd117lo CD29−, CD44−, CD73− Oct4+, Rex1+, Nanog+ Thy-1+, CD13+, CD49b+, CD73+, CD44+, HLA−I+, CD29+, CD105+, Flk-1+, CD49a+ CD14−, CD45−, CD38−, HLA-II−, CD133−, CD117−, CD34− Oct4+, Rex-1+, Nanog+, SSEA-1+ Sca-1+,Thy-1+ CD73+, CD105+, CD44+, CD34−, CD45− Oct4+, Rex1+, Nanog+, SSEA-1+ Sca-1+ CD45− Thy-1+, CD105+,CD13+,CD29+, CD44+, CD49e+ Class-II−, CD34−, CD45−, CD117−, CD33− Oct3/4+,SSEA-4+ Thy-1+, CD44+, CD73+, CD117+, Class-IIlo CD34−, CD45− Oct4+, Rex-1+, Nanog+, SSEA-1+ Thy-1+, Sca-1+lo, CD117+lo, Ter119−, CD34−

Osteoblasts, chondrocytes, adipocytes, pancreatic islets, neural cells

Cardiomyocytes, endothelium, smooth muscle

Osteoblasts, myocytes, endothelium, hepatocytes, neurons, glia

Osteoblasts, chrondrocytes, adipocytes, hematopoietic cells, endothelium, hepatocytes, astrocytes Cardiomyocytes, pancreas, neural cells

Osteoblasts, chrondrocytes, adipocytes, hematopoietic cells, neural cells Osteoblasts, hepatocytes, neural cells

Cardiomyocytes, smooth muscle, endothelium, hepatocyte-like cells, neurons

AFS, amniotic fluid-derived stem cell; BM, bone marrow; hBMSC, human BM-derived multipotent stem cell; maGSC, multipotent adult germline stem cell; MAPC, multipotent adult progenitor cell; MASC, multipotent adult stem cell; MIAMI, marrow-isolated adult multilineage inducible cell; SSEA-1, stage-specific embryonic antigen-1; USSC, unrestricted somatic stem cell; VSEL, very small embryonic-like cell.

82

Chapter 7

from BM, UCB, and other tissues. The first report was in 2002, when multipotent adult progenitor cells (MAPCs) were described. MAPCs are isolated from adherent BM cultures under conditions that may not support MSCs, specifically low concentrations of fetal bovine serum (FBS) (2%). Interestingly, isolation of MAPCs from rat and mouse BM requires leukemia inhibitory factor, a cytokine that is also a prerequisite for the isolation of murine ESCs [90]. MAPCs isolated from rodents, swine, and human BM are a clonal population of cells that can be expanded ex vivo without signs of cell senescence. They express the ESC transcription factor Oct4, but not appreciable levels of Nanog or Sox2. By FACS, human MAPCs are characterized as CD13−CD34− CD45−HLA-II−, Thy-1+, and CD45loHLA-Ilo. Under specific in vitro differentiation conditions, they can differentiate into typical mesenchymal cell types as well as endothelium, hepatocyte-like endodermal cells, and neuron and glia-like neuroectodermal cells [90,176–184]. Differentiated cell types including smooth muscle cells, osteoblasts, endothelium (arterial or venous specified endothelium), hepatocyte-like cells, and neuroectodermal-like cells were characterized based on morphology, phenotype, expressed protein, and gene profile, as well as extensive functional characterization. When transplanted in vivo, murine MAPCs can regenerate a complete lymphohematopoietic system [90,185], and some cell lines also contribute to endodermal tissues such as liver, gastrointestinal, and pulmonary endoderm [90]. Undifferentiated or endothelially committed MAPCs also contribute to neovascularization in tumors, or in Matrigel assays [178,183]. Some, but definitely not all, cell lines also contributed in variable degrees to multiple somatic tissues when injected in the blastocyst, although germline transmission has not been observed [90]. In 2004, D’Ippolito et al. described marrow-isolated adult multilineage inducible (MIAMI) cells [186]. These cells are derived from human BM from vertebral bodies by plating on fibronectin at 3% oxygen and 15% FBS for the first 2 weeks; FBS is then decreased to 2%. MIAMI cells, like MAPCs, express Oct3a and Rex1, and can be expanded for 30–50 cell doublings without evidence of senescence or loss of differentiation capacity. By FACS, MIAMI cells are negative for CD34, CD36, CD45, CD117 and HLA-II. In vitro, MIAMI cells can be induced to differentiate to mesodermal cell types as well as cells with phenotypic features of neuroectodermal-like cells and endodermal features (transcripts of pancreatic endoderm), although no functional studies have been done to prove differentiation to ectoderm and endoderm. Yoon et al. purified cells from human BM that could be expanded from single cells for greater than 140 population doublings without senescence and loss of differentiation, named human BM-derived multipotent stem cells (hBMSCs) [97]. By FACS, hBMSCs are negative for CD29, CD44, and CD73, and express low levels of CD105, CD90, and CD117, all surface markers used to characterize mesenchymal cells. In contrast to most cells with apparent greater potency, BMSCs do not express Oct4. They can differentiate into cells with phenotypic features of endothelium, smooth muscle, neural cells, and hepatocytes. However, the investigators did not evaluate the functional properties of the lineage differentiated progeny. When co-cultured with cardiomyocytes in vitro, cells appeared to differentiate into cardiomyocytes, although fusion is, at least in part, responsible for this observation. When hBMSCs labeled with the dye DiL were given as an intracardiac graft in an infarcted heart, multilineage differentiation (endothelium, smooth muscle and cardiac muscle) was seen, which resulted in favorable remodeling and improved cardiac function. Whether the functional improvement was due to apparent transdifferentiation versus trophic factors secreted by BMSCs, as has been seen for hematopoietic cells and MSCs, is not clear. As discussed earlier, the use of DiL for in vivo tracking is not ideal. The authors also did not conclusively demonstrate that the resulting differentiation was

cell autonomous versus fusion between hBMSCs and resident cells in the heart. Multipotent adult stem cells (MASCs) were isolated from adult human BM, liver or heart tissue [93]. These cells were cultured in Mesencult (a medium optimized for human MSC cultures) and subsequently on fibronectin-coated dishes and above stromal cells with 2% FBS. The cell-surface phenotype is that of MSCs, but the cells express Oct4, Rex1, and Nanog transcripts, as well as Oct4 and Nanog protein. Clonally isolated cells differentiate into cells with phenotypic and functional features of osteoblasts, myocytes, endothelial cells, hepatocytes, neurons and glia. Unrestricted somatic stem cells, isolated from human UCBs, are CD45−, HLA class II-negative mononuclear cells with fibroblastic morphology and extended telomere length that can be expanded for 30–50 population doublings [187]. The cells express Oct4 and Rex1 and, in vitro/in vivo, demonstrate differentiation potential to osteoblasts, chondroblasts, adipocytes, and hematopoietic, neural and hepatic cells. As is true for many studies, limited proof exists for acquisition of functional features of the unexpected endodermal and ectodermal cell types. Similarly potent cells have been found in amniotic fluid [188]. In addition, Guan et al. demonstrated that cells with pluripotent features, including teratoma formation and contribution to somatic and germline cells when injected in the blastocyst, could be generated from murine testicular tissue [189]. The latter cells are therefore the only ones that have capabilities similar to those of ESCs. One question that has not been answered is whether the cell populations described above (MAPCs, MIAMI cells, BSSCs, and MASCs) exist in vivo or are created in culture as the result of dedifferentiation. Two recent studies may start to shed light on this. AnjosAfonso and Bonnet have shown that cells expressing stage-specific embryonic antigen-1 (SSEA-1), found on mouse ESCs, can be used to isolate cells from MSC cultures following one passage in vitro [91]. These cells express high levels of Oct4 as well as Nanog and Sox2, and can differentiate following expansion into cells with phenotypic and in vitro functional characteristics of multiple mesodermal cell types, including endothelium, as well as endodermal hepatocyte-like cells and ectodermal lineage cells such as astrocytes. When grafted in the tibia in vivo, pre-MSCs contribute to endothelium, osteoblasts, adipocytes, chrondrocytes, and hematopoietic cells. They further demonstrated that when SSEA-1− cells are isolated that do not express Oct4 and Nanog, these cells cannot be induced to express SSEA-1 or the ESC transcription factors. Kucia et al. recently demonstrated that a homogeneous population of Sca-1+Lin−CD45−CXCR4+ cells can be selected directly from murine BM or human UCBs that express, like the cells identified by AnjosAfonso and like ESCs, SSEA-1, Oct4, Nanog and Rex-1, or SSEA-4 in human UCBs [92,190]. These cells are difficult to culture ex vivo, but by performing co-cultures, phenotypic changes were seen consistent with differentiation to endothelium and possibly pancreatic β cells. No functional confirmation of such differentiation was demonstrated, nor was fusion excluded. The latter two studies suggest that rare cells exist in murine BM with phenotypic features of MAPCs, MIAMI cells, hBMSCs, unrestricted somatic stem cells, and MASCs. It is unknown whether the differentiation ability ascribed to MAPCs, MIAMI cells, hBMSCs, unrestricted somatic stem cells, MASCs, pre-MSCs, multipotent adult germline stem cells or amniotic fluid-derived stem cells is already present in the freshly isolated cells, and hence these cells represent cells with greater potency persisting in vivo into postnatal life, or whether the multilineage differentiation ability is acquired once cells are cultured in vitro, and therefore represent the dedifferentiation of rare Oct4+ cells.

Cellular Biology of Hematopoiesis

Conclusions and future directions This chapter has summarized the significant progress that has been made in defining the properties of stem cells within BM, including HSCs, MSCs, and endothelial progenitor cells. There are continuing advances in understanding the unique biologic properties of these cells and their progeny. This information is leading to increasing numbers of clinical applications for these cells, not only with HSCs, but trials are being initiated with MSCs and EPCs as well. New information highlighting the importance of the BM microenvironment demonstrates that the beneficial effect of these cells extends beyond their direct contribution to the host tissue. The trophic effects of MSCs can be harnessed to increase the potential of existing pharmacologic therapies, especially in ischemic disorders. The immunomodulatory characteristics of MSCs are also being tested in the setting of GVHD, graft rejection, and other immune-mediated disorders. The limitations and potential toxicity of these treatments should, however, be carefully assessed in animal models and clinical trials as translation to humans may reveal unsuspected complications. Many studies have suggested that BM-derived stem cells may differentiate into not only cells of the tissue of origin, but also cells of other

83

tissues, a process known as stem cell plasticity. The evidence for transdifferentiation is often confusing, with some studies providing evidence of phenotypic switch and others failing to demonstrate such phenomena. There may be technical reasons contributing to the differences between studies. In addition, the mechanisms underlying the apparent lineage switch are not always clear. Undoubtedly, fusion occurs, especially between cells that are more “fusigenic” by nature. Aside from fusion, there is mounting evidence for rare events of cell intrinsic lineage switch. Studies aimed at understanding the conditions that allow such transdifferentiation may ultimately yield optimization of local signals to induce such fate change. There is also more and more evidence that cells can be cultivated that have (or acquire) greater potency. Further characterization of the potential of these cells to be used for treatment of not only hematopoietic disorders, but also diseases of the central nervous system and liver, among others, may demonstrate that BM cells could be used outside hematologic diseases. Stem cell potential as we know it presently is in its infancy. The huge strides that have been made since the pioneering use of HSCs in human HCT in the 1960s are small steps along the way to fulfilling the possibly much broader, but latent potential of BM-derived cells.

References 1. Balsam LB, Robbins RC. Haematopoietic stem cells and repair of the ischaemic heart. Clin Sci 2005; 109: 483–92. 2. Gatti RA, Meuwissen HJ, Allen HD, Hong R, Good RA. Immunological reconstitution of sexlinked lymphopenic immunological deficiency. Lancet 1968; 2: 1366–9. 3. Copelan EA. Hematopoietic stem-cell transplantation. N Engl J Med 2006; 354: 1813–26. 4. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH. Viable offspring derived from fetal and adult mammalian cells. Nature 1997; 385: 810–13. 5. Eglitis MA, Mezey E. Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice. PNAS 1997; 94: 4080– 5. 6. Gussoni E, Soneoka Y, Strickland CD et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 1999; 401: 390–4. 7. Lagasse E, Connors H, Al-Dhalimy M et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000; 6: 1229– 34. 8. Theise ND, Badve S, Saxena R et al. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 2000; 31: 235–40. 9. Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. 2001; 410: 701–5. 10. Bhattacharya V, McSweeney PA, Shi Q et al. Enhanced endothelialization and microvessel formation in polyester grafts seeded with CD34(+) bone marrow cells. Blood 2000; 95: 581–5. 11. Kucia M, Ratajczak J, Reca R, JanowskaWieczorek A, Ratajczak MZ. Tissue-specific muscle, neural and liver stem/progenitor cells reside in the bone marrow, respond to an SDF-1 gradient and are mobilized into peripheral blood during stress and tissue injury. Blood Cells Mol Dis 2004; 32: 52–7.

12. Willenbring H, Bailey AS, Foster M et al. Myelomonocytic cells are sufficient for therapeutic cell fusion in liver. Nat Med 2004; 10: 744–8. 13. Harrison DE, Stone M, Astle CM. Effects of transplantation on the primitive immunohematopoietic stem cell. J Exp Med 1990; 172: 431– 7. 14. Alvarez-Silva M, Belo-Diabangouaya P, Salaun J, Dieterlen-Lievre F. Mouse placenta is a major hematopoietic organ. Development 2003; 130: 5437–44. 15. Gekas C, Dieterlen-Lievre F, Orkin SH, Mikkola HKA. The placenta is a niche for hematopoietic stem cells. Dev Cell 2005; 8: 365–75. 16. Wang JC, Doedens M, Dick JE. Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay. Blood 1997; 89: 3919–24. 17. Goodman JW, Hodgson GS. Evidence for stem cells in the peripheral blood of mice. Blood 1962; 19: 702–14. 18. Socinski MA, Cannistra SA, Elias A, Antman KH, Schnipper L, Griffin JD. Granulocyte-macrophage colony stimulating factor expands the circulating haemopoietic progenitor cell compartment in man. Lancet 1988; 1: 1194–8. 19. To LB, Shepperd KM, Haylock DN et al. Single high doses of cyclophosphamide enable the collection of high numbers of hemopoietic stem cells from the peripheral blood. Exp Hematol 1990; 18: 442–7. 20. Szilvassy SJ, Humphries RK, Lansdorp PM, Eaves AC, Eaves CJ. Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy. Proc Natl Acad Sci U S A 1990; 87: 8736–40. 21. Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science 1988; 241: 58–62. 22. Waller E, Olweus J, Lund-Johansen F et al. The “common stem cell” hypothesis reevaluated:

23.

24.

25.

26.

27.

28.

29.

30.

31.

human fetal bone marrow contains separate populations of hematopoietic and stromal progenitors. Blood 1995; 85: 2422–35. Zijlmans JMJM, Visser JWM, Laterveer L et al. The early phase of engraftment after murine blood cell transplantation is mediated by hematopoietic stem cells. PNAS 1998; 95: 725–9. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996; 183: 1797–806. Sutherland H, Lansdorp P, Henkelman D, Eaves A, Eaves C. Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. PNAS 1990; 87: 3584–8. Ploemacher R, van der Sluijs J, van Beurden C, Baert M, Chan P. Use of limiting-dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colonyforming hematopoietic stem cells in the mouse. Blood 1991; 78: 2527–33. Punzel M, Wissink SD, Miller JS, Moore KA, Lemischka IR, Verfaillie CM. The myeloid–lymphoid initiating cell (ML-IC) assay assesses the fate of multipotent human progenitors in vitro. Blood 1999; 93: 3750–6. Larochelle A, Vormoor J, Hanenberg H et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat Med 1996; 2: 1329–37. Zanjani ED, Pallavicini MG, Ascensao JL et al. Engraftment and long-term expression of human fetal hemopoietic stem cells in sheep following transplantation in utero. J Clin Invest 1992; 89: 1178–88. Baume C, Weissman I, Tsukamoto A, Buckle A, Peault B. Isolation of a candidate human hematopoietic stem-cell population. PNAS 1992; 89: 2804–8. Bhatia M, Wang JCY, Kapp U, Bonnet D, Dick JE. Purification of primitive human hematopoietic

84

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

Chapter 7

cells capable of repopulating immune-deficient mice. PNAS 1997; 94: 5320–5. Liu H, Verfaillie CM. Myeloid–lymphoid initiating cells (ML-IC) are highly enriched in the rhodamine-c-kit(+)CD33(−)CD38(−) fraction of umbilical cord CD34(+) cells. Exp Hematol 2002; 30: 582–9. Uchida N, Fujisaki T, Eaves AC, Eaves CJ. Transplantable hematopoietic stem cells in human fetal liver have a CD34+ side population (SP)phenotype. J Clin Invest 2001; 108: 1071–7. Baum CM, Weissman IL, Tsukamoto AS, Buckle AM, Peault B. Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci U S A 1992; 89: 2804–8. Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol 1999; 181: 67–73. Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284: 143–7. Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol 1976; 4: 267–74. Campagnoli C, Fisk N, Overton T, Bennett P, Watts T, Roberts I. Circulating hematopoietic progenitor cells in first trimester fetal blood. Blood 2000; 95: 1967–72. In’t Anker PS, Scherjon SA, Kleijburg-van der Keur C et al. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells 2004; 22: 1338–45. In’t Anker PS, Noort WA, Scherjon SA et al. Mesenchymal stem cells in human secondtrimester bone marrow, liver, lung, and spleen exhibit a similar immunophenotype but a heterogeneous multilineage differentiation potential. Haematologica 2003; 88: 845–52. In’t Anker PS, Scherjon SA, Kleijburg-van der Keur C et al. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood 2003; 102: 1548–9. Miura M, Gronthos S, Zhao M et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A 2003; 100: 5807–12. Zuk PA, Zhu M, Ashjian P et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002; 13: 4279–95. Digirolamo CM, Stokes D, Colter D, Phinney DG, Class R, Prockop DJ. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol 1999; 107: 275–81. Trivedi P, Hematti P. Simultaneous generation of CD34+ primitive hematopoietic cells and CD73+ mesenchymal stem cells from human embryonic stem cells cocultured with murine OP9 stromal cells. Exp Hematol 2007; 35: 146–54. Aslan H, Zilberman Y, Kandel L et al. Osteogenic differentiation of noncultured immunoisolated bone marrow-derived CD105+ cells. Stem Cells 2006; 24: 1728–37. Simmons PJ, Torok-Storb B. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood 1991; 78: 55–62. El-Badri NS, Wang BY, Cherry, Good RA. Osteoblasts promote engraftment of allogeneic

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60. 61.

62.

63.

hematopoietic stem cells. Exp Hematol 1998; 26: 110–16. Nolta JA, Hanley MB, Kohn DB. Sustained human hematopoiesis in immunodeficient mice by cotransplantation of marrow stroma expressing human interleukin-3: analysis of gene transduction of long-lived progenitors. Blood 1994; 83: 3041–51. Noort WA, Kruisselbrink AB, In’t Anker PS et al. Mesenchymal stem cells promote engraftment of human umbilical cord blood-derived CD34(+) cells in NOD/SCID mice. Exp Hematol 2002; 30: 870–8. Lazarus HM, Koc ON, Devine SM et al. Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transplant 2005; 11: 389–98. Horwitz EM, Prockop DJ, Fitzpatrick LA et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999; 5: 309–13. Cilloni D, Carlo-Stella C, Falzetti F et al. Limited engraftment capacity of bone marrow-derived mesenchymal cells following T-cell-depleted hematopoietic stem cell transplantation. Blood 2000; 96: 3637–43. Gao J, Dennis JE, Muzic RF, Lundberg M, Caplan AI. The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion. Cells Tissues Organs 2001; 169: 12– 20. Aizawa S, Nakano M, Iwase O et al. Bone marrow stroma from refractory anemia of myelodysplastic syndrome is defective in its ability to support normal CD34-positive cell proliferation and differentiation in vitro. Leuk Res 1999; 23: 239–46. Flores-Figueroa E, Arana-Trejo RM, GutierrezEspindola G, Perez-Cabrera A, Mayani H. Mesenchymal stem cells in myelodysplastic syndromes: phenotypic and cytogenetic characterization. Leuk Res 2005; 29: 215–24. Blau O, Hofmann W-K, Baldus CD et al. Chromosomal aberrations in bone marrow mesenchymal stroma cells from patients with myelodysplastic syndrome and acute myeloblastic leukemia. Exp Hematol 2007; 35: 221–9. Bhatia R, McGlave P, Dewald G, Blazar B, Verfaillie C. Abnormal function of the bone marrow microenvironment in chronic myelogenous leukemia: role of malignant stromal macrophages. Blood 1995; 85: 3636–45. Gunsilius E, Duba HC, Petzer AL et al. Evidence from a leukaemia model for maintenance of vascular endothelium by bone-marrow-derived endothelial cells. Lancet 2000; 355: 1688–91. Luttun A, Verfaillie CM. Will the real EPC please stand up? Blood 2007; 109: 1795–6. Tolar J, Nauta AJ, Osborn MJ et al. Sarcoma derived from cultured mesenchymal stem cells. Stem Cells 2007; 25: 371–9. Semont A, Francois S, Mouiseddine M et al. Mesenchymal stem cells increase self-renewal of small intestinal epithelium and accelerate structural recovery after radiation injury. Adv Exp Med Biol 2006; 585: 19–30. Caplan JED, Dennis JE. Mesenchymal stem cells as trophic mediators. J Cell Biochem 2006; 98: 1076–84.

64. Togel F, Hu Z, Weiss K, Isaac J, Lange C, Westenfelder C. Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms. Am J Physiol Renal Physiol 2005; 289: F31–42. 65. Li Y, Chen J, Chopp M. Adult bone marrow transplantation after stroke in adult rats. Cell Transplant 2001; 10: 31–40. 66. Mangi AA, Noiseux N, Kong D et al. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts. Nat Med 2003; 9: 1195–201. 67. Breitbach M, Bostani T, Roell W et al. Potential risks of bone marrow cell transplantation into infarcted hearts. Blood 2007; 110: 1362–9. 68. Meyer GP, Wollert KC, Lotz J et al. Intracoronary bone marrow cell transfer after myocardial infarction: eighteen months’ follow-up data from the randomized, controlled BOOST (Bone Marrow transfer to enhance ST-elevation infarct regeneration) Trial. Circulation 2006; 113: 1287–94. 69. Welt FGP, Losordo DW. Cell therapy for acute myocardial infarction: curb your enthusiasm? Circulation 2006; 113: 1272–4. 70. Le Blanc K, Tammik C, Rosendahl K, Zetterberg E, Ringden O. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp Hematol 2003; 31: 890–6. 71. Spaggiari GM, Capobianco A, Becchetti S, Mingari MC, Moretta L. Mesenchymal stem cellnatural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation. Blood 2006; 107: 1484–90. 72. Krampera M, Glennie S, Dyson J et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood 2003; 101: 3722–9. 73. Corcione A, Benvenuto F, Ferretti E et al. Human mesenchymal stem cells modulate B-cell functions. Blood 2006; 107: 367–72. 74. Augello A, Tasso R, Negrini SM et al. Bone marrow mesenchymal progenitor cells inhibit lymphocyte proliferation by activation of the programmed death 1 pathway. Eur J Immunol 2005; 35: 1482–90. 75. Deng W, Han Q, Liao L, You S, Deng H, Zhao RC. Effects of allogeneic bone marrow-derived mesenchymal stem cells on T and B lymphocytes from BXSB mice. DNA Cell Biol 2005; 24: 458– 63. 76. Chung NG, Jeong DC, Park SJ et al. Cotransplantation of marrow stromal cells may prevent lethal graft-versus-host disease in major histocompatibility complex mismatched murine hematopoietic stem cell transplantation. Int J Hematol 2004; 80: 370–6. 77. Le Blanc K, Rasmusson I, Sundberg B et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004; 363: 1439–41. 78. Krause DS, Theise ND, Collector MI et al. Multiorgan, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001; 105: 369– 77. 79. Wagers AJ, Sherwood RI, Christensen JL, Weissman IL. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 2002; 297: 2256–9.

Cellular Biology of Hematopoiesis 80. Borue X, Lee S, Grove J et al. Bone marrowderived cells contribute to epithelial engraftment during wound healing. Am J Pathol 2004; 165: 1767–72. 81. Odelberg SJ. Cellular plasticity in vertebrate regeneration. Anat Rec B New Anat 2005; 287: 25–35. 82. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature 2007; 448: 313–17. 83. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126: 663–76. 84. Lapidot T, Dar A, Kollet O. How do stem cells find their way home? Blood 2005; 106: 1901– 10. 85. Terada N, Hamazaki T, Oka M et al. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002; 416: 542–5. 86. Ying QL, Nichols J, Evans EP, Smith AG. Changing potency by spontaneous fusion. Nature 2002; 416: 545–8. 87. Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM et al. Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature 2003; 425: 968–73. 88. Camargo FD, Finegold M, Goodell MA. Hematopoietic myelomonocytic cells are the major source of hepatocyte fusion partners. J Clin Invest 2004; 113: 1266–70. 89. Vassilopoulos G, Wang PR, Russell DW. Transplanted bone marrow regenerates liver by cell fusion. Nature 2003; 422: 901–4. 90. Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002; 418: 41–9. 91. Anjos-Afonso F, Bonnet D. Nonhematopoietic/ endothelial SSEA-1+ cells define the most primitive progenitors in the adult murine bone marrow mesenchymal compartment. Blood 2007; 109: 1298–306. 92. Kucia M, Reca R, Campbell FR et al. A population of very small embryonic-like (VSEL) CXCR4(+) SSEA-1(+)Oct-4+ stem cells identified in adult bone marrow. Leukemia 2006; 20: 857–69. 93. Beltrami AP, Cesselli D, Bergamin N et al. Multipotent cells can be generated in vitro from several adult human organs (heart, liver and bone marrow). Blood 2007; 110: 3438–46. 94. Brazelton TR, Blau HM. Optimizing techniques for tracking transplanted stem cells in vivo. Stem Cells 2005; 23: 1251–65. 95. Burns TC, Ortiz-Gonzalez XR, Gutierrez-Perez M et al. Thymidine analogs are transferred from prelabeled donor to host cells in the central nervous system after transplantation: a word of caution. Stem Cells 2006; 24: 1121–7. 96. Coyne TM, Marcus AJ, Woodbury D, Black IB. Marrow stromal cells transplanted to the adult brain are rejected by an inflammatory response and transfer donor labels to host neurons and glia. Stem Cells 2006; 24: 2483–92. 97. Yoon YS, Wecker A, Heyd L et al. Clonally expanded novel multipotent stem cells from human bone marrow regenerate myocardium after myocardial infarction. J Clin Invest 2005; 115: 326–38. 98. Spotl L, Sarti A, Dierich MP, Most J. Cell membrane labeling with fluorescent dyes for the demonstration of cytokine-induced fusion between

99.

100.

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

111.

112.

113.

114.

115.

116.

monocytes and tumor cells. Cytometry 1995; 21: 160–9. Stroh A, Faber C, Neuberger T et al. In vivo detection limits of magnetically labeled embryonic stem cells in the rat brain using high-field (17.6 T) magnetic resonance imaging. Neuroimage 2005; 24: 635–45. Cohen RB, Tsou KC, Rutenburg SH, Seligman AM. The colorimetric estimation and histochemical demonstration of beta-d-galactosidase. J Biol Chem 1952; 195: 239–49. Sanchez-Ramos J, Song S, Dailey M et al. The X-gal caution in neural transplantation studies. Cell Transplant 2000; 9: 657–67. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. “Green mice” as a source of ubiquitous green cells. FEBS Lett 1997; 407: 313– 19. Hadjantonakis AK, Macmaster S, Nagy A. Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal. BMC Biotechnol 2002; 2: 11. McTaggart RA, Feng S. An uncomfortable silence em leader while we all search for a better reporter gene in adult stem cell biology. Hepatology 2004; 39: 1143–6. Moore JC, Spijker R, Martens AC et al. A P19Cl6 GFP reporter line to quantify cardiomyocyte differentiation of stem cells. Int J Dev Biol 2004; 48: 47–55. Goldman S, Roy N. Reply to “Human neural progenitor cells: better blue than green?” Nat Med 2000; 6: 483–4. Oh H, Bradfute SB, Gallardo TD et al. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci U S A 2003; 100: 12313–18. Reinecke H, Minami E, Poppa V, Murry CE. Evidence for fusion between cardiac and skeletal muscle cells. Circ Res 2004; 94: e56–60. Ianus A, Holz GG, Theise ND, Hussain MA. In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J Clin Invest 2003; 111: 843–50. Ferrari G, Cusella G, Angelis D et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998; 279: 1528–30. Bittner RE, Schofer C, Weipoltshammer K et al. Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice. Anat Embryol (Berl) 1999; 199: 391– 6. LaBarge MA, Blau HM. Biological progression from adult bone marrow to mononucleate muscle stem cell to multinucleate muscle fiber in response to injury. Cell 2002; 111: 589–601. Makino S, Fukuda K, Miyoshi S et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 1999; 103: 697–705. Corbel SY, Lee A, Yi L et al. Contribution of hematopoietic stem cells to skeletal muscle. Nat Med 2003; 9: 1528–32. Gussoni E, Bennett RR, Muskiewicz KR et al. Long-term persistence of donor nuclei in a Duchenne muscular dystrophy patient receiving bone marrow transplantation. J Clin Invest 2002; 110: 807–14. Klug MG, Soonpaa MH, Koh GY, Field LJ. Genetically selected cardiomyocytes from differ-

117.

118.

119.

120.

121.

122.

123.

124.

125.

126.

127.

128.

129.

130.

131.

132.

133.

85

entiating embryonic stem cells form stable intracardiac grafts. J Clin Invest 1996; 98: 216–24. Kolossov E, Bostani T, Roell W et al. Engraftment of engineered ES cell-derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium. J Exp Med 2006; 203: 2315–27. Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation 2002; 105: 93–8. Mangi AA, Noiseux N, Kong D et al. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts. Nat Med 2003; 9: 1195–1201. Gnecchi M, He H, Noiseux N et al. Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J 2006; 20: 661–9. Jackson KA, Majka SM, Wang H et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 2001; 107: 1395–402. Murry CE, Soonpaa MH, Reinecke H et al. Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature 2004; 428: 664–8. Petersen BE, Bowen WC, Patrene KD et al. Bone marrow as a potential source of hepatic oval cells. Science 1999; 284: 1168–70. Balsam LB, Wagers AJ, Christensen JL, Kofidis T, Weissman IL, Robbins RC. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature 2004; 428: 668– 673. Janssens S, Dubois C, Bogaert J et al. Autologous bone marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction: double-blind, randomised controlled trial. Lancet 2006; 367: 113–21. Lunde K, Solheim S, Aakhus S et al. Intracoronary injection of mononuclear bone marrow cells in acute myocardial infarction. N Engl J Med 2006; 355: 1199–209. Schachinger V, Erbs S, Elsasser A et al. Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N Engl J Med 2006; 355: 1210–21. Assmus B, Honold J, Schachinger V et al. Transcoronary transplantation of progenitor cells after myocardial infarction. N Engl J Med 2006; 355: 1222–32. Theise ND, Nimmakayalu M, Gardner R et al. Liver from bone marrow in humans. Hepatology 2000; 32: 11–16. Harris RG, Herzog EL, Bruscia EM, Grove JE, Van Arnam JS, Krause DS. Lack of a fusion requirement for development of bone marrowderived epithelia. Science 2004; 305: 90–3. Alison MR, Poulsom R, Jeffery R et al. Hepatocytes from non-hepatic adult stem cells. Nature 2000; 406: 257. Korbling M, Katz RL, Khanna A et al. Hepatocytes and epithelial cells of donor origin in recipients of peripheral-blood stem cells. N Engl J Med 2002; 346: 738–46. Zanjani ED, Porada CD, Crapnell KB et al. Production of human hepatocytes by human Lin−,CD34+/− cells in vivo. Blood 2000; 96 Suppl: 494a.

86

Chapter 7

134. Mathews V, Hanson PT, Ford E, Fujita J, Polonsky KS, Graubert TA. Recruitment of bone marrow-derived endothelial cells to sites of pancreatic beta-cell injury. Diabetes 2004; 53: 91–8. 135. Wang X, Ge S, Gonzalez I et al. Formation of pancreatic duct epithelium from bone marrow during neonatal development. Stem Cells 2006; 24: 307–14. 136. Hess D, Li L, Martin M et al. Bone marrowderived stem cells initiate pancreatic regeneration. Nat Biotechnol 2003; 21: 763–70. 137. Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000; 290: 1779–82. 138. Brazelton TR, Rossi FMV, Keshet GI, Blau HM. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 2000; 290: 1775–9. 139. Castro RF, Jackson KA, Goodell MA, Robertson CS, Liu H, Shine HD. Failure of bone marrow cells to transdifferentiate into neural cells in vivo. Science 2002; 297: 1299. 140. Deng W, Obrocka M, Fischer I, Prockop DJ. In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem Biophys Res Commun 2001; 282: 148– 52. 141. Woodbury D, Schwarz EJ, Prockop DJ, Black IB. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 2000; 61: 364–70. 142. Sanchez-Ramos SR. Neural cells derived from adult bone marrow and umbilical cord blood. J Neurosci Res 2002; 69: 880–93. 143. Barry FP, Murphy JM. Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol 2004; 36: 568–84. 144. Tondreau T, Lagneaux L, Dejeneffe M et al. Bone marrow-derived mesenchymal stem cells already express specific neural proteins before any differentiation. Differentiation 2004; 72: 319–26. 145. Neuhuber B, Gallo G, Howard L, Kostura L, Mackay A, Fischer I. Reevaluation of in vitro differentiation protocols for bone marrow stromal cells: disruption of actin cytoskeleton induces rapid morphological changes and mimics neuronal phenotype. J Neurosci Res 2004; 77: 192– 204. 146. Kohyama J, Abe H, Shimazaki T et al. Brain from bone: efficient “meta-differentiation” of marrow stroma-derived mature osteoblasts to neurons with Noggin or a demethylating agent. Differentiation 2001; 68: 235–44. 147. Wislet-Gendebien S, Hans G, Leprince P, Rigo JM, Moonen G, Rogister B. Plasticity of cultured mesenchymal stem cells: switch from nestinpositive to excitable neuron-like phenotype. Stem Cells 2005; 23: 392–402. 148. Dezawa M, Kanno H, Hoshino M et al. Specific induction of neuronal cells from bone marrow stromal cells and application for autologous transplantation. J Clin Invest 2004; 113: 1701–10. 149. Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. PNAS 1999; 96: 10711–16. 150. Hofstetter CP, Schwarz EJ, Hess D et al. Marrow stromal cells form guiding strands in the injured

151.

152.

153.

154.

155. 156.

157.

158.

159.

160.

161.

162.

163.

164.

165.

166.

167.

168.

spinal cord and promote recovery. Proc Natl Acad Sci U S A 2002; 99: 2199–204. Ankeny DP, McTigue DM, Jakeman LB. Bone marrow transplants provide tissue protection and directional guidance for axons after contusive spinal cord injury in rats. Exp Neurol 2004; 190: 17–31. Borlongan CV, Lind JG, Dillon-Carter O et al. Bone marrow grafts restore cerebral blood flow and blood brain barrier in stroke rats. Brain Res 2004; 1010: 108–16. Neuhuber B, Timothy Himes B, Shumsky JS, Gallo G, Fischer I. Axon growth and recovery of function supported by human bone marrow stromal cells in the injured spinal cord exhibit donor variations. Brain Res 2005; 1035: 73–85. Behrstock S, Svendsen CN. Combining growth factors, stem cells, and gene therapy for the aging brain. Ann N Y Acad Sci 2004; 1019: 5–14. Yin T, Li L. The stem cell niches in bone. J Clin Invest 2006; 116: 1195–201. Kopp HG, Avecilla ST, Hooper AT, Rafii S. The bone marrow vascular niche: home of HSC differentiation and mobilization. Physiology (Bethesda) 2005; 20: 349–56. Li W, Johnson SA, Shelley WC, Yoder MC. Hematopoietic stem cell repopulating ability can be maintained in vitro by some primary endothelial cells. Exp Hematol 2004; 32: 1226–37. Cancelas JA, Williams DA. Stem cell mobilization by beta2-agonists. Nat Med 2006; 12: 278– 9. Kiel MJ, Yilmaz OH, Iwashita T, Terhorst C, Morrison SJ. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 2005; 121: 1109–21. Sabin F. Studies on the origin of blood vessels and of red blood corpuscles as seen in the living blastoderm of chicks during the second day of incubation. Contrib Embryol 1920; 9: 213–62. Murray P. The development in vitro of the blood of the early chick embryo. Proc Roy Soc London 1932; 11: 497–521. Pardanaud L, Yassine F, Dieterlen-Lievre F. Relationship between vasculogenesis, angiogenesis and haemopoiesis during avian ontogeny. Development 1989; 105: 473–85. Choi K, Kennedy M, Kazarov A, Papadimitriou J, Keller G. A common precursor for hematopoietic and endothelial cells. Development 1998; 125: 725–32. Wood HB, May G, Healy L, Enver T, MorrissKay GM. CD34 expression patterns during early mouse development are related to modes of blood vessel formation and reveal additional sites of hematopoiesis. Blood 1997; 90: 2300–11. Jaffredo T, Gautier R, Eichmann A, DieterlenLievre F. Intraaortic hemopoietic cells are derived from endothelial cells during ontogeny. Development 1998; 125: 4575–83. Orkin SH, Zon LI. Hematopoiesis and stem cells: plasticity versus developmental heterogeneity. Nat Immunol 2002; 3: 323–8. Kennedy M, Firpo M, Choi K et al. A common precursor for primitive erythropoiesis and definitive haematopoiesis. Nature 1997; 386: 488–93. Nishikawa SI, Nishikawa S, Hirashima M, Matsuyoshi N, Kodama H. Progressive lineage analysis by cell sorting and culture identifies FLK1+VE–cadherin+ cells at a diverging point of

169.

170.

171. 172.

173.

174.

175.

176.

177.

178.

179.

180.

181.

182.

183.

184.

185.

186.

endothelial and hemopoietic lineages. Development 1998; 125: 1747–57. Yao H, Liu B, Wang X et al. Identification of high proliferative potential precursors with hemangioblastic activity in the mouse AGM region. Stem Cells 2007; 25: 1423–30. Tavian M, Coulombel L, Luton D, Clemente HS, Dieterlen-Lievre F, Peault B. Aorta-associated CD34+ hematopoietic cells in the early human embryo. Blood 1996; 87: 67–72. Keller GM. In vitro differentiation of embryonic stem cells. Curr Opin Cell Biol 1995; 7: 862–9. Shi Q, Rafii S, Wu MH et al. Evidence for circulating bone marrow-derived endothelial cells. Blood 1998; 92: 362–7. Lin Y, Weisdorf DJ, Solovey A, Hebbel RP. Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest 2000; 105: 71–7. Grant MB, May WS, Caballero S et al. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med 2002; 8: 607–12. Yoder MC, Mead LE, Prater D et al. Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals. Blood 2007; 109: 1801–9. Reyes M, Lund T, Lenvik T, Aguiar D, Koodie L, Verfaillie CM. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 2001; 98: 2615–25. Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, Verfaillie CM. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest 2002; 109: 337–46. Schwartz RE, Reyes M, Koodie L et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 2002; 109: 1291–302. Qi H, Aguiar DJ, Williams SM, La Pean A, Pan W, Verfaillie CM. Identification of genes responsible for osteoblast differentiation from human mesodermal progenitor cells. Proc Natl Acad Sci U S A 2003; 100: 3305–10. Jiang Y, Henderson D, Blackstad M, Chen A, Miller RF, Verfaillie CM. Neuroectodermal differentiation from mouse multipotent adult progenitor cells. Proc Natl Acad Sci U S A 2003; 100(Suppl 1): 11854–60. Muguruma Y, Reyes M, Nakamura Y et al. In vivo and in vitro differentiation of myocytes from human bone marrow-derived multipotent progenitor cells. Exp Hematol 2003; 31: 1323– 30. Aranguren XL, Luttun A, Clavel C et al. In vitro and in vivo arterial differentiation of human multipotent adult progenitor cells. Blood 2007; 109: 2634–42. Zeng L, Rahrmann E, Hu Q et al. Multipotent adult progenitor cells from swine bone marrow. Stem Cells 2006; 24: 2355–66. Ross JJ, Hong Z, Willenbring B et al. Cytokineinduced differentiation of multipotent adult progenitor cells into functional smooth muscle cells. J Clin Invest 2006; 116: 3139–49. Serafini M, Dylla SJ, Oki M et al. Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells. J Exp Med 2007; 204: 129–39. D’Ippolito G, Diabira S, Howard GA, Menei P, Roos BA, Schiller PC. Marrow-isolated

Cellular Biology of Hematopoiesis adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential. J Cell Sci 2004; 117: 2971– 81. 187. Kogler G, Sensken S, Airey JA et al. A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J Exp Med 2004; 200: 123–35.

188. De Coppi P, Bartsch G, Siddiqui MM et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 2007; 25: 100– 6. 189. Guan K, Nayernia K, Maier LS et al. Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 2006; 440: 1199–1203. 190. Kucia M, Halasa M, Wysoczynski M et al. Morphological and molecular characterization of

87

novel population of CXCR4+ SSEA-4+ Oct-4+ very small embryonic-like cells purified from human cord blood – preliminary report. Leukemia 2006; 21: 297–303. 191. Smith A. A glossary for stem-cell biology. Nature 2006; 441: 1060. 192. Verfaillie CM. Adult stem cells: assessing the case for pluripotency. Trends Cell Biol 2002; 12: 502–8.

8

Colleen Delaney & Irwin Bernstein

Expansion of Hematopoietic Stem Cells

Introduction Hematopoietic stem cells (HSCs) are useful in a variety of clinical settings and are routinely available in sufficient numbers from a variety of sources, including bone marrow and mobilized peripheral blood. However, there are instances when the number of available HSCs is insufficient for adequate and/or timely engraftment of the host, in particular when cord blood is used as the source of HSCs for transplantation or in the case of gene therapy, where the frequency of transduced cells is too low. One potential solution to the problem of low stem cell numbers is ex vivo proliferation of the cells prior to transplantation. Extensive research has been done to define the optimal conditions necessary for ex vivo expansion of HSCs, and various expansion techniques have been developed for this purpose [1–7]. However, while there is still a lack of convincing data that pluripotent stem cells increase in number and retain an ability to engraft and sustain multilineage hematopoiesis in a myeloablated recipient following ex vivo expansion, more recent advances in the field, particularly with respect to molecular mechanisms that control HSC amplification, make this goal more realistic. In order to maintain hematopoietic function throughout the life of the organism, pluripotent HSCs are thought to undergo self-renewal, where at least one, possibly identical, progeny remains undifferentiated and capable of long-term, multilineage hematopoietic repopulation, thus preserving the stem cell pool [8]. The differentiated progeny of longterm repopulating cells are thought to include pluripotent, short-term repopulating cells, which in turn generate precursors committed to lymphoid or myeloid differentiation [9,10]. Whereas maintenance of the HSC pool in vivo requires asymmetric division in which only one of the progeny maintains stem cell properties, HSC expansion requires symmetric divisions giving rise to two HSCs (Fig. 8.1). The ability of HSCs to undergo symmetric divisions and expand in numbers has been demonstrated in transplantation studies. However, serial transplant studies have also demonstrated that HSC life span is limited and self-renewal potential diminishes with the age of the animal [11–13]. While several recent studies challenge our concepts of HSC senescence [14–16], the central tenet remains that, in the absence of transforming events, HSCs have finite, though very lengthy, life spans in vivo [17–19]. These “aging” or senescence processes appear to be accelerated in ex vivo

cultures [20]. Nevertheless, studies suggest that HSC progeny formed after several divisions retain the ability to engraft recipients long term, but may actually be somewhat more differentiated with less proliferative capacity that is detectable only under stress conditions. Thus, cells with HSC properties may themselves be heterogeneous, and their self-renewal capacity may depend on the source from which they are derived, with fetal liver HSCs having greater self-renewal capacity than cord blood HSCs, which in turn have greater self-renewal capacity than cells from adult bone marrow [16,21–23]. Substantial effort has focused on the exogenous signals that may be used to favor stem cell self-renewal versus differentiation in order to develop optimal conditions for the ex vivo expansion of stem cells. Most studies have evaluated using combinations of cytokines and/or bone marrow stroma and have met with limited success (reviewed in [3,6,24,25]). The effect of cytokines that support hematopoietic cell survival, proliferation, and differentiation has been extensively studied in vitro, but a significant role for these cytokines in enhancing selfrenewal has not been shown. Consequently, a stochastic model of cellular determination has been suggested in which the fate of hematopoietic precursors is not instructed by soluble cytokines [26,27], but rather by specific interactions between stem and other cells within a particular microenvironment or “stem cell niche.” These interactions are mediated by extrinsic regulators of stem cell fate and are likely to play a key role maintaining numbers of stem cells by regulating their self-renewal and differentiation, now demonstrated by the work of several groups [28–30]. Thus, more recent studies are aimed at identifying intrinsic and extrinsic factors that regulate HSC fate. Clinical trials, which have also mainly evaluated cytokine-driven expansion systems, have not yet provided evidence for stem cell expansion, but have demonstrated the feasibility and safety of ex vivo culturing of stem cells. However, a newer generation of clinical trials, as discussed below, is currently underway evaluating the use of extrinsic regulators of stem cell fate (Notch) and co-culture systems utilizing nonhematopoietic components (mesenchymal stromal cells) of the stem cell niche. Here, we address studies of ex vivo HSC expansion in animals and humans, as well as promising approaches under development.

Preclinical studies: growth factor and stroma-induced expansion Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

88

In this section, we review work in mice and large-animal models designed to address the ex vivo expansion of HSCs. We will focus on studies that have used in vivo repopulating assays to evaluate stem cell

Expansion of Hematopoietic Stem Cells

HSC

HSC HSC

HSC STR

HSC

HSC

HSC

HSC

STR STR

HSC

STR

STR HSC

Symmetric division

STR Asymmetric division

Fig. 8.1 Maintenance versus expansion of hematopoietic stem cell (HSC) numbers. Symmetric divisions will expand stem cell numbers, whereas asymmetric divisions maintain numbers. The number of HSCs generated may also be affected by cell loss due to apoptosis and, for transplantable stem cells, loss of homing properties. STR, short-term repopulating cell.

expansion as in vitro assays have not always proven informative of HSC function. We then briefly review factors that regulate HSC fate in ex vivo expansion cultures. Murine studies The discovery of transplantable HSCs in long-term cultures of mouse marrow cells (LTMCs) initially described by Dexter [31–33] suggested that HSCs might possibly expand in ex vivo cultures. The HSCs in these LTMCs were dependent on the stromal cells for survival, and their cell cycle status was influenced both positively and negatively by growth factors [34,35], as well as by cellular and noncellular components of the stromal cell layers [36]. However, it was unclear whether HSCs in LTMCs divided or simply remained quiescent. Subsequent studies using gene marking to follow the progeny of individual HSCs demonstrated that HSCs were able to undergo several divisions and still maintain HSC function measured in vivo [37,38]. Evidence for a limited number of in vitro divisions before loss of HSC function also came from studies demonstrating that the progeny of isolated HSCs, formed after one to three in vitro divisions, maintained in vivo repopulating function [39]. However, none of these and other studies [40] demonstrated net gains of HSCs number in these cultures, indicating that divisions were mainly asymmetric. HSC cultured for purposes of ex vivo expansion are altered in obvious as well as subtle ways, including the expression of various cell surface antigens such as receptors that function in cell adhesion and motility. Research is ongoing to better define HSC markers on freshly isolated and subsequently cultured HSCs [41]. Moreover, the impact of stem cell death, loss of homing properties [42,43], and change of cell-cycle status [44–46] that occur during in vitro culture of HSCs and how these influence in vivo repopulating ability have not yet been fully elucidated. For example, both mouse HSCs and human hematopoietic precursors appear to have diminished repopulating function if transplanted during the G1 and S phases of the cell cycle rather than in G0 phase. The combination of cytokines is also critical, with specific hematopoietic growth factors involved in promoting or inhibiting HSC function during ex vivo expansion. Elegant single-cell manipulation studies have demonstrated that interleukin-3 (IL-3) and IL-1 can abrogate the selfrenewal of murine HSCs in vitro [47], in contrast to stem cell factor

89

(SCF) and Flt3 ligand (Flt3L), which maintain HSCs [48]. Further, the addition of exogenous thrombopoietin (TPO) to murine LTMCs led to an expansion of marrow repopulating cells [49]. Members of the transforming growth factor (TGF) family and cooperating factors have been demonstrated to inhibit the cell-cycle progression of HSCs in vitro [50–52]. The type of culture medium used also can influence the ex vivo expansion of HSCs. Whereas numerous studies using serum-containing medium have shown maintenance or loss of HSC activity following culture with cytokines, more recent studies using serum-free medium, which allows for a more careful dissection of the interaction of HSCs with defined ligands and avoids the introduction of inhibitory molecules such as TGF-β, have demonstrated enhanced repopulating ability, with some studies documenting up to an eightfold increase of murine marrow HSCs [41].

Large-animal studies To date, human trials of gene therapy and HSC manipulation have not been nearly as successful as experiments in mice might have predicted, and important differences in HSC behavior in vivo in large animals are beginning to be appreciated [53–55]. Thus, studies of stem cell expansion using clinically feasible procedures in larger animals to better mimic human physiology that are now being carried out may provide important insights needed for human clinical application. Thus far, the use of ex vivo cultured cells has mainly led to an enhanced rate of engraftment, but this may have occurred at the expense of long-term repopulating cells. The possibility of enhancing short-term repopulating activity by ex vivo expansion was suggested by two studies of lethally irradiated baboons engrafted with ex vivo, expanded CD34enriched peripheral blood progenitor cells (PBPCs) collected after granulocyte colony-simulating factor (G-CSF) mobilization [56–58]. Significant expansion of precursor cells with in vitro colony-forming activities was documented in both studies, and in one study severe combined immunodeficiency disease (SCID) mouse repopulating cells were also expanded twofold. Animals transplanted with ex vivo expanded cells experienced significantly shorter periods of post-transplant neutropenia, but the recovery of platelet and red cell production was significantly slower, and administration of TPO had no effect on platelet recovery. Although these studies were not designed to assess effects on long-term reconstituting stem cells, the possibility that cytokine-driven expansion may deplete long-term repopulating HSCs in primate models has been suggested by gene-marking studies, in which prolonging the period of in vitro culture worsened rather than improved gene transfer efficiency [59,60]. An increase in the number of precursors at the expense of in vivo marrow reconstituting function has also been observed in cats following ex vivo expansion of marrow cells [61]. In dogs, HSCs and progenitors have been cultured ex vivo for brief periods to promote gene transfer into marrow repopulating cells. The detection of gene-marked progeny in vivo suggests that at least a limited number of cells with HSC function divided in the ex vivo culture [62].

Xenogeneic animal models: transplantation of human cells Immunodeficient mouse xenogeneic transplant models In preclinical studies, xenogeneic transplant models have been developed in which human hematopoietic cells can engraft in immunodeficient mice and then be serially passaged in vivo [21,22,63–69]. These engrafting cells are referred to as SCID repopulating cells (SRCs). At

90

Chapter 8

present, the relationship of SRCs to HSCs in humans remains to be clarified, but the SRC is generally accepted as the most reliable surrogate marker of an HSC currently available. Nevertheless, human SRC expansion has been assayed in various immune-deficient mouse strains. The necessity for in vivo assessment of ex vivo HSC expansion was emphasized by several studies in which ex vivo cultures of CD34+ cells have resulted in the dramatic increase in numbers of colony-forming cell (CFC) or even long-term culture-initiating cell activity, but with a concomitant decrease in SRCs [70–73]. In contrast, several other studies have suggested a modest increase in engraftment of immunodeficient mice following short-term culture, in which cells were cultured with multiple cytokines or in the presence of stromal cells or stromal cellconditioned medium [6,74–80]. Quantitative studies using limiting dilution SRC assays have confirmed modest two- to fourfold cytokine-induced increases in SRCs. For example, Bhatia et al. and Conneally et al. have cultured CD34+ CD38− cord blood cells in serum-free medium supplemented with SCF, Flt3L, G-CSF, IL-3, and IL-6 and showed expansion of SRCs [65,74]. The study by Bhatia et al., in which cells were cultured for 4 days, found a fourfold increase in CD34+ CD38− cord blood cells, a 10-fold increase in CFCs, and a two- to fourfold increase in SRCs [74] . However, all SRCs were lost after 9 days of culture, despite continued expansion in the total number of cells throughout the culture period. In a similar study, Conneally et al. confirmed that this culture system produced significant increases in CFCs (100-fold) and long-term culture-initiating cells (fourfold), as well as a modest twofold increase in SRCs. Similarly, studies using an IL-6/IL-6-receptor chimera induced significantly higher levels of CD34+CD38− cells and a fourfold increase in SRCs by limiting dilution methods [79,81–84]. Cytokine-induced expansion systems have also utilized several cloned stromal feeder cells and mesenchymal stem cell layers as sources of growth factors, and have been shown to support long-term repopulating cells [85,86]. However, because the cultured cells are in contact with the stromal feeder cells in this type of culture system, it is less advantageous to use this system for clinical application, and noncontact stromalbased culture systems are also being explored as HSC expansion systems [4,87–91]. An example of this type of expansion system was reported by Shih and colleagues [89], in which a murine bone marrow-derived stromal cell line, AC6.21, treated with leukemia inhibitory factor, secreted a factor that resulted in expansion of human HSCs when the HSCs were cultured with the conditioned medium. The expansion occurred in the absence of the stromal cells, and the magnitude of expansion was dependent on the level of activity of the secreted factor. In another example, Lewis et al. cultured umbilical cord blood (UCB) CD34+ with IL-7, SCF, Flt3L, and TPO in a Transwell (where stromal cells are separated from CD34+ cells by a transmembrane) above the murine fetal liver cell stromal feeder line, AFT024, for 7 days and demonstrated the maintenance of long-term repopulating cells in culture using secondary transplants into NOD/SCID mice, as well as secondary and tertiary transplants into fetal sheep [92]. Additionally, the use of conditioned medium from the HS-5 cell line, an immortalized stromal cell line derived from human marrow after transduction with a replication-defective retrovirus containing the human papilloma virus E6/E7 gene, has been shown to support the proliferation of hematopoietic progenitors and maintain CFCs for up to 8 weeks in culture [88,93]. While the above studies documented the expansion of SRCs in shortterm culture conditions only, Piacibello et al. [94] reported maintenance of cord blood CD34+ cell growth for up to 12 weeks in serum-containing medium supplemented with Flt3L, SCF, and IL-6. Additionally, limiting dilution transplants demonstrated a more than 70-fold expansion of SRCs. Although it is not clear whether this approach will be reproducible, Tanavde et al. recently reported the ability to expand and maintain

UCB SRCs for up to 4 weeks [95] using CD34+ from UCB and adult mobilized PBPCs cultured in serum-free medium supplemented with Flt3L, SCF, and TPO with or without IL-6/IL-6-receptor chimera. UCB cells demonstrated extensive in vitro expansion and maintained repopulating ability throughout the 4-week culture period, whereas mobilized PBPCs showed less expansion and no engrafting potential of cultured cells (only fresh cells being engrafted). While the ability to maintain and expand repopulating cells in vitro for extended periods would be beneficial for gene therapy, augmentation of clinical transplantation studies with expanded cells will require shorter expansion times, as time to transplant is critical in the clinical setting. Moreover, there is always the concern that long-term culture of cells may in fact induce transformation of the cultured cells. Fetal sheep xenogeneic transplant model When primitive hematopoietic cells isolated from both cord blood and adult bone marrow are expanded ex vivo and transplanted into fetal sheep, only short-term engraftment of the animals is seen. In their initial study attempting ex vivo expansion of enriched adult human bone marrow cells with in vivo engrafting capabilities, Shimizu and colleagues were able to document the maintenance of cells with engrafting capabilities in fetal sheep for up to 2 months when cultured with KIT ligand, Flt3L, IL-6, and erythropoietin with or without IL-3 [96]. Engraftment was noted in secondary recipients of fresh human cells, but not in recipients of cultured cells, suggesting that the culture conditions used were unable to support or expand long-term engrafting cells. Similar results were demonstrated by McNiece et al. using a two-step culture procedure in which cord blood progenitors cultured in the presence of SCF, megakaryocyte growth and development factor, and GCSF for 7 or 14 days prior to transplant provided no long-term engraftment of fetal sheep [97]. While the expanded cells were capable of early rapid engraftment (threefold over fresh cells), they lacked secondary and tertiary engrafting potential compared with fresh cells, again suggesting that the culture conditions described resulted in the loss of long-term repopulating cells and led to an increase of more mature shortterm repopulating cells. Overall, preclinical studies of human HSC expansion assayed based on repopulating ability in xenogeneic transplant models show promise but must be interpreted with caution, as human NOD/SCID or fetal sheep repopulating ability has not been correlated with non-human primate or human in vivo repopulating ability. In fact, studies that compared repopulation by gene-marked cells from non-human primates in autologous recipients and NOD/SCID mice revealed substantially greater repopulation in mice [98]. Although this finding might reflect detection in mice of a more mature precursor, such as a short-term repopulating cell, other studies have demonstrated serial repopulation by expanded human cells in murine recipients, a function consistent with self-renewing stem cells. Further, while these studies have shown in vitro expansion of SRCs, the results are highly variable, and include numerous reports of loss of stem cell activity after ex vivo culture [73,97,99–101]. Finally, while studies of cytokine-induced expansion suggest the potential for enhancement of short- and long-term repopulating activity, only a few cell divisions at most have been observed. Thus, current efforts are now focused on identifying and exploiting other extrinsic and intrinsic regulators of stem cell fate. Novel approaches for ex vivo HSC expansion using extrinsic and intrinsic regulators of stem cell fate Optimization of cytokine-driven expansion systems has not led to clinically significant HSC expansion, perhaps due to a predominantly permissive, rather than directed, role in determining stem cell fate. The

Expansion of Hematopoietic Stem Cells

search for extrinsic and intrinsic regulators that act directly on human HSCs in regulating cell fate and self-renewal has suggested a role for regulatory molecules active in early development that are important in HSC maintenance and regulation. More recent studies have focused on the regulation of intrinsic signaling pathways by retrovirus-mediated transduction of HSCs, for example expression of homeobox genes. However, culture of cells for clinical application requires the use of extrinsic regulators of cell development including bone morphogenic proteins (BMPs), angiopoietin-like proteins, and Sonic hedgehog (Shh), Wnt, and Notch ligands. Retrovirally mediated overexpression of Hox transcription factors, in particular HoxB4, has led to extensive ex vivo HSC expansion in vitro (3 logs over control cultures) without loss of full in vivo lymphomyeloid repopulating ability [102–105]. Although methods to alter homeobox gene expression in the absence of transducing cells are not available, the self-renewal induced by HoxB4 has suggested the exploration of extrinsic regulators of cell fate involved in embryonic development, such as BMP-4, a member of the TGF-β superfamily, and Shh of the Hedgehog family of proteins, both having been implicated in early hematopoietic development [106,107]. In ex vivo expansion of human cord blood, soluble human BMP-4 has been shown to increase the survival of repopulating blood cells in ex vivo culture [106], while Shh has been shown to induce a few-fold increase in repopulating cells via mechanisms that are dependent on downstream BMP signals [108]. Wnt proteins, which are involved in the growth and differentiation of a variety of primitive tissues, have also been implicated in the regulation of hematopoiesis, possibly exerting their effects through stromal cells [109], and have been shown to stimulate the proliferation of hematopoietic precursor cells [110]. There are also data supporting the presence of an interactive relationship between the various signaling pathways. For example, Notch-1 receptors have been shown to be upregulated in response to Wnt signaling in HSCs [111]. The most extensively studied and successfully utilized extrinsic regulators have been ligands that activate the Notch pathway, with growing data supporting a role of Notch signaling in maintenance and/or self-renewal of HSCs. All four Notch receptors (Notch-1, -2, -3, and -4) identified in vertebrates have been detected in hematopoietic cells [112], and several investigators have reported the expression of Notch-1 and Notch-2 in human CD34+ or CD34+ lineage-negative (Lin−) precursors [112–114], and of the Notch ligands Delta-1 and Jagged-1 in human bone marrow stromal cells and human hematopoietic precursors [115– 118] . Moreover, expression of a constitutively active, truncated form of Notch-1 in murine hematopoietic precursors inhibited differentiation and enhanced self-renewal, leading to the establishment of an immortal cell line that phenotypically resembles primitive hematopoietic precursors [119]. This cell line, depending upon the cytokine context, can differentiate along the lymphoid or myeloid lineage. More recently, expression of constitutively active Notch in murine precursors transplanted in vivo led to an increased stem cell number that was evident in secondary transplantation studies [120]. While these findings point to a role for Notch signaling in the regulation of stem cell self-renewal, expression of activated Notch-1 in human CD34+ cord blood precursors induced only a modest increase in the number of progenitors [121]. In order to affect nontransduced cells, exogenous Notch ligands have been used to induce endogenous Notch signaling in HSCs. Initial studies in mice and humans using soluble or cell-bound Notch ligand revealed limited increases in precursor cell numbers [116,117,121]. However, Varnum-Finney et al. and others [122,123] have demonstrated a requirement for ligand immobilization to induce Notch signaling. Studies performed with hematopoietic precursors cultured in the presence of immobilized Delta-1 demonstrated profound effects on the differentiation of isolated murine marrow precursors, with a multi-log increase in

91

the number of stem cell antigen (Sca-1)+ Gr-1− cells with short-term lymphoid and myeloid repopulating ability [124]. Similarly, studies with human cord blood CD34+CD38− precursors cultured in serum-free conditions with immobilized ligand and cytokines resulted in an approximately 100–200-fold increase in the number of CD34+ cells generated compared with control cultures, and included cells capable of repopulating immunodeficient mice [125,126]. Moreover, more recent studies with both murine and human hematopoietic progenitors have demonstrated density-dependent effects of Delta-1 on fate decisions of hematopoietic progenitors, and suggest that optimal ligand densities are required to promote expansion of cells that retain repopulating ability [125,127]. Other investigators have shown similar outcomes. Kertesz et al. demonstrated that culture of murine Lin− hematopoietic progenitor cells with immobilized Jagged-1 resulted in expansion of serially transplantable HSCs, with superior expansion dependent on the combinatorial effect of Notch and cytokine-induced signaling pathways [128]. These data indicate that manipulation of Notch signaling can enhance stem cell self-renewal ex vivo and thereby increase HSC numbers for transplantation. Despite limited evidence of ex vivo HSC expansion with current methodologies, including stroma and stroma-free systems, clinical trials have been conducted with autologous marrow, cytokine-mobilized peripheral blood cells or cord blood cells, and their ability to shorten engraftment periods has been determined. Stroma-free systems provide a clear advantage for clinical use, since they are easier to standardize and maintain for cyclic guanosine monophosphate. However, some studies have used noncontact systems in which stromal cells serve to condition the medium. These trials will be discussed in the next section.

Clinical trials of ex vivo expanded HSCs The first report of a clinical trial using in vitro cultured hematopoietic progenitor cells appeared in 1992. Since then, a number of trials have been carried out with the primary goal of enhancing hematopoietic recovery after high-dose chemotherapy with the infusion of expanded stem cell grafts derived from bone marrow, mobilized peripheral blood or cord blood. Clinical trials using bone marrow and mobilized peripheral blood Table 8.1 summarizes select trials with autologous bone marrow or mobilized PBPCs cultured ex vivo with cytokines and/or stromal elements [129–138]. Studies of expanded mobilized PBPCs were all done in the autologous setting, and included only those patients with sufficient stem cell harvests to allow the use of an aliquot of the apheresis product for expansion. This excluded “poor mobilizers” who clearly stand to benefit from expansion procedures allowing them to undergo treatment with high-dose therapy. However, it is not known whether HSCs derived from such patients are equivalent to those derived from patients with adequate mobilization of HSCs. These trials, primarily in breast cancer patients, assessed enhanced short-term repopulation as a function of decreased time to neutrophil and/or platelet engraftment. With one exception, these studies were unable to address the expansion of longterm repopulating HSCs because patients received nonmyeloablative chemotherapy and/or infusion of noncultured cells. Overall, although the safety and feasibility of stem cell expansion approaches were demonstrated, more rapid engraftment was not observed in patients receiving reduced-intensity preparative regimens. There have, however, been suggestions of ex vivo expansion of short-term repopulating cells in patients who have received myeloablative therapy.

92

Chapter 8

Table 8.1 Selected bone marrow and mobilized peripheral blood clinical expansion trials

Author/ reference

Conditioning regimen

Infusion expanded cells

Patients

Stem cell source

Cytokines/serum

Notes

Williams et al. (1996) [138]

n=9 Metastatic breast cancer

MPBPC

PIXY321, human serum albumin 1% × 12 days

NM1

Day +1

↑ TNC 26-fold CD15+ post expansion average 29%

Alcorn et al. (1996) [129]

n = 10 Nonmyeloid malignant

MPBPC

SCF, IL-1β, IL-3, IL-6, EPO + autologous plasma × 8 days

M/NM2

Day 0

↑ TNC 21-fold, CFU-GM 139-fold, BFU-E 114-fold (mean) No change in engraftment compared with controls

Bertolini et al. (1997) [131]

n = 10 8 breast cancer, 2 NHL

MPBPC

MGDF, SCF, IL-3, IL-6, IL-11, Flt3L, MIP-1α × 7 days

NM3

Day 0

Study aimed at looking at generating megakaryocytic progenitors

Holyoake et al. (1997) [133]

n=4 2 NHL, 2 MM

MPBPC Expanded cells only

SCF, IL-1β, IL-3, IL-6, EPO + autologous plasma × 8 days

M4

Day 0

No long-term engraftment seen ↑ TNC 8–27-fold, CFU-GM 17–130-fold

Reiffers et al. (1999) [130]

n = 14 MM

MPBPC

SCF, G-CSF, MGDF

M5

Day 0

↑ TNC 34.4-fold, CD34+ cells 2.6-fold, CFU-GM 14.1-fold (median)

McNiece et al. (2000) [134]

2 cohorts 1) n = 10 2) n = 11 Breast CA

MPBPC Cohort 2 – expanded cells only

SCF, G-CSF, MGDF ×10 days

NM6

Day 0

Cohort 1:

Stiff et al. (2000) [137]

n = 19 Breast cancer

BM (~75 mL) – expanded cells only

AastromReplicell Bioreactor Flt3L, EPO, PIXY321, 10% FBS, 10% horse serum × 12 days

NM1

Day 0

TNC 4.8-fold, CFU-GM 4.2-fold, CD34+Lin− cells 0.9-fold, LTC-IC 1.2fold (median) Engraftment times similar to autologous bone marrow transplantation Correlation: cell dose and engraftment

Pecora et al. (2001) [136]

n = 34 Breast cancer

BM (50–100 ml) for expansion + low-dose MPBPC

AastromReplicell Bioreactor Flt3L, EPO, PIXY321 (+TPO in seven cases), 10% FBS, 10% horse serum × 12 days

NM1

Day 0

CD34+Lin− cell number and quantity of stromal progenitors contained in the expanded product correlated with engraftment outcome

Engelhardt et al. (2001) [132]

n = 10 Breast cancer

BM (median 97 mL) – expanded cells only 2 patients: irradiated PBPCs

AastromReplicell Bioreactor Flt3L, EPO, PIXY321, 10% FBS, 10% horse serum × 12 days

NM1

Day 0

↑ TNC 4.5-fold, CFU-GM 18-fold (median) Correlation: CD34+Lin− cells and engraftment

Paquette et al. (2002) [135]

n = 43 Breast cancer

MPBPC

GCSF, SCF, MGDF, 1% human albumin, transferrin × 9–14 days

NM7

Day 0

Varied duration of cultures and starting cell density

↑ TNC 20-fold (median) ↑ CD34+ cells 1.8-fold Cohort 2: ↑ TNC 14-fold (median) ↑ CD34+ cells 2.0-fold TNC/kg post expansion best predictor of time to neutrophil engraftment

BFU-E, burst-forming unit-erythroid; BM, bone marrow; CFU-GM, colony-forming unit-granulocyte–macrophage; EPO, erythropoietin; FBS, fetal bovine syndrome; LTC-IC, longterm culture-initiating cells; M, myeloablative; MGDF, megakaryocyte growth and development factor; MIP, macrophage inhibitory protein; MPBPC, mobilized peripheral blood progenitor cells; MM, multiple myeloma; NHL, non-Hodgkin’s lymphoma; NM, nonmyeloablative; TNC, total nucleated cells. See text for other abbreviations. 1 Cyclophosphamide/carbo/thiotepa (STAMP V). 2 Melphalan/total body irradiation, BEAM, thiotepa/cyclophosphamide, carboplatin/etoposide/melphalan, cyclophosphamide/TBI. 3 Thiotepa/melphalan (breast cancer); mitoxantrone/melphalan (NHL). 4 Cyclophosphamide/total body irradiation (NHL) and busulfan/melphalan (MM). 5 Melphalan ± total body irradiation. 6 Cyclophosphamide/cisplatin/BCNU or taxol/cyclophosphamide/cisplatin or taxotere/melphalan/carboplatin. 7 Cyclophosphamide/carmustine/cisplatin.

Expansion of Hematopoietic Stem Cells

For example, in a study by Holyoake et al. [133], the gain of short-term repopulating cells at the expense of long-term repopulating cells was suggested. In that study, mobilized PBPCs cultured ex vivo with SCF, IL-1β, IL-3, IL-6, erythropoietin, and autologous plasma for 8 days were infused in the absence of noncultured cells into patients that had received myeloablative chemotherapy. None of the four patients showed evidence of long-term hematopoietic recovery, and all required infusions of unmanipulated cryopreserved autologous back-up mobilized PBPCs, after which full hematopoietic recovery was seen. Three patients showed initial neutrophil engraftment that was unsustained, suggesting that short-term repopulating cells may have been generated ex vivo at the expense of long-term repopulating ones, possibly by inducing their differentiation. A shortening of the neutropenic period following high-dose chemotherapy in 21 patients with breast cancer was also shown in a study in which autologous mobilized PBPCs were cultured with SCF, G-CSF, and megakaryocyte growth and development factor for 10 days. The patients in this study were divided into two cohorts: those who received expanded cells only, and those who received both expanded and unmanipulated cells in tandem [134]. Expanded PBPCs resulted in a more rapid neutrophil engraftment (p = 0.02 for cohorts 1 and 2 versus the historical controls), the best predictor of time to neutrophil engraftment was the total number of cells harvested after expansion, and patients receiving more than 4 × 107 cells/kg engrafted by day 8. Reiffers et al. also reported a decreased time to neutrophil engraftment using the same culture conditions to expand autologous mobilized PBPCs for infusion with unmanipulated cells into patients with multiple myeloma after high-dose conditioning. The use of continuous perfusion methods for the expansion of bone marrow has recently been evaluated in three trials utilizing the automated perfusion bioreactor system AastromReplicell [132,136,137]. Notably, these are the only trials that used bone marrow (unselected for CD34+ cells) as the source of stem cells for ex vivo expansion, thereby perhaps including accessory or stromal cells of unclear significance in expansion cultures, and perhaps enhancing the engraftment ability of this expanded cell population. In these studies, breast cancer patients underwent the same conditioning regimen, and cells derived from autologous bone marrow were placed directly into the automated perfusion bioreactor system for expansion with Flt3L, erythropoietin, and PIXY321 cytokines, as well as fetal bovine serum and horse serum. In all trials, cultures were initiated with small amounts of harvested marrow (75– 100 mL). The trials by Stiff et al. and Engelhardt et al. used expanded cells only, while Pecora et al. also infused mobilized PBPCs in low doses. Engraftment was not enhanced but did occur, suggesting the maintenance of HSCs in the bone marrow cultures, and all trials reported a correlation between the time to neutrophil and platelet engraftment with CD34+Lin− cell dose/kg. Stiff et al. reported that only 2 × 105 CD34+ cells/kg of the expanded cells were needed to produce optimal platelet engraftment. This cell dose was lower than the number of cells required for predictable platelet engraftment by day 28 in breast cancer patients undergoing PBPC transplantation with unmanipulated PBPCs, in which the minimal number of CD34+ cells required was reported to be 2–5 × 106/kg [139]. Clinical trials using UCB cells Rationale for use of UCB stem cells for ex vivo expansion With greater than 6000 UCB transplants performed to date since 1988 [140], UCB has emerged as an alternative source of HSC for transplantation. This is especially important for minority patients and patients of mixed ethnicity, for whom UCB is a particularly attractive alternative

93

donor stem cell source as it is readily available with no donor attrition and allows reduced stringency in human leukocyte antigen (HLA) matching without an increase in graft-versus-host disease (GVHD). Numerous clinical studies have consistently shown that the total nucleated cell and CD34+ cell doses in cord blood grafts are highly correlated with the rate of neutrophil and platelet engraftment, as well as the incidence of graft failure and early transplant-related complication [141– 149]. Based on these studies, critical cell dose thresholds have been established, and outcomes for patients receiving less than the generally accepted threshold of more than 2.5 × 107 total nucleated cells/kg are significantly inferior in terms of engraftment, transplant-related mortality and overall survival. For patients weighing greater than 35–40 kg, obtaining an adequate cell dose from a single UCB unit may be an impossible hurdle. Delayed engraftment has also been correlated with HLA mismatch at more than two loci, but recent studies have suggested that the impact of HLA disparity on survival can be partially overcome by increasing cell dose [148,150]. Efforts to overcome the obstacle of small cell numbers in UCB grafts by ex vivo cytokine-mediated expansion have not yet demonstrated improved time to engraftment. Infusion of multiple cord blood units as an alternative approach to increase cell numbers is also under investigation. The initial results from this approach are encouraging and have demonstrated the safety of this approach in adults, with improved engraftment and decreased transplant-related mortality, but the significant delay in engraftment has not been abrogated in these patients [151]. Thus, the collective data point to improved outcome following UCB transplantation in patients receiving a higher cell dose, in terms of both CD34+ and total nucleated cell doses, an observation leading to the hypotheses that ex vivo expansion of CD34+ cell numbers or the infusion of more than one unit of cord blood might improve the rate of cord blood engraftment and overall survival. Several lines of evidence suggest that UCB contains a higher frequency of primitive hematopoietic progenitor cells and early committed progenitors than adult bone marrow or peripheral blood [21,68]. There is also increasing evidence that the stem cells isolated from UCB survive longer in culture [95] and may be less mature and have greater proliferative capacity [78,152–154]. Phenotypic analyses of UCB have shown that the more primitive cell population which expresses the CD34 antigen, but not the CD38 antigen, is fourfold more prevalent than in bone marrow or PBPCs, and that this subpopulation has a higher in vitro cloning efficiency than the same population isolated from adult bone marrow [155]. These findings correlate with data from studies using in vitro with bone marrow or PBPCs [156–158]. Second, there are numerous reports of the increased proliferative potential of UCB cells in response to cytokine stimulation. For example, using IL-11, SCF, and G-CSF or granulocyte–macrophage colonystimulating factor, Cairo and colleagues demonstrated an 80-fold increase after a 14-day expansion of UCB versus adult bone marrow [159]. Moreover, compared with adult bone marrow, UCB has been shown to have increased serial in vitro replating efficiency [153] and increased culture life span with increased progenitor cell production [94,156]. Finally, in vivo assays of UCB versus bone marrow have shown that HSCs from UCB, but not adult bone marrow, can engraft NOD/SCID mice without the use of exogenous cytokines [67]. More recently, Rosler et al. showed that expanded cord blood had a competitive repopulating advantage compared with expanded adult bone marrow using an in vivo assay with NOD/SCID mice [22]. A potential contribution to the differences observed between UCB and adult HSCs may arise from the differential response of HSCs from different sources in cytokine-driven expansion systems, leading to variations in cell-cycle status and homing ability of the expanded cells. Other possible sources of stem cells may have even greater proliferative

94

Chapter 8

potential. For example, murine fetal liver cells have greater proliferation and repopulation potential than HSCs isolated from adult murine bone marrow or peripheral blood [160,161]. Overall, these results suggest that UCB progenitor cells are functionally superior to adult bone marrow, with greater proliferative potential and possibly greater self-renewal capacity. Thus, UCB may represent a more viable target for ex vivo stem cell expansion, a possibility that has led to several clinical studies on the ex vivo expansion of cord blood cells to augment conventional UCB transplantation. Clinical ex vivo expansion trials with UCB UCB expansion trials (summarized in Table 8.2) [162–166] were undertaken to determine whether the delayed engraftment associated with UCB transplantation could be overcome if a portion of the cells from the UCB unit were expanded ex vivo. Like the trials using PBPCs and bone marrow, these initial trials utilized cytokine-based expansion systems as well as newer automated perfusion systems. The choice of exogenous cytokines used for culture has varied, reflecting the still undefined optimal conditions for expansion of the stem/progenitor cell. In all of the initial studies, only a portion of the single cord blood graft was used for expansion, and the expanded cells were infused in addition to unmanipulated cells, as there is a lack of definitive data to suggest that cultured cells retain HSC properties and do not differentiate. There have been no adverse toxicities associated with infusion of the expanded cell product, nor has there been any change in engraftment kinetics. However, only two of the studies have enrolled more than a handful of patients, making it difficult to draw any definitive conclusions. More recently, novel culture systems for the ex vivo expansion of UCB progenitors have begun patient accrual, including a Notch-mediated ex vivo expansion trial at the Fred Hutchinson Cancer Research Center, a trial utilizing a copper-chelating agent and an alternative strategy of coculture of UCB cells with bone marrow mesenchymal stem cells. These will be discussed below. In one of the larger studies to date, Jaroscak et al. reported preliminary data from a phase I trial at Duke University Medical Center undertaken to assess augmentation of UCB transplantation with cells expanded ex vivo in the AastromReplicell Cell Production System [162]. This trial included 28 patients with both malignant and inherited disorders who were conditioned with one of three regimens (Table 8.2), depending on diagnosis. On day 0, a portion of a single unrelated UCB unit was expanded ex vivo in medium supplemented with fetal bovine serum, horse serum, and cytokines (erythropoietin, PIXY321, and Flt3L) for 12 days. The expanded cells were then infused to augment the conventional transplant on day 12 after transplantation. Although expansion of total nucleated cells and CFCs occurred in vitro in all cases, no increase in CD34+Lin− cell number was achieved. In vivo, significant effects on engraftment kinetics were not observed with median time to neutrophil engraftment (absolute neutrophil count [ANC] > 500/μL) of 22 (range 13–40) days. No adverse reactions were observed due to infusion of the cultured cells. This phase I trial was an important contribution to the concept and development of ex vivo expansion of UCB CD34+ cells for use in the clinical setting, since it demonstrates both the safety of reserving an aliquot of an already small cord blood unit for expansion and the feasibility of expansion in a clinical setting. It is also possible that, although the numbers of CD34+Lin- and CD3+ cells were not expanded with this culture system and may have explained the failure to improve engraftment, the delayed infusion (day 10–12 post transplant) of expanded cells may have masked or prevented the benefit of a more rapid engraftment. Newer-generation clinical cord blood expansion trials, discussed below, are currently underway based on a double cord blood model, in which one unit is infused unmanipulated on the day of transplant with a second unit that has been expanded ex vivo.

Shpall and colleagues are currently conducting a number of cord blood expansion trials at the MD Anderson Cancer Center: a cytokinemediated expansion trial and a triethylenephosphoramide (TEPA)-based ex vivo expansion trial. The cytokine-mediated expansion trial is the successor trial to an earlier published trial performed at the University of Colorado [165], with some important changes. In this currently accruing trial, patients are now being randomized to receive either two unmanipulated cord blood units or one unmanipulated unit and one ex vivo expanded unit following high-dose therapy, compared with the previous trial in which only a portion of a single cord blood unit was used for expansion. For those patients randomized to receive expanded cells, the unit identified for ex vivo expansion is thawed, CD133 selected, and placed into a cytokine-mediated expansion system 14 days prior to the day of transplant. Accrual on this trial continues, but analysis of the first cohort of 22 patients enrolled has been performed (Shpall, personal communication). The median time to neutrophil engraftment is 20 and 21 days for patients receiving a conventional unmanipulated double UCB transplant and those receiving an unmanipulated unit and ex vivo expanded unit, respectively. Overall, 30% of patients developed acute grade III–IV GVHD. Based on preclinical data in which Peled et al. demonstrated enhanced expansion of CD34+ UCB progenitors, an additional ex vivo clinical expansion trial is enrolling at the MD Anderson Cancer Center using the copper chelator TEPA (Shpall, personal communication) [167]. Similar to the cytokine-mediated expansion trial, patients with hematologic malignancies are eligible, but all patients in this trial must have identified a single unit of cord blood that has been cryopreserved in two fractions. The smaller of the two UCB fractions is CD133 selected, and expanded ex vivo in the presence of cytokines and TEPA starting 21 days prior to infusion. Ten patients with high-risk malignancies have been treated thus far. The average fold expansion of total nucleated cells placed in culture was 219 (range 2–616), and the median time to engraftment was 28 (range 16–46) days for neutrophils and 48 (range 27–96) days for platelets. Evidence of GVHD during the 180-day study period was found in five of the 10 evaluable patients. Three patients experienced acute GVHD, all grade II, with skin involvement, and two patients developed chronic GVHD, one chronic extensive and one mild limited chronic. One patient with acute GVHD persisted to chronic limited GVHD with skin involvement. All cases were resolved with steroid therapy. There were no incidences of engraftment failure or grades III–IV acute GVHD, and 100-day survival was 90%. Thirty percent of the patients are alive and free of disease at a median 18 months post transplant. At the Fred Hutchinson Cancer Research Center, a phase I trial assessing the potential efficacy of cord blood progenitors cultured in the presence of an engineered form of the Notch ligand Delta-1 in contributing to rapid early engraftment in patients undergoing a high-dose UCB transplant is currently accruing patients. As discussed above, there is accumulating evidence on the role of the Notch signaling pathway in hematopoietic cell fate decisions. This trial has accrued only three patients to date, and while conclusions cannot be made as to the efficacy of this approach, the early results are encouraging (Delaney, unpublished data). Three patients with acute myeloid leukemia have enrolled and received cord blood progenitors that were cultured with Delta-1 following infusion of a second noncultured and unrelated donor cord blood graft. Upon harvest of the cells at day 16 post culture initiation, there was an average fold expansion of CD34+ cells of 116 (±49) and an average fold expansion of total cell numbers of 565 (±179) (Fig. 8.2(a)). The infused CD34 cell dose (defined as the number of CD34+ cells/kg recipient body weight) derived from the ex vivo expanded unit ranged from 1 to 13 million CD34+ cells/kg. In the first two patients, early myeloid recovery was observed, occurring at a time when engraftment due to the infusion of two noncultured

Expansion of Hematopoietic Stem Cells

95

Table 8.2 Selected umbilical cord blood clinical expansion trials Author/ reference

Conditioning regimen

Infusion expanded cells

AastromReplicell system Epo, Pixy321, Flt3L × 12 days

CY/TBI or BU/ CY/ATG

Day +12

No significant change in neutrophil engraftment No infusional toxicities 2 deaths prior to engraftment

UCB – sibling 12.5% expanded + 87.5% unmanipulated

G-CSF, TPO, Flt3L, 10% autologous cord blood plasma × 10 days

TBI/thiotepa/CY

Day +1

ANC 310/μL by day +8 Event-free survival now greater than 1 year

n=2 CML (blast crisis and accelerated phase)

UCB (unrelated) 11%, 17% expanded + 89%, 83% unmanipulated cells

AastromReplicell system Epo, Pixy321, Flt3L × 12 days

(1) BU/ATG CY (2) CY/TBI/ATG

Day +12

No infusional toxicities Improved platelet engraftment No change in neutrophil engraftment Lin−, CD34+ cells decreased in one patient, increased 1.7-fold in other patient

Shpall et al. (2002) [165]

n = 37 (25 adult) Stratum A = 25 Stratum B = 12 (split unit) Dx: hematologic malignancy (34), breast cancer (3)

UCB (unrelated) Expanded fraction Cohort A = 40% Cohort B = 60% + unmanipulated cells

G-CSF, SCF, MGDF expanded in Teflon bags × 10 days

Adults: High-dose melphalan/ ATG/TBI Or High-dose melphalan/ ATG/BU Children: CY/TBI/ arabinoside C/ ATG

Stratum (A) Day +10 (B) Day 0

No infusional toxicities No change in engraftment seen No neutrophil engraftment failures

Jaroscak et al. (2003) [162]

n = 28 (26 children) Varied Dx: both malignancies and inherited disorders

UCB (unrelated, one sibling) Expanded + unmanipulated cells

AastromReplicell system PIXY321, Epo, Flt3L, 10% horse serum, 10% FBS × 12 days

Hematologic malignancy TBI/melphalan/ ATG Or BU/melphalan/ ATG Inherited disorders BU/CY/ATG

Day +12

No infusional toxicities No alteration in time to engraftment ↑ TNC 2.4-fold, CFU-GM 82.7-fold, CD34+ 0.5-fold (median)

Shpall et al. (personal communication; see text)

n = 22 (accrual continues)

UCB – double unrelated Randomization: (A) Double unmanipulated versus (B) Expanded + unmanipulated

CD133+ cells G-CSF, SCF, MGDF × 14 days

Myeloid malignancy Flu/BU/ATG Lymphoid malignancy Flu/melphalan/ thiotepa/ATG

Day 0

No infusional toxicities Fold expansion N/A Average time ANC > 500/μL (A) 20 days (B) 21 days

Shpall et al. MDACC (personal communication; see text)

n = 10 (accrual continues)

Single cord blood unit cryopreserved in two fractions: 80%–20%, 60%–40% or 50%– 50% (smaller fraction ex vivo expanded)

CD133+ cells TEPA + Flt3L, TPO, SCF, IL6 × 21 days

Myeloid malignancy Flu/BU/ATG Lymphoid malignancy Flu/melphalan/ thiotepa/ATG

Day +1

No infusional toxicities Fold expansion TNC 219 (mean), range 2–616 Average time to ANC > 500/μL 28 days (16–46 days)

Delaney et al. (personal communication; see text)

n=3 (accrual continues)

UCB – two units Unmanipulated unit Ex vivo expanded unit

CD34+ cells TPO, SCF, Flt3L, IL-3, IL-6 Serum free

CY/TBI/Flu

Day 0

No infusional toxicities Fold expansion TNC 565 (mean), range 210–791 Fold expansion CD34 115 (mean), range 41–210 Average time to ANC > 500/μL 15 days (9–20 days)

Patients

Stem cell source

Cytokines/serum

Stiff et al. (1998) [166]

n=9 Dx: advanced hematologic disorders

UCB (unrelated) Expanded + unmanipulated

Kögler et al. (1999) [163]

n=1 High-risk leukemia

Pecora et al. (2000) [164]

Notes

ATG, antithymocyte globulin; BU, busulfan; CML, chronic myeloid leukemia; CY, cyclophosphamide; Dx, diagnosis; EFS, event-free survival; FBS, fetal bovine serum; TBI, total body irradiation. For other abbreviations, see Table 8.1 and text.

96

Chapter 8

Patient 1 Patient 2 Patient 3

09

1.0 × 10

08

1.0 × 10

1.0 × 1007 1000000 0

5

10

1.0 × 10 10 1.0 × 10 1.0 × 10

Fold expansion 800

Patient 1 Patient 2 Patient 3

09

700

Fold expansion

Absolute CD34 cell number

1.0 × 1010

Total number of cells

CD34 cell growth

Total cell growth

(a)

08

1.0 × 10 07

600

TNC fold expansion CD34 fold expansion

500 400 300 200 100 0

1000000

15

0

Days in culture

5

10

15

TNC

CD34

Days in culture

Peripheral blood chimerism

(b)

Patient 1

Patient 2

Patient 3

100

100

100

75

75

75

50

50

50

25

25

25

0

0

0 7

14 21 28 42 56 80 100

7

7

14 21 28 42 56 80 100

100

100

100

75

75

75

50

50

50

25

25

25

Not done

0 7

Not done

0

14 21 28 42 56 80 100

CD33

7

14

21

28

CD14

0 7

14 21 28 42 56 80 100

100

100

100

75

75

75

50

50

50

25

25

14

21

28

CD56

25 QNS

7

Percent

0

0

0 14 21 28 42 56 80 100

7

7

14 21 28 42 56 80 100

100

100

100

75

75

75

50

50

50

25

25

25

7

14 21 28 42 56 80 100

21

28

CD3

0

0

0

14

7

14 21 28 42 56 80 100

7

14

21

28

Days post transplant Fig. 8.2 Clinical grade culture of umbilical cord blood (UCB) progenitors with Delta-1ext-IgG for therapeutic application results in significant in vitro expansion of both total nucleated and CD34+ cells. CD34+ cord blood progenitors were selected using the clinical grade Isolex 300i and placed into culture with Delta-1ext-IgG as described. (a) In vitro growth characteristics are presented for three patients enrolled to date, including absolute growth of total nucleated cells (TNC), CD34+ cells, and the resultant fold expansion of both total nucleated and CD34+ cells. Data shown in the bar graph are the mean ± standard error of the mean for the three patients. (b) Peripheral blood was sorted by fluorescence-activated cell sorting into CD3+, CD33+, CD14+, and CD56+ cell fractions to assess for contribution to engraftment from the ex vivo expanded and unmanipulated units (chimerism). Chimerism testing was performed by amplified fragment length polymorphism analysis. The y-axis represents the percent engraftment as contributed by the ex vivo expanded cord blood unit (black), the unmanipulated unit (grey), and the host (white). The x-axis represents time post transplant.

Expansion of Hematopoietic Stem Cells

units would not be expected. Both patients achieved an ANC of 100/μL by day 7 post transplant, a threshold that has been shown to be strongly associated with a survival benefit post allogeneic HCT [168], and an ANC of 500/μL by days 9 and 16 post transplant. A third patient received a more limited CD34 cell dose (1 × 106 cells/kg) due to a less robust in vitro expansion and did not show engraftment before 2 weeks. The CD34 fold expansion (at 41-fold expansion) for this patient was equivalent to the one outlier out of 19 in our preclinical validation runs that resulted in a CD34 fold expansion of less than 50. It is anticipated that a portion of cord blood units will result in less than expected expansion of CD34+ cells based on experience to date. Prediction of units that will expand poorly is not possible at this point prior to ex vivo expansion and suggests a benefit in having pre-expanded units cryopreserved for future use. Nonetheless, the average time to engraftment for all patients (defined as the first of three consecutive days with an ANC > 500/μL) was 15 days, contrasting the expected time to engraftment following a double unmanipulated cord blood transplantation of 23 days, as reported by Barker and colleagues [151]. The kinetics of hematopoietic recovery and the relative contribution of the expanded and unmanipulated cord blood units to engraftment over time were determined by a DNA-based assay for short tandem repeat loci on cells obtained from peripheral blood sorted by fluorescenceactivated cell sorting for CD3+, CD33+, CD56+, CD14+, and CD19+ cell fractions, beginning on day 7 post transplant. These studies revealed that initial myelomonocytic engraftment in these patients was derived from the cultured cells, with subsequent sustained engraftment primarily from the noncultured unit. Analysis of peripheral blood sorted cell fractions revealed loss of contribution to engraftment in all cell populations from the expanded cells by day 21 in two out of three patients transplanted to date; however, there was persistent contribution to engraftment from both the cultured and noncultured cells in the CD14, CD56, and CD19 (data not shown for CD19) sorted cell fractions at day 100 post transplant in one patient (Fig. 8.2(b)). Accrual continues on this study. It is not yet known whether loss of the cultured cord blood unit in marrow and peripheral blood engraftment resulted from loss of stem cell self-renewal capacity due to maturation of the stem cell during culture or yet-to-be-defined immune-mediated mechanisms that allow only cells from a single unit to survive in a double cord blood transplant setting. Our findings are consistent with

97

results seen in most patients undergoing conventional cord blood transplant with two unmanipulated units, where only one unit contributes to sustained donor engraftment, and donor dominance is usually observed early in the majority of patients. However, a clear difference in the expansion setting versus the conventional double cord blood transplant setting is the infusion of two unmanipulated, immunocompetent units versus infusion of an immunocompetent, noncultured unit and a T-celldepleted ex vivo cultured unit, which may allow for rejection of the cultured cells by the noncultured unit.

Conclusion Although optimal conditions for expanding HSC numbers remain undefined, the collective results of studies outlined above suggest that enhanced short-term and long-term repopulating ability may be achievable with cytokine-induced expansion systems. However, these studies further suggest that cytokine-induced effects on HSC self-renewal and expansion are limited, and clinically significant expansion has not been achieved. It is likely that we have not yet identified critical factors and combinations required to induce symmetric HSC self-renewal, and thus current efforts are now focused on identifying and exploiting factors previously shown to regulate stem cell fate in other developing organ systems or during embryogenesis. This approach is beginning to yield promising results, and improved methods for HSC expansion are emerging. Identification of genes and regulatory elements that are responsible for preventing stem cell senescence and driving symmetric divisions of the HSC, and a better understanding of how culturing affects the homing ability of stem cells and of the complex cellular interactions in the bone marrow microenvironment, are all essential to the ultimate success of ex vivo expansion. These areas are discussed in more detail in other chapters. Clinically, ex vivo expansion studies of primitive cells from bone marrow, PBPCs, and UCB have demonstrated the feasibility and safety of ex vivo expansion, and have suggested the enhancement of short-term repopulating cells perhaps at the expense of long-term repopulating cells. Continued development of methods using novel stem cell regulators that are shown to expand HSCs in preclinical studies is necessary, and current trials are underway to assess the safety and efficacy of these approaches, which hold promise for the future of expanding HSC numbers ex vivo.

References 1. Attar EC, Scadden DT. Regulation of hematopoietic stem cell growth. Leukemia 2004; 18: 1760– 8. 2. Hofmeister CC, Zhang J, Knight KL et al. Ex vivo expansion of umbilical cord blood stem cells for transplantation: growing knowledge from the hematopoietic niche. Bone Marrow Transplant 2007; 39: 11–23. 3. McNiece I, Briddell R. Ex vivo expansion of hematopoietic progenitor cells and mature cells. Exp Hematol 2001; 29: 3–11. 4. Robinson SN, Ng J, Niu T et al. Superior ex vivo cord blood expansion following co-culture with bone marrow-derived mesenchymal stem cells. Bone Marrow Transplant 2006; 37: 359–66. 5. Sauvageau G, Iscove NN, Humphries RK. In vitro and in vivo expansion of hematopoietic stem cells. Oncogene 2004; 23: 7223–32. 6. Verfaillie C. Ex vivo expansion of hematopoietic stem cells. In: Zon L, editor. Hematopoiesis: A Developmental Approach. New York: Oxford University Press; 2001. pp. 119–29.

7. Sorrentino BP. Clinical strategies for expansion of haematopoietic stem cells. Nat Rev Immunol 2004; 4: 878–88. 8. Ogawa M. Differentiation and proliferation of hematopoietic stem cells. Blood 1993; 81: 2844– 53. 9. Morrison SJ, Wandycz AM, Hemmati HD et al. Identification of a lineage of multipotent hematopoietic progenitors. Development 1997; 124: 1929–39. 10. Weissman IL. Stem cells: units of development, units of regeneration, and units in evolution. Cell 2000; 100: 157–68. 11. Ogden DA, Mickliem HS. The fate of serially transplanted bone marrow cell populations from young and old donors. Transplantation 1976; 22: 287–93. 12. Ross EA, Anderson N, Micklem HS. Serial depletion and regeneration of the murine hematopoietic system. Implications for hematopoietic organization and the study of cellular aging. J Exp Med 1982; 155: 432–44.

13. Harrison DE, Astle CM. Loss of stem cell repopulating ability upon transplantation. Effects of donor age, cell number, and transplantation procedure. J Exp Med 1982; 156: 1767–79. 14. Morrison SJ, Wandycz AM, Akashi K et al. The aging of hematopoietic stem cells. Nat Med 1996; 2: 1011–16. 15. Iscove NN, Nawa K. Hematopoietic stem cells expand during serial transplantation in vivo without apparent exhaustion. Curr Biol 1997; 7: 805–8. 16. Sudo K, Ema H, Morita Y, Nakauchi H. Age-associated characteristics of murine hematopoietic stem cells. J Exp Med 2000; 192: 1273– 80. 17. Allsopp RC, Weissman IL. Replicative senescence of hematopoietic stem cells during serial transplantation: does telomere shortening play a role? Oncogene 2002; 21: 3270–3. 18. Geiger H, Van Zant G. The aging of lymphohematopoietic stem cells. Nat Immunol 2002; 3: 329–33.

98

Chapter 8

19. Rosendaal M, Hodgson GS, Bradley TR. Organization of hematopietic stem cell: the generation age hypothesis. Cell Tissue Kinet 1979; 12: 17– 29. 20. Joseph NM, Morrison SJ. Toward an understanding of the physiological function of Mammalian stem cells. Dev Cell 2005; 9: 173–83. 21. Holyoake TL, Nicolini FE, Eaves CJ. Functional differences between transplantable human hematopoietic stem cells from fetal liver, cord blood, and adult marrow. Exp Hematol 1999; 27: 1418– 27. 22. Rosler ES, Brandt JE, Chute J, Hoffman R. An in vivo competitive repopulation assay for various sources of human hematopoietic stem cells. Blood 2000; 96: 3414–21. 23. Weekx SF, Van Bockstaele DR, Plum J et al. CD34++ CD38− and CD34+ CD38+ human hematopoietic progenitors from fetal liver, cord blood, and adult bone marrow respond differently to hematopoietic cytokines depending on the ontogenic source. Exp Hematol 1998; 26: 1034– 42. 24. Scheding S, Kratz-Albers K, Meister B et al. Ex vivo expansion of hematopoietic progenitor cells for clinical use. Semin Hematol 1998; 35: 232– 40. 25. Verfaillie CM. Hematopoietic stem cells for transplantation. Nat Immunol 2002; 3: 314–17. 26. Nakahata T, Gross AJ, Ogawa M. A stochastic model of self-renewal and commitment to differentiation of the primitive hemopoietic stem cells in culture. J Cell Physiol 1982; 113: 455– 8. 27. Socolovsky M, Lodish HF, Daley GQ. Control of hematopoietic differentiation: lack of specificity in signaling by cytokine receptors. Proc Natl Acad Sci U S A 1998; 95: 6573–5. 28. Yin T, Li L. The stem cell niches in bone. J Clin Invest 2006; 116: 1195–201. 29. Wilson A, Trumpp A. Bone-marrow haematopoietic-stem-cell niches. Nat Rev Immunol 2006; 6: 93–106. 30. Adams GB, Scadden DT. The hematopoietic stem cell in its place. Nat Immunol 2006; 7: 333–7. 31. Dexter TM, Moore MA, Sheridan AP. Maintenance of hemopoietic stem cells and production of differentiated progeny in allogeneic and semiallogeneic bone marrow chimeras in vitro. J Exp Med 1977; 145: 1612–16. 32. Fraser CC, Eaves CJ, Szilvassy SJ, Humphries RK. Expansion in vitro of retrovirally marked totipotent hematopoietic stem cells. Blood 1990; 76: 1071–6. 33. Miller CL, Eaves CJ. Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability. Proc Natl Acad Sci U S A 1997; 94: 13648–53. 34. Dexter TM, Allen TD, Lajtha LG. Conditions controlling the proliferation of haemopoietic stem cells in vitro. J Cell Physiol 1977; 91: 335– 44. 35. Roberts R, Gallagher J, Spooncer E et al. Heparan sulphate bound growth factors: a mechanism for stromal cell mediated haemopoiesis. Nature 1988; 332: 376–8. 36. Spooncer E, Gallagher JT, Krizsa F, Dexter TM. Regulation of haemopoiesis in long-term bone marrow cultures. IV. Glycosaminoglycan synthesis and the stimulation of haemopoiesis by betaD-xylosides. J Cell Biol 1983; 96: 510–14.

37. Ando K, Yahata T, Sato T et al. Direct evidence for ex vivo expansion of human hematopoietic stem cells. Blood 2006; 107: 3371–7. 38. Fraser CC, Szilvassy SJ, Eaves CJ, Humphries RK. Proliferation of totipotent hematopoietic stem cells in vitro with retention of long-term competitive in vivo reconstituting ability. Proc Natl Acad Sci U S A 1992; 89: 1968–72. 39. Trevisan M, Yan XQ, Iscove NN. Cycle initiation and colony formation in culture by murine marrow cells with long-term reconstituting potential in vivo. Blood 1996; 88: 4149–58. 40. Ema H, Takano H, Sudo K, Nakauchi H. In vitro self-renewal division of hematopoietic stem cells. J Exp Med 2000; 192: 1281–8. 41. Zhang CC, Lodish HF. Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion. Blood 2005; 105: 4314–20. 42. Giet O, Van Bockstaele DR, Di Stefano I et al. Increased binding and defective migration across fibronectin of cycling hematopoietic progenitor cells. Blood 2002; 99: 2023–31. 43. Berrios VM, Dooner GJ, Nowakowski G et al. The molecular basis for the cytokine-induced defect in homing and engraftment of hematopoietic stem cells. Exp Hematol 2001; 29: 1326–35. 44. Gothot A, Pyatt R, McMahel J et al. Assessment of proliferative and colony-forming capacity after successive in vitro divisions of single human CD34+ cells initially isolated in G0. Exp Hematol 1998; 26: 562–70. 45. Gothot A, van der Loo JC, Clapp DW, Srour EF. Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34(+) cells in non-obese diabetic/severe combined immune-deficient mice. Blood 1998; 92: 2641– 9. 46. Habibian HK, Peters SO, Hsieh CC et al. The fluctuating phenotype of the lymphohematopoietic stem cell with cell cycle transit. J Exp Med 1998; 188: 393–8. 47. Yonemura Y, Ku H, Hirayama F et al. Interleukin 3 or interleukin 1 abrogates the reconstituting ability of hematopoietic stem cells. Proc Natl Acad Sci U S A 1996; 93: 4040–4. 48. Yonemura Y, Ku H, Lyman SD, Ogawa M. In vitro expansion of hematopoietic progenitors and maintenance of stem cells: comparison between FLT3/FLK-2 ligand and KIT ligand. Blood 1997; 89: 1915–21. 49. Yagi M, Ritchie KA, Sitnicka E et al. Sustained ex vivo expansion of hematopoietic stem cells mediated by thrombopoietin. Proc Natl Acad Sci U S A 1999; 96: 8126–31. 50. Cashman JD, Eaves AC, Raines EW et al. Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. I. Stimulatory role of a variety of mesenchymal cell activators and inhibitory role of TGF-beta. Blood 1990; 75: 96–101. 51. Cashman JD, Eaves CJ, Sarris AH, Eaves AC. MCP-1, not MIP-1alpha, is the endogenous chemokine that cooperates with TGF-beta to inhibit the cycling of primitive normal but not leukemic (CML) progenitors in long-term human marrow cultures. Blood 1998; 92: 2338–44. 52. Eaves CJ, Cashman JD, Kay RJ et al. Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. II. Analysis of positive and negative regulators produced by stromal cells

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

within the adherent layer. Blood 1991; 78: 110– 17. Abkowitz JL, Catlin SN, McCallie MT, Guttorp P. Evidence that the number of hematopoietic stem cells per animal is conserved in mammals. Blood 2002; 100: 2665–7. Cheshier SH, Morrison SJ, Liao X, Weissman IL. In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells. Proc Natl Acad Sci U S A 1999; 96: 3120–5. Mahmud N, Devine SM, Weller KP et al. The relative quiescence of hematopoietic stem cells in nonhuman primates. Blood 2001; 97: 3061– 8. Andrews RG, Briddell RA, Hill R et al. Engraftment of primates with G-CSF mobilized peripheral blood CD34+ progenitor cells expanded in G-CSF, SCF and MGDF decreases the duration and severity of neutropenia. Stem Cells 1999; 17: 210–18. Drouet M, Herodin F, Norol F et al. Cell cycle activation of peripheral blood stem and progenitor cells expanded ex vivo with SCF, FLT-3 ligand, TPO, and IL-3 results in accelerated granulocyte recovery in a baboon model of autologous transplantation but G0/G1 and S/G2/M graft cell content does not correlate with transplantability. Stem Cells 2001; 19: 436–42. Norol F, Drouet M, Mathieu J et al. Ex vivo expanded mobilized peripheral blood CD34+ cells accelerate haematological recovery in a baboon model of autologous transplantation. Br J Haematol 2000; 109: 162–72. Kiem HP, Rasko JE, Morris J et al. Ex vivo selection for oncoretrovirally transduced green fluorescent protein-expressing CD34-enriched cells increases short-term engraftment of transduced cells in baboons. Hum Gene Ther 2002; 13: 891– 9. Tisdale JF, Hanazono Y, Sellers SE et al. Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished longterm repopulating ability. Blood 1998; 92: 1131– 41. Abkowitz JL, Taboada MR, Sabo KM, Shelton GH. The ex vivo expansion of feline marrow cells leads to increased numbers of BFU-E and CFUGM but a loss of reconstituting ability. Stem Cells 1998; 16: 288–93. Goerner M, Horn PA, Peterson L et al. Sustained multilineage gene persistence and expression in dogs transplanted with CD34(+) marrow cells transduced by RD114-pseudotype oncoretrovirus vectors. Blood 2001; 98: 2065–70. Bhatia M, Bonnet D, Murdoch B et al. A newly discovered class of human hematopoietic cells with SCID-repopulating activity. Nat Med 1998; 4: 1038–45. Bhatia M, Wang JC, Kapp U et al. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci U S A 1997; 94: 5320–5. Conneally E, Cashman J, Petzer A, Eaves C. Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice. Proc Natl Acad Sci U S A 1997; 94: 9836–41. Larochelle A, Vormoor J, Hanenberg H et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse

Expansion of Hematopoietic Stem Cells

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

bone marrow: implications for gene therapy. Nat Med 1996; 2: 1329–37. Vormoor J, Lapidot T, Pflumio F et al. Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice. Blood 1994; 83: 2489–97. Wang JC, Doedens M, Dick JE. Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay. Blood 1997; 89: 3919–24. Glimm H, Eisterer W, Lee K et al. Previously undetected human hematopoietic cell populations with short-term repopulating activity selectively engraft NOD/SCID-beta2 microglobulin-null mice. J Clin Invest 2001; 107: 199–206. Dorrell C, Gan OI, Pereira DS et al. Expansion of human cord blood CD34(+)CD38(−) cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function. Blood 2000; 95: 102–10. Gan OI, Murdoch B, Larochelle A, Dick JE. Differential maintenance of primitive human SCIDrepopulating cells, clonogenic progenitors, and long-term culture-initiating cells after incubation on human bone marrow stromal cells. Blood 1997; 90: 641–50. Danet GH, Lee HW, Luongo JL et al. Dissociation between stem cell phenotype and NOD/SCID repopulating activity in human peripheral blood CD34(+) cells after ex vivo expansion. Exp Hematol 2001; 29: 1465–73. Mobest D, Goan SR, Junghahn I et al. Differential kinetics of primitive hematopoietic cells assayed in vitro and in vivo during serum-free suspension culture of CD34+ blood progenitor cells. Stem Cells 1999; 17: 152–61. Bhatia M, Bonnet D, Kapp U et al. Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture. J Exp Med 1997; 186: 619–24. Gilmore GL, DePasquale DK, Lister J, Shadduck RK. Ex vivo expansion of human umbilical cord blood and peripheral blood CD34(+) hematopoietic stem cells. Exp Hematol 2000; 28: 1297–305. Luens KM, Travis MA, Chen BP et al. Thrombopoietin, kit ligand, and flk2/flt3 ligand together induce increased numbers of primitive hematopoietic progenitors from human CD34+Thy1+Lin– cells with preserved ability to engraft SCID-hu bone. Blood 1998; 91: 1206–15. Novelli EM, Cheng L, Yang Y et al. Ex vivo culture of cord blood CD34+ cells expands progenitor cell numbers, preserves engraftment capacity in nonobese diabetic/severe combined immunodeficient mice, and enhances retroviral transduction efficiency. Hum Gene Ther 1999; 10: 2927–40. Piacibello W, Sanavio F, Severino A et al. Engraftment in nonobese diabetic severe combined immunodeficient mice of human CD34(+) cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells. Blood 1999; 93: 3736–49. Ueda T, Tsuji K, Yoshino H et al. Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL6, and soluble IL-6 receptor. J Clin Invest 2000; 105: 1013–21.

80. da Silva CL, Goncalves R, Crapnell KB et al. A human stromal-based serum-free culture system supports the ex vivo expansion/maintenance of bone marrow and cord blood hematopoietic stem/ progenitor cells. Exp Hematol 2005; 33: 828– 35. 81. Kimura T, Sakabe H, Tanimukai S et al. Simultaneous activation of signals through gp130, c-kit, and interleukin-3 receptor promotes a trilineage blood cell production in the absence of terminally acting lineage-specific factors. Blood 1997; 90: 4767–78. 82. Kimura T, Wang J, Minamiguchi H et al. Signal through gp130 activated by soluble interleukin (IL)-6 receptor (R) and IL-6 or IL-6R/IL-6 fusion protein enhances ex vivo expansion of human peripheral blood-derived hematopoietic progenitors. Stem Cells 2000; 18: 444–52. 83. Kollet O, Aviram R, Chebath J et al. The soluble interleukin-6 (IL-6) receptor/IL-6 fusion protein enhances in vitro maintenance and proliferation of human CD34(+)CD38(−/low) cells capable of repopulating severe combined immunodeficiency mice. Blood 1999; 94: 923–31. 84. Tajima S, Tsuji K, Ebihara Y et al. Analysis of interleukin 6 receptor and gp130 expressions and proliferative capability of human CD34+ cells. J Exp Med 1996; 184: 1357–64. 85. Emmons RV, Doren S, Zujewski J et al. Retroviral gene transduction of adult peripheral blood or marrow-derived CD34+ cells for six hours without growth factors or on autologous stroma does not improve marking efficiency assessed in vivo. Blood 1997; 89: 4040–6. 86. Hanania EG, Giles RE, Kavanagh J et al. Results of MDR-1 vector modification trial indicate that granulocyte/macrophage colony-forming unit cells do not contribute to posttransplant hematopoietic recovery following intensive systemic therapy. Proc Natl Acad Sci U S A 1996; 93: 15346–51. 87. Bhatia R, McGlave PB, Miller JS et al. A clinically suitable ex vivo expansion culture system for LTC-IC and CFC using stroma-conditioned medium. Exp Hematol 1997; 25: 980–91. 88. Roecklein B, Almaida-Porada G, Torok-Storb B et al. Serial xenogeneic transplantation of ex vivo expanded human CD34+ cells. Blood 1997; 90: 1750a. 89. Shih CC, Hu MC, Hu J et al. A secreted and LIFmediated stromal cell-derived activity that promotes ex vivo expansion of human hematopoietic stem cells. Blood 2000; 95: 1957–66. 90. Verfaillie CM, Catanzarro PM, Li WN. Macrophage inflammatory protein 1 alpha, interleukin 3 and diffusible marrow stromal factors maintain human hematopoietic stem cells for at least eight weeks in vitro. J Exp Med 1994; 179: 643–9. 91. Yildirim S, Boehmler AM, Kanz L, Mohle R. Expansion of cord blood CD34+ hematopoietic progenitor cells in coculture with autologous umbilical vein endothelial cells (HUVEC) is superior to cytokine-supplemented liquid culture. Bone Marrow Transplant 2005; 36: 71–9. 92. Lewis ID, Almeida-Porada G, Du J et al. Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system. Blood 2001; 97: 3441–9. 93. Roecklein BA, Torok-Storb B. Functionally distinct human marrow stromal cell lines immortal-

94.

95.

96.

97.

98.

99.

100.

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

99

ized by transduction with the human papilloma virus E6/E7 genes. Blood 1995; 85: 997–1005. Piacibello W, Sanavio F, Garetto L et al. Extensive amplification and self-renewal of human primitive hematopoietic stem cells from cord blood. Blood 1997; 89: 2644–53. Tanavde VM, Malehorn MT, Lumkul R et al. Human stem-progenitor cells from neonatal cord blood have greater hematopoietic expansion capacity than those from mobilized adult blood. Exp Hematol 2002; 30: 816–23. Shimizu Y, Ogawa M, Kobayashi M et al. Engraftment of cultured human hematopoietic cells in sheep. Blood 1998; 91: 3688–92. McNiece IK, Almeida-Porada G, Shpall EJ, Zanjani E. Ex vivo expanded cord blood cells provide rapid engraftment in fetal sheep but lack long-term engrafting potential. Exp Hematol 2002; 30: 612–16. Horn PA, Thomasson BM, Wood BL et al. Distinct hematopoietic stem/progenitor cell populations are responsible for repopulating NOD/SCID mice compared with nonhuman primates. Blood 2003; 102: 4329–35. Ballen K, Becker PS, Greiner D et al. Effect of ex vivo cytokine treatment on human cord blood engraftment in NOD-scid mice. Br J Haematol 2000; 108: 629–40. Glimm H, Eaves CJ. Direct evidence for multiple self-renewal divisions of human in vivo repopulating hematopoietic cells in short-term culture. Blood 1999; 94: 2161–8. Guenechea G, Segovia JC, Albella B et al. Delayed engraftment of nonobese diabetic/severe combined immunodeficient mice transplanted with ex vivo-expanded human CD34(+) cord blood cells. Blood 1999; 93: 1097–105. Antonchuk J, Sauvageau G, Humphries RK. HOXB4 overexpression mediates very rapid stem cell regeneration and competitive hematopoietic repopulation. Exp Hematol 2001; 29: 1125– 34. Antonchuk J, Sauvageau G, Humphries RK. HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell 2002; 109: 39–45. Sauvageau G, Thorsteinsdottir U, Eaves CJ et al. Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes Dev 1995; 9: 1753–65. Thorsteinsdottir U, Sauvageau G, Humphries RK. Enhanced in vivo regenerative potential of HOXB4-transduced hematopoietic stem cells with regulation of their pool size. Blood 1999; 94: 2605–12. Bhatia M, Bonnet D, Wu D et al. Bone morphogenetic proteins regulate the developmental program of human hematopoietic stem cells. J Exp Med 1999; 189: 1139–48. Zon LI. Self-renewal versus differentiation, a job for the mighty morphogens. Nat Immunol 2001; 2: 142–3. Bhardwaj G, Murdoch B, Wu D et al. Sonic hedgehog induces the proliferation of primitive human hematopoietic cells via BMP regulation. Nat Immunol 2001; 2: 172–80. Yamane T, Kunisada T, Tsukamoto H et al. Wnt signaling regulates hemopoiesis through stromal cells. J Immunol 2001; 167: 765–72. Austin TW, Solar GP, Ziegler FC et al. A role for the Wnt gene family in hematopoiesis: expansion

100

111.

112. 113.

114.

115.

116.

117.

118.

119.

120.

121.

122.

123.

124.

125.

126.

127.

Chapter 8

of multilineage progenitor cells. Blood 1997; 89: 3624–35. Reya T, Duncan AW, Ailles L et al. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 2003; 423: 409–14. Kojika S, Griffin JD. Notch receptors and hematopoiesis. Exp Hematol 2001; 29: 1041–52. Milner LA, Kopan R, Martin DI, Bernstein ID. A human homologue of the Drosophila developmental gene, Notch, is expressed in CD34+ hematopoietic precursors. Blood 1994; 83: 2057–62. Ohishi K, Varnum-Finney B, Flowers D et al. Monocytes express high amounts of Notch and undergo cytokine specific apoptosis following interaction with the Notch ligand, Delta-1. Blood 2000; 95: 2847–54. Jones P, May G, Healy L et al. Stromal expression of Jagged 1 promotes colony formation by fetal hematopoietic progenitor cells. Blood 1998; 92: 1505–11. Karanu FN, Murdoch B, Gallacher L et al. The notch ligand jagged-1 represents a novel growth factor of human hematopoietic stem cells. J Exp Med 2000; 192: 1365–72. Karanu FN, Murdoch B, Miyabayashi T et al. Human homologues of Delta-1 and Delta-4 function as mitogenic regulators of primitive human hematopoietic cells. Blood 2001; 97: 1960–7. Li L, Milner LA, Deng Y et al. The human homolog of rat Jagged1 expressed by marrow stroma inhibits differentiation of 32D cells through interaction with Notch1. Immunity 1998; 8: 43–55. Varnum-Finney B, Xu L, Brashem-Stein C et al. Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nat Med 2000; 6: 1278–81. Stier S, Cheng T, Dombkowski D et al. Notch1 activation increases hematopoietic stem cell selfrenewal in vivo and favors lymphoid over myeloid lineage outcome. Blood 2002; 99: 2369–78. Carlesso N, Aster JC, Sklar J, Scadden DT. Notch1-induced delay of human hematopoietic progenitor cell differentiation is associated with altered cell cycle kinetics. Blood 1999; 93: 838– 48. Varnum-Finney B, Wu L, Yu M et al. Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling. J Cell Sci 2000; 113(Pt 23): 4313–18. Vas V, Szilagyi L, Paloczi K, Uher F. Soluble Jagged-1 is able to inhibit the function of its multivalent form to induce hematopoietic stem cell self-renewal in a surrogate in vitro assay. J Leukoc Biol 2004; 75: 714–20. Varnum-Finney B, Brashem-Stein C, Bernstein ID. Combined effects of Notch signaling and cytokines induce a multiple log increase in precursors with lymphoid and myeloid reconstituting ability. Blood 2003; 101: 1784–9. Delaney C, Varnum-Finney B, Aoyama K et al. Dose-dependent effects of the Notch ligand Delta1 on ex vivo differentiation and in vivo marrow repopulating ability of cord blood cells. Blood 2005; 106: 2693–9. Ohishi K, Varnum-Finney B, Bernstein ID. Delta1 enhances marrow and thymus repopulating ability of human CD34(+)CD38(−) cord blood cells. J Clin Invest 2002; 110: 1165–74. Dallas MH, Varnum-Finney B, Delaney C et al. Density of the Notch ligand Delta1 determines

128.

129.

130.

131.

132.

133.

134.

135.

136.

137.

138.

139.

140.

141.

142.

generation of B and T cell precursors from hematopoietic stem cells. J Exp Med 2005; 201: 1361– 6. Kertesz Z, Vas V, Kiss J et al. In vitro expansion of long-term repopulating hematopoietic stem cells in the presence of immobilized Jagged-1 and early acting cytokines. Cell Biol Int 2006; 30: 401–5. Alcorn MJ, Holyoake TL, Richmond L et al. CD34-positive cells isolated from cryopreserved peripheral-blood progenitor cells can be expanded ex vivo and used for transplantation with little or no toxicity. J Clin Oncol 1996; 14: 1839–47. Reiffers J, Cailliot C, Dazey B et al. Abrogation of post-myeloablative chemotherapy neutropenia by ex-vivo expanded autologous CD34-positive cells. Lancet 1999; 354: 1092–3. Bertolini F, Battaglia M, Pedrazzoli P et al. Megakaryocytic progenitors can be generated ex vivo and safely administered to autologous peripheral blood progenitor cell transplant recipients. Blood 1997; 89: 2679–88. Engelhardt M, Douville J, Behringer D et al. Hematopoietic recovery of ex vivo perfusion culture expanded bone marrow and unexpanded peripheral blood progenitors after myeloablative chemotherapy. Bone Marrow Transplant 2001; 27: 249–59. Holyoake TL, Alcorn MJ, Richmond L et al. CD34 positive PBPC expanded ex vivo may not provide durable engraftment following myeloablative chemoradiotherapy regimens. Bone Marrow Transplant 1997; 19: 1095–101. McNiece I, Jones R, Bearman SI et al. Ex vivo expanded peripheral blood progenitor cells provide rapid neutrophil recovery after high-dose chemotherapy in patients with breast cancer. Blood 2000; 96: 3001–7. Paquette RL, Dergham ST, Karpf E et al. Culture conditions affect the ability of ex vivo expanded peripheral blood progenitor cells to accelerate hematopoietic recovery. Exp Hematol 2002; 30: 374–80. Pecora AL, Stiff P, LeMaistre CF et al. A phase II trial evaluating the safety and effectiveness of the AastromReplicell system for augmentation of low-dose blood stem cell transplantation. Bone Marrow Transplant 2001; 28: 295–303. Stiff P, Chen B, Franklin W et al. Autologous transplantation of ex vivo expanded bone marrow cells grown from small aliquots after high-dose chemotherapy for breast cancer. Blood 2000; 95: 2169–74. Williams SF, Lee WJ, Bender JG et al. Selection and expansion of peripheral blood CD34+ cells in autologous stem cell transplantation for breast cancer. Blood 1996; 87: 1687–91. Glaspy JA, Shpall EJ, LeMaistre CF et al. Peripheral blood progenitor cell mobilization using stem cell factor in combination with filgrastim in breast cancer patients. Blood 1997; 90: 2939–51. Gebbie K, Aubry W, Champlin RE et al. Cord Blood: Establishing a National Hematopoietic Stem Cell Bank Program. Washington, DC: National Academies Press; 2005. Barker JN. Who should get cord blood transplants? Biol Blood Marrow Transplant 2007; 13(Suppl 1): 78–82. Brunstein CG, Wagner JE. Umbilical cord blood transplantation and banking. Annu Rev Med 2006; 57: 403–17.

143. Gluckman E. Current status of umbilical cord blood hematopoietic stem cell transplantation. Exp Hematol 2000; 28: 1197–205. 144. Gluckman E, Rocha V, Boyer-Chammard A et al. Outcome of cord-blood transplantation from related and unrelated donors. Eurocord Transplant Group and the European Blood and Marrow Transplantation Group. N Engl J Med 1997; 337: 373–81. 145. Kurtzberg J, Laughlin M, Graham ML et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 1996; 335: 157–66. 146. Laughlin MJ, Barker J, Bambach B et al. Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors. N Engl J Med 2001; 344: 1815–22. 147. Rocha V, Gluckman E. Clinical use of umbilical cord blood hematopoietic stem cells. Biol Blood Marrow Transplant 2006; 12(1 Suppl 1): 34– 41. 148. Rubinstein P, Carrier C, Scaradavou A et al. Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N Engl J Med 1998; 339: 1565–77. 149. Wagner JE, Rosenthal J, Sweetman R et al. Successful transplantation of HLA-matched and HLA-mismatched umbilical cord blood from unrelated donors: analysis of engraftment and acute graft-versus-host disease. Blood 1996; 88: 795–802. 150. Wagner JE, Barker JN, DeFor TE et al. Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant diseases: influence of CD34 cell dose and HLA disparity on treatment-related mortality and survival. Blood 2002; 100: 1611–18. 151. Barker JN, Weisdorf DJ, DeFor TE et al. Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood 2005; 105: 1343–7. 152. Broxmeyer HE, Hangoc G, Cooper S et al. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl Acad Sci U S A 1992; 89: 4109–13. 153. Lu L, Xiao M, Shen RN et al. Enrichment, characterization, and responsiveness of single primitive CD34 human umbilical cord blood hematopoietic progenitors with high proliferative and replating potential. Blood 1993; 81: 41– 8. 154. Mayani H, Dragowska W, Lansdorp PM. Characterization of functionally distinct subpopulations of CD34+ cord blood cells in serum-free longterm cultures supplemented with hematopoietic cytokines. Blood 1993; 82: 2664–72. 155. Hao QL, Thiemann FT, Petersen D et al. Extended long-term culture reveals a highly quiescent and primitive human hematopoietic progenitor population. Blood 1996; 88: 3306–13. 156. Hows JM, Bradley BA, Marsh JC et al. Growth of human umbilical-cord blood in longterm haemopoietic cultures. Lancet 1992; 340: 73–6. 157. Mayani H, Lansdorp PM. Thy-1 expression is linked to functional properties of primitive hematopoietic progenitor cells from human umbilical cord blood. Blood 1994; 83: 2410–17. 158. Steen R, Tjonnfjord GE, Egeland T. Comparison of the phenotype and clonogenicity of normal

Expansion of Hematopoietic Stem Cells CD34+ cells from umbilical cord blood, granulocyte colony-stimulating factor-mobilized peripheral blood, and adult human bone marrow. J Hematother 1994; 3: 253–62. 159. van de Ven C, Ishizawa L, Law P, Cairo MS. IL-11 in combination with SLF and G-CSF or GM-CSF significantly increases expansion of isolated CD34+ cell population from cord blood vs. adult bone marrow. Exp Hematol 1995; 23: 1289– 95. 160. Rebel VI, Miller CL, Eaves CJ, Lansdorp PM. The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their liver adult bone marrow counterparts. Blood 1996; 87: 3500–7. 161. Szilvassy SJ, Meyerrose TE, Ragland PL, Grimes B. Differential homing and engraftment properties of hematopoietic progenitor cells from murine

bone marrow, mobilized peripheral blood, and fetal liver. Blood 2001; 98: 2108–15. 162. Jaroscak J, Goltry K, Smith A et al. Augmentation of umbilical cord blood (UCB) transplantation with ex vivo-expanded UCB cells: results of a phase 1 trial using the AastromReplicell System. Blood 2003; 101: 5061–7. 163. Kögler G, Nurnberger W, Fischer J et al. Simultaneous cord blood transplantation of ex vivo expanded together with non-expanded cells for high risk leukemia. Bone Marrow Transplant 1999; 24: 397–403. 164. Pecora AL, Stiff P, Jennis A et al. Prompt and durable engraftment in two older adult patients with high risk chronic myelogenous leukemia (CML) using ex vivo expanded and unmanipulated unrelated umbilical cord blood. Bone Marrow Transplant 2000; 25: 797–9.

101

165. Shpall E, Quinines R, Giller R et al. Transplantation of ex vivo expanded cord blood. Biol Blood Marrow Transplant 2002; 8: 368–76. 166. Stiff P, Pecora A, Parthasarathy M, Preti R, Chen B. Umbilical cord blood transplants in adults using a combination of unexpanded and ex vivo expanded cells: preliminary clinical observations. Blood 1998: 92(Suppl 1): 646a. 167. Peled T, Landau E, Mandel J et al. Linear polyamine copper chelator tetraethylenepentamine augments long-term ex vivo expansion of cord blood-derived CD34+ cells and increases their engraftment potential in NOD/SCID mice. Exp Hematol 2004; 32: 547–55. 168. Offner F, Schoch G, Fisher LD et al. Mortality hazard functions as related to neutropenia at different times after marrow transplantation. Blood 1996; 88: 4058.

9

Edwin M. Horwitz

Mesenchymal Stromal Cells and Hematopoietic Cell Transplantation

Introduction Hematopoietic cell transplantation (HCT) is well established as therapy for hematologic malignancies as well as many nonmalignant disorders. Over the last several years, the spectrum of diseases that may be treated with HCT has dramatically expanded, increasing the importance of this therapeutic modality and extending HCT beyond the traditional bounds of hematology and oncology. HCT is founded on the principle that hematopoietic stem cells (HSCs) can be intravenously infused, will home to and engraft in the stem cell niche within the bone marrow microenvironment, and will then proliferate and differentiate to repopulate all lineages of the blood. The cells that comprise the microenvironment are thought to support, and possibly regulate, hematopoiesis. However, one cellular constituent of the microenvironment, the marrow mesenchymal stromal cells (MSCs), may have quite complex biologic functions that can be exploited in the development of primary or adjunct cell therapy with HCT or in regenerative medicine. MSCs are spindle-shaped, plastic-adherent cells isolated from bone marrow as well as other tissue sources [1]. First recognized nearly 40 years ago [2], MSCs have emerged as one of the most rapidly evolving areas of research in HCT. The trilineage differentiation of MSCs, capable of giving rise to osteoblasts, chondrocytes, and adipocytes in vitro, led investigators to hypothesize a vast differentiation potential and focus research on the regenerative capacity of MSCs in an effort to repair diseased or damaged tissue [3]. More recently, MSCs have been recognized to possess striking immunomodulatory properties, and it is this characteristic of MSCs that has generated the greatest interest among physicians and investigators in the field of HCT [4,5]. Indeed, MSCs produce an extensive array of immunomodulatory cytokines as well as a variety of other, known and likely undiscovered, biochemical mediators. The secretion of these biologic response modifiers may well prove to be the most biologically and clinically important property of MSCs. While MSC research continues along many avenues, this chapter will focus on the biology and early clinical applications of MSCs in close relation to HCT.

“MSC” [2]. He showed that these cells were precursors for osteoblasts [2] and comprised the hematopoietic microenvironment [6], and that a fraction of the population, cells he termed “CFU-F”, possessed a high proliferative potential demonstrated by forming clonal colonies when plated at low density [7]. Friedenstein went on to demonstrate the extensive expansion potential, showing that single cells could regenerate an entire population of cells, suggesting that individual cells were precursors for fibroblasts in vivo [8,9]. However, Friedenstein did not suggest that the marrow stromal fibroblasts that he identified were stem cells. The first formal presentation of the concept that a nonhematopoietic stem cell might reside within the bone marrow microenvironment was by Maureen Owen [10]. She proposed that the marrow stroma consisted of a lineage of cells analogous to the hematopoietic system. She then expanded this theory and proposed a stromal stem cell, with fibroblastic, osteogenic, and adipocytic cells being the terminally differentiated phenotypes [11,12]. The term “mesenchymal stem cell” was proposed by Arnold Caplan, who hypothesized that this cell could differentiate to a wide variety of mesenchymal tissues and proposed a broad lineage “tree” suggesting that the mesenchymal stem cell could give rise to bone, cartilage, muscle, hematopoietic-supportive stroma, tendon, ligament, adipose, and other connective tissues [13]. Data demonstrating the identification of MSCs by reactivity with the monoclonal antibodies SH2 and SH3, the considerable self-renewal capacity, and the multilineage differentiation potential in vitro supported the notion that these cells were a homogeneous population of stem cells [14]. However, more recent data demonstrated that the adherent cells isolated from marrow are a heterogeneous population of cells [15,16], although a nonhematopoietic stem cell may exist within this cell population [17]. Many other investigators, too numerous to recount here, have made important contributions to the scientific development of MSCs. At present, MSCs are generally accepted to be a heterogeneous population of cells with diverse biologic properties suggesting a remarkable potential as cell therapy. While the expansion and differentiation potential of MSCs has yet to make a substantial impact in regenerative medicine, the cytokine-generating and immunomodulatory properties promise to contribute to HCT.

Brief history of MSCs The notion that a mesenchymal progenitor cell with osteogenic differentiation potential resided within bone marrow was considered in the latter 19th century. However, it was the pioneering work of Alexander Friedenstein that first identified the cells that now bear the designation Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

102

Nomenclature The nomenclature of these cells merits clarification. MSCs have been designed by a wide variety of terms including “marrow stromal cells,” “marrow stromal fibroblasts,” and most often “mesenchymal stem cells,” which was the basis for the abbreviation MSC. While the specific properties of MSCs studied in the laboratory may vary depending on the methods of isolation and culture expansion, the assortment of terms, in general, refer to the same heterogeneous population of cells.

Mesenchymal Stromal Cells and Hematopoietic Cell Transplantation

The term “stem cell,” however, implies specific biologic properties. Our general concept of a stem cell evolved from our understanding of the HSC. Till and McCulloch suggested that the stem cell could be defined as a cell with extensive self-renewal capacity and the potential to terminally differentiate into two or more lineages [18]. While MSCs can, indeed, undergo extensive self-renewal and trilineage differentiation to osteoblasts, chondrocytes, and adipocytes in vitro, true “stemness” is certainly much more complex and may be best defined operationally. As this idea has become increasingly recognized, many investigators have suggested that convincing data are lacking to support the notion that the unfractionated population of plastic-adherent cells from bone marrow designated as MSCs are properly considered as stem cells [19]. These thoughts are not intended to dispel the notion of a mesenchymal stem cell. Such a stem cell most assuredly exists and may, indeed, reside within the adherent population of cells that we designate MSCs or some other cell population. Notably, the antibody STRO-1, which identifies CFU-Fs from freshly harvested human bone marrow, was generated from CD34+ nonadherent cells [20]. Recently, Anjos-Afonso et al. showed that a subset of murine MSCs that expressed stage-specific embryonic antigen-1 (SSEA-1) was capable of single-cell expansion followed by multilineage differentiation in vivo, suggesting that SSEA1+ MSCs may be primitive stem/progenitor cells [17]. These data notwithstanding, the entire unfractionated population of MSCs isolated by plastic adherence does not seem to meet the criteria of a homogeneous population of stem cells. Therefore, in 2005, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy recommended that the term “mesenchymal stromal cell” was a more appropriate designation for this heterogeneous population of cells, maintaining the abbreviation MSC while reserving the term “mesenchymal stem cell” for a subset of these (or other) cells that demonstrate stem cell activity in vivo by clearly stated criteria [1].

103

Table 9.1 Summary of criteria to identify mesenchymal stromal cells 1. Adherence to plastic in standard culture conditions 2. Phenotype Positive (≥95% +) CD105 CD73 CD90

3. Differentiation: (by staining of in vitro cell culture)

Negative (80) 85 (45) 78 (24)

77 (45) 73 (53) 96 (39) 88 (35) 42 (58) 75 (39)

0 (>80) 8 (>80) 8 (>80) 0 (>80) 7 (>80) 0 (>80)

Data were pooled from more than 20 experiments with totals of 15–39 mice for each strain combination. Donor marrow cells (4 × 106) were T-cell depleted and injected intravenously along with unseparated (whole) T cells or T-cell subsets (1 × 106 cells) into appropriate lethally irradiated (8–10 Gy) recipient mice. (Adapted from [56].)

182

Chapter 14

intensity of GVHD [91], nor does injecting the host mice with anti-CD4 antibody [97]. These findings imply that the capacity of CD8+ cells to mediate GVHD to mHAs does not necessarily require help from CD4+ cells. Yet, in some strain combinations, like B6 → BALB.B, the CD8+ cells will only mediate GVHD in the presence of CD4+ cells [98]. The criteria which dictate CD8+ cell dependency on exogenous help appear to involve the cytokines available to APCs and their ability to express the 4-1BB ligand molecule, which can bind to its counterpart (4-1BB; CD137) on CD8+ T cells and serve as a co-stimulator for activation and survival [99]. In an effort to investigate the heterogeneity of the T-cell response across a donor–recipient pair disparate at multiple mHA loci, the repertoire of responding CD8+ T cells in the B6 → BALB.B (8.25Gy) GVHD model was analyzed by TCR Vβ CDR3-size polymerase chain reaction spectratyping. This response was found to be oligoclonal in nature, involving T cells from seven Vβ families [79]. With the exception of a single Vβ family, these responses were also found after transplantation of B6 CD8+ T cells into irradiated CXBE mice, a recombinant inbred strain that is derived from a cross between B6 and BALB/c mice and therefore expresses a subset of the mHA found in the BALB.B strain. This overlap in the CD8+ T-cell response repertoire suggested that the phenomenon of competitive immunodominance was not a factor in the recognition of mHAs involved in the development of GVHD. In addition, it was demonstrated that lethal GVHD could be induced by the transplantation of T cells from a single purified B6 CD8+ Vβ family into either irradiated BALB.B or CXBE mice, supporting their involvement in disease pathogenesis and suggesting a possible hierarchy of GVHDmediating specificities within the responding T-cell population [79].

Role of CD4+ cells In the case of the CBA → B10.BR combination and certain other combinations, CD4+ cells play no obvious role in GVHD. The helper function of these cells is not required, and transferring even high doses of purified CD4+ cells generally fails to cause GVHD. Nevertheless, in two of six combinations tested from this panel, namely B10.D2 → BALB/c and B10.D2 → DBA/2, purified CD4+ cells do cause a high incidence of lethal GVHD [90,100] (Table 14.1). This is also true in the B6 → BALB.B combination, in which CD4+ T cells are required for CD8mediated GVHD and can cause GVHD on their own account [98]. Why CD4+ cells mediate GVHD in only a limited number of mHA-different strain combinations is obscure. Insight into this problem has been generated from studying the B6 CD4+-mediated GVHD response in BALB.B versus CXBE recipients, where BALB.B, but not CXBE, mice succumb to lethal GVHD despite sharing multiple mHAs. A comparison of the responding CD4+ TCR Vβ repertoires from the thoracic duct lymph of irradiated BALB.B and CXBE recipients was made at the early stages of GVHD development (5–6 days) [101]. The spectratype analysis revealed overlapping utilization of nine Vβ families. In addition, the unique skewing of two Vβ families in the B6 → BALB.B response suggested the recognition of BALB.B mHAs not expressed in the CXBE recipients. Interestingly, the B6 → CXBE strain combination also exhibited unique skewing of two other Vβ families, which may hint at “immunodominance effects,” since one would have expected them to also be present in the B6 → BALB.B response. Direct evidence that Vβ families found skewed by spectratype analysis were involved in the pathogenesis of GVHD came from immunohistochemical staining of lingual epithelial tissue from BALB.B recipients, which revealed a high correlation between several of the skewed Vβ families and those with increased representation in the epithelial tissue infiltrates [101]. Further proof that the skewed Vβ families were indeed involved with GVHD was provided upon transplantation of those CD4+

Vβ families into irradiated BALB.B recipients, which resulted in a significant level of lethal GVHD. In contrast, mice that received transplants of the unskewed Vβ families survived with minimal symptoms of disease [101]. Applying the above observations concerning differences in responding Vβ repertoires, the capacity of B6 CD4+ T cells to induce lethal GVHD in BALB.B but not CXBE recipients was further examined. Transplantation of highly enriched populations of the two B6 CD4+ Vβ families, that were uniquely skewed in the B6 → BALB.B response, i.e. Vβ2 and Vβ11, were indeed able to induce lethal GVHD in BALB.B recipients. These data suggested that the BALB.B unique responses contribute in a way that either qualitatively or quantitatively enhances the severity of GVHD. Subsequent data suggested that these cells might preferentially infiltrate the small bowel of BALB.B recipients, thus increasing their importance to the systemic severity of GVHD (Fig. 14.3) [102]. Further studies revealed that the B6 Vβ11 CD4+ T cells, alone, could mediate severe GVHD, and immunohistochemical staining of host lingual and intestinal epithelial tissues supported their capacity to infiltrate typical GVHD-associated target areas [80]. To further characterize the specific CD4+ Vβ11+ T cells involved in this anti-mHA response, TCR Vβ spectratype analysis was performed, and indicated that six Vβ chains were used by this reactive population [80]. Probing deeper into this limited Vβ response, using a nonpalindromic adaptor polymerase chain reaction method, it was found that there was dominant use of the TRAV13–TRAJ16 transcript combination [103]. Then, using laser capture microdissection, the identical TRAV– TRAJ nucleotide sequence was found in areas dominated by the infiltrating Vβ11(+) CD4(+) T cells during the development of GVHD, both in the rete-like prominences of the dorsal lingual epithelium and the ileal crypts of the small intestine [103]. These combined results provided evidence that a restricted repertoire of T-cell specificities, presumably recognizing a correspondingly low number of mHAs, was sufficient for the induction of severe GVHD. In order to investigate whether the scope of the mHA-driven alloresponse changes during the development of GVHD, the responding B6 T-cell repertoire was examined between days 7 and 40 post HCT in the B6 → BALB.B strain combination. The results indicated that for the CD8 response, eight Vβ families were consistently skewed throughout the period of observation. It is interesting that skewing in four of those Vβ families had not been detected earlier in the thoracic duct population when examining the single day 5 time point [79]. In addition, skewing of three other Vβ families appeared to develop at later stages post transplant, which could indicate weaker anti-mHA responses, responses to antigens revealed via tissue destruction, or responses to nonalloantigen, such as to infectious agents. Similar findings were obtained for the CD4 analysis. Eight Vβ families were skewed throughout the course of GVHD development, all of which had been detected earlier in the thoracic duct lymph at day 6 post transplant [101]. Four additional Vβ families were transiently skewed at later time points. Furthermore, elimination of the eight consistently skewed Vβ families from the CD4+ T-cell donor inoculum delayed the onset in the appearance of most of the remaining four responsive Vβ families. The reason for this delayed appearance is unclear but would be consistent with them resulting from tissue damage, which would likely be slowed in the absence of dominant GVHD effector cells. These complex relationships in the development of anti-mHA GVHD are in obvious need of further investigation. One further aspect of the Vβ repertoire studies that remains unclear is the redundant capacity of a single mHA to drive the response of multiple Vβ families via cross-reactive TCR-binding sites. Insight gained into this issue will provide clues as to the minimum number of mHAs required to elicit GVHD in an MHC-matched combination and

Murine Models of Graft-versus-Host Disease and Graft-versus-Tumor Effect A

183

100

100

80 % survival

% survival

80

60

40

ATBM – MMB3.19 CD4+ T cells – MMB3.19 Vβ7+ CD4+ T cells –MMB3.19

60 40 20

20

0 0

20

30

40

50

60

70

Day

0 0

10

20

30

40

50

60

70

80

90

Days post HCT

Unseparated CD4→BALB.B (5) Vβ2/11–→BALB.B (9) Vβ2/11Enr→BALB.B (10) ATBM alone→BALB.B (7) Vβ2/11Enr→CXB-2 (4) ATBM alone→CXB-2 (4) B 110 Mean % initial body weight

10

100 90

80 70 60 0

2

4

6 8 Weeks post HCT

10

12

Fig. 14.3 GVHD potential of B6 anti-BALB.B Vβ2+ and 11+ CD4+ T cell responses. BALB.B and CXB-2 mice were lethally irradiated and injected with 2 × 106 B6 anti-thy1-depleted bone marrow (ATBM) cells alone, or in combination with either 2 × 107 unseparated B6 CD4+ T cells, an equal number of Vβ2- and 11-depleted (Vβ2/11−) CD4+ T cells, or 1.1 × 106 Vβ2and 11-enriched (Vβ2/11+) CD4+ T cells (mean purity 91%). (a) Survival of transplanted recipients. (BALB.B CD4 versus BALB.B Vβ2/11+; p ≥ 0.14). (b) Body weights for each group normalized as the mean ± standard error of the mean % initial body weight during sequential 1-week periods. HCT, hematopoietic cell transplantation. (Adapted from [84], with permission.)

why some mHA dominate the responses. Overall, the study of Vβ representation in a heterogeneously responding T-cell population in this acute GVHD model has begun to provide valuable insight into the scope of the allogeneic anti-mHA T-cell response.

Approaches for the separation of GVHD and graft-versus-leukemia responses The particular goal of any effort in this area is to provide a mechanism for reducing GVHD potential without sacrificing GVL or the more generalized graft-versus-tumor capability of donor T cells. Various

Fig. 14.4 Vβ7-enriched CD4+ T cells mediate a graft-versus-leukemia response with minimal induction of graft-versus-host disease in an allogeneic bone marrow transplantation model. Lethally irradiated (B6 × DBA/2)F1 mice were injected with 2 × 106 B6 anti-thy1-depleted bone marrow (ATBM) cells and 2.5 × 105 MMB3.19-primed Vβ7-enriched (82%) B6 CD4+ T cells or an equal number of unfractionated CD4+ T cells. The mice received MMB3.19 challenge (2 × 104; intraperitoneally) 1 day post bone marrow transplantation. Spectratype analysis had previously indicated that B6 Vβ7+ T cells were reactive to the MMB3.19 tumor cells, but not to stimulation by host (B6 × DBA/2)F1 alloantigen. (Adapted from [86], with permission).

strategies have been utilized to minimize GVHD in murine models, such as focusing on providing only CD8+ [104–107] or CD4+ [108, 90] T-cell subsets, with or without retention of GVL activity. The subset approach for separation of the two processes has also been studied for donor lymphocyte infusion models [109]. Restricting donor T cells to effector/memory subpopulations, either CD62L− or CD44hi, has also proved capable of preventing GVHD and often without impairment of GVL responses [110–113]. Likewise, and possibly related to the induction of memory cells, allogeneic sensitization of donor T cells to either leukemia cells or host cells can amplify GVL responses with reduced GVHD [114]. Results for all of these investigations may vary, depending upon the histoincompatibility involved and the specific tumor type used. Other approaches involve ex vivo tolerization of alloreactive T cells [115,116], separation of donor T cells based on functional phenotypes [117,118], and cytolytic effector mechanisms [119,120]. Cytokines, their promoters or their inhibitors have also been administered to tip the balance of donor T-cell responses towards GVL [121–127]. Establishing mixed chimerism with nonmyeloablative conditioning regimens has also been used as a means of reducing GVHD effects and allowing GVL responses with either donor-derived [128] or recipient-derived [129] delayed donor lymphocyte infusion. The alloreactive specificity of the donor T cells, as well as tissuespecific expression of host mHA or tumor-specific antigens, can also lead to a more targeted immunotherapeutic approach for minimizing GVHD development [130–132]. Vβ spectratype analysis has also been used to identify antihost alloreactive and antileukemia-specific responses and guide selected immunotherapy for the latter with GVHD minimization (Fig. 14.4) [133]. The high inverse correlation between the clinical incidence of GVHD and the rate of leukemic relapse has long suggested that antihost alloreactivity may provide the dominant GVL effect [134]. Therefore, strategies that would involve permitting the initiation of GVHD, presumably with an associated concurrent GVL response that could then be brought under control to avoid full pathologic development of disease, would be highly desirable. As mentioned earlier, several studies using CD4+CD25+ Treg or other suppressor type cells have shown some promise in accomplishing this goal [44,46–48].

184

Chapter 14

Immunopathology As for anti-H-2 GVHD, the effector mechanisms involved in GVHD to mHAs are still not fully understood. The histopathology of GVHD mediated by CD4+ cells and CD8+ cells is quite similar in the major target tissues of the skin, liver, and intestinal tract, although gut pathology is more prominent in recipients of CD4+ cells [96]. In the skin, epidermal cytotoxicity is induced by both subsets, albeit by different pathways, but dermal fibrosis seems to be a unique aspect of CD8-mediated GVHD. Mast cell degranulation occurs in the skin early after transplantation of either subset, even before lymphocyte infiltration is detected [135]. In addition, TNF-α (from either mast cells or T cells) does not appear to play a role in initial keratinocyte damage associated with early GVHD stages, but is involved in later CD4-mediated pathology, once the cells have infiltrated into the epidermal layers. In the case of CD8+ cells, it would seem that tissue damage, at least in the skin, reflects primarily direct CTL activity, although cytokines other than TNF-α may contribute to pathology. It has also been established that apoptosis has a central role in epithelial injury in acute GVHD [136], and that the primary targets of attack are phenotypically and antigenically distinctive epithelial cells within the basal layer of the skin and squamous mucosa [137,138], potentially corresponding to cytokeratin-15-expressing follicular stem cells (Plate 14.1) [139]. These cells are confined to rete ridges in the skin and

rete-like prominences in the dorsal tongue, and their discovery raises questions about their central role in driving the GVHD response forward. In all, the course and kinetics of pathogenesis in GVHD to mHAs may depend on several variables, including the strength of the individual CD4+ and CD8+ cell responses, the levels of expression of antigens in target tissue and the fluctuations of cytokines, both locally and systemically.

Summary Studies in murine models have provided many important insights into the pathophysiology of GVHD directed across MHC and/or mHA disparities. We now have a workable understanding of the T-cell subsets that can be involved in any given situation of GVHD and clues as to their mechanisms of action. These and future studies in the mouse can help guide us in the development of novel approaches to selectively reduce the risk of GVHD while allowing for the generation of GVL responses that can counteract the relapse of leukemia.

Acknowledgment The writing of this chapter was supported by grants HL55593 and HL75622 from the United States Public Health Service.

References 1. Ferrara JL, Levy R, Chao NJ. Pathophysiologic mechanisms of acute graft-vs.-host disease. Biol Blood Marrow Transplant 1999; 5: 347–56. 2. Ferrara J, Deeg JH, Burakoff SJ, editors. Graftvs.-Host Disease. New York: Marcel Dekker; 1997. 3. Korngold R. Biology of graft-vs.-host disease. Am J Ped Hematol/Oncol 1993; 15: 18–27. 4. Sprent J, Webb SR. Function and specificity of T-cell subsets in the mouse. Adv Immunol 1987; 41: 39–133. 5. Hedrick SM, Eidelman FJ. T lymphocyte antigen receptors. In: Paul WE, editor. Fundamental Immunology, 3rd edn. New York: Raven Press; 1993. pp. 383–420. 6. Shevach EM. Accessory molecules. In: Paul WE, editor. Fundamental Immunology, 3rd edn. New York: Raven Press; 1993. pp. 531–76. 7. Carbone FR, Bevan MJ. Major histocompatibility complex control of T-cell recognition. In: Paul WE, editor. Fundamental Immunology, 2nd edn. New York: Raven Press; 1989. pp. 541–70. 8. Sprent J. T lymphocytes and the thymus. In: Paul WE, editor. Fundamental Immunology, 3rd edn. New York: Raven Press; 1993. pp. 75–110. 9. von Boehmer H. Developmental biology of T cells in T-cell receptor transgenic mice. Annu Rev Immunol 1990; 8: 531–56. 10. Whitelegg A, Barber LD. The structural basis of T-cell allorecognition. Tissue Antigens 2004: 63: 101–8. 11. Sprent J, Schaefer M, Lo D, Korngold R. Properties of purified T-cell subsets. II. In vivo responses to class I vs. class II H2 differences. J Exp Med 1986; 163: 998–1011. 12. Shlomchik WD, Couzens MS, Tang CB et al. Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science 1999; 285: 412–15.

13. Matte CC, Liu J, Cormier J et al. Donor APCs are required for maximal GVHD but not for GVL. Nat Med 2004; 10: 987–92. 14. Teshima T, Ordemann R, Reddy P et al. Acute graft-versus-host disease does not require alloantigen expression on host epithelium. Nat Med 2002; 8: 575–81. 15. Korngold R, Sprent J. Features of T cells causing H2-restricted lethal graft-versus-host disease across minor histocompatibility barriers. J Exp Med 1982; 155: 872–83. 16. Jones SC, Murphy GF, Friedman TM, Korngold R. Importance of minor histocompatibility antigen expression by nonhematopoietic tissues in a CD4+ T cell-mediated graft-versus-host disease model. J Clin Invest 2003; 112: 1880– 6. 17. Sprent J, Korngold R. A comparison of lethal graft-versus-host disease to minor-versus-major differences in mice: implications for marrow transplantation in man. Prog Immunol 1983; 5: 1461–75. 18. Bennett M. Biology and genetics of hybrid resistance. Adv Immunol 1987; 41: 333–445. 19. Murphy WJ, Kumar V, Bennett M. Rejection of bone marrow allografts by mice with severe combined immunodeficiency (SCID): evidence that natural killer (NK) cells can mediate the specificity of marrow graft rejection. J Exp Med 1987; 165: 1212–17. 20. Bix M, Liao NS, Zijlstra M, Loring J, Jaenisch R, Raulet D. Rejection of class I MHC-deficient hemopoietic cells by irradiated MHC-matched mice. Nature 1991; 349: 329–31. 21. Jones M, Komatsu M, Levy RB. Cytotoxically impaired transplant recipients can efficiently resist major histocompatibility complex-matched bone marrow allografts. Biol Blood Marrow Transplant 2000; 6: 456–64.

22. Moller G, editor. Elimination of allogeneic lymphoid cells. Immunol Rev 1983; 73: 1–126. 23. Ruggeri L, Aversa F, Martelli MF, Velardi A. Allogeneic hematopoietic transplantation and natural killer cell recognition of missing self. Immunol Rev 2006; 214: 202–18. 24. Vallera DA, Soderling CCB, Kersey JH. Bone marrow transplantation across major histocompatibility barriers in mice. III. Treatment of donor grafts with monoclonal antibodies directed against Lyt determinants. J Immunol 1982; 128: 871–5. 25. Korngold R, Sprent J. Surface markers of T cells causing lethal graft-versus-host disease to class I vs. class II H2 differences. J Immunol 1985; 135: 3004–10. 26. Korngold R, Sprent J. Purified T-cell subsets and lethal graft-versus-host disease in mice. In: Gale RP, Champlin R, editors. Progress in Bone Marrow Transplantation. New York: Liss; 1987. pp. 213–18. 27. Cobbold S, Martin G, Waldmann H. Monoclonal antibodies for the prevention of graft-versus-host disease and marrow graft rejection. Transplantation 1986; 42: 239–47. 28. Piguet P-F. GVHR elicited by products of class I or class II loci of the MHC: analysis of the response of mouse T lymphocytes to products of class I and class II loci of the MHC in correlation with GVHR-induced mortality, medullary aplasia, and enteropathy. J Immunol 1985; 135: 1637– 43. 29. Blazar BR, Taylor PA, Snover DC, Sehgal SN, Vallera DA. Murine recipients of fully mismatched donor marrow are protected from lethal graft-versus-host disease by the in vivo administration of rapamycin but develop an autoimmunelike syndrome. J Immunol 1993; 151: 5726–41. 30. Vallera DA, Taylor PA, Panoskaltsis-Mortari A, Blazar BR. Therapy for ongoing graft-versus-host

Murine Models of Graft-versus-Host Disease and Graft-versus-Tumor Effect

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

disease induced across the major or minor histocompatibility barrier in mice with antiCD3F(ab′)2-ricin toxin A chain immunotoxin. Blood 1995; 86: 4367–75. Panoskaltsis-Mortari A, Lacey DL, Vallera DA, Blazar BR. Keratinocyte growth factor administered before conditioning ameliorates graftversus-host disease after allogeneic bone marrow transplantation in mice. Blood 1998; 92: 3960– 7. Sprent J, Schaefer M, Lo D, Korngold R. Function of purified L3T4+ and Lyt-2+ cells in vitro and in vivo. Immunol Rev 1986; 91: 195–218. Sprent J, Schaefer M, Korngold R. Role of T-cell subsets in lethal graft-versus-host disease (GVHD) directed to class I versus class II H2 differences. II. Protective effects of L3T4+ cells in anti-class II H2 differences. J Immunol 1990; 144: 2946– 54. Guy-Grand D, Vassalli P. Gut injury in mouse graft-versus-host reactions. J Clin Invest 1986; 77: 1584–95. Mowat AM, Sprent J. Induction of intestinal graftversus-host reactions across mutant major histocompatibility antigens by T lymphocyte subsets in mice. Transplantation 1989; 47: 857–63. Piguet PF, Grau GE, Allet B, Vassalli P. Tumor necrosis factor/cachectin is an effector of skin and gut lesions of the acute phase of graft-vs.-host disease. J Exp Med 1987; 166: 1280–9. Korngold R, Marini JC, de Baca ME, Murphy GF, Giles-Komar J. Role of tumor necrosis factoralpha in graft-versus-host disease and graftversus-leukemia responses. Biol Blood Marrow Transplant 2003; 9: 292–303. Vossen JM, Heidt PJ. Gnotobiotic measures for prevention of acute graft-versus-host disease. In: Burakoff SJ, Deeg HJ, Ferrara JLM, Atkinson K, editors. Graft-Versus-Host Disease: Immunology, Pathophysiology, and Treatment. New York: Marcel Dekker; 1990. pp. 403–13. Asano M, Toda M, Sakaguchi N, Sakaguchi S. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J Exp Med 1996; 184: 387–96. Kuniyasu Y, Takahashi T, Itoh M, Shimizu J, Toda G, Sakaguchi S. Naturally anergic and suppressive CD4+CD25+ T cells as a functionally and phenotypically distinct immunoregulatory T cell subpopulation. Int Immunol 2000; 12: 1145–55. Taylor PA, Lees CJ, Blazar BR. The infusion of ex vivo activated and expanded CD4(+)CD25(+) immune regulatory cells inhibits graft-versus-host disease lethality. Blood 2002; 99: 3493–9. Hoffmann P, Ermann J, Edinger M, Fathman CG, Strober S. Donor-type CD4(+)CD25(+) regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation. J Exp Med 2002; 196: 389–99. Cohen JL, Trenado A, Vasey D, Klatzmann D, Salomon BL. CD4(+)CD25(+) immunoregulatory T cells: new therapeutics for graft-versus-host disease. J Exp Med 2002; 196: 401–6. Jones SC, Murphy GF, Korngold R. Posthematopoietic cell transplantation control of graftversus-host disease by donor CD4+25+ T cells to allow an effective graft-versus-leukemia response. Biol Blood Marrow Transplant 2003; 9: 243–56. Johnson BD, Becker EE, LaBelle JL, Truitt RL. Role of immunoregulatory donor T cells in suppression of graft-versus-host disease following

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

donor leukocyte infusion therapy. J Immunol 1999; 163: 6479–87. Edinger M, Hoffmann P, Ermann J et al. CD4+CD25+ regulatory T cells preserve graftversus-tumor activity while inhibiting graftversus-host disease after bone marrow transplantation. Nat Med 2003; 9: 1144–50. Xia G, Truitt RL, Johnson BD. Graft-versusleukemia and graft-versus-host reactions after donor lymphocyte infusion are initiated by hosttype antigen-presenting cells and regulated by regulatory T cells in early and long-term chimeras. Biol Blood Marrow Transplant 2006; 4: 397–407. Trenado A, Charlotte F, Fisson S et al. Recipienttype specific CD4+CD25+ regulatory T cells favor immune reconstitution and control graftversus-host disease while maintaining graftversus-leukemia. J Clin Invest 2003; 112: 1688–96. Steiner D, Brunicki N, Blazar BR, Bachar-Lustig E, Reisner Y. Tolerance induction by third-party “off-the-shelf” CD4+CD25+ Treg cells. Exp Hematol 2006; 34: 66–71. Taylor PA, Panoskaltsis-Mortari A, Swedin JM et al. L-Selectin(hi) but not the L-selectin(lo) CD4+25+ T-regulatory cells are potent inhibitors of GVHD and BM graft rejection. Blood 2004; 104: 3804–12. Ermann J, Hoffmann P, Edinger M et al. Only the CD62L+ subpopulation of CD4+CD25+ regulatory T cells protects from lethal acute GVHD. Blood 2005; 105: 2220–6. Nguyen VH, Shashidhar S, Chang DS et al. The impact of regulatory T cells on T-cell immunity following hematopoietic cell transplantation. Blood 2008; 111: 945–53. Giorgini A, Noble A. Blockade of chronic graftversus-host disease by alloantigen-induced CD4+CD25+Foxp3+ regulatory T cells in nonlymphopenic hosts. J Leukoc Biol 2007; 82: 1053–61. Chen X, Vodanovic-Jankovic S, Johnson B, Keller M, Komorowski R, Drobyski WR. Absence of regulatory T-cell control of TH1 and TH17 cells is responsible for the autoimmune-mediated pathology in chronic graft-versus-host disease. Blood 2007; 110: 3804–13. Zeiser R, Nguyen VH, Beilhack A et al. Inhibition of CD4+CD25+ regulatory T-cell function by calcineurin-dependent interleukin-2 production. Blood 2006; 108: 390–9. Coenen JJ, Koenen HJ, van Rijssen E et al. Rapamycin, not cyclosporine, permits thymic generation and peripheral preservation of CD4+ CD25+ FoxP3+ T cells. Bone Marrow Transplant 2007; 39: 537–45. Baker J, Verneris MR, Ito M, Shizuru JA, Negrin RS. Expansion of cytolytic CD8(+) natural killer T cells with limited capacity for graft-versus-host disease induction due to interferon gamma production. Blood 2001; 97: 2923–31. Margalit M, Ilan Y, Ohana M et al. Adoptive transfer of small numbers of DX5+ cells alleviates graft-versus-host disease in a murine model of semiallogeneic bone marrow transplantation: a potential role for NKT lymphocytes. Bone Marrow Transplant 2005; 35: 191–7. Hashimoto D, Asakura S, Miyake S et al. Stimulation of host NKT cells by synthetic glycolipid regulates acute graft-versus-host disease by

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

185

inducing Th2 polarization of donor T cells. J Immunol 2005; 174: 551–6. Pillai AB, George TI, Dutt S, Teo P, Strober S. Host NKT cells can prevent graft-versus-host disease and permit graft antitumor activity after bone marrow transplantation. J Immunol 2007; 178: 6242–51. Ilan Y, Ohana M, Pappo O et al. Alleviation of acute and chronic graft-versus-host disease in a murine model is associated with glucocerebroside-enhanced natural killer T lymphocyte plasticity. Transplantation 2007; 83: 458–67. Nathenson SG, Geliebter J, Pfaffenbach GM, Zeff RA. Murine major histocompatibility complex class-I mutants. Molecular analysis and structure–function implications. Ann Rev Immunol 1986; 4: 471–502. Sprent J, Schaefer M, Gao E-K, Korngold R. Role of T-cell subsets in lethal graft-versus-host disease (GVHD) directed to class I versus class II H2 differences. I. L3T4+ cells can either augment or retard GVHD elicited by Lyt-2+ cells in class Idifferent hosts. J Exp Med 1988; 167: 556– 9. Simonsen M. Graft-versus-host reactions. Their natural history and applicability as tools of research. Progr Allergy 1962; 6: 349–467. Rolink AG, Pals ST, Gleichmann E. Allosuppressor and allohelper T cells in acute and chronic graft-vs.-host disease. II. F1 recipients carrying mutations at H2K and/or I-A. J Exp Med 1983; 157: 755–71. Gleichmann E, Pals ST, Rolink AG, Radaszkiewicz T, Gleichmann H. Graft-versus-host reactions. Clues to the etiopathology of a spectrum of immunological diseases. Immunol Today 1984; 5: 324–32. Rolink AG, Gleichmann E. Allosuppressor and allohelper T cells in acute and chronic graft-vs.host disease. III. Different Lyt subsets of donor T cells induce different pathological syndromes. J Exp Med 1983; 158: 546–58. Rolink AG, Radaszkiewicz T, Pals ST, van der Meer WGJ, Gleichmann E. Allosuppressor and allohelper T cells in acute and chronic graft-vs.host disease. I. Alloreactive suppressor cells rather than killer T cells appear to be the decisive cells in lethal graft-vs.-host disease. J Exp Med 1982; 155: 1501–22. van Elven EH, Rolink AG, van der Veen F, Gleichmann E. Capacity of genetically different T lymphocytes to induce lethal graft-versus-host disease correlates with their capacity to generate suppression but not with their capacity to generate anti-F1 killer cells. A non-H2 locus determines the inability to induce lethal graft-versus-host disease. J Exp Med 1981; 153: 1474–88. Via CS, Sharrow SO, Shearer GM. Role of cytotoxic T lymphocytes in the prevention of lupuslike disease occurring in a murine model of graft-vs.-host disease. J Exp Med 1987; 139: 1840–9. Wang W, Meadows LR, den Haan JM et al. Human H-Y: a male-specific histocompatibility antigen derived from the SMCY protein. Science 1995; 269: 1588–90. den Haan JM, Meadows LM, Wang W et al. The minor histocompatibility antigen HA-1: a diallelic gene with a single amino acid polymorphism. Science 1998; 279: 1054–7.

186

Chapter 14

73. Goulmy E. Minor histocompatibility antigens: from T cell recognition to peptide identification. Hum Immunol 1997; 54: 8–14. 74. Korngold R, Sprent J. Lethal graft-versus-host disease after bone-marrow transplantation across minor histocompatibility barriers in mice. Prevention by removing mature T cells from marrow. J Exp Med 1978; 148: 1687–98. 75. Hamilton BL, Bevan MJ, Parkman R. Antirecipient cytotoxic T lymphocyte precursors are present in the spleens of mice with acute graftversus-host disease due to minor histocompatibility antigens. J Immunol 1981; 126: 621–5. 76. Korngold R, Leighton C, Mobraaten LE, Berger MA. Inter-strain graft-versus-host disease T cell responses to immunodominant minor histocompatibility antigens. Biol Blood Marrow Transplant 1997; 3: 57–64. 77. Blazar BR, Roopenian DC, Taylor PA, Christianson GJ, Panoskaltsis-Mortari A, Vallera DA. Lack of GVHD across classical, single minor histocompatibility (miH) locus barriers in mice. Transplantation 1996; 61: 619–24. 78. Perreault C, Jutras J, Roy DC, Filep JG, Brochu S. Identification of an immunodominant mouse minor histocompatibility antigen (MiHA). T cell response to a single dominant MiHA causes graft-versushost disease. J Clin Invest 1996; 98: 622–8. 79. Friedman TM, Gilbert M, Briggs C, Korngold R. Repertoire analysis of CD8+ T cell responses to minor histocompatibility antigens involved in graft-versus-host disease. J Immunol 1998; 161: 41–8. 80. DiRienzo CG, Murphy GF, Jones SC, Korngold R, Friedman TM. T-cell receptor Valpha spectratype analysis of a CD4-mediated T-cell response against minor histocompatibility antigens involved in severe graft-versus-host disease. Biol Blood Marrow Transplant 2006; 12: 818–27. 81. Berger MA, Korngold R. Immunodominant CD4+ T cell receptor Vb repertoire involved in graftversus-host disease responses to minor histocompatibility antigens. J Immunol 1997; 159: 77–85. 82. Howell CD, Li J, Roper E, Kotzin BL. Biased liver T cell receptor Vb repertoire in a murine graft-versus-host disease model. J Immunol 1995; 155: 2350–8. 83. Brochu S, Baron C, Hetu F, Roy DC, Perreault C. Oligoclonal expansion of CTLs directed against a restricted number of dominant minor histocompatibility antigens in hemopoietic chimeras. J Immunol 1995; 155: 5104–14. 84. Perreault C, Roy DC, Fortin C. Immunodominant minor histocompatibility antigens: the major ones. Immunol Today 1998; 19: 69–74. 85. Korngold R, Wettstein PJ. Immunodominance in the graft-vs-host disease T-cell response to minor histocompatibility antigen. J Immunol 1990; 145: 4079–88. 86. Nevala WK, Wettstein PJ. H4 and CTT-2 minor histocompatibility antigens: concordant genetic linkage and migration in two-dimensional peptide separation. Immunogenetics 1996; 44: 400–4. 87. Malarkannan S, Shih PP, Eden PA et al. The molecular and functional characterization of a dominant minor H antigen, H60. J Immunol 1998; 161: 3501–9. 88. Malarkannan S, Horng T, Eden PA et al. Differences that matter: major cytotoxic T cellstimulating minor histocompatibility antigens. Immunity 2000; 3: 333–44.

89. Wettstein PJ. Immunodominance in the T-cell response to multiple non-H2 histocompatibility antigens. II. Observation of a hierarchy among dominant antigens. Immunogenetics 1986; 24: 24–31. 90. Korngold R, Sprent J. Variable capacity of L3T4+ T cells to cause lethal graft-versus-host disease across minor histocompatibility barriers in mice. J Exp Med 1987; 165: 1522–64. 91. Korngold R, Sprent J. Lethal GVHD across minor histocompatibility barriers: nature of the effector cells and role of the H2 complex. Immunol Rev 1983; 71: 5–29. 92. Jaffee BD, Claman HN. Chronic graft-versus-host disease (GVHD) as a model for scleroderma. I. Description of model systems. Cell Immunol 1983; 77: 1–12. 93. Ferrara J, Guillen FJ, Sleckman B, Burakoff SJ, Murphy GF. Cutaneous acute graft-versus-host disease to minor histocompatibility antigens in a murine model. Histologic analysis and correlation to clinical disease. J Invest Dermatol 1986; 123: 401–6. 94. Rappaport H, Khalil A, Halle-Pannenko O, Pritchard L, Dantcher D, Mathe G. Histopathologic sequence of events in adult mice undergoing lethal graft-vs-host reaction developed across H2 and/or non H2 histocompatibility barriers. Am J Pathol 1979; 96: 121–43. 95. Charley MR, Bangert JL, Hamilton BL, Gilliam JN, Sontheimer RD. Murine graft-versus-host skin disease. A chronologic and quantitative analysis of two histologic patterns. J Invest Dermatol 1983; 81: 412–17. 96. Murphy GF, Whitaker D, Sprent J, Korngold R. Characterization of target injury of murine acute graft-versus-host disease directed to multiple minor histocompatibility antigens elicited by either CD4+ or CD8+ effector cells. Am J Path 1991; 138: 983–90. 97. Korngold R. Lethal graft-versus-host disease in mice directed to multiple minor histocompatibility antigens. Features of CD8+ and CD4+ T-cell responses. Bone Marrow Transplant 1992; 9: 355–64. 98. Berger M, Wettstein PJ, Korngold R. T cell subsets involved in lethal graft-versus-host disease directed to immunodominant minor histocompatibility antigens. Transplantation 1994; 57: 1095–102. 99. Kwon B, Moon CH, Kang S, Seo SK, Kwon BS. 4-1BB: still in the midst of darkness. Mol Cells 2000; 10: 119–26. 100. Hamilton BL. L3T4-positive T cells participate in the induction of graft-vs-host disease in response to minor histocompatibility antigens. J Immunol 1987; 139: 2511–15. 101. Friedman TM, Statton D, Jones SC, Berger MA, Murphy GF, Korngold R. Vb spectratype analysis reveals heterogeneity of CD4+ T-cell responses to minor histocompatibility antigens involved in graft-versus-host disease: correlations with epithelial tissue infiltrate. Biol Blood Marrow Transplant 2001; 7: 2–13. 102. Jones SC, Friedman TM, Murphy GF, Korngold R. Specific donor Vbeta-associated CD4 T-cell responses correlate with severe acute graft-versushost disease directed to multiple minor histocompatibility antigens. Biol Blood Marrow Transplant 2004; 10: 91–105. 103. DiRienzo CG, Murphy GF, Friedman TM, Korngold R. T-cell receptor V(alpha) usage by

104.

105.

106.

107.

108.

109.

110.

111.

112.

113.

114.

115.

116.

117.

effector CD4+Vbeta11+ T cells mediating graftversus-host disease directed to minor histocompatibility antigens. Biol Blood Marrow Transplant 2007; 13: 265–76. Truitt RL, Atasoylu AA. Contribution of CD4+ and CD8+ T cells to graft-versus-host disease and graft-versus-leukemia reactivity after transplantation of MHC-compatible bone marrow. Bone Marrow Transplant 1991; 8: 51–8. Palathumpat V, Dejbakhsh-Jones S, Strober S. The role of purified CD8+ T cells in graft-versusleukemia activity and engraftment after allogeneic bone marrow transplantation. Transplantation 1995; 60: 355–61. Zhang C, Lou J, Li N et al. Donor CD8+ T cells mediate graft-versus-leukemia activity without clinical signs of graft-versus-host disease in recipients conditioned with anti-CD3 monoclonal antibody. J Immunol 2007; 178: 838–50. Fowler DH, Gress RE. CD8+ T cells of Tc2 phenotype mediate a GVL effect and prevent marrow rejection. Vox Sang 1998; 74(Suppl 2): 331–40. Suzuki Y, Adachi Y, Minamino K et al. A new strategy for treatment of malignant tumor: intrabone marrow-bone marrow transplantation plus CD4-donor lymphocyte infusion. Stem Cells 2005; 23: 365–70. Johnson BD, Becker EE, Truitt RL. Graft-vs.-host and graft-vs.-leukemia reactions after delayed infusions of donor T-subsets. Biol Blood Marrow Transplant 1999; 5: 123–32. Anderson BE, McNiff J, Yan J et al. Memory CD4+ T cells do not induce graft-versus-host disease. J Clin Invest 2003; 112: 101–8. Giver CR, Montes RO, Mittelstaedt S et al. Ex vivo fludarabine exposure inhibits graft-versushost activity of allogeneic T cells while preserving graft-versus-leukemia effects. Biol Blood Marrow Transplant 2003; 9: 616–32. Chen BJ, Cui X, Sempowski GD, Liu C, Chao NJ. Transfer of allogeneic CD62L- memory T cells without graft-versus-host disease. Blood 2004; 103: 1534–41. Zhang Y, Joe G, Zhu J et al. Dendritic cell-activated CD44hiCD8+ T cells are defective in mediating acute graft-versus-host disease but retain graft-versus-leukemia activity. Blood 2004; 103: 3970–8. Ji YH, Weiss L, Zeira M et al. Allogeneic cellmediated immunotherapy of leukemia with immune donor lymphocytes to upregulate antitumor effects and downregulate antihost responses. Bone Marrow Transplant 2003; 32: 495–504. Blazar BR, Taylor PA, Noelle RJ, Vallera DA. CD4(+) T cells tolerized ex vivo to host alloantigen by anti-CD40 ligand (CD40L:CD154) antibody lose their graft-versus-host disease lethality capacity but retain nominal antigen responses. J Clin Invest 1998; 102: 473–82. O’Shaughnessy MJ, Chen ZM, Gramaglia I et al. Elevation of intracellular cyclic AMP in alloreactive CD4(+) T cells induces alloantigen-specific tolerance that can prevent GVHD lethality in vivo. Biol Blood Marrow Transplant 2007; 13: 530–42. Fowler DH, Kurasawa K, Smith R, Eckhaus MA, Gress RE. Donor CD4-enriched cells of Th2 cytokine phenotype regulate graft-versus-host disease without impairing allogeneic engraftment in sublethally irradiated mice. Blood 1994; 84: 3540– 9.

Murine Models of Graft-versus-Host Disease and Graft-versus-Tumor Effect 118. Fowler DH, Breglio J, Nagel G, Eckhaus MA, Gress RE. Allospecific CD8+ Tc1 and Tc2 populations in graft-versus-leukemia effect and graftversus-host disease. J Immunol 1996; 157: 4811–21. 119. Hsieh MH, Varadi G, Flomenberg N, Korngold R. Leucyl-leucine methyl ester-treated haploidentical donor lymphocyte infusions can mediate graft-versus-leukemia activity with minimal graftversus-host disease risk. Biol Blood Marrow Transplant 2002; 8: 303–15. 120. Schmaltz C, Alpdogan O, Horndasch KJ et al. Differential use of Fas ligand and perforin cytotoxic pathways by donor T cells in graft-versushost disease and graft-versus-leukemia effect. Blood 2001; 97: 2886–95. 121. Sykes M, Harty MW, Szot GL, Pearson DA. Interleukin-2 inhibits graft-versus-host diseasepromoting activity of CD4+ cells while preserving CD4- and CD8-mediated graft-versus-leukemia effects. Blood 1994; 83: 2560–9. 122. Krijanovski OI, Hill GR, Cooke KR et al. Keratinocyte growth factor separates graft-versusleukemia effects from graft-versus-host disease. Blood 1999; 94: 825–31. 123. Hill GR, Teshima T, Gerbitz A et al. Differential roles of IL-1 and TNF-alpha on graft-versus-host disease and graft versus leukemia. J Clin Invest 1999; 104: 459–67. 124. Teshima T, Hill GR, Pan L et al. IL-11 separates graft-versus-leukemia effects from graft-versushost disease after bone marrow transplantation. J Clin Invest 1999; 104: 317–25. 125. Reddy P, Teshima T, Hildebrandt G et al. Pretreatment of donors with interleukin-18 attenuates acute graft-versus-host disease via STAT6 and preserves graft-versus-leukemia effects. Blood 2003; 101: 2877–85.

126. Korngold R, Marini JC, de Baca ME, Murphy GF, Giles-Komar J. Role of tumor necrosis factoralpha in graft-versus-host disease and graftversus-leukemia responses. Biol Blood Marrow Transplant 2003; 9: 292–303. 127. Durakovic N, Radojcic V, Powell J, Luznik L. Rapamycin promotes emergence of IL-10-secreting donor lymphocyte infusion-derived T cells without compromising their graft-versusleukemia reactivity. Transplantation 2007; 83: 631–40. 128. Pelot MR, Pearson DA, Swenson K et al. Lymphohematopoietic graft-vs.-host reactions can be induced without graft-vs.-host disease in murine mixed chimeras established with a cyclophosphamide-based nonmyeloablative conditioning regimen. Biol Blood Marrow Transplant 1999; 5: 133–43. 129. Rubio MT, Kim YM, Sachs T, Mapara M, Zhao G, Sykes M. Antitumor effect of donor marrow graft rejection induced by recipient leukocyte infusions in mixed chimeras prepared with nonmyeloablative conditioning: critical role for recipient-derived IFN-gamma. Blood 2003; 102: 2300–7. 130. Anderson LD Jr, Savary CA, Mullen CA. Immunization of allogeneic bone marrow transplant recipients with tumor cell vaccines enhances graft-versus-tumor activity without exacerbating graft-versus-host disease. Blood 2000; 95: 2426– 33. 131. Fontaine P, Roy-Proulx G, Knafo L, Baron C, Roy DC, Perreault C. Adoptive transfer of minor histocompatibility antigen-specific T lymphocytes eradicates leukemia cells without causing graftversus-host disease. Nat Med 2001; 7: 789–94. 132. Meunier MC, Delisle JS, Bergeron J, Rineau V, Baron C, Perreault C. T cells targeted against a

133.

134.

135.

136.

137.

138.

139.

187

single minor histocompatibility antigen can cure solid tumors. Nat Med 2005; 11: 1222–9. Patterson AE, Korngold R. Infusion of select leukemia-reactive TCR Vb+ T cells provides graftversus-leukemia responses with minimization of graft-versus-host disease following murine hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2001; 7: 187–96. Kernan NA. T-cell depletion for the prevention of graft-versus-host disease. In: Thomas ED, Blume KG, Forman SJ, editors. Hematopoietic Cell Transplantation, 2nd edn. Malden, MA: Blackwell Science; 1999. pp. 186–96. Murphy GF, Sueki H, Teuscher C, Whitaker D, Korngold R. Role of mast cells in early epithelial target cell injury in experimental acute graftversus-host disease. J Invest Dermatol 1994; 102: 451–61. Gilliam AC, Whitaker-Menezes D, Korngold R, Murphy GF. Apoptosis is the predominant form of epithelial target cell injury in acute experimental graft-versus-host disease. J Invest Dermatol 1996; 107: 377–83. Murphy GF, Lavker RM, Whitaker D, Korngold R. Cytotoxic folliculitis in acute graft-versus-host disease. Evidence of follicular stem cell injury and recovery. J Cutan Pathol 1991; 18: 309– 14. Sale GE, Beauchamp M. The parafollicular hair bulge in human GVHD. A stem cell rich primary target. Bone Marrow Transplant 1993; 11: 223– 5. Whitaker-Menezes D, Jones SC, Friedman TM, Korngold R, Murphy GF. An epithelial target site in experimental graft-versus-host disease and cytokine-mediated cytotoxicity is defined by cytokeratin 15 expression. Biol Blood Marrow Transplant 2003; 9: 559–70.

15

Megan Sykes

Mechanisms of Tolerance

Introduction Immune tolerance to a set of antigens can be defined as a state in which the immune system does not mount a destructive response to organs or tissues expressing those antigens but is capable of responding normally to foreign antigens. Tolerance may occur in several different immunologic states. Tolerance can be “operational,” in which, for example, a graft is accepted without immunosuppressive therapy but a second graft from the same donor is rejected. In a slightly more robust form of tolerance, a second graft of the same type from the same donor would be accepted, but a more immunogenic graft from that donor would be rejected. In an even more robust type of tolerance, any organ bearing the same set of alloantigens as the original graft would be accepted. Finally, tolerance may be systemic, meaning that the animal’s entire immune system is tolerant to that set of antigens, so that a response cannot be induced to them either in vivo or in vitro. In allogeneic hematopoietic cell transplantation (HCT), persistent graft survival and the absence of graft-versus-host disease (GVHD) depend on a state of mutual tolerance of the donor to the host (graftversus-host, or GVH, tolerance), and of the host to the donor (hostversus-graft, or HVG, tolerance). Unfortunately, this state is not always achieved, and GVHD or failure of engraftment, respectively, ensues. Development of tolerance in the HVG direction requires host conditioning that either eliminates mature host immune cells, creating an immunologic “clean slate,” or permits pre-existing T cells to be rendered tolerant by the donor hematopoietic cells. Since newly developing T and B lymphocytes have a unique capacity to be rendered tolerant by antigens they encounter during their maturation, especially on hematopoietic cells, these lymphocytes can be educated to recognize an engrafted donor and host antigens as “self,” so that a state of donor- and hostspecific tolerance results. The ability of hematopoietic stem cells (HSCs) to induce a state of donor-specific tolerance suggests an additional potential application for HCT, namely the induction of organ allograft acceptance without the need for chronic immunosuppressive therapy. Such a state of immune tolerance would circumvent several major limitations to the practice of organ and tissue transplantation. In particular, improvements in immunosuppressive therapy have greatly augmented early graft survival, but these have had little impact on late graft loss, which is due in large part

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

188

to chronic rejection. Moreover, a high incidence of malignancies and opportunistic infections as well as drug-specific metabolic and other toxicities severely limit the tolerability of long-term chronic immunosuppressive therapy. The induction of donor-specific immune tolerance would avoid these complications while also preventing chronic rejection. Reliable, nontoxic methods of achieving allogeneic HSC engraftment across major histocompatibility complex (MHC) barriers will be required before HCT can be routinely used for the induction of organ or tissue allograft tolerance. However, recent progress in experimental systems has brought this approach into pilot clinical trials. A shortage of allogeneic donors has become another major limitation to solid organ transplantation, resulting in considerable interest in xenotransplantation (i.e. transplantation of organs from other species) to augment organ and tissue availability. However, it has become increasingly clear that the immunologic barriers to xenografts are even stronger than those resisting allografts, making it unlikely that an acceptable amount of nonspecific immunosuppressive therapy could be used to prevent xenograft rejection. Thus, it will be especially critical to develop methods of inducing donor-specific tolerance across xenogeneic barriers in order to make xenotransplantation efficacious. Because of its demonstrated efficacy in experimental models, and because of recent developments in our understanding of the mechanisms by which HCT can induce allo- and xenotolerance, this approach may have the greatest potential to make tolerance induction a routine part of clinical organ transplantation. This chapter will first provide a general discussion of mechanisms of T-cell tolerance, and then discuss these mechanisms in the context of specific models associated with induction of HVG tolerance in the absence or presence of HCT. The mechanisms by which both mature T cells pre-existing in allogeneic HCT inocula as well as T cells developing de novo from donor hematopoietic cells can be rendered tolerant of host antigens will also be reviewed.

Mechanisms of T-cell tolerance Three major mechanisms have been proposed to explain the induction and/or maintenance of T-cell tolerance to self or alloantigens: clonal deletion, clonal anergy, and active suppression. For developing T cells, these processes take place in the thymus, which is the central organ for T-cell development. Hence, induction of tolerance among developing thymocytes is referred to as “central,” as distinguished from the “peripheral” tolerance that may develop among already mature T cells when they encounter antigen in the peripheral tissues. It will become apparent to the reader from the discussion that follows that overlapping

Mechanisms of Tolerance

mechanisms have been implicated in models involving both central and peripheral tolerance induction. Clonal deletion T-cell receptors (TCR), consisting of uniquely rearranged α and β chains, known as αβTCR, are the cell surface heterodimers that recognize complexes of MHC antigens plus peptide. The thymus plays a critical role in the development of tolerance by a clonal deletion mechanism. Deletion occurs during T-cell maturation when the avidity of an interaction between an immature thymocyte and an antigen-presenting cell (APC) in the thymus is sufficiently high to induce apoptotic cell death [1]. The concept of avidity includes variations in the intensity of T-cell signaling resulting from variations in TCR affinity and density and ligand density, and from interactions of accessory molecules with their ligands on APCs. High avidity of the overall T cell–APC interaction is due, at least in part, to a relatively high affinity interaction between a rearranged αβTCR and a self-peptide–MHC complex presented by an APC in the thymus. Several marrow-derived cell types, including dendritic cells (DCs), B cells, and thymocytes, as well as nonhematopoietic cells of the thymic stroma, have the capacity to induce intrathymic tolerance by both deletional and nondeletional mechanisms, but DCs may be the most potent deletional APCs in the thymus (reviewed in [2]). Evidence for clonal deletion as a mechanism of tolerance was originally obtained by using monoclonal antibodies (mAbs) that are specific for certain V-gene products contributing to the β chain of the αβTCR [3]. Certain Vβ gene products selectively recognize “superantigens” encoded by endogenous retroviruses in the genome of mammalian species. Superantigens bind to a unique site on the class II MHC molecule. If a particular Vβ is capable of recognizing a superantigen, developing thymocytes with TCR using these Vβ undergo deletion [3]. These observations allowed the original demonstration that self-tolerance occurs largely through an intrathymic deletional mechanism, and that tolerance to marrow donors also occurs through a similar mechanism when allogeneic bone marrow transplantation (BMT) is performed in mice [3]. Later data supporting clonal deletion as a major mechanism for induction of self-tolerance utilized TCR transgenic mice expressing TCRs specific for antigens expressed naturally in the animal or by bone marrow grafts. Deletion is not unique to immature stages of T-cell development. Peripheral deletion has been described for mature T cells upon exposure to antigen in vivo [4]. Activation-induced cell death, which leads to partial deletion of expanded T-cell clones, is a normal physiologic consequence of high levels of T-cell activation. Deletion is also the natural consequence of cross-presentation by lymph node DCs of self-antigen expressed in parenchymal tissues to CD8+ cytotoxic lymphocytes (CTLs), under noninflammatory conditions [5]. Peripheral CD8 cells may undergo clonal deletion through “exhaustion” in the presence of a large, persistent antigen load, which may explain the anergy and deletion observed for host-reactive CTLs in the context of GVHD [6]. As discussed below, exposure to alloantigens in the presence of co-stimulatory blockade can lead to peripheral deletion of alloreactive T cells by mechanisms that are incompletely understood. In addition, veto cells and “double-negative” T cells (see below) can delete alloreactive CTL precursors in the peripheral tissues. T-cell anergy Studies on mechanisms of tolerance often include functional data, such as limiting dilution analyses to quantify CTLs, helper T cells or T cells proliferating in response to antigen. However, the failure to detect T

189

cells with a particular allospecificity using this approach does not distinguish between clonal deletion and functional inactivation, or so-called “anergy.” Anergy may result when T cells encounter antigens without receiving adequate accessory or co-stimulatory signals. Anergic lymphocytes cannot be fully activated by encounter with the antigen that its clonally distributed surface receptor (surface immunoglobulin [Ig] in the case of the B-cell receptor, and the TCR in the case of the T cell) specifically recognizes. In the case of T cells, anergy is associated with a lack of proliferation and interleukin-2 (IL-2) production, and can usually, but not always, be overcome by providing exogenous IL-2. T-cell anergy is associated with altered signaling and tyrosine phosphorylation patterns [7]. Numerous methods of inducing T-cell anergy in vitro have been described, many of which involve blocking of molecular interactions between T-cell surface molecules and their ligands on APCs. Some of these interactions are termed “co-stimulatory signals” because they provide signals to the responding T cell, other than those transmitted through the TCR–CD3 complex, that are essential to obtaining a full state of activation [7,8]. These molecules, including CD28, inducible co-stimulator, OX40, 4-1BB, and several additional co-stimulatory pathways, have been targets of attempts to induce tolerance by presenting allo- or xenoantigens without adequate co-stimulation (see below). T-cell anergy has, in some instances, been associated with TCR downregulation [4,9], and a similar phenomenon has been associated with B-cell anergy [10]. In addition to the encounter of antigen without co-stimulation, T cells may also be rendered anergic by an encounter with peptide ligands for which they have low affinity [1]. An overall low avidity interaction between a T cell and an APC may also result in anergy [11]. It appears that, in addition to mature peripheral T cells, thymocytes are also susceptible to anergy induction, and this can be induced by antigens presented on both hematopoietic and nonhematopoietic stromal [12] cells. Anergy can be reversed in vivo and can be overcome by infection or by removal of antigen. Anergy can be induced in vivo by nonhematopoietic cells. In hematopoietic chimeras prepared with recipient myeloablation followed by allogeneic BMT, all marrow-derived cells are of donor origin, so that clonal deletion of thymocytes recognizing host-specific antigens is incomplete [12]. T cells bearing these host-reactive TCRs can be anergized by thymic epithelial cells [13] or by antigens encountered extrathymically [14–16]. T-cell clones with specificity for particular alloantigens can exist without causing destruction of tissue bearing those antigens [11,13,14,16]. In some instances, T cells with receptors specific for the in vivo tolerated antigen can be stimulated to respond to the antigen in vitro [13,14,17] or may even induce a state of inflammation that does not lead to organ destruction in the absence of additional stimuli [11]. In other systems, the T cells are fully or partially refractory to stimulation through their TCRs in vitro [11,14]. However, anergy is not the only mechanism that can account for the failure of T cells with self-reactive or alloreactive TCRs to cause autoimmunity or graft rejection, respectively. In the situation where T cells respond to antigens in vitro but not in vivo, they seem to be able simply to ignore antigens presented in the periphery [9]. This may be due to the presentation of these antigens by “nonprofessional APCs” that are unable to activate T cells, or it may reflect a failure of recipient T cells to migrate to the antigen-bearing tissue [18]. The level of peripheral antigen expression, the recency of T-cell emergence from the thymus [9], and the presence or absence of proinflammatory cytokines and costimulatory molecules within peripheral tissues may all influence the decision of a T cell to ignore or respond to peripheral antigens. T-cell activation may even occur within a tissue without initiating the whole cascade of organ damage [11]. However, proinflammatory stimuli, as

190

Chapter 15

may be present due to infection, may overcome such precarious states [9]. Some experimental models in which transplantation tolerance has been induced include T-cell anergy in association with a persistent suppressive T-cell population [19]. However, in most of these models, including those involving co-stimulatory blockade and other approaches designed to render APCs non-costimulatory, anergy has not been shown clearly to be the sole mechanism maintaining tolerance in vivo. Suppression A third mechanism, active suppression, has also been implicated in the induction and maintenance of self- and allotolerance. Suppressive activity has been attributed to both T cells and non-T cells, and may be antigen-specific or nonspecific. In general, the models in which suppressor T cells have been implicated involve exposure to antigen in the presence of a pre-existing immune response to the antigen. When, on the other hand, all mature lymphocytes are eliminated prior to administration of the antigen, tolerance may be due mainly to clonal deletion or anergy mechanisms and not to active suppression [20]. In the ensuing discussion, we categorize mechanisms of suppression in terms of the degree of specificity of their ability to suppress immune responses. Specific suppression While functional evidence for the existence of specific suppressor cells had been obtained in several transplantation models [21,22], until the last decade there had been little success in identifying or cloning suppressive cells, or in understanding the mechanisms by which they suppressed. In early studies, anti-idiotypic recognition, which is the recognition of determinants specifically expressed by a rearranged T cell or Ig receptor, was implicated [21,22]. However, apparent idiotypic specificity can be difficult to distinguish from suppression by T cells with the same specificity as the T cell being suppressed. The importance of regulatory phenomena in self-tolerance and regulation of autoimmunity, and in certain transplantation models, has recently become very clear. Some of these suppressive cell types are briefly described in the ensuing paragraphs. T cells comprise several major subsets of regulatory cells. Regulatory T cells (Tregs) can be classified as “naturally arising” and “antigen driven” [23,24]. Naturally arising Tregs are generated in the postnatal thymus and constitutively express CD25 (IL-2 receptor α chain) as well as CTLA4 [25]. A deficiency in “Treg” numbers in neonatally thymectomized mice permits the development of multiorgan autoimmune disease in susceptible strains [25]. Tregs require nonhematopoietic thymic stromal cells for their selection, and expression of the forkhead winged helix transcription factor 3 (FoxP3) is essential for full differentiation and maintenance of their regulatory cell features, including failure to produce IL-2 and dependence on paracrine IL-2 [26]. While it has been suggested that Tregs are positively selected on cortical epithelial cells of the thymus [27], recent studies suggest that the thymic medulla may be the major site of their positive selection [28]. Once in the periphery, these Tregs can be expanded in the presence of the positively selecting peptide antigen [29]. While some studies have suggested that the TCR repertoire of Tregs leaving the thymus is skewed toward greater affinity for self-antigens than that of conventional T cells, recent studies challenge this conclusion and suggest that the repertoire and specificities of both subsets may be quite similar [30]. Tregs play an important role in preventing autoimmune disease throughout adult life, as illustrated by the development of severe autoimmunity when FoxP3+ cells are selectively depleted in vivo. Their importance in preventing autoimmunity in humans is illustrated by congenital FoxP3 deficiency, which results in immune dysregulation, polyendocri-

nopathy, enteropathy, X-linked (IPEX) syndrome [31]. The regulatory cells are themselves hyporesponsive to TCR-mediated stimulation alone, and require IL-2 for their development and expansion [32]. IL-15 can also promote their expansion [33]. Transforming growth factor-β (TGFβ) also plays an important role in maintenance of their function via maintenance of FoxP3 expression [26]. Suppression seems to require cell-to-cell contact [25]. CD25+CD4+ Tregs express the memory T cellassociated CD45 isoform (CD45RC+ in the rat, CD45RBlow in the mouse). Although CD4 cells have been the best-studied targets of regulation by these cells, they can also suppress CD8 T-cell reactivity. While the Tregs show specificity for the antigen to which they are inducing suppression, the effector mechanism of suppression is non-antigen specific [25]. The immunosuppressive drug rapamycin appears to allow Treg survival and expansion while suppressing effector T-cell responses [34]. Antigen-driven Tregs include murine and human CD4 cells stimulated through their TCRs in the presence of IL-10. These “Tr1” cells produce high levels of IL-10 and TGF-β without making IL-4, distinguishing them from T helper cells type 2 (Th2) [35]. These cells, which do not constitutively express FoxP3, suppress antigen-specific responses in vitro and can suppress inflammatory bowel disease syndrome induced by naïve CD4 cells in mice. Immature or specialized IL-10-secreting DCs promote Tr1 differentiation [35]. In addition to Tr1 cells, antigen exposure can also result in the differentiation of CD25−CD4 cells to FoxP3+ Tregs [36–38]. This differentiation appears to be critically dependent on TGF-β and is inhibited by the presence of IL-6, which, in combination with TGF-β, promotes the differentiation of proinflammatory IL-17-producing Th17 effector cells. Recent studies showing that the transcription factor nuclear factor of activated T cells (NFAT), which has long been known to interact with the activating transcription complex activator protein-1 upon TCR stimulation, can alternatively interact with FoxP3 to turn on the “Treg” program that includes CD25 and CTLA4 upregulation, suppression of IL-2 production and suppressive function [39]. Thus, the choice of transcription partner by NFAT may determine the regulatory versus effector differentiation pathway of a T cell. It has previously been suggested that T-cell polarization to the Th2 subset that secretes IL-4 and IL-10 could promote allograft acceptance [40]. For practical purposes, an immune response that is itself nondestructive and inhibits the development of destructive Th1 responses could provide a powerful means of ensuring graft acceptance. However, only limited data exist to implicate an active role for Th2 cells in tolerance induction [41,42]. It is clear that Th2 responses are not always benign, as Th2 cells and Th2-associated cytokines can mediate or contribute to allograft rejection [43,44]. The original T cells associated with a suppressive network were CD8+. Recently, several groups have reported that FoxP3+ CD8 cells, which may also express CD25, can mediate suppressive activities [37]. A subset of suppressive human CD8+CD28− T cells that upregulate the inhibitory ligands immuoglobulin-like transcript-3 and -4 expressed on APCs, rendering them tolerogenic, has also been described [45]. NKT cells (T cells that express natural killer [NK] cell-associated markers and may utilize an invariant TCR-α chain) are another subset of T cells with regulatory activity, which may be mediated in part by Th2-type cytokines. These cells have been reported to suppress autoimmune diseases, apparently by suppressing interferon-γ (IFN-γ) production via an IL-4-dependent mechanism. The role of these cells in various transplantation models is discussed below. A double-negative, NK1.1− T-cell population that suppresses skin graft rejection by CD8 T cells with the same TCR has been described in a TCR transgenic mouse model [46], but the importance of this cell population in the setting of polyclonal T-cell responses remains to be determined.

Mechanisms of Tolerance

Veto cells Alloresponses can be downregulated by “veto” activity, which may be mediated by several different T-cell types. “Veto” cells inactivate CTLs recognizing antigens expressed on the veto cell surface [47]. Several types of cell have been reported to have veto activity, including CTLs [47] and various bone marrow subpopulations, including CD34+ cells and their progeny [48]. CD34 cells mediate this activity through a tumor necrosis factor-α (TNF-α)-dependent mechanism, whereas CD8 T cells may act through a Fas–Fas ligand-dependent pathway [49]. Veto activity has also been ascribed to marrow cells with characteristics of NK or lymphokine-activated killer cells, which can be used to promote GVH tolerance and facilitate allogeneic marrow engraftment in murine BMT models [50]. Murine studies also suggest that veto cells might contribute to the donor-specific transfusion (DST) effect [51], wherein prior administration of donor blood induces subsequent hyporesponsiveness to that donor. One suggested mechanism for the veto effect involves triggering of a CTL through a surface class I MHC molecule while it is also activated through its TCR, resulting in apoptosis of the responding CTL [52]. This class I-mediated signal could result from binding of a T-cell’s class I molecules to CD8 molecules on the veto cell. However, not all veto cells express CD8, so this mechanism cannot explain all veto phenomena. Veto activity has been also suggested to involve the immunosuppressive cytokine TGF-β [53]. Other suppressive cell populations Non-antigen-specific suppressive activity has been previously attributed to “natural suppressor” cells, which were originally described as suppressive non-T, non-B cells that were detected in sites of hematopoiesis after BMT, in lymphoid tissues in the presence of GVHD, and in normal bone marrow [54]. Such suppressive populations proved to be heterogeneous, including NKT cells [55,56] and hematopoietic progenitors [57]. Overall, the abundance of suppressive populations results in a strong inhibitory effect of bone marrow on immune responses in vitro [54,58]. Suppressive populations found in lymphoid tissues in association with GVH responses include a myeloid progenitor cell population whose activity depends on IFN-γ and nitric oxide [59]. Suppressive myeloid progenitor cells have also been detected in the setting of sepsis and traumatic stress, and immature myeloid cells have been associated with cancer-induced immunosuppression in humans and mice. Other cell types with the capacity to downregulate T cell responses, such as mesenchymal stem cells and specially manipulated DCs, are discussed in more detail elsewhere in this textbook. Thus, while a plethora of phenotypes and activities for regulatory cells has been described in recent years, much remains to be learned about the relative importance of each of these and the circumstances in which they can be optimally generated and utilized to improve transplant outcomes.

Mechanisms of B-cell tolerance The above discussion has focused mainly on the induction of T-cell tolerance. If T-cell tolerance is achieved, T-cell-dependent antibody responses do not occur. In this setting, tolerance of mature B cells may occur via a deletional process associated with lack of T-cell help [60]. However, T-cell-independent B-cell responses are also important in settings where natural antibodies (NABs) are present (i.e. antibodies that are present in the absence of known antigen exposure), such as in ABO blood group-mismatched transplantation and xenotransplantation. Several mechanisms of B-cell tolerance have been described. Autoreactive developing B cells undergo developmental arrest, and recombination activating gene-dependent light-chain receptor editing then occurs. Formation of a non-autoreactive Ig receptor allows the B cell to survive;

191

if such a rearrangement does not occur, the B cell undergoes apoptosis [61]. Encounter of immature B cells with soluble antigen expressed at relatively low levels leads to B-cell anergy [10]. The induction of B-cell anergy versus activation may be dependent on antigen concentration [10]. Antigen expressed only on cell membranes in the periphery has, in some instances, been associated with a deletion of B cells [10,62]. Anergy is a major mechanism of self-tolerance of newly formed B cells [63]. However, interaction with membrane-bound self-antigen can activate anergic B cells, breaking their tolerance [64]. Cells of the B-1 subset, which produce NABs responsible for xenograft hyperacute rejection [65], may undergo apoptosis when their surface Ig is crosslinked by cell-bound antigen [66].

Approaches to inducing HVG transplantation tolerance The potential benefits of inducing HVG tolerance in the setting of organ transplantation were described in the introductory section. To recapitulate briefly, these benefits include the avoidance of chronic immunosuppressive therapy, the prevention of chronic rejection, and facilitation of the ability to use xenogeneic (i.e. from other species) organ and tissue sources, such as the pig. Thus, induction of HVG tolerance has become a major goal of research in transplantation. In this section, we will discuss some experimental approaches to inducing tolerance and will divide the discussion into efforts to induce peripheral tolerance versus those aimed at achieving HSC engraftment and central T-cell tolerance. However, it should become apparent by the end of the discussion that tolerance cannot always be readily characterized as peripheral versus central, and that a combination of central and peripheral tolerance mechanisms may ultimately prove to be most practical and effective for the induction of HVG tolerance. Approaches to peripheral tolerance induction Tolerance of pre-existing, peripheral T cells has been induced in numerous animal models, often by mechanisms that are not fully understood. Evidence for anergy has been obtained in a few models [67], and recently, a role for regulatory cells has been found in many (see below). Peripheral deletion of donor-reactive T cells has been implicated [68] and directly demonstrated [69–72] in some models. It is important to note that many of the strategies for inducing tolerance to primarily vascularized rodent allografts such as hearts and kidneys rely heavily on the capacity of the organ graft itself to control the immune response, and that the tolerance thereby achieved is not systemic. Most of these tolerance protocols are not effective when tested with less “tolerogenic” grafts, such as primary skin allografts across full MHC barriers. Strategies that are effective for primarily vascularized allografts in rodents are often ineffective in large animals and man. Thus, a thorough understanding of the mechanisms involved in tolerance induction and successful extension to large-animal models are important criteria to fulfill before these approaches can be attempted in humans as a substitute for chronic immunosuppressive therapy. Some experimental approaches to tolerance induction are summarized below. DST, modified APCs or administration of autologous cells transduced with allogeneic MHC genes In rodents, infusion of donor leukocytes around the time of transplant can lead to graft prolongation and, in some cases, operational tolerance [73]. In other species, DST has been less effective as the only graftprolonging therapy, but it has shown beneficial effects, even in humans [74]. However, infusion of donor cells is also associated with the risk of sensitizing the recipient to donor alloantigens. Moreover, improve-

192

Chapter 15

ments in graft outcomes with modern immunosuppression, combined with the ability to treat uremia-associated anemia with erythropoietin instead of transfusion, have ended the practice of transfusion prior to transplantation. B cells and T cells in DST, both of which can present antigen in a non-costimulatory manner, may contribute to their immunomodulatory effect [75]. Administration of autologous bone marrow transduced with a single donor MHC antigen promotes tolerance to other antigens expressed by the cardiac or renal allograft donor in mice [76] and miniature swine models, respectively [77]. Several mechanisms of tolerance have been implicated in murine DST recipients, including veto activity leading to deletion of donor-reactive T cells [51], regulatory CD4 cells [78], and a role for the Th2 cytokines IL-4 [79] and IL-10 [78]. The extension of tolerance in these models from the antigens transduced into autologous cells to additional antigens expressed on the donor organ is a form of “linked suppression” (see below). The introduction of alloantigen on tolerogenic APCs can contribute to the acceptance of allografts [80]. Stimulation with immature DCs, or with DCs modified in certain ways, for example with cytokines [81], with transduced genes that promote T-cell death or tolerance [82] or with rapamycin [83], can favor the activation of Tregs over effector T cells. Attempts to intentionally immunize with “tolerogenic” DCs have been associated with prolonged heart, islet, and kidney allograft survival in rodent models, usually without permanent survival [84]. The combination of rapamycin-treated DCs and a short course of posttransplant rapamycin has recently permitted long-term survival of vascularized cardiac allografts in mice [83]. Thus, both mature and immature DCs, under various conditions, can favor graft acceptance. The mechanisms by which DCs promote unresponsiveness after differentiation under various conditions vary. Indoleamine 2,3 dioxygenease is an enzyme that catalyses tryptophan catabolism. Its production is induced by a signal transducer and activator of transcription-1 (STAT1)-dependent signal mediated by IFN-γ, which may, paradoxically, be produced by Tregs [85], which thereby promote DC-induced tryptophan depletion [86]. Plasmacytoid (type I IFN-producing) DCs have been implicated in the generation of IL-10-producing Tregs [87] via a mechanism associated with plasmacytoid DC expression of the costimulatory molecule inducible costimulator [88]. Costimulatory blockade, with or without infusion of donor lymphocytes Additional signals besides those resulting from TCR stimulation are required for T-cell activation. This costimulatory signal, or “signal 2,” must be provided to prevent the development of T-cell anergy [7]. While the B7 (CD80, CD86)–CD28 costimulatory interaction was the first to be defined, it is now known that multiple molecular pathways may costimulate T cells, many through Ig and TNF receptor family members [89]. Efforts to induce tolerance by blocking the B7–CD28 co-stimulatory pathway have enjoyed success in rodents receiving vascularized allografts under cover of CTLA4Ig [90], a fusion protein containing the B7-binding portion of CTL antigen-4 (CTLA4), a high-affinity T-cell ligand for B7. CTLA4Ig blockade of CD28 binding to both CD80 and CD86, combined with the tolerogenic capacity of primarily vascularized organ allografts in rodents, can lead to long-term graft acceptance. Signaling through the interaction of CD40 on APCs and B cells with its T-cell ligand (CD40L, CD154, which is expressed transiently on activated T cells) plays an important role in activating APCs so that they can optimally co-stimulate T cells. Signaling through CD40 leads to upregulation of B7 expression, as well as other costimulatory molecules, class II MHCs, and cytokines such as IL-12, and induces Ig class switching by B cells. This APC activation via CD40 is a major facet of CD4 T cell-mediated “help” for CTL generation [91]. Administration of

DST under cover of anti-CD154 mAb can prevent islet allograft rejection [92]. In thymectomized mice, the combination of DST and antiCD154 permits long-term skin graft survival in fully MHC-mismatched recipients, but this approach is less successful in euthymic mice [93]. DST in combination with anti-CD154 permits deletion of peripheral donor-reactive CD8 T cells [94]. Although CD4+ regulatory cells promote CD8 tolerance in this model [95], they are of insufficient potency or duration to prevent rejection by donor-reactive new thymic emigrants [94]. Xenogeneic skin graft prolongation is also observed with DST and anti-CD40L [96]. Cardiac allograft survival is markedly prolonged in the presence of anti-CD40L [97], but chronic graft arteriosclerosis still occurs [98]. Graft prolongation with anti-CD40L or CD28 blockade has been associated with a Th2-dominant immune response [99], which may promote graft arteriosclerosis [98]. The combination of CTLA4Ig and anti-CD40L antibody can markedly prolong the survival of primary skin allografts [100], renal allografts in large animals [101], and xenografts [102], but without inducing tolerance. Functional tolerance has been achieved with this combination only in rodent models of cardiac or islet allografts [103] or islet xenografts [104], but not in the more stringent models of primary skin grafting or large-animal vascularized organ or islet allografts [105–107]. Linked suppression and “infectious tolerance,” in which tolerant CD4+ T cells can render naïve T cells unresponsive in secondary recipients, have been demonstrated in several models involving co-stimulatory blockade. These phenomena are largely attributable to Tregs [108]. Costimulatory blockade more effectively suppresses CD4-mediated than CD8 cell-mediated alloreactivity [105,109], although the extent of CD8 cell-mediated resistance is genetically determined. A short course of rapamycin with anti-CD40L and CTLA4Ig, has, quite remarkably, permitted long-term acceptance of primary skin allografts across full MHC barriers in mice in certain strain combinations [110]. The combination of rapamycin, anti-CD154, and DST has also led to markedly prolonged islet allograft survival in non-human primates [111]. The beneficial effect of rapamycin may relate to its ability to allow the selective expansion of Tregs [34,112]. A recent protocol involving the combination of rapamycin, cytolytic IL-15–Fc fusion protein, and agonist IL-2 protein has been reported to lead to graft survival in stringent models by causing the death of activated effector T cells while sparing Tregs [113]. The mechanisms of hyporesponsiveness induced by organ transplantation with costimulatory blockade are only partly understood. The greater success in achieving acceptance of primarily vascularized allografts in rodents using a short course of costimulatory blockade or any of a number of different immunosuppressive agents suggests that these types of graft are highly tolerogenic. As noted above, comparable results usually have not been achieved in large-animal models of organ grafting, limiting the clinical applicability of these approaches. This disparity between results in large and small animals may reflect, in part, the inability of CD8 T cells to reject vascularized allografts in rodents without CD4 T-cell help, combined with the tendency of such grafts to activate Tregs, which then predominate over the suppressed effector T-cell response. Thus, many different forms of relatively mild and shortterm immunosuppression have successfully allowed the long-term survival of such grafts in rodents but not in large animals or man. Calcineurin inhibitors such as cyclosporine and FK506 can block graft prolongation induced by costimulatory blockade [100], possibly by preventing IL-2-induced priming for activation-induced cell death [110] and by preventing de novo T cell conversion to Tregs [112]. Consistently, IL-2 has been shown to play an essential role in the induction of tolerance with CTLA4Ig and cardiac allografts [114]. However, Fas and TNF-α receptors do not appear to be required for tolerance induction with this method.

Mechanisms of Tolerance

Despite the observation that Th1-associated cytokines are associated with graft rejection, IFN-γ plays a critical role in graft prolongation induced with combined CD40/CD28 co-stimulatory blockade [114], DST with CD28 blockade [114], and DST with CD40 blockade [95]. IFN-γ has several known mechanisms for downmodulating T-cell responses, including the promotion of activation-induced cell death, the blockade of T-cell differentiation to a proinflammatory Th17 phenotype, and the induction of indoleamine 2,3 dioxygenease production by DCs (see above). Antibodies to T cells and adhesion molecules Nondepleting anti-T cell mAbs, with or without T-cell-depleting mAbs, have been used to induce skin graft tolerance across minor or MHC barriers, and this has led to infectious tolerance mediated by Tregs [108]. Combination of such mAbs with DST can lead to permanent cardiac allograft survival in mice. Similar results have been achieved by blockade of 1eukocyte function-associated antigen-1 and intercellular adhesion molecule-1 (ICAM-1) interactions, which, in addition to promoting cell–cell adhesion, transmit costimulatory signals to T cells. LFA-1/ ICAM-1 blockade has also been combined with anti-CD154 or blockade of very late antigen-4 and vascular cell adhesion molecule (VCAM) interactions to achieve cardiac allograft prolongation. Other mAbs that have been used in rodent heart graft models include combinations of anti-CD2 and anti-CD3 mAbs [115], anti-CD2 alone [116], and antiCD2 plus anti-CD28 [117]. Various mechanisms, including Th2 deviation, have been implicated. Anti-CD45RB mAb, especially in combination with anti-CD154 or rapamycin, can promote allograft survival through a mechanism that is dependent on CTLA4 upregulation by T cells and Tregs [118]. Total lymphoid irradiation Total lymphoid irradiation (TLI) has also been evaluated as immunosuppression for organ transplantation and has been successful in about one third of baboons studied. In clinical transplantation, TLI has been reported to be beneficial, although it is cumbersome and toxic, especially in the case of cadaver-donor transplantation. Donor-specific tolerance has been demonstrated in a small number of patients in whom immunosuppressive therapy was terminated following kidney transplantation under cover of TLI. One of these patients was studied 12 years after immunosuppression withdrawal, and shown to have active anti-donor mixed lymphocyte reaction and no evidence for microchimerism [119]. TLI used in conjunction with antithymocyte globulin has been shown to induce tolerance to organs in several large-animal models [120]. The relationship between peripheral T-cell tolerance and central tolerance In addition to inducing central tolerance, the thymus can promote tolerance of T cells that are already in the periphery at the time of organ grafting. In a pig model in which class I MHC-mismatched kidneys are accepted under cover of a short course of cyclosporine, the thymus appears to play a role in the induction of tolerance among pre-existing peripheral T cells [121]. The thymus also promotes peripheral tolerance when soluble donor MHC antigens are injected intrathymically or intravenously in a rat model, as removal of the thymus before or within the first few days of allografting results in rejection of the allograft [122]. Migration of DCs from the periphery to the thymus can lead to deletion of thymocytes recognizing antigens these DCs present [123]. While this mechanism would explain the tolerization of newly developing T cells, it does not explain the role of the thymus in tolerizing pre-existing peripheral T cells. There are several possible explanations for the latter phenomenon:

193

1 T cells that are activated in the periphery by the organ allograft may recirculate to the thymus, as has been described [124], and encounter donor antigen there, perhaps on graft-derived DCs, which may then inactivate the T cells. This mechanism has been implicated in the rat soluble antigen injection model described above [125]. 2 The migration of donor antigen to the thymus may result in the development of Tregs that specifically recognize the donor antigen, migrate to the periphery and downregulate the activity of destructive alloreactive T cells. The role of the thymus in the development of Tregs has already been discussed. However, recent evidence suggests that only antigens produced by thymic epithelial cells, and not those produced by hematopoietic cells, promote the production of Tregs recognizing them [28]. Nevertheless, active regulatory cell populations have been implicated in both the rat and pig models discussed above. 3 Recent thymic emigrants that encounter antigen in the periphery may differentiate into suppressive rather than rejecting T cells. The role of the allograft itself in inducing tolerance in the above models in which a pre-existing peripheral T-cell repertoire must be rendered tolerant should again be emphasized. Transferable tolerance is not induced by intrathymic marrow injection alone without an organ allograft in rats [126]. In contrast to models involving intrathymic injections or short-term immunosuppressive treatments and immediate solid organ transplantation, pure intrathymic deletional tolerance induced by mixed allogeneic chimerism (discussed below) is not dependent on the continued presence of an organ allograft [127]. In fact, this tolerance does not depend on the continual presence of any donor antigen in the periphery, as long as there is a continuous supply of donor antigen to the thymus [128].

Achieving HVG tolerance with HCT Resistance to engraftment of HSCs Two major factors influence the engraftment of HSCs. The first is sometimes referred to as “space” in the hematopoietic system. The mechanism by which myelosuppressive host treatment promotes marrow engraftment is not fully understood, and could include both the creation of physical niches and the upregulation of cytokines and other molecules that transmit signals to promote hematopoiesis. It is probably the species specificity of some of these interactions that accounts for the competitive advantage enjoyed by recipient marrow over xenogeneic marrow. An understanding of these physiologic barriers is likely to be critical to the success of the approach of using xenogeneic HCT to induce xenotolerance. Once these are understood, genetically modified donors of other species, such as pigs, could be engineered such that their HSCs would be better able to compete for hematopoietic function in a human marrow microenvironment. In syngeneic BMT recipients, a mild myelosuppressive treatment such as a low dose of total body irradiation (TBI) is required to make physiologic “space” for the engraftment of marrow cells given in numbers similar to those which could be obtained from marrow of living human allogeneic marrow donors [129]. However, this requirement can be overcome by the administration of very high doses of marrow [130,131]. While HSCs injected intravenously readily home to marrow niches [132], the efficiency of this process may be enhanced by direct injection into the bone marrow cavity [133]. The second factor limiting alloengraftment is the host immune system. Because of this immune resistance, allogeneic HCT can be performed successfully only in immunosuppressed recipients. Not surprisingly, T cells of the CD4 and CD8 subsets resist engraftment of class II- and class I-mismatched marrow grafts, respectively. It is also noteworthy that CD4 cells weakly resist engraftment of class I-disparate marrow and

194

Chapter 15

that CD8 cells quite markedly resist class II-disparate marrow grafts in mice [134,135]. NK cells, which may kill target cells due to the absence of self-MHC molecules on allogeneic marrow cells [136], resist allogeneic marrow engraftment in mice, and are responsible for the ability of A × B F1 recipients to resist engraftment of AA or BB parental marrow. NK cells of both humans and mice express clonally distributed surface receptors that recognize specific class I molecules. This recognition transmits an inhibitory signal to the NK cell, thereby preventing the NK cell from killing “self” class I-bearing targets [136]. While the ability of NK cells to resist the engraftment of pluripotent HSCs in mice is rather limited [137,138], NK cells might pose a more significant barrier to human leukocyte antigen (HLA)-mismatched human marrow engraftment, in which stem cell and progenitor cell numbers in the donor inoculum may be more limited. Such a role might become apparent in the future as more reduced-intensity conditioning regimens are used in the setting of HLA-mismatched transplantation. However, a clear role for NK cells in resisting alloengraftment has not yet been established for large animals or man. GVH-reactive NK cells have been reported to promote donor engraftment in mice and to mediate graft-versus-leukemia (GVL) effects against myeloid leukemias in man [139], and the presence or absence of NK cells in patients with severe combined immunodeficiency may determine whether or not cytotoxic host conditioning is needed in order to achieve engraftment of haploidentical HCT [140]. NK cells are tolerized by the induction of mixed chimerism [141], consistent with the observed long-term stability of the chimerism. Such tolerance requires that each functional NK cell receives adequate inhibitory signals from two completely disparate sets of MHC molecules on donor and host cells. This “tolerance” cannot be explained by changes in the expression of inhibitory or activating MHC-recognizing (Ly49) receptors [142]. Several observations suggest that anergy of NK cells chronically encountering cells lacking inhibitory ligands is responsible for their tolerance in mixed chimeras. Tolerance is broken when donor and host NK cells are separated in vitro [143]. Moreover, tolerance is specific for the donor and recipient in the allogeneic situation [142], but is associated with global unresponsiveness in the presence of mixed xenogeneic chimerism [144]. Inhibitory Ly49 receptors are quite broad in their class I allorecognition, and recognition of even fully allogeneic class I molecules can confer some protection from NK-mediated marrow destruction compared with that observed for cells lacking class I MHC. Because of the increased disparity of xenogeneic compared with allogeneic MHC molecules, NK cells likely receive less inhibitory signal from xenogeneic than allogeneic cells. Thus, “tolerance” that reflects anergy induction by repeated encounter with cells lacking inhibitory ligand would be associated in this situation with a global functional NK defect, as has been observed. Consistently, this global lack of NK function is similar to that observed in the presence of chimerism from class Ideficient marrow [145]. Humoral mechanisms can also mediate resistance to marrow engraftment. Antibodies that inhibit engraftment can exist without prior immunization (so-called “natural” antibodies, or NABs). For example, NABs that recognize ABO blood group determinants or xenoantigens, and that exist in normal sera, can resist the engraftment of hematopoietic cells that express the determinants recognized by the NAB. Antibodies against donor antigens can also be present due to presensitization, usually from prior blood product infusions in HCT recipients. Animal studies suggest that the effect of NABs on engraftment can be overcome by the administration of sufficiently high marrow doses [146], and in man, deleterious consequences of ABO-incompatible BMT can usually be avoided with adequate red blood cell depletion from the marrow product. Occasionally, plasmapheresis may be needed to remove such antibodies if they are present at particularly high titer [147]. The presence of antibodies

resulting from presensitization to donor alloantigens, on the other hand, is associated with a high incidence of marrow graft failure in humans and mice. A permissive environment for engraftment of allogeneic marrow can exist under several physiologic and artificially induced conditions, as outlined below. The mechanisms by which HCT induces tolerance in each of these models is discussed. HCT in developmentally immunodeficient recipients Hematopoietic engraftment has been achieved without conditioning in developmentally immunoincompetent recipients. This was first observed in Freemartin cattle (fraternal twins sharing a placental circulation) [148]. Since the prenatal diagnosis of a number of congenital diseases has become possible, the injection of HSCs to preimmune fetuses is an attractive approach to inducing tolerance for postnatal organ transplantation from the same allogeneic or xenogeneic donor. While renal allograft tolerance has recently been achieved with this approach in a largeanimal model [149], the timing and purity of HSC injection are critical in balancing the tendencies for rejection and GVHD, and chimerism and tolerance have been variable in other animal models [150]. Animals can also be rendered tolerant by injection of alloantigens shortly after birth. Several mechanisms have been invoked to explain the observed HVG tolerance (reviewed in [151]). Lasting microchimerism has been detected in some studies and may be essential for the maintenance of tolerance. Evidence to support an intrathymic deletional mechanism has been obtained in some, but not other, strain combinations. However, the presence of microchimerism does not always predict skin graft tolerance in recipients of allogeneic lymphocytes perinatally, and nontolerant animals can still maintain microchimerism following rejection of donor skin grafts. Thus, it is not surprising that several additional mechanisms have been implicated in rodents in which neonatal tolerance has been induced. These include the following: 1 The action of specific suppressor T cells [151]. Tolerance cannot be easily broken by the infusion of nontolerant host-type lymphocytes in animals rendered tolerant at birth, and this has been attributed to the presence of suppressive T-cell populations. Specific suppressor T cells have been detected, and Tregs have been implicated [152]. 2 Neonatal mice have a tendency to produce Th2 responses, which have been implicated in donor-specific skin graft acceptance [41,153]. 3 The ability of allogeneic spleen cell infusions to induce tolerance may reflect the high ratio of non-costimulatory APCs (resting T and B cells) in donor inocula to recipient T cells in the neonate, rather than any particular susceptibility to tolerance induction at this time of life [154]. HCT in immunocompetent recipients following high-dose conditioning Marrow chimerism and tolerance can be achieved in otherwise normal adult animals if immunodeficiency and “space” are artificially created by lethal TBI. While the toxicity associated with this approach makes it unusable for the purpose of organ allograft tolerance induction in humans, studies in animals have provided important information on the immunobiology of HCT and tolerance induction. In murine recipients of T-cell-depleted (TCD) allogeneic BMT, lymphohematopoietic repopulation is largely (95–99%) allogeneic; a small population of host cells (mainly T cells) survives permanently in such animals. If TCD allogeneic and TCD syngeneic marrow are co-administered, mixed allogeneic chimerism ensues, i.e. lymphohematopoietic cells of both donor and recipient type coexist permanently in the survivors [155]. These mixed chimeras show no evidence of GVHD and show specific tolerance, both

Mechanisms of Tolerance

in vivo and in vitro, to donor antigens [155], i.e. “systemic” tolerance is achieved. Animals reconstituted with fully MHC-mismatched TCD allogeneic marrow alone demonstrate a similar pattern of specific unresponsiveness but tend to show poorer immunocompetence both in vivo and in vitro, possibly due to a mismatch between the MHC restriction imposed by the host thymus and the MHCs of donor-type APCs in full chimeras across complete MHC barriers. Another potential advantage of mixed chimerism over full allogeneic chimerism is that hematopoietic cells are most efficient at inducing clonal deletion of T cells in the thymus [3]; thus, a continuous source of host-type hematopoietic cells assures that host-reactive T-cell clones will not emerge from the thymus [127], whereas such cells can emerge in animals reconstituted with TCD allogeneic marrow alone [12]. The importance of antigen on hematopoietic cells for the induction of tolerance among newly developing T cells in humans is illustrated by studies in patients with severe combined immunodeficiency receiving HLAmismatched BMT and in whom donor T-cell, but not B-cell or myeloid, engraftment develops initially. Newly developing donor T cells from these patients exhibit reactivity toward donor class II MHC antigens. This reactivity disappears if the engraftment of donor class II-bearing cells later occurs [156]. Clonal deletion is the predominant mechanism of HVG tolerance in lethal TBI-treated recipients of TCD allogeneic BMT [3,12]. In vivo evidence suggests that suppressor cells are not involved in the maintenance of tolerance to donor antigens, regardless of whether TCD allogeneic marrow cells are given alone or with TCD syngeneic cells. Tolerance can be readily broken in such chimeras by administering a relatively small number of nontolerant recipient-type spleen cells [157], indicating the absence of any potent suppressive activity. Results of in vitro co-culture studies supported this conclusion. As discussed earlier, one important difference between TBI/BMT recipients and neonatally tolerized animals is that TBI leads to the elimination of most pre-existing T-cells. Thus, the number of remaining alloreactive T cells may be too small to provide an adequate stimulus for the activation and expansion of regulatory T-cell populations. In other words, activation of Tregs may require the presence of alloreactive effector cells, which is not the case if there is complete T-cell ablation followed by the intrathymic deletion of donor-reactive cells. In peripheral tolerance models, in contrast, mature donor-reactive T cells already exist in the periphery, and the observed difficulty in breaking tolerance by the infusion of nontolerant host-type cells is consistent with the presence of potent suppressive mechanisms. When, on the other hand, “pure” deletional tolerance is induced in animals in which the pre-existing peripheral T-cell response has been fully ablated, the absence of suppressive cell populations makes it easy to abolish tolerance by the infusion of nontolerant host-type lymphocytes [128,157,158]. In contrast to the above chimeras prepared with lethal TBI followed by reconstitution with TCD allogeneic marrow, animals receiving nonTCD allogeneic cells are highly resistant to the breaking of tolerance by the administration of nontolerant host-type lymphocytes [157]. This resistance is dependent upon the GVH reactivity of those T cells [157]. Rather than implying the existence of regulatory mechanisms, this finding suggests that GVH-reactive T cells persist long-term in these chimeras and eliminate administered nontolerant host-type lymphocytes before they can “take” and eliminate donor cells. Studies in animal models have addressed the possibility that, following lethal irradiation and reconstitution with exhaustively TCD autologous marrow, tolerance might be induced by expression of antigen on nonhematopoietic organs grafted simultaneously with the TCD autologous marrow. This approach was, however, insufficient to induce tolerance to MHC mismatched skin allografts in rodents or to MHC-mismatched vascularized grafts in large-animal studies. Similarly, in vivo treatment

195

with depleting anti-T-cell mAbs alone does not permit induction of skin allograft tolerance across MHC barriers in mice. Thus, alloantigen seems to be required within the lymphohematopoietic system for the most complete tolerization of newly developing T cells, probably reflecting the unique ability of alloantigen expressed on marrow-derived cells to induce clonal deletion in the thymus. An alternative to the induction of mixed chimerism in lethally irradiated recipients has been to reinfuse autologous marrow that is genetically modified to express an allogeneic MHC gene. This approach has led to donor-specific hyporesponsiveness in a class I-mismatched mouse model and with class II gene transduction in a pig model [77]. While it would be technically cumbersome to modify each individual transplant recipient’s marrow with all of the MHC genes of any given cadaveric organ donor, the tolerance-inducing capacity of the organ graft itself can induce spreading to other graft antigens of the tolerance that is induced by a single donor MHC gene introduced into the recipient marrow [77]. The discussion so far would suggest that induction of mixed chimerism may be the most straightforward way of ensuring donor- and hostspecific tolerance across MHC barriers. However, the original methods for producing mixed allogeneic chimeras in mice were not directly applicable to man, because of the toxicity of lethal TBI and the high risk of GVHD and graft failure encountered when HCT is attempted across MHC barriers in man. HCT following lethal TBI would not be justifiable as a means of inducing tolerance for the purpose of organ transplantation, for which less toxic chronic immunosuppressive therapy is available. If HCT, therefore, is to be used as a means of inducing donorspecific organ allograft or xenograft tolerance, it will be essential to develop less toxic and more effective methods of rendering recipients permissive for engraftment than those which are currently available. Conditioning regimens are needed which specifically eliminate the host elements that resist alloengraftment without producing generalized toxic effects. Some approaches to achieving this goal are discussed below. HCT in immunocompetent recipients following conditioning with immunosuppressive but nonmyeloablative protocols Recently, several HCT protocols have been developed that involve myelotoxic and/or immunosuppressive, but not myeloablative, conditioning regimens. The term “nonmyeloablative” is used in this chapter to describe regimens that are known to leave sufficient host hematopoiesis intact to avoid significant cytopenia without a bone marrow graft or if the donor graft fails. These criteria have been demonstrated for certain regimens used in animals and patients, but not for all reduced-intensity regimens. Since host HSCs survive after nonmyeloablative conditioning, mixed chimerism develops when allogeneic marrow is administered. Such regimens include TLI [159], sublethal TBI [160], administration of cyclophosphamide following sensitization with allogeneic donor antigens [161], and the use of mAbs against host T cells in combination with other modalities [162]. HCT has also been performed in unconditioned patients receiving organ transplantation under conventional immunosuppressive drug therapy. Low levels of chimerism have been achieved, but withdrawal of immunosuppressive therapy has not been made possible with this approach [163,164]. However, the incidence of chronic rejection may be decreased in this setting [164]. A brief description of some nonmyeloablative or reduced-intensity conditioning approaches follows. Sublethal TBI Induction of mixed chimerism using fractionated sublethal TBI and allogeneic BMT can lead to donor-specific tolerance across complete

196

Chapter 15

MHC barriers in mice [160]. Donor T cells that need not have alloreactivity against the recipient promote alloengraftment in this model, and donor T cells maintain tolerance [160]. In dogs, a low dose of TBI, in combination with post-transplant cyclosporine and mycophenolate mofetil, has been associated with mixed chimerism and tolerance across minor, but not major, histocompatibility barriers [165].

Recently, combinations of chemotherapeutic agents have been used as reduced-intensity conditioning in patients with hematologic malignancies who were considered to be at high risk from complications of conventional high-dose conditioning. Thus far, these approaches have not been combined with organ transplantation in man. Anti-T-cell antibodies

TLI Another early approach to achieving mixed hematopoietic chimerism and donor-specific tolerance involved the use of TLI plus BMT. The long bones are shielded during the radiation preparative regimen, so BMT results in mixed chimerism rather than full donor reconstitution, and recipient T cells are not fully ablated. Mice treated in this way have been shown to be both resistant to GVHD and tolerant to donor skin grafts (reviewed in [159]). NKT cells producing Th2-type responses predominate following TLI, and this Th1 to Th2 shift may play a role in permitting graft acceptance and preventing GVHD. TLI as the sole conditioning modality for BMT has only variably permitted the induction of mixed chimerism and tolerance in large animals, and this has often been associated with considerable toxicity. A trial of donor BMT with fractionated TLI in high-risk patients receiving renal allografts was not associated with chimerism, and significant toxicity was also observed [166]. A recent trial of HLA-mismatched allogeneic HCT in patients conditioned with TLI and antithymocyte globulin has demonstrated transient induction of chimerism, but withdrawal of immunosuppressive therapy was associated with rejection of donor kidneys grafted at the same time (reviewed in [120]). Nevertheless, remarkably low rates of acute GVHD have been observed in patients receiving HLA-identical HCT for treatment of hematologic malignancies using TLI plus antithymocyte globulin-based conditioning [167]. Pharmacologic agents Cyclophosphamide has long been a staple of high-dose conditioning regimens and of less-intense conditioning regimens for HCT to treat aplastic anemia. Pre-HCT cyclophosphamide has more recently been used in combination with other agents in reduced-intensity conditioning regimens for HCT to treat hematologic malignancies. As is discussed below, a nonmyeloablative HCT/kidney allograft regimen involving cyclophosphamide conditioning has been the first successful attempt at the intentional induction of organ allograft tolerance in humans [168]. In rodents, high doses of cyclophosphamide administered several days following inoculation with large numbers of allogeneic marrow and/or spleen cells permit microchimerism and donor-specific tolerance across multiple minor histoincompatibilities and selected MHC disparities. Additional host treatment with a monoclonal anti-T-cell antibody [162], with sublethal TBI or with fludarabine and low-dose TBI [169], permits the induction of tolerance across complete MHC barriers. Sensitization followed by administration of cyclophosphamide causes selective destruction of host T cells driven into cell cycle in response to alloantigen, so that pre-existing donor-reactive T-cell clones are eliminated [162], and newly developing T cells can mature in the presence of donor marrow-derived antigen in the thymus. While thymic and peripheral chimerism can be demonstrated in animals receiving the regimen involving cyclophosphamide alone, the levels of such chimerism are extremely low, reflecting the fact that cyclophosphamide is relatively ineffective at producing marrow “space.” Evidence has been obtained for intrathymic clonal deletion as a mechanism of tolerance in this model. However, chimerism was shown to disappear with time, and anergy was reported as a mechanism of longer-term tolerance. Post-transplant cyclophosphamide has recently been applied clinically in a reduced-intensity regimen for haploidentical HCT [170].

Early experimental efforts to combine BMT with T-cell depletion to eliminate host cells that mediate alloresistance involved polyclonal sera (antilymphocyte serum). A fraction of mice receiving antilymphocyte serum and skin allografts several days prior to administration of marrow from the same allogeneic donor could be rendered specifically tolerant of donor antigens in the presence of certain MHC disparities [171]. The addition of rapamycin to the regimen significantly increases the incidence of tolerance [172]. Complex mechanisms, including suppressor cells of donor origin, were implicated in this model, and more recent studies have demonstrated that T-cell depletion by antilymphocyte serum is associated with sparing of Tregs [172,173]. A similar approach has been reported to prolong renal allograft survival in large animals, with the best results being achieved when the donor and recipient share a DR class II MHC allele [174]. In the primate studies, a veto cell population has been implicated. Conventional marrow doses are administered in this model without host myelosuppression, so that high levels of donor HSC engraftment are not achieved. Moreover, T-cell depletion is incomplete, so the involvement of regulatory cells in tolerance is therefore not surprising. In vivo depletion of host CD4+ and CD8+ T cells along with TBI (at least 6 Gy) permitted engraftment of allogeneic marrow and induction of skin graft tolerance across complete MHC barriers [17]. Adding a high dose of thymic irradiation (7 Gy) to the regimen permitted engraftment of fully MHC-mismatched allogeneic marrow in animals receiving only 3 Gy TBI. Thymic irradiation is needed because thymocytes become coated with mAbs but, unlike T cells in the peripheral lymphoid compartment, are not eliminated. The low dose (3 Gy) of TBI is necessary for the creation of marrow “space.” Permanent mixed chimerism and donor- and host-specific tolerance are reliably induced across complete MHC barriers using this regimen (Fig. 15.1) (reviewed in [20]). This approach has been extended to a xenogeneic (rat→mouse) combination by adding anti-NK1.1 and anti-Thy1.2 mAbs to the conditioning regimen [175]. These mAbs are needed to deplete recipient NK cells and γδ T cells [176], both of which pose a much more significant barrier to engraftment of xenogeneic than allogeneic HCT. Allogeneic tolerance has been achieved in a non-human primate model using this nonmyeloablative approach to inducing mixed chimerism [177]. In both the allogeneic [128](reviewed in [20]) and the xenogeneic (reviewed in [179]) rodent systems, intrathymic clonal deletion, rather than peripheral suppression or anergy, is the major mechanism inducing and maintaining long-term donor-specific tolerance. Accordingly, donor class IIhigh cells are present in the thymuses of these animals at all times, including 10 days post-BMT, when the first wave of thymopoiesis is underway. Furthermore, tolerance can be broken by depleting donor cells with mAbs after stable chimerism has been established, and this loss of tolerance correlates with the de novo appearance in the periphery of T cells bearing donor-reactive TCRs [128]. However, if the host thymus is removed before depletion of donor hematopoietic cells, or if donor-depleted spleen cells from chimeras are transferred to syngeneic athymic mice, donor-specific tolerance is maintained, and cells with donor-reactive TCR do not appear in the periphery [128]. These results demonstrate that intrathymic chimerism is essential and sufficient to maintain ongoing deletional tolerance in long-term mixed allogeneic chimeras, whereas peripheral chimerism is not required for the maintenance of tolerance. Since persistent antigen is required to maintain

197

Mechanisms of Tolerance

3 Gy TBI

CyA Pt 1

creat Pt 2

CyA Pt 2

MIXED ALLOGENEIC

Day –5 B

Anti-CD8 mAbs

Specifically tolerant to donor in vivo (skin grafting) and in vitro (MLR, CML)

BMT from donor A

creatinine (mg/dl)

CHIMERA (A+B)

6

600 500

4

300 2

200 100

0

0 0

Fig. 15.1 Nonmyeloablative conditioning regimen allowing the induction of mixed allogeneic chimerism and specific transplantation tolerance across complete major histocompatability barriers in mice. BMT, bone marrow transplantation; CML, chronic myeloid leukemia; mAb, monoclonal antibody; MLR, mixed lymphocyte reaction; TBI, total body irradiation.

anergy, tolerance cannot be explained by a peripheral anergy mechanism. Moreover, the ease with which new thymic emigrants break tolerance after donor cell depletion by mAbs or with infusion of nontolerant recipient lymphocytes [128] is strong evidence that suppression does not play a significant role in maintaining long-term tolerance. Thus, this is a relatively pure model of ablation of pre-existing peripheral and intrathymic mature T cells followed by lifelong central, deletional tolerance. The potential toxicity of the regimen shown in Fig. 15.1 can be further minimized by replacing thymic irradiation with a second injection of anti-donor mAbs on day –1, and deletional tolerance is again attained. These thymus-directed host treatments are needed to eliminate pre-existing donor-alloreactive thymocytes and also to create “thymic space.” Thymic engraftment is regulated independently of marrow engraftment, and specific measures are required to permit donor progenitors to repopulate the recipient thymus at high levels, even in syngeneic mice (reviewed in [180]). In this allogeneic BMT model, TBI can be eliminated from host conditioning by giving a very high dose of donor marrow cells, still achieving durable multilineage mixed chimerism and systemic donorspecific tolerance, all in association with the lasting presence of donor APCs in the thymus and intrathymic deletion of donor-reactive host thymocytes. Thus, high levels of allogeneic hematopoietic repopulation and central deletional tolerance may be achieved with a conditioning regimen that excludes myelosuppressive treatment. Hematopoietic cell engraftment and donor-specific tolerance have been achieved across a full haplotype barrier using a similar nonmyelosuppressive regimen plus high stem cell doses in a large animal porcine model [180]. Based on the mouse model in Fig. 15.1, a primate model for tolerance induction has recently been developed. Since effective T-cell-depleting mAbs are not available for use in primates, polyclonal antithymocyte globulin and a short (28-day) course of cyclosporine are used in their place. A high percentage of cynomolgus monkeys receiving class I- and II-mismatched marrow with this protocol develop transient mixed chimerism and donor kidney allograft acceptance [177]. Several other regimens involving various combinations of anti-T-cell antibodies, irradiation, and immunosuppressive drugs have also permitted the achievement of mixed chimerism in mice (reviewed in [181]). Depleting and nondepleting anti-T-cell mAbs can also be used to induce tolerance in association with conventional-dose BMT without irradiation, although tolerance is then only reliably achieved across minor

400

CyA discontinued

CyA level (ng/dl)

7 Gy thymic irradiation

Anti-CD4

creat Pt 1

100

200

300 500

700

900

Days Fig. 15.2 Renal function (measured by serum creatinine [creat]) and cyclosporine levels (measured by a monoclonal antibody assay) in two patients (Pt 1, Pt 2) with multiple myeloma who received combined kidney and bone marrow transplantation from human leukocyte antigen-identical sibling donors following nonmyeloablative conditioning with cyclophosphamide, pre- and post-transplant antithymocyte globulin, and thymic irradiation. Cyclosporine (CyA) was the only pharmacologic immunosuppressive agent given post-transplant, and it was discontinued in the third month post-transplant. Both patients have sustained normal creatinine levels (now at 7 years and 9 years) following discontinuation of cyclosporine, indicating the presence of tolerance to their kidney allografts. Partial and complete remissions of the multiple myeloma were achieved in the two patients, and no GVHD has occurred. (Reproduced from Buhler LH, Spitzer TR, Sykes M et al. Induction of kidney allograft tolerance after transient lymphohematopoietic chimerism in patients with multiple myeloma and end-stage renal disease Transplantation 2002; 74: 1405–9, with permission.)

histocompatibility barriers, and lasting high levels of chimerism are not achieved [17]. In this setting, clonal anergy rather than deletion may be the major mechanism of tolerance [17]. The successful induction of tolerance in a primate model using nonmyeloablative conditioning to induce mixed chimerism [177], combined with murine studies demonstrating the utility of mixed chimerism followed by delayed donor leukocyte infusions (DLIs) as an approach to achieving GVL effects without GVHD [182–184], has been used as the basis for a clinical trial of mixed chimerism induction followed by DLI for the treatment of patients with hematologic malignancies [185,186]. The experience with this relatively nontoxic protocol provided an opportunity to evaluate the potential of this approach to induce transplantation tolerance in patients with a hematologic malignancy, multiple myeloma, and consequent renal failure. Six patients received a simultaneous nonmyeloablative bone marrow transplant and renal allograft from an HLA-identical sibling, and have accepted their kidney graft without any immunosuppression for follow-ups as long as 9 years. This study describes the first intentional achievement of organ allograft tolerance in humans. Three of the six patients have had prolonged complete remissions of their myelomas [168] (Fig. 15.2). This is especially surprising since chimerism in four of these six patients (including two with prolonged complete remissions of their myelomas) was only transient [168]. These data raise the possibility that transient chimerism followed by marrow rejection, which was evidenced by sensitized anti-donor T-cell responses in some of these patients [168], could lead to antitumor responses, and this hypothesis has been supported by data in a mouse model [187]. The renal allograft tolerance achieved in patients who lost chimerism suggests that the kidney graft itself may participate in tolerance induc-

198

Chapter 15

tion and/or maintenance after chimerism has played its initial role. The detection of sensitized T-cell responses to donor minor histocompatibility antigens expressed on hematopoietic cells with unresponsiveness to donor renal tubular epithelial cells in these patients [168] raises the possibility that tolerance may be specific for minor antigens expressed on the kidney graft itself. In the primate model described above, chimerism is also transient, but both BMT and early renal transplantation have been shown to be essential for tolerance induction [177]. Because T-cell depletion is only partial in these models, it is clear that the pure central, deletional tolerance described above in murine models has not yet been achieved with nonmyeloablative conditioning in large animals or humans. Nevertheless, the promising results obtained in these patients have provided an important proof of principle. Recently, this approach has been extended to a pilot study in patients without malignant disease with renal failure from other causes, who receive HLA-mismatched combined kidney and BMT. A systemic state of donor-specific tolerance was achieved in 4 out of 5 patients. These four patients have been off all immunosuppression for periods of more than 1 year to almost 5 years, providing the first successful intentional achievement of organ allograft tolerance across HLA barriers (178). The mechanisms of T-cell elimination using antibody treatments are not entirely clear. Animals treated with T-cell-depleting mAbs after thymectomy show a partial but significant recovery of CD4+ and CD8+ T cells over a period of several weeks, suggesting that some T cells escape depletion and expand due to lymphopenia and/or antigenic stimuli. Many studies have shown that the initially recovering T cells in large animals and humans predominantly express the “memory” phenotype, consistent with lymphopenia-driven expansion, but also consistent with selective resistance of memory cells to antibody-mediated depletion. T-cell depletion with at least some mAbs is independent of complement, but this pathway seems to be involved in T- and B-cell depletion with other mAbs. Co-stimulatory blockade with HCT Both thymic irradiation and host T-cell depleting mAbs in the conditioning regimen shown in Fig. 15.1 can be replaced by co-stimulatory blockade [69]. All preconditioning can be eliminated by giving a high dose of fully MHC-mismatched donor marrow followed by a single injection of each of two costimulatory blockers [188] or repeated injections of anti-CD40 ligand [189]. This ability to replace recipient Tcell depletion with costimulatory blockade to allow bone marrow engraftment is encouraging for several reasons. First, it has been dif-ficult to achieve T-cell depletion with antibodies in large animals and humans that is as exhaustive as that achieved in the above rodent models, perhaps due to the use of inadequate doses or suboptimal reagents. Second, if sufficiently exhaustive T-cell depletion could be achieved in humans, T-cell recovery from the thymus might be dangerously slow, especially in older individuals, since thymic function diminishes with age (reviewed in [190]). The ability to minimize the degree and duration of T-cell depletion by replacing some [109] or all [69,188,189] of the T-cell depleting antibodies with co-stimulatory blockers is therefore encouraging. As in other protocols achieving sustained mixed chimerism, long-term tolerance is maintained by intrathymic deletion in mixed chimeras prepared with costimulatory blockade [69,109,188]. However, since alloreactive T cells are abundant in the peripheral repertoire at the time of BMT with these regimens, these peripheral T cells must be rendered tolerant by BMT under cover of costimulatory blockade. Initial tolerance of peripheral T cells involves specific deletion of donor-reactive CD4 [71] and CD8 [72] T cells. This specific peripheral deletion of CD4 cells only requires failure of the CD154–CD40 interaction and does not require antibody-mediated clearance of CD154+ cells [191]. In mice receiving BMT with anti-CD154 and a low dose of TBI, CD8 deletion

occurs within 1–2 weeks and requires CD4 cells that do not have characteristics of “natural” Tregs [72]. Unlike models involving “adaptive” Tregs, CD4 cells are not required for maintenance of tolerance after this initial 2-week period [72]. Thus, while CD4 cells are clearly required for CD8 tolerance in this model, there is currently no evidence that this regulatory function of CD4 cells involves a specific subset of cells that is differentiated to mediate suppression. Deletion of donor-reactive CD4 cells occurs more slowly, over 4–5 weeks, and is preceded by a state of anergy [71,191]. Regulatory cells do not appear to play a major role in maintaining the long-term tolerance induced by anti-CD154 with BMT, since tolerance and chimerism are obliterated by the infusion of relatively small numbers of nontolerant recipient-type spleen cells in this model and linked suppression is not observed [71]. Since HSC engraftment ensures complete central deletional tolerance in these long-term chimeras [69,70,109,188], and specific peripheral deletion is quite complete, there may be insufficient donor-reactive T cells present to induce the expansion and maintenance of specific regulatory cells. However, some models using CTLA4Ig and anti-CD154 in combination with allogeneic BMT may be associated with less-complete deletion of donor-reactive T cells and involve regulatory mechanisms [192,193]. GVHD does not occur in the rodent models discussed above, despite the use of unmodified donor bone marrow cells. This is most readily explained by the continued presence of the T-cell-depleting or costimulatory blocking antibodies in the serum of the hosts at the time of BMT. These levels are sufficient to prevent alloreactivity by the relatively small number of mature T cells in the donor marrow. There are similarities and differences in the mechanisms of tolerance induced by anti-CD154 in combination with BMT versus DST. Only the BMT model allows engraftment of HSCs and intrathymic deletion as a mechanism of long-term tolerance [109,188]. Thus, in contrast to BMT, DST and anti-CD40L allows long-term skin graft survival only in thymectomized mice [95]. The inability to maintain tolerance in the presence of a thymus indicates the failure to establish central tolerance in the absence of substantial chimerism. In addition, the inability to resist breaking of tolerance by new thymic emigrants in this model argues that powerful peripheral regulatory mechanisms are not operative in these animals, even though regulatory mechanisms may play a role in the initial suppression of CD4-mediated alloresponses [95]. Concurrent viral infections have a deleterious effect on the induction of mixed chimerism under cover of co-stimulatory blockade [194]. Another concern is that successful induction of allogeneic tolerance in the presence of viral infections may also lead to tolerance to the virus and failure to clear it. These results suggest that de novo systemic viral infections should be avoided during the period of BMT and tolerance induction, and have important implications for the practice of BMT with this approach. Application of mixed chimerism for the induction of xenogeneic tolerance Considerable difficulties have been encountered in achieving high levels of engraftment and hematopoietic function from highly disparate species. Xenogeneic hematopoiesis can be enhanced by the administration of donor-specific cytokines, and spontaneous seeding of the thymus with tolerance-inducing cells from the donor has been detected in pig cytokine-transgenic mice receiving porcine HCT, resulting in the achievement of donor-specific tolerance [195]. An alternative approach involves the removal of the recipient thymus and its replacement with a xenogeneic thymus. Donor-specific skin graft tolerance across the discordant pig to mouse species barrier has been achieved with this approach [196]. Normal, immunocompetent mice that are thymectomized and treated with T-cell-depleting mAbs before porcine thymus grafting recover T cells in the xenogeneic thymic grafts. These cells repopulate the periphery and are competent to resist infection [197]. Tolerance to both donor

Mechanisms of Tolerance

and host by intrathymic deletional mechanisms is observed, and this deletion reflects the presence of class IIhigh cells from both species within the graft [196,198]. Since MHC restriction is determined by the MHC of the thymus, and porcine MHC is entirely responsible for positive selection of murine T cells in this model [199], it was surprising that T cells that differentiated in xenogeneic thymus grafts were able to respond to peptide antigens presented by host MHC [197]. However, the excellent immune function achieved in humans receiving HLA-mismatched allogeneic thymic transplantation for the treatment of congenital thymic aplasia (DiGeorge syndrome) suggests that this “restriction incompatibility” may not be a major obstacle to the achievement of adequate immune function [200]. Perhaps this high level of crossreactivity, even between species, reflects the fact that MHC reactivity is inherent in unselected TCR sequences [201]. Importantly, it has been demonstrated that human T cells can also develop in porcine thymic grafts (in immunodeficient mice), and that these T cells show specific tolerance toward the MHC of the xenogeneic thymus donor [202]. Induction of B-cell tolerance using HCT As mentioned above, mixed chimerism can also induce tolerance across xenograft barriers. Pigs are widely believed to be the most suitable xenogeneic donor species for transplantation to humans, but transplantation from this species is impeded by the presence in human sera of NABs that cause hyperacute rejection of porcine vascularized xenografts. In humans, the major specificity recognized by NABs on porcine tissues is a ubiquitous carbohydrate epitope, Galα1-3Galβ1-4GlcNAc-R(αGal). Humans lack a functional α1-3Gal transferase (galactosyltransferase; GalT) enzyme, as do GalT knockout mice, which also make anti-αGal NABs. Both pre-existing and newly developing B cells producing anti-αGal antibodies are tolerized by the induction of mixed chimerism in GalT knockout mice receiving αGal-expressing allogeneic or xenogeneic marrow. The induction of mixed xenogeneic chimerism prevents hyperacute rejection, acute vascular rejection, and cell-mediated rejection of primarily vascularized cardiac xenografts. Long-term chimeras produced in GalT knockout mice lack anti-Gal surface Ig-bearing cells in the spleen, and show tolerance in enzyme-linked immunosorbent spot assays, suggesting that clonal deletion and/or receptor editing tolerizes B cells developing after BMT. However, tolerance develops by 2 weeks post-BMT, even in mice that are presensitized to Gal antigens, despite the fact that the conditioning regimen does not deplete antibodyproducing cells. In GalT−/− mice rendered tolerant by the induction of mixed chimerism 2 weeks earlier, cells with anti-Gal receptors are present in the spleen but do not produce anti-Gal (reviewed in [203]). This rapid tolerance of NAB-producing cells in mice rendered mixed chimeric with BMT is due to an anergy mechanism [204]. The cells producing anti-Gal and most IgM NABs in mice belong to the B-1b subset [65], and their tolerization requires the expression of complement receptors on recipient nonhematopoietic cells [205] by mechanisms that are incompletely understood. An alternative approach for tolerizing B cells toward the Gal epitope involves retroviral transduction of the GalT gene into autologous HSC. Administration of marrow transduced in this manner to lethally irradiated GalT knockout mice led to tolerance to the Gal epitope [206]. Establishing molecular chimerism involves modification of autologous BM stem cells, avoiding the difficulties in achieving engraftment of pig HSCs in primates. However, this approach has only been evaluated in lethally irradiated mice, and it is not yet known whether or not a transduced, transplanted BM population could compete sufficiently with host hematopoietic cells to give significant long-term Gal expression in recipients of a more clinically relevant, nonmyeloablative conditioning regimen. Even if this approach were to be successfully applied, non-

199

Gal-reactive xenoreactive NABs and cell-mediated rejection would remain to be overcome. The significance of these non-Gal antibodies has become apparent since the recent development of αGal knockout pigs using nuclear transfer [207–209]. In contrast, all types of rejection can be avoided with a single treatment by induction of mixed xenogeneic hematopoietic chimerism.

Preventing GVHD by depleting or tolerizing mature donor T cells in the HCT inoculum: is GVH tolerance a desirable goal? In order to avoid GVHD, the de novo development of host-reactive T cells from progenitors in the marrow must be prevented, and the GVH reactivity of pre-existing mature T cells in the marrow inoculum must be inhibited. While avoidance of GVH alloreactivity is not always desirable (see below), many strategies for preventing GVHD induced by mature T cells in the HCT inoculum involve depletion or tolerization of pre-existing GVH-reactive T cells. Tolerization of mature T cells in donor marrow could have both beneficial and harmful effects. The beneficial effects of GVH alloreactivity include GVL/graft-versus-lymphoma effects and promotion of alloengraftment. However, T cells that lack GVH reactivity also have the potential, if given in sufficient numbers, to promote alloengraftment (see Chapter 11), possibly by the veto mechanism described above [210]. Additionally, non-GVH-reactive T-cell populations can confer immunity to pathogens, thereby avoiding the high risk of opportunistic infection associated with global T-cell depletion. Induction of global GVH tolerance is highly desirable in the use of HCT for the treatment of nonmalignant diseases, such as the induction of transplantation tolerance (discussed above), and the treatment of inborn hematologic, immunologic, and metabolic disorders. However, when hematologic malignancy is present, GVH tolerance can be associated with reduced GVL effects, since GVH alloresponses are potent and largely responsible for the GVL response. One way to circumvent this deleterious aspect of GVH tolerance would be to preserve tumor antigenspecific responses while responses to normal host antigens were inhibited. Several candidate tumor-specific antigens have recently been identified (see Chapter 18), and CTL activity can be generated against them, generating interest in the idea of tolerizing GVH-reactive T cells while maintaining tumor-specific responses. However, the frequency of tumor antigen-specific T cells pre-existing in a given T-cell repertoire is even lower than that against multiple minor histocompatibility antigens, and the generation of meaningful tumor-specific responses is likely to necessitate donor presensitization along with in vitro expansion of tumor-specific effector cells, which might limit the homing capacity of injected cells. Such prolonged cultures are impractical for routine use in the setting of HCT, in which leukemia-reactive cells must eliminate exponentially expanding leukemic cells. In theory, the less risky strategy of generating tumor-specific responses from autologous T cells could achieve similar outcomes. However, T-cell immunity may be markedly impaired in the tumor-bearing host, and the use of immunologically unimpaired allogeneic donors is therefore attractive. Minor histocompatibility alloantigens expressed by lymphohematopoietic cells (including leukemias and lymphomas) but not by the epithelial GVHD target tissues may also be targeted using in vitro expanded CTLs in order to achieve GVL without GVHD. Several human minor histocompatibility antigens have demonstrated this pattern of expression (see Chapters 12 and 18]. However, GVH disparities for some of these same minor antigens (e.g. HA-1) have been associated with an increased incidence of GVHD. Recently, it was shown that administration of primed T cells specific for a single immunodominant class I-restricted minor histocompatibility antigen could mediate GVL without GVHD.

200

Chapter 15

Avoidance of GVHD was dependent on the absence of GVH-reactive T cells with additional specificities in the donor inoculum [211]. If such T cells are present, the response to immunodominant minor histocompatability antigens may induce APC activation and cytokine production, which activates T-cell responses to additional minor histoincompatibilities that are shared by GVHD target organs. However, the potency of antitumor effects in HCT is greatest in the setting of MHC disparity [212–215], in which GVH alloresponses are most potent. The potent ability of the anti-MHC GVH alloresponse to mediate GVL effects reflects the extraordinarily high frequency of T cells recognizing MHC alloantigens that is present in the unprimed Tcell repertoire. T cells reactive with an allogeneic MHC determinant may represent as many as 2% or more of the total T-cell population, whereas T cells reactive with a peptide–self-MHC complex are several orders of magnitude less frequent. Several reasons for this high frequency of allogeneic MHC-reactive T cells have been identified. One is the large number of different “nonself” peptide–MHC complexes presented by an allogeneic MHC molecule (i.e. high number of different determinants), resulting in recognition by a large number of different T-cell clones. The second reason is the large number of MHC molecules on an allogeneic cell. This may lead to recognition by large numbers of TCRs that are sufficiently independent of the peptide component for their recognition of the MHC– peptide complex that they bind with relatively low affinity to the MHC molecules regardless of the peptides to which they are complexed. In these instances, the high density of MHC determinants compensates for the low affinity of the TCRs recognizing them. In contrast, since minor histocompatibility antigens are peptides, the number of minor antigen– MHC complexes on a given APC will be more limited, perhaps resulting in recognition only by TCRs with relatively high affinity. As is discussed later in this section, a number of experimental strategies have been developed that permit exploitation of the GVH alloresponse for GVL effects while avoiding GVHD. Specific depletion or tolerization of mature GVH-reactive T-cells present in the donor inoculum at the time of HCT Several approaches have been developed in efforts to tolerize GVHreactive mature T cells within hematopoietic cell grafts, including blocking of adhesive and co-stimulatory interactions of T cells with recipient APCs. One of these approaches was evaluated in a pilot clinical trial with some success at reducing acute GVHD [216], but insufficient numbers of patients have been transplanted with this approach to allow an assessment of protective immunity from infection or tumor relapse. Additional approaches involve the selective elimination, rather than tolerization, of GVH-reactive T cells. These approaches preserve T-cell populations that may react to tumor-specific antigens and pathogens and perhaps promote engraftment. This goal can be achieved by stimulating donor T cells with host antigens, then depleting the activated cells using antibodies or immunotoxins directed against cell surface markers associated with recent activation, such as CD25 and CD69. Early clinical results with this approach suggest that GVHD is reduced, though not eliminated, and that such inocula may confer some anti-infectious immunity to the recipients [217]. Use of suppressive T cell populations to induce GVH tolerance CD25+CD4+ T cells generated by in vitro culture with alloantigen and anti-CD154 have been shown to suppress GVHD and to tolerize naïve and presensitized T cells via a regulatory cell population [218]. Additionally, donor Tregs can inhibit GVHD while preserving some GVL activity [219], although it is unclear whether or not the Tregs reduce the magnitude of GVL effects. While several groups are now exploring the

possibility of infusing fresh or expanded Tregs clinically, expanding the cells ex vivo has proved to be difficult. One important benefit of Treg infusion in an experimental model is protection of the recipient lymphoid tissues from GVH-associated damage, resulting in improved antiinfectious immunity [220]. In mice, NKT cells in the marrow or in the lymphoid tissues after TLI treatment also have the ability to suppress GVHD, apparently via an IL-4-dependent mechanism [221], while GVL activity is preserved. As is discussed above, this approach has recently been applied in patients receiving HLA-identical HCT with strikingly low rates of acute GVHD [167]. However, it remains to be determined to what extent GVL effects are preserved with this approach. In addition to the NKT cells, T cells lacking NK-cell markers that are resident in murine bone marrow have a reduced capacity to produce GVHD, with preserved ability to mediate GVL [222]. Approaches to preventing GVHD that do not involve induction of tolerance Cytokine manipulation Considerable interest was generated in the 1990s by the notion that GVHD-inducing T cells had a Th1 (IFN-γ- and IL-2-producing) phenotype and that Th2 (IL-4-, IL-5-, and IL-13-producing cells) cells could suppress GVHD, and some evidence supported this possibility. Similarly, CD8 cells producing type 1 cytokines mediated severe GVHD in MHC class I-mismatched or parent into F1 hybrid combinations, whereas type 2 cytokines did not [223]. Meanwhile, Th2 cytokines mediated GVL and promoted donor marrow engraftment [223]. However, infusion of ex vivo polarized donor Th2 cells did not produce a measurable reduction in GVHD in a clinical trial [224]. In fact, the role of Th1- and Th2-associated cytokines in HCT and transplantation in general is rather complex. Systemic administration of Th1-type cytokines such as IL-2, IL-12 or IFN-γ shortly after BMT in mice can actually reduce the severity of GVHD [225–227] while preserving the GVL effect [228,229]. Furthermore, T cells from IFN-γ-deficient donors have an increased capacity to cause acute GVHD in lethally irradiated recipients (reviewed in [230]). While the mechanism by which IFN-γ inhibits graft rejection and GVHD is not fully understood, possible mechanisms have already been discussed above. Further studies have shown that Th2 and Th2-associated cytokines are also capable of contributing significantly to GVHD pathology of particular tissues. Other approaches A number of additional approaches to ameliorating GVHD do not involve initial tolerance induction of donor T cells in the HCT graft. One such approach is to transduce donor T cells with suicide genes, such as herpes simplex virus thymidine kinase, so that the alloresponse can be turned off at will, hopefully after tumor has been eradicated [231]. Initial clinical success with this approach has been somewhat limited, in part due to the fact that ganciclovir, the drug used to kill thymidine kinase-transduced T cells, is also needed clinically to treat cytomegalovirus reactivation, which is a common and serious complication of HCT. Furthermore, the transduction of GVH-reactive T cells may be incomplete, resulting in escape of some clones from suicide after treatment has been initiated. Finally, the manipulations involved in the transduction process itself might render the donor T cells less effective at eliminating leukemias when transferred back to the patient. The development of new vectors and the use of genes other than thymidine kinase may lead to further improvements in the utility of this approach. Another approach is to attempt to block the early inflammatory mediators of GVHD, such as IL-1 or TNF-α. While some of these approaches have been evaluated in pilot clinical trials, none has yet been shown to have

Mechanisms of Tolerance

a durable effect on GVHD. Recently, keratinocyte growth factor has been shown to ameliorate GVHD without blocking GVL, perhaps by reducing the disruption of the intestinal barrier associated with myeloablative conditioning and by preventing some of the thymic injury and consequent immune damage associated with GVHD (see Chapter 16). Delayed donor T-cell administration DLIs can mediate powerful GVL effects in patients with chronic myeloid leukemia, lymphomas, and multiple myeloma who have relapsed following allogeneic HCT (see Chapter 72). In established mixed allogeneic chimeras, which are immunologically tolerant of their original marrow donor’s antigens, a GVH reaction takes place after administration of DLI, resulting in conversion of the state of mixed hematopoietic chimerism to a state of full donor chimerism. This conversion to full chimerism is due to a GVH reaction directed primarily against recipient MHC alloantigens. Remarkably, this powerful GVH alloreaction is not associated with any clinically significant GVHD, even though recipient alloantigens are expressed on many cell types and donor T cells are given in numbers that would cause rapidly lethal GVHD in freshly irradiated recipients [182,183,215,232]. This demonstration that GVH reactions could be confined to the lymphohematopoietic system suggested an approach to separating GVHD from GVL effects. Since hematologic malignancies reside largely in the lymphohematopoietic system, the result suggested that GVH reactions might be confined to this system and eliminate tumor cells without entering the epithelial GVHD target tissues. Indeed, GVL is achieved without GVHD when delayed DLIs are given to established murine mixed allogeneic chimeras [184,215]. Recent studies have revealed mechanisms by which delayed DLI may mediate GVL without GVHD. Antihost MHC alloreactivity clearly mediates the most potent GVL effects [184,215]. Moreover, GVHreactive donor T cells become activated and proliferate markedly in mixed chimeric recipients of DLI [215,232]. However, these cells do not migrate to GVHD target tissues (epithelial tissues such as skin, intestines, and liver) unless there are inflammatory signals in those tissues [232]. The recipient’s recovery from conditioning-induced tissue injury markedly reduces the tendency of GVH-reactive donor T cells that are activated by host alloantigens in the lymphoid tissues to leave the lymphohematopoietic system and cause GVHD [232]. Provision of a local toll-like receptor stimulus promotes the entry of such cells into the skin and induces localized GVHD [232], indicating that tissue inflammation provides a critical checkpoint for T-cell recruitment to GVHD target tissues. Such inflammatory signals can be provided by the tissue injury induced by conditioning therapy and by toll-like receptor activation [232], which may be induced by infection or by conditioning itself. Both high-dose and reduced-intensity conditioning are associated with very early production of chemokines in the GVHD target tissues, which is followed by immigration of T cells that then elicit a further cascade of chemokines that amplifies the response [233]. Adhesion molecules that promote leukocyte infiltration through the microvasculature of these tissues are also likely to be upregulated by conditioning. Lethal TBI and cyclophosphamide are known to upregulate the proinflammatory cytokines IL-1, IL-6, and TNF-α [234–236], which can upregulate endothelial cell E-selectin, P-selectin, ICAM-1, and VCAM1 [237,238]. Proof of principle has been obtained that delayed DLI can mediate GVH responses confined to the lymphohematopoietic system in patients receiving nonmyeloablative HCT with in vivo or ex vivo T-cell depletion of the initial donor inoculum, who achieve mixed chimerism without GVHD [186,239]. However, some patients achieving initial mixed chimerism from a TCD donor inoculum nevertheless develop GVHD following administration of delayed DLI. In general, delayed DLI is somewhat variably associated with GVHD in patients, but to a lesser

201

degree than would be expected in freshly conditioned recipients of similar cell numbers [240,241]. The greater ease in avoiding GVHD in mice compared with humans receiving delayed DLI after TCD HCT may reflect the more robust thymic T-cell recovery that occurs in mice than in heavily pretreated human adults receiving TCD HCT. This thymic recovery confers protection from infection, avoiding toll-like receptor stimuli, and also allows the recovery of regulatory T-cell populations that downregulate GVH reactions. The ability of Treg to inhibit DLI-induced GVHD has been demonstrated in mouse models [242– 244]. The potent antitumor responses seen in patients with advanced lymphoid malignancies who received the transplant approach described above (mixed chimerism induction using TCD HCT and nonmyeloablative conditioning, followed by delayed DLI [245]) led to the hypothesis that an initial state of mixed chimerism might permit more powerful graft-versus-tumor effects to be achieved from the GVH alloresponse than is possible in full allogeneic chimeras. To test this hypothesis, the GVL effects of DLI were compared in mixed versus full allogeneic chimeras inoculated with a host-type T-cell lymphoma in a murine model. Remarkably, DLI led to only a slight prolongation of survival in leukemic full allogeneic chimeras, whereas they led to almost complete protection, with 80–100% survival, in mixed chimeras receiving the same tumor inoculum (Fig. 15.3). This markedly more potent GVT effect of DLI in mixed compared with full chimeras was shown to be dependent on the expression of host class I and class II MHC molecules on recipient-type hematopoietic cells [184,215]. GVHD was not observed in the mixed chimeras with these potent GVL effects from DLI [184]. These data are not inconsistent with an earlier report showing a critical role for recipient hematopoietic APCs in initiating GVHD [246]; in that report, the donor T cells were given to freshly lethally irradiated mice, whereas the donor T cells were given as delayed DLI in the studies comparing mixed with full chimeras [184]. The complete absence of GVHD in mixed chimeras enjoying potent GVL effects of DLI dramatically illustrates the power to separate GVHD and GVT effects by confining the anti-MHC alloresponse to the lymphohematopoietic system. GVH tolerance of T-cells developing from progenitors in the marrow As has been discussed above, the mechanism of GVH tolerance in lethally irradiated recipients of TCD allogeneic marrow alone (i.e. “full” chimeras) appears mainly to involve T-cell anergy rather than clonal deletion [12]. In contrast, mixed chimeras show excellent clonal deletion of T cells reacting against host antigens [127,247], undoubtedly due to the intrathymic presence of host-type marrow-derived cells in mixed chimeras [127]. However, partial clonal deletion of host-reactive T cells has been described in lethally irradiated mice receiving allogeneic BMT alone [248,249], which may reflect the weaker ability of thymic epithelial cells (compared with hematopoietic cells) to induce intrathymic deletion [250–252]. Perturbations in the thymic environment, such as the destruction of thymic epithelial elements observed in GVHD [253,254], or thymic injury induced by treatment with cyclosporine [255,256], can impair processes leading to tolerance [257,258]. Defective production of Tregs may contribute to autoimmunity in this setting.

Summary and future directions It is clear that HCT has not yet met its full potential to provide a cure for lymphohematopoietic malignancies or to treat nonmalignant diseases. There is a need for improved methods of exploiting the immuno-

202

Chapter 15

% Survival

75

Mix

Mix+DLI

Full

Full + DLI

100

100

100

Mix+DLI+EL4

75

Full + EL4

75

50

50

50

25

Full +DLI+EL4

0

25

25

0

28

56

84

112

140

FULL+DLI+EL4

Mix+EL4 0

168

(a)

Mix+DLI+EL4

0 0

28

56

(b)

84

112

140

168

0

28

56

84

112

140

168

(c)

Fig. 15.3 Superior donor leukocyte infusion (DLI)-mediated graft-versus-leukemia effects in mixed compared with full allogeneic chimeras. Mixed and full chimeras were prepared by lethally irradiating B6 mice and reconstituting them with either T-cell-depleted allogeneic (B10.A) bone marrow cells alone (to produce full chimeras), or a mixture of T-cell-depleted B6 plus B10.A marrow (to produce mixed [Mix] chimeras). DLIs were administered on day 56 postbone marrow transplantation, and this step was followed by intravenous injection of 500 EL4 (a B6 T-cell lymphoma) cells, on day 63 (↓). (a) Full chimeras receiving DLI and EL4 cells (䊏; n = 20) showed a slight but statistically significant prolongation of survival compared with full chimeras receiving tumor cells only (䊐; n = 12; p < 0.001). Full chimeras receiving (䉲; n = 14) or not receiving DLI (䉮; n = 7) showed no treatment-related mortality. (b) Mixed chimeras receiving (◊; n = 11) or not receiving (䉫; n = 8) DLI showed no treatment-related mortality. Mixed chimeras receiving DLI and EL4 cells (䊉; n = 25) had a significantly improved survival compared with mixed chimeras receiving EL4 alone (䊊; n = 14; p < 0.0001). (c) Mixed chimeras receiving DLI and EL4 cells (䊉; n = 25) had a significantly improved survival compared with full chimeras receiving EL4 +DLI (䊏; n = 20; p < 0.0001). (Reproduced from Mapara MY, Kim YM, Wang SP, Bronson R, Sachs DH, Sykes M. Donor lymphocyte infusions mediate superior graft-versus-leukemia effects in mixed compared to fully allogeneic chimeras: a critical role for host antigen-presenting cells. Blood 2002; 100: 1903–9, with permission.)

therapeutic aspects of HCT without causing GVHD. In view of the new strategies and improved understanding of these phenomena that are developing, it seems likely that this challenge can be met and that HCT will ultimately be made available to all who could benefit from it, including those lacking an HLA-matched or closely matched donor. In addition, advances in the ability to achieve engraftment of hematopoietic and thymic tissue without ablative treatment of the host, and the demonstration of efficacy in primate models, have brought these approaches to initial clinical application for the induction of solid organ graft tolerance. Improvements in the ability to overcome HVG resistance to HLAmismatched HSC engraftment without increasing host toxicity should

further broaden the applicability of this approach. It will also be essential to extend this ability to xenogeneic marrow and organ transplantation, which presents additional immunologic and physiologic hurdles, to overcome the critical organ shortage that currently limits the number of life-saving organ transplants that can be performed. A major goal of transplant immunologists therefore now will be to extend new laboratory findings, first to additional preclinical models in large animals, and then to clinical protocols. In addition, it will be important to continue basic research into the mechanisms by which tolerance is induced and maintained in rodent model systems, so that these mechanisms can be further exploited.

References 1. Alam SM, Travers PJ, Wung JL et al. T-cellreceptor affinity and thymocyte positive selection. Nature 1996; 381: 616–20. 2. Palmer E. Negative selection – clearing out the bad apples from the T-cell repertoire. Nat Rev Immunol 2003; 3: 383–91. 3. Pullen AM, Kappler JW, Marrack P. Tolerance to self antigens shapes the T-cell repertoire. Immunol Rev 1989; 107: 125–39. 4. Rocha B, Von Boehmer H. Peripheral selection of the T cell repertoire. Science 1991; 251: 1225–8. 5. Carbone FR, Kurts C, Bennett SRM, Miller JFAP, Heath WR. Cross-presentation: a general mechanism for CTL immunity and tolerance. Immunol Today 1998; 19: 368–72. 6. Dey B, Yang Y-G, Preffer F et al. The fate of donor TCR transgenic T cells with known host antigen specificity in a GVHD model. Transplantation 1999; 68: 141–9. 7. Appleman LJ, Boussiotis VA. T cell anergy and costimulation. Immunol Rev 2003; 192: 161–80. 8. Rudd CE. Upstream-downstream: CD28 cosignaling pathways and T cell function. Immunity 1996; 4: 527–34. 9. Arnold B, Schonrich G, Hammerling GJ. Multiple levels of peripheral tolerance. Immunol Today1993; 14: 12–14.

10. Goodnow CC. Transgenic mice and analysis of B-cell tolerance. Ann Rev Immunol 1992; 10: 489–518. 11. Akkaraju S, Ho WY, Leong D, Canaan K, Davis MM, Goodnow CC. A range of CD4 T cell tolerance: partial inactivation to organ-specific antigen allows nondestructive thyroiditis or insulitis. Immunity 1997; 7: 255–71. 12. Ramsdell F, Fowlkes BJ. Clonal deletion versus clonal anergy: the role of the thymus in inducing self tolerance. Science 1990; 248: 1342–8. 13. Salaun J, Bandeira A, Khazaal I, et al. Thymic epithelium tolerizes for histocompatibility antigens. Science 1990; 247: 1471–4. 14. Burkly LC, Lo D, Flavell RA. Tolerance in transgenic mice expressing major histocompatibility molecules extrathymically on pancreatic cells. Science 1990; 248: 1364–8. 15. Morahan G, Allison J, Miller JFAP. Tolerance of class I histocompatibility antigens expressed extrathymically. Nature 1989; 339: 622–4. 16. Morahan G, Hoffman MW, Miller JFAP. A nondeletional mechanism of peripheral tolerance in T-cell receptor transgenic mice. Proc Natl Acad Sci U S A 1991; 88: 11421–5. 17. Cobbold SP, Qin S, Waldmann H. Reprogramming the immune system for tolerance with

18.

19.

20.

21.

22.

23.

24.

monoclonal antibodies. Semin Immunol 1990; 2: 377–87. Ochsenbein AF, Sierro S, Odermatt B et al. Roles of tumour localization, second signals and cross priming in cytotoxic T-cell induction. Nature 2001; 411: 1058–64. Quezada SA, Bennett K, Blazar BR, Rudensky AY, Sakaguchi S, Noelle RJ. Analysis of the underlying cellular mechanisms of anti-CD154induced graft tolerance: the interplay of clonal anergy and immune regulation. J Immunol 2005; 175: 771–9. Sykes M. Mechanisms of tolerance induced via mixed chimerism. Front Biosci 2007; 12: 2922– 34. Roser BJ. Cellular mechanisms in neonatal and adult tolerance. Immunol Rev 1989; 107: 179– 202. Wilson DB. Idiotypic regulation of T cells in graft-versus-host disease and autoimmunity. Immunol Rev 1989; 107: 159–76. O’Garra A, Vieira P. Regulatory T cells and mechanisms of immune system control. Nat Med 2004; 10: 801–5. Fontenot JD, Dooley JL, Farr AG, Rudensky AY. Developmental regulation of Foxp3 expression during ontogeny. J Exp Med 2005; 202: 901–6.

Mechanisms of Tolerance 25. Shevach EM. Certified professionals: CD4(+) CD25(+) suppressor T cells. J Exp Med 2001; 193: F41–6. 26. Zheng Y, Rudensky AY. Foxp3 in control of the regulatory T cell lineage. Nat Immunol 2007; 8: 457–62. 27. Bensinger SJ, Bandeira A, Jordan MS, Caton AJ, Laufer TM. Major histocompatibility complex class II-positive cortical epithelium mediates the selection of CD4(+)25(+) immunoregulatory T cells. J Exp Med 2001; 194: 427–38. 28. Aschenbrenner K, D’Cruz LM, Vollmann EH et al. Selection of Foxp3(+) regulatory T cells specific for self antigen expressed and presented by Aire(+) medullary thymic epithelial cells. Nat Immunol 2007; 8: 351–8. 29. Cozzo C, Larkin J III, Caton AJ. Self-peptides drive the peripheral expansion of CD4+CD25+ regulatory T cells. J Immunol 2003; 171: 5678– 82. 30. Stephens GL, Shevach EM. Foxp3+ regulatory T cells: selfishness under scrutiny. Immunity 2007; 27: 417–19. 31. Bacchetta R, Passerini L, Gambineri E et al. Defective regulatory and effector T cell functions in patients with FOXP3 mutations. J Clin Invest 2006; 116: 1713–22. 32. Malek TR, Bayer AL. Tolerance, not immunity, crucially depends on IL-2. Nat Rev Immunol 2004; 4: 665–74. 33. Clark RA, Kupper TS. IL-15 and dermal fibroblasts induce proliferation of natural regulatory T cells isolated from human skin. Blood 2007; 109: 194–202. 34. Battaglia M, Stabilini A, Migliavacca B, HorejsHoeck J, Kaupper T, Roncarolo MG. Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients. J Immunol 2006; 177: 8338–47. 35. Roncarolo MG, Gregori S, Battaglia M, Bacchetta R, Fleischhauer K, Levings MK. Interleukin-10secreting type 1 regulatory T cells in rodents and humans. Immunol Rev 2006; 212: 28–50. 36. Vigouroux S, Yvon E, Biagi E, Brenner MK. Antigen-induced regulatory T cells. Blood 2004; 104: 26–33. 37. Hoglund P. Induced peripheral regulatory T cells: the family grows larger. Eur J Immunol 2006; 36: 264–6. 38. Long E, Wood KJ. Understanding FOXP3: progress towards achieving transplantation tolerance. Transplantation 2007; 84: 459–61. 39. Wu Y, Borde M, Heissmeyer V et al. FOXP3 controls regulatory T cell function through cooperation with NFAT. Cell 2006; 126: 375–87. 40. Lowry RP, Takeuchi T. The Th1, Th2 Paradigm and Transplantation Tolerance. Austin: RG Landes; 1994. 41. Donckier V, Wissing M, Bruyns C et al. Critical role of interleukin 4 in the induction of neonatal transplantation tolerance. Transplantation 1995; 59: 1571–6. 42. Onodera K, Hancock WW, Graser E et al. Type 2 helper T cell-type cytokines and the development of “infectious” tolerance in rat cardiac allograft recipients. J Immunol 1997; 158: 1572– 81. 43. Piccotti JR, Chan SY, VanBuskirk AM, Eichwald EJ, Bishop DK. Are Th2 helper T lymphocytes beneficial, deleterious, or irrelevant in promoting

44.

45.

46.

47. 48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

allograft survival? Transplantation 1997; 63: 619–24. Le Moine A, Goldman M, Abramowicz D. Multiple pathways to allograft rejection. Transplantation 2002; 73: 1373–81. Chang CC, Ciubotariu R, Manavalan JS et al. Tolerization of dendritic cells by T(S) cells: the crucial role of inhibitory receptors ILT3 and ILT4. Nat Immunol 2002; 3: 237–43. Zhang ZX, Yang L, Young KJ, DuTemple B, Zhang L. Identification of a previously unknown antigen-specific regulatory T cell and its mechanism of suppression [In process citation]. Nat Med 2000; 6: 782–9. Miller RG. The veto phenomenon and T-cell regulation. Immunol Today 1986; 7: 112–14. Gur H, Krauthgamer R, Berrebi A et al. Tolerance induction by megadose hematopoietic progenitor cells: expansion of veto cells by short-term culture of purified human CD34(+) cells. Blood 2002; 99: 4174–81. Reich-Zeliger S, Gan J, Bachar-Lustig E, Reisner Y. Tolerance induction by veto CTLs in the TCR transgenic 2C mouse model. II. Deletion of effector cells by Fas-Fas ligand apoptosis. J Immunol 2004; 173: 6660–6. Azuma E, Yamamoto H, Kaplan J. Use of lymphokine-activated killer cells to prevent bone marrow graft rejection and lethal graft-vs-host disease. J Immunol 1989; 143: 1524–9. Heeg K, Wagner H. Induction of peripheral tolerance to class I major histocompatibility complex (MHC) alloantigens in adult mice: transfused class I MHC-incompatible splenocytes veto clonal response of antigen-reactive Lyt-2+ T cells. J Exp Med 1990; 172: 719. Sambhara SR, Miller RG. Programmed cell death of T cells signaled by the T cell receptor and the alpha-3 domain of class I MHC. Science 1991; 252: 1424–7. Verbanac KM, Carver FM, Haisch CE, Thomas JM. A role for transforming growth factor-beta in the veto mechanism in transplant tolerance. Transplantation 1994; 57: 893–900. Dorshkind K, Rosse C. Physical, biologic, and phenotypic properties of natural regulatory cells in murine bone marrow. Am J Anat 1982; 164: 1–17. Sykes M, Hoyles KA, Romick ML, Sachs DH. In vitro and in vivo analysis of bone marrow derived CD3+, CD4−, CD8−, NK1.1+ cell lines. Cell Immunol 1990; 129: 478–93. Hertel-Wulff B, Lindsten T, Schwadron R, Gilbert DM, Davis MM, Strober S. Rearrangement and expression of T cell receptor genes in cloned murine natural suppressor cell lines. J Exp Med 1987; 166: 1168–73. Sugiura K, Ikehara S, Gengozian N et al. Enrichment of natural suppressor activity in a wheat germ agglutinin positive hematopoietic progenitor-enriched fraction of monkey bone marrow. Blood 1990; 75: 1125–31. Mathew JM, Carreno M, Fuller L et al. Regulation of alloimmune responses (GvH reactions) in vitro by autologous donor bone marrow cell preparation used in clinical organ transplantation. Transplantation 2002; 74: 846–55. Billiau AD, Fevery S, Rutgeerts O, Landuyt W, Waer M. Transient expansion of Mac1+Ly6– G+Ly6–C+ early myeloid cells with suppressor activity in spleens of murine radiation marrow

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

203

chimeras: possible implications for the graftversus-host and graft-versus-leukemia reactivity of donor lymphocyte infusions. Blood 2003; 102: 740–8. Li Y, Ma L, Shen J, Chong AS. Peripheral deletion of mature alloreactive B cells induced by costimulation blockade. Proc Natl Acad Sci U S A 2007 104: 12093–8. Ferry H, Leung JC, Lewis G, Nijnik A, Silver K, Lambe T, Cornall RJ. B-cell tolerance. Transplantation 2006; 81: 308–15. Klinman NR. The “clonal selection hypothesis” and current concepts of B cell tolerance. Immunity 1996; 5: 189–95. Merrell KT, Benschop RJ, Gauld SB et al. Identification of anergic B cells within a wild-type repertoire. Immunity 2006; 25: 953–62. Cooke MP, Heath AW, Shokat KM et al. Immunoglobulin signal transduction guides the specificity of B cell-T cell interactions and is blocked in tolerant self-reactive B cells. J Exp Med 1994; 179: 425–38. Ohdan H, Swenson KG, Kruger-Gray HW et al. Mac-1-negative B-1b phenotype of natural antibody-producing cells, including those responding to Gala1,3Gal epitopes in a1,3-galactosyltransferase deficient mice. J Immunol 2000; 165: 5518– 29. Murakami M, Tsubata T, Okamoto M et al. Antigen-induced apoptotic death of Ly-1 B cells responsible for autoimmune disease in transgenic mice. Nature 1992; 357: 77–80. Waldmann H, Cobbold S. The use of monoclonal antibodies to achieve immunological tolerance. Immunol Today 1993; 14: 247–51. Li XC, Wells AD, Strom TB, Turka LA. The role of T cell apoptosis in transplantation tolerance. Curr Opin Immunol 2000; 12: 522–7. Wekerle T, Sayegh MH, Hill J et al. Extrathymic T cell deletion and allogeneic stem cell engraftment induced with costimulatory blockade is followed by central T cell tolerance. J Exp Med 1998; 187: 2037–44. Wekerle T, Sayegh MH, Chandraker A, Swenson KG, Zhao Y, Sykes M. Role of peripheral clonal deletion in tolerance induction with bone marrow transplantation and costimulatory blockade. Transplant Proc 1999; 31: 680. Kurtz J, Shaffer J, Anosova N, Benichou G, Sykes M. Mechanisms of early peripheral CD4 T cell tolerance induction by anti-CD154 monoclonal antibody and allogeneic bone marrow transplantation: evidence for anergy and deletion, but not regulatory cells. Blood 2004; 103: 4336–43. Fehr T, Takeuchi Y, Kurtz J, Sykes M. Early regulation of CD8 T cell alloreactivity by CD4+CD25− T cells in recipients of anti-CD154 antibody and allogeneic BMT is followed by rapid peripheral deletion of donor-reactive CD8+ T cells, precluding a role for sustained regulation. Eur J Immunol 2005; 35: 2679–90. Fabre JW, Morris PJ. The effect of donor strain blood pretreatment on renal allograft rejection in rats. Transplantation 1972; 14: 608–17. Persijn GG, Hendriks GFJ, Van Rood JJ. HLA matching, blood transfusion and renal transplantation. Clin Immunol All 1984; 4: 535–64. Eynon EE, Parker DC. Small B cells as antigenpresenting cells in the induction of tolerance to soluble protein antigens. J Exp Med 1992; 175: 131–8.

204

Chapter 15

76. Wong W, Morris PJ, Wood KJ. Syngeneic bone marrow expressing a single donor class I MHC molecule permits acceptance of a fully allogeneic cardiac allograft. Transplantation 1996; 62: 1462–8. 77. Sonntag KC, Emery DW, Yasumoto A et al. Tolerance to solid organ transplants through transfer of MHC class II genes. J Clin Invest 2001; 107: 65–71. 78. Hara M, Kingsley CI, Niimi M et al. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J Immunol 2001; 166: 3789–96. 79. Bushell A, Niimi M, Morris PJ, Wood KJ. Evidence for immune regulation in the induction of transplantation tolerance: a conditional but limited role for IL-4. J Immunol 1999; 162: 1359–66. 80. Barratt-Boyes SM, Thomson AW. Dendritic cells: tools and targets for transplant tolerance. Am J Transplant 2005; 5: 2807–13. 81. Rutella S, Danese S, Leone G. Tolerogenic dendritic cells: cytokine modulation comes of age. Blood 2006; 108: 1435–40. 82. Wang Q, Liu Y, Wang J et al. Induction of allospecific tolerance by immature dendritic cells genetically modified to express soluble TNF receptor. J Immunol 2006; 177: 2175–85. 83. Turnquist HR, Raimondi G, Zahorchak AF, Fischer RT, Wang Z, Thomson AW. Rapamycinconditioned dendritic cells are poor stimulators of allogeneic CD4+ T cells, but enrich for antigenspecific Foxp3+ T regulatory cells and promote organ transplant tolerance. J Immunol 2007; 178: 7018–31. 84. Hackstein H, Morelli AE, Thomson AW. Designer dendritic cells for tolerance induction: guided not misguided missiles. Trends Immunol 2001; 22: 437–42. 85. Sawitzki B, Kingsley CI, Oliveira V, Karim M, Herber M, Wood KJ. IFN-{gamma} production by alloantigen-reactive regulatory T cells is important for their regulatory function in vivo. J Exp Med 2005; 201: 1925–35. 86. Zanin-Zhorov A, Tal-Lapidot G, Cahalon L et al. Cutting edge: T cells respond to lipopolysaccharide innately via TLR4 signaling. J Immunol 2007; 179: 41–4. 87. Gilliet M, Liu YJ. Generation of human CD8 T regulatory cells by CD40 ligand-activated plasmacytoid dendritic cells. J Exp Med 2002; 195: 695–704. 88. Ito T, Yang M, Wang YH et al. Plasmacytoid dendritic cells prime IL-10-producing T regulatory cells by inducible costimulator ligand. J Exp Med 2007; 204: 105–15. 89. Greenwald RJ, Freeman GJ, Sharpe AH. The B7 family revisited. Annu Rev Immunol 2005; 23: 515–48. 90. Pearson TC, Alexander DZ, Winn KJ, Linsley PS, Lowry RP, Larsen CP. Transplantation tolerance induced by CTLA4Ig. Transplantation 1994; 57: 1701–6. 91. Lanzavecchia A. Licence to kill. Nature 1998; 393: 413–14. 92. Parker DC, Greiner DL, Phillips NE et al. Survival of mouse pancreatic islet allografts in recipients treated with allogeneic small lymphocytes and antibody to CD40 ligand. Proc Natl Acad Sci U S A 1995; 92: 9560–4. 93. Markees TG, Phillips NE, Noelle RJ et al. Prolonged survival of mouse skin allografts in recip-

94.

95.

96.

97.

98.

99.

100.

101.

102.

103.

104.

105.

106.

107.

108.

ients treated with donor splenocytes and antibody to CD40 ligand. Transplantation 1997; 64: 329– 35. Iwakoshi NN, Markees TG, Turgeon N et al. Skin allograft maintenance in a new synchimeric model system of tolerance. J Immunol 2001; 167: 6623– 30. Markees TG, Phillips NE, Gordon EJ et al. Longterm survival of skin allografts induced by donor splenocytes and anti-CD154 antibody in thymectomized mice requires CD4+ T cells, Interferon-g, and CTLA4. J Clin Invest 1998; 101: 2446–55. Gordon EJ, Woda BA, Shultz LD, Rossini AA, Greiner DL, Mordes JP. Rat xenograft survival in mice treated with donor-specific transfusion and anti-CD154 antibody is enhanced by elimination of host CD4+ cells. Transplantation 2001; 71: 319–27. Larsen CP, Alexander DZ, Hollenbaugh D et al. CD40-GP39 interactions play a critical role during allograft rejection: suppression of allograft rejection by blockade of the CD40-gp39 pathway. Transplantation 1996; 61: 4–9. Ensminger SM, Spriewald BM, Witzke O et al. Intragraft interleukin-4 mRNA expression after short-term CD154 blockade may trigger delayed development of transplant arteriosclerosis in the absence of CD8+ T cells [In process citation]. Transplantation 2000; 70: 955–63. Sayegh MH, Akalin E, Hancock WW et al. CD28-B7 blockade after alloantigenic challenge in vivo inhibits Th1 cytokines but spares Th2. J Exp Med 1995; 181: 1869–74. Larsen CP, Elwood ET, Alexander DZ et al. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 1996; 381: 434–8. Kirk AD, Harlan DM, Armstrong NN et al. CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates. Proc Natl Acad Sci U S A 1997; 94: 8789–98. Elwood ET, Larsen CP, Cho HR et al. Prolonged acceptance of concordant and discordant xenografts with combined CD40 and CD28 pathway blockade. Transplantation 1998; 65: 1422–8. Li Y, Zheng XX, Li XC, Zand MS, Strom TB. Combined costimulation blockade plus rapamycin but not cyclosporine produces permanent engraftment. Transplantation 1998; 66: 1387–8. Lehnert AM, Yi S, Burgess JS, O’Connell PJ. Pancreatic islet xenograft tolerance after shortterm costimulation blockade is associated with increased CD4+ T cell apoptosis but not immune deviation. Transplantation 2000; 69: 1176–85. Tramblay J, Bingaman AW, Lin A et al. Asialo GM1+ CD8+ T cells play a critical role in costimulation blockade-resistant allograft rejection. J Clin Invest 1999; 104: 1715–22. Kenyon NS, Chatzipetrou M, Masetti M et al. Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154. Proc Natl Acad Sci U S A 1999; 96: 8132–7. Kirk AD, Burkly LC, Batty DS et al. Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates. Nature Med 1999; 5: 686– 93. Waldmann H, Cobbold S. Regulating the immune response to transplants. A role for CD4(+) regulatory cells? Immunity 2001; 14: 399–406.

109. Ito H, Kurtz J, Shaffer J, Sykes M. CD4 T cellmediated alloresistance to fully MHC-mismatched allogeneic bone marrow engraftment is dependent on CD40-CD40L interactions, and lasting T cell tolerance is induced by bone marrow transplantation with initial blockade of this pathway. J Immunol 2001; 166: 2981. 110. Li Y, Li XC, Zheng XX, Wells AD, Turka LA, Strom TB. Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance. Nature Med 1999; 5: 1298–302. 111. Koulmanda M, Smith RN, Qipo A, Weir G, Auchincloss H, Strom TB. Prolonged survival of allogeneic islets in cynomolgus monkeys after short-term anti-CD154-based therapy: nonimmunologic graft failure? Am J Transplant 2006; 6: 687–96. 112. Gao W, Lu Y, El Essawy B, Oukka M, Kuchroo VK, Strom TB. Contrasting effects of cyclosporine and rapamycin in de novo generation of alloantigen-specific regulatory T cells. Am J Transplant 2007; 7: 1722–32. 113. Zheng XX, Sanchez-Fueyo A, Sho M, Domenig C, Sayegh MH, Strom TB. Favorably tipping the balance between cytopathic and regulatory T cells to create transplantation tolerance. Immunity 2003; 19: 503–14. 114. Lakkis FG, Dai Z. The role of cytokines, CTLA4 and costimulation in transplant tolerance and rejection. Curr Opin Immunol 1999; 11: 504–8. 115. Chavin KD, Qin L, Lin J, Yagita H, Bromberg JS. Combined anti-CD2 and anti-CD3 receptor monoclonal antibodies induce donor-specific tolerance in a cardiac transplant model. J.Immunol 1993; 151: 7249–59. 116. Krieger NR, Most D, Bromberg JS et al. Coexistence of Th1- and Th2-type cytokine profiles in anti-CD2 monoclonal antibody-induced tolerance. Transplantation 1996; 62: 1285–92. 117. Woodward JE, Qin L, Chavin KD et al. Blockade of multiple costimulatory receptors induces hyporesponsiveness: inhibition of CD2 plus CD28 pathways. Transplantation 1996; 62: 1011–18. 118. Salvalaggio PR, Camirand G, Ariyan CE et al. Antigen exposure during enhanced CTLA-4 expression promotes allograft tolerance in vivo. J Immunol 2006; 176: 2292–8. 119. Strober S, Benike C, Krishnaswamy S, Engleman EG, Grumet FC. Clinical transplantation tolerance twelve years after prospective withdrawal of immunosuppressive drugs: studies of chimerism and anti-donor reactivity. Transplantation 2000; 69: 1549–54. 120. Strober S, Lowsky RJ, Shizuru JA, Scandling JD, Millan MT. Approaches to transplantation tolerance in humans. Transplantation 2004; 77: 932– 6. 121. Yamada K, Gianello PR, Iereno FL et al. Role of the thymus in transplantation tolerance in miniature swine. I. Requirement of the thymus for rapid and stable induction of tolerance to class Imismatched renal allografts. J Exp Med 1997; 186: 497–506. 122. Sayegh MH, Perico N, Gallon L et al. Mechanisms of acquired thymic unresponsiveness to renal allografts. Thymic recognition of immunodominant allo-MHC peptides induces peripheral T cell anergy. Transplantation 1994; 58: 125– 32.

Mechanisms of Tolerance 123. Bonasio R, Scimone ML, Schaerli P, Grabie N, Lichtman AH, von Andrian UH. Clonal deletion of thymocytes by circulating dendritic cells homing to the thymus. Nat Immunol 2006; 7: 1092–100. 124. Agus DB, Surh CD, Sprent J. Reentry of T cells to the adult thymus is restricted to activated T cells. J Exp Med 1991; 173: 1039–46. 125. Ali A, Garrovillo M, Oluwole OO et al. Mechanisms of acquired thymic tolerance: induction of transplant tolerance by adoptive transfer of in vivo allomhc peptide activated syngeneic T cells. Transplantation 2001; 71: 1442– 48. 126. Odorico JS, O’Connor T, Campos L, Barker CF, Posselt AM, Naji A. Examination of the mechanisms responsible for tolerance induction after intrathymic inoculation of allogeneic bone marrow. Ann Surg 1993; 218: 525–31. 127. Tomita Y, Khan A, Sykes M. Role of intrathymic clonal deletion and peripheral anergy in transplantation tolerance induced by bone marrow transplantation in mice conditioned with a nonmyeloablative regimen. J Immunol 1994; 153: 1087–98. 128. Khan A, Tomita Y, Sykes M. Thymic dependence of loss of tolerance in mixed allogeneic bone marrow chimeras after depletion of donor antigen. Peripheral mechanisms do not contribute to maintenance of tolerance. Transplantation 1996; 62: 380–87. 129. Tomita Y, Sachs DH, Sykes M. Myelosuppressive conditioning is required to achieve engraftment of pluripotent stem cells contained in moderate doses of syngeneic bone marrow. Blood 1994; 83: 939–48. 130. Ramshaw HS, Crittenden RB, Dooner M, Peters SO, Rao SS, Quesenberry PJ. High levels of engraftment with a single infusion of bone marrow cells into normal unprepared mice. Biol Blood Marrow Transplant 1995; 1: 74–80. 131. Sykes M, Szot GL, Swenson K, Pearson DA, Wekerle T. Separate regulation of hematopietic and thymic engraftment. Exp Hematol 1997; 26: 457–65. 132. Wright DE, Wagers AJ, Gulati AP, Johnson FL, Weissman IL. Physiological migration of hematopoietic stem and progenitor cells. Science 2001; 294: 1933–6. 133. Ikehara S. A novel strategy for allogeneic stem cell transplantation: perfusion method plus intrabone marrow injection of stem cells. Exp Hematol 2003; 31: 1142–6. 134. Sharabi Y, Sachs DH, Sykes M. T cell subsets resisting induction of mixed chimerism across various histocompatibility barriers. In: Gergely J, Benczur M, Falus A, Fust Gy, Medgyesi G, Petranyi Gy, Rajnavolgyi E, editors. Progress in Immunology VIII. Proceedings of the Eighth International Congress of Immunology, Budapest, 1992. Heidelberg: Springer-Verlag; 1992. p. 801– 5. 135. Hayashi H, LeGuern C, Sachs DH, Sykes M. Alloresistance to K locus mismatched bone marrow engraftment is mediated entirely by CD4+ and CD8+ T cells. Bone Marrow Transplant 1996; 18: 285–92. 136. Moretta L, Ciccone E, Moretta A, Hoglund P, Ohlen C, Karre K. Allorecognition by NK cells: nonself or no self? Immunol Today 1992; 13: 300–6.

137. Lee LA, Sergio JJ, Sykes M. Natural killer cells weakly resist engraftment of allogeneic long-term multilineage-repopulating hematopoietic stem cells. Transplantation 1996; 61: 125–32. 138. Aguila HL, Weissman IL. Hematopoietic stem cells are not direct cytotoxic targets of natural killer cells. Blood 1996; 87: 1225–31. 139. Ruggeri L, Capanni M, Urbani E et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002; 295: 2097–100. 140. O’Reilly RJ, Brochstein J, Collins N et al. Evaluation of HLA-haplotye disparate parental marrow grafts depleted of T lymphocytes by differential agglutination with a soybean lectin and E-rosette depletion for the treatment of severe combined immunodeficiency. Vox Sang 1986; 51(Suppl 2): 81–6. 141. Zhao Y, Ohdan H, Manilay JO, Sykes M. NK cell tolerance in mixed allogeneic chimeras. J Immunol 2003; 170: 5398–405. 142. Manilay JO, Waneck GL, Sykes M. Levels of Ly-49 receptor expression are determined by the frequency of interactions with MHC ligands: evidence against receptor calibration to a “useful” level. J Immunol 1999; 163: 2628–33. 143. Manilay JO, Waneck GL, Sykes M. Altered expression of Ly-49 receptors on natural killer cells developing in mixed allogeneic bone marrow chimeras. Int Immunol 1998; 10: 1943–55. 144. Kawahara T, Rodriguez-Barbosa JI, Zhao Y, Zhao G, Sykes M. Global unresponsiveness as a mechanism of natural killer cell tolerance in mixed xenogeneic chimeras. Am J Transplant 2007; 7: 2090–7. 145. Raulet DH, Vance RE. Self-tolerance of natural killer cells. Nat Rev Immunol 2006; 6: 520–31. 146. Ohdan H, Swenson KG, Kitamura H, Yang Y-G, Sykes M. Tolerization of Gala1,3Gal-reactive B cells in presensitized a1,3-galactosyltransferasedeficient mice by nonmyeloablative induction of mixed chimerism. Xenotransplantation 2002; 8: 227–38. 147. Seebach JD, Stussi G, Passweg JR et al. ABO Blood group barrier in allogeneic bone marrow transplantation revisited. Biol Blood Marrow Transplant 2005; 11: 1006–13. 148. Owen RD. Immunogenetic consequences of vascular anastomoses between bovine twins. Science 1945; 102: 400–1. 149. Mathes DW, Solari MG, Randolph MA et al. Long-term acceptance of renal allografts following prenatal inoculation with adult bone marrow. Transplantation 2005; 80: 1300–8. 150. Carrier E, Lee TH, Busch MP, Cowan MJ. Induction of tolerance in nondefective mice after in utero transplantation of major histocompatibility complex-mismatched fetal hematopoietic stem cells. Blood 1995; 86: 4681–90. 151. Streilein JW. Neonatal tolerance of H-2 alloantigens. Transplantation 1991; 52: 1–10. 152. Gao Q, Rouse TM, Kazmerzak K, Field EH. CD4+CD25+ cells regulate CD8 cell anergy in neonatal tolerant mice. Transplantation 1999; 68: 1891–7. 153. Chen N, Field EH. Enhanced type 2 and diminished type 1 cytokines in neonatal tolerance. Transplantation 1995; 59: 933–41. 154. Ridge JP, Fuchs EJ, Matzinger P. Neonatal tolerance revisited: Turning on newborn T cells with dendritic cells. Science 1996; 271: 1723–6.

205

155. Ildstad ST, Sachs DH. Reconstitution with syngeneic plus allogeneic or xenogeneic bone marrow leads to specific acceptance of allografts or xenografts. Nature 1984; 307: 168–70. 156. De Villartay J-P, Griscelli C, Fischer A. Selftolerance to host and donor following HLAmismatched bone marrow transplantation. Eur J Immunol 1986; 16: 117–22. 157. Sykes M, Sheard MA, Sachs DH. Effects of T cell depletion in radiation bone marrow chimeras. II. Requirement for allogeneic T cells in the reconstituting bone marrow inoculum for subsequent resistance to breaking of tolerance. J Exp Med 1988; 168: 661–73. 158. Kurtz J, Wekerle T, Sykes M. Tolerance in mixed chimerism – a role for regulatory cells? Trends Immunol 2004; 25: 518–23. 159. Slavin S. Total lymphoid irradiation. Immunol Today 1987; 3: 88–92. 160. Pierce GE. Allogeneic versus semiallogeneic F1 bone marrow transplantation into sublethally irradiated MHC-disparate hosts. Effects on mixed lymphoid chimerism, skin graft tolerance, host survival, and alloreactivity. Transplantation 1990; 49: 138–44. 161. Eto M, Mayumi H, Tomita Y, Yoshikai Y, Nomoto K. Intrathymic clonal deletion of V beta 6+ T cells in cyclophosphamide-induced tolerance to H-2-compatible, Mls-disparate antigens. J Exp Med 1990; 171: 97–113. 162. Mayumi H, Good RA. Long-lasting skin allograft tolerance in adult mice induced across fully allogeneic (multimajor H-2 plus multiminor histocompatibility) antigen barriers by a toleranceinducing method using cyclophosphamide. J Exp Med 1989; 169: 213–38. 163. Fontes P, Rao AS, Demetris AJ et al. Bone marrow augmentation of donor-cell chimerism in kidney, liver, heart and pancreas islet transplantation. Lancet 1994; 344: 151–5. 164. Ciancio G, Miller J, Garcia-Morales RO et al. Six-year clinical effect of donor bone marrow infusions in renal transplant patients. Transplantation 2001; 71: 827–35. 165. Kuhr CS, Allen MD, Junghanss C et al. Tolerance to vascularized kidney grafts in canine mixed hematopoietic chimeras. Transplantation 2002; 73: 1487–92. 166. Najarian JS, Ferguson RM, Sutherland DER et al. Fractionated total lymphoid irradiation as preparative immunosuppression in high risk renal transplantation. Ann Surg 1982; 196: 442– 51. 167. Lowsky R, Takahashi T, Liu YP et al. Protective conditioning for acute graft-versus-host disease. N Engl J Med 2005; 353: 1321–31. 168. Fudaba Y, Spitzer TR, Shaffer J et al. Myeloma responses and tolerance following combined kidney and nonmyeloablative marrow transplantation: in vivo and in vitro analyses. Am J Transplant 2006; 6: 2121–33. 169. Luznik L, Jalla S, Engstrom LW, Iannone R, Fuchs EJ. Durable engraftment of major histocompatibility complex-incompatible cells after nonmyeloablative conditioning with fludarabine, low-dose total body irradiation, and posttransplantation cyclophosphamide. Blood 2001; 98: 3456–64. 170. O’Donnell PV, Luznik L, Jones RJ et al. Nonmyeloablative bone marrow transplantation from partially HLA-mismatched related donors using

206

171.

172.

173.

174.

175.

176.

177.

178.

179.

180. 181.

182.

183.

184.

185.

Chapter 15

posttransplantation cyclophosphamide. Biol Blood Marrow Transplant 2002; 8: 377–86. Wood ML, Monaco AP, Gozzo JJ, Liegois A. Use of homozygous allogeneic bone marrow for induction of tolerance with anti-lymphocyte serum: dose and timing. Transplant Proc 1970; 3: 676. Kanamoto A, Monaco AP, Maki T. Active role of chimerism in transplantation tolerance induced by antilymphocyte serum, sirolimus, and bonemarrow-cell infusion. Transplantation 2004; 78: 825–30. Minamimura K, Gao W, Maki T. CD4+ regulatory T cells are spared from deletion by antilymphocyte serum, a polyclonal anti-T cell antibody. J Immunol 2006; 176: 4125–32. Thomas JM, Verbanac KM, Smith JP et al. The facilitating effect of one-DR antigen sharing in renal allograft tolerance induced by donor bone marrow in rhesus monkeys. Transplantation 1995; 59: 245–55. Sharabi Y, Aksentijevich I, Sundt TM III, Sachs DH, Sykes M. Specific tolerance induction across a xenogeneic barrier: production of mixed rat/ mouse lymphohematopoietic chimeras using a nonlethal preparative regimen. J Exp Med 1990; 172: 195–202. Nikolic B, Cooke DT, Zhao G, Sykes M. Both g/d T cells and NK cells inhibit the engraftment of xenogeneic rat bone marrow cells in mice. J Immunol 2001; 166: 1398–404. Kawai T, Cosimi AB, Colvin RB et al. Mixed allogeneic chimerism and renal allograft tolerance in cynomologus monkeys. Transplantation 1995; 59: 256–62. Kawai T, Cosimi AB, Spitzer TR et al. HLAmismatched renal transplantation without mantenance immunosuppression. New Engl J Med 2008; 358: 353–361. Yang YG, Sykes M. Xenotransplantation: current status and a perspective on the future. Nat Rev Immunol 2007; 7: 519–31. Sykes M. Mixed chimerism and transplant tolerance. Immunity 2001; 14: 417–424. Wekerle T, Sykes M. Mixed chimerism and transplantation tolerance. Annu Rev Med 2001; 52: 353–70. Sykes M, Sheard MA, Sachs DH. Graft-versushost-related immunosuppression is induced in mixed chimeras by alloresponses against either host or donor lymphohematopoietic cells. J Exp Med 1988; 168: 2391–6. Pelot MR, Pearson DA, Swenson K et al. Lymphohematopoietic graft-vs-host reactions can be induced without graft-vs-host disease in murine mixed chimeras established with a cyclophosphamide-based non-myeloablative conditioning regimen. Biol Blood Marrow Transplant 1999; 5: 133–43. Mapara MY, Kim Y-M, Wang S-P, Bronson R, Sachs DH, Sykes M. Donor lymphocyte infusions mediate superior graft-versus-leukemia effects in mixed compared to fully allogeneic chimeras: a critical role for host antigen-presenting cells. Blood 2002; 100: 1903–9. Sykes M, Preffer F, McAffee S et al. Mixed lymphohematopoietic chimerism and graft-vslymphoma effects are achievable in adult humans following non-myeloablative therapy and HLAmismatched donor bone marrow transplantation. Lancet 1999; 353: 1755–9.

186. Spitzer TR, MCafee S, Sackstein R et al. The intentional induction of mixed chimerism and achievement of anti-tumor responses following non-myeloablative conditioning therapy and HLA-matched and mismatched donor bone marrow transplantation for refractory hematologic malignancies. Biol Blood Marrow Transplant 2000; 6: 309–20. 187. Rubio MT, Kim YM, Sachs T, Mapara M, Zhao G, Sykes M. Anti-tumor effect of donor marrow graft rejection induced by recipient leukocyte infusions in mixed chimeras prepared with nonmyeloablative conditioning: critical role for recipient-derived IFN-{gamma}. Blood 2003; 102: 2300–7. 188. Wekerle T, Kurtz J, Ito H et al. Allogeneic bone marrow transplantation with costimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment. Nature Med 2000; 6: 464–9. 189. Durham MM, Bingaman AW, Adams AB et al. Cutting edge: administration of anti-CD40 ligand and donor bone marrow leads to hemopoietic chimerism and donor-specific tolerance without cytoreductive conditioning. J Immunol 2000; 165: 1–4. 190. Haynes BF, Markert ML, Sempowski GD, Patel DD, Hale LP. The role of the thymus in immune reconstiutution in aging, bone marrow transplantation, and HIV-1 infection. Ann Rev Immunol 2000; 18: 529–60. 191. Kurtz J, Ito H, Wekerle T, Shaffer J, Sykes M. Mechanisms involved in the establishment of tolerance through costimulatory blockade and BMT: lack of requirement for CD40L-mediated signaling for tolerance or deletion of donor-reactive CD4+ cells. Am J Transplant 2001; 1: 339–49. 192. Bigenzahn S, Blaha P, Koporc Z et al. The role of non-deletional tolerance mechanisms in a murine model of mixed chimerism with costimulation blockade. Am J Transplant 2005; 5: 1237– 47. 193. Domenig C, Sanchez-Fueyo A, Kurtz J et al. Roles of deletion and regulation in creating mixed chimerism and allograft tolerance using a nonlymphoablative irradiation-free protocol. J Immunol 2005; 175: 51–60. 194. Williams MA, Tan JT, Adams AB et al. Characterization of virus-mediated inhibition of mixed chimerism and allospecific tolerance. J Immunol 2001; 167: 4987–95. 195. Abe M, Qi J, Sykes M, Yang Y-G. Mixed chimerism induces donor-specific T cell tolerance across a highly disparate xenogeneic barrier. Blood 2002; 99: 3823–9. 196. Zhao Y, Swenson K, Sergio JJ, Arn JS, Sachs DH, Sykes M. Skin graft tolerance across a discordant xenogeneic barrier. Nature Med 1996; 2: 1211– 16. 197. Zhao Y, Fishman JA, Sergio JJ et al. Immune restoration by fetal pig thymus grafts in T celldepleted, thymectomized mice. J Immunol 1997; 158: 1641–9. 198. Zhao Y, Sergio JJ, Swenson KA, Arn JS, Sachs DH, Sykes M. Positive and negative selection of functional mouse CD4 cells by porcine MHC in pig thymus grafts. J Immunol 1997; 159: 2100–7. 199. Zhao Y, Swenson K, Sergio JJ, Sykes M. Pig MHC mediates positive selection of mouse CD4+ T cells with a mouse MHC-restricted TCR in pig thymus grafts. J Immunol 1998; 161: 1320–6.

200. Markert ML, Kostyu DD, Ward FE et al. Successful formation of a chimeric human thymus allograft following transplantation of cultured postnatal human thymus. J Immunol 1997; 158: 998–1005. 201. Zerrahn J, Held W, Raulet DH. The MHC reactivity of the T cell repertoire prior to positive and negative selection. Cell 1997; 88: 627–36. 202. Nikolic B, Gardner JP, Scadden DT, Arn JS, Sachs DH, Sykes M. Normal development in porcine thymus grafts and specific tolerance of human T cells to porcine donor MHC. J Immunol 1999; 162: 3402–7. 203. Ohdan H, Sykes M. B cell tolerance to xenoantigens. Xenotransplantation 2003; 10: 98–106. 204. Kawahara T, Shimizu I, Ohdan H, Zhao G, Sykes M. Differing mechanisms of early and late B cell tolerance induced by mixed chimerism. Am J Transplant 2005; 5: 2821–9. 205. Shimizu I, Kawahara T, Haspot F, Bardwell PD, Carroll MC, Sykes M. B-cell extrinsic CR1/CR2 promotes natural antibody production and tolerance induction of anti-alpha-Gal-producing B-1 cells. Blood 2006; 109: 1773–81. 206. Bracy JL, Iacomini J. Induction of B-cell tolerance by retroviral gene therapy. Blood 2000; 96: 3008–15. 207. Phelps CJ, Koike C, Vaught TD et al. Production of alpha 1,3-galactosyltransferase-deficient pigs. Science 2003; 299: 411–14. 208. Kolber-Simonds D, Lai L, Watt SR et al. Production of {alpha}-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations. Proc Natl Acad Sci U S A 2004; 101: 7335–40. 209. Chen G, Qian H, Starzl T et al. Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys. Nat Med 2005; 11: 1295–8. 210. Reich-Zeliger S, Zhao Y, Krauthgamer R, BacharLustig E, Reisner Y. Anti-third party CD8+ CTLs as potent veto cells: coexpression of CD8 and FasL is a prerequisite. Immunity 2000; 13: 507– 15. 211. Fontaine P, Roy-Proulx G, Knafo L, Baron C, Roy DC, Perreault C. Adoptive transfer of minor histocompatibility antigen-specific T lymphocytes eradicates leukemia cells without causing graft-versus-host disease. Nat Med 2001; 7: 789–94. 212. Sykes M, Sachs DH. Genetic analysis of the antileukemic effect of mixed allogeneic bone marrow transplantation. Transplant Proc 1989; 21: 302224. 213. Aizawa S, Sado T. Graft-versus-leukemia effect in MHC-compatible and -incompatible allogeneic bone marrow transplantation of radiation-induced, leukemia-bearing mice. Transplantation 1991; 52: 885–9. 214. Beatty PG, Anasetti C, Hansen JA et al. Marrow transplantation from unrelated donors for treatment of hematologic malignancies: effect of mismatching for one HLA locus. Blood 1993; 81: 249–53. 215. Chakraverty R, Eom HS, Sachs J et al. Host MHC Class II+ antigen-presenting cells and CD4 cells are required for CD8-mediated graft-versusleukemia responses following delayed donor leukocyte infusions. Blood 2006; 108: 2106–13. 216. Guinan EC, Boussiotis VA, Neuberg D et al. Transplantation of anergic histoincompatible

Mechanisms of Tolerance

217.

218.

219.

220.

221.

222.

223.

224.

225.

226.

227.

228.

229.

bone marrow allografts. New Engl J Med 1999; 340: 1704–14. Amrolia PJ, Muccioli-Casadei G, Huls H et al. Adoptive immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical stem cell transplantation. Blood 2006; 108: 1797–808. Taylor PA, Noelle RJ, Blazar BR. CD4(+)CD25(+) immune regulatory cells are required for induction of tolerance to alloantigen via costimulatory blockade. J Exp Med 2001; 193: 1311–18. Edinger M, Hoffmann P, Ermann J et al. CD4(+)CD25(+) regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation. Nat Med 2003; 9: 1144–50. Nguyen VH, Shashidhar S, Chang DS et al. The impact of regulatory T cells on T cell immunity following hematopoeitic cell transplantation. Blood 2008; 111: 945–53. Lan F, Zeng D, Higuchi M, Huie P, Higgins JP, Strober S. Predominance of NK1.1(+)TCRalphabeta(+) or DX5(+)TCRalphabeta(+) T cells in mice conditioned with fractionated lymphoid irradiation protects against graft-versus-host disease: “natural suppressor” cells. J Immunol 2001; 167: 2087– 96. Zeng D, Hoffmann P, Lan F, Huie P, Higgins J, Strober S. Unique patterns of surface receptors, cytokine secretion, and immune functions distinguish T cells in the bone marrow from those in the periphery: impact on allogeneic bone marrow transplantation. Blood 2002; 99: 1449–57. Fowler DH, Breglio J, Nagel G, Eckhaus MA, Gress RE. Allospecific CD8+ Tc1 and Tc2 populations in graft-versus-leukemia effect and graftversus-host disease. J Immunol 1996; 157: 4811–21. Fowler DH, Odom J, Steinberg SM et al. Phase I clinical trial of costimulated, IL-4 polarized donor CD4+ T cells as augmentation of allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 2006; 12: 1150–60. Sykes M, Romick ML, Hoyles KA, Sachs DH. In vivo administration of interleukin 2 plus T celldepleted syngeneic marrow prevents graft-versushost disease mortality and permits alloengraftment. J Exp Med 1990; 171: 645–58. Brok HPM, Heidt PJ, Van der Meide PH, Zurcher C, Vossen JM. Interferon-gamma prevents graftversus-host disease after allogeneic bone marrow transplantation in mice. J Immunol 1993; 151: 6451–9. Sykes M, Szot GL, Nguyen PL, Pearson DA. Interleukin-12 inhibits murine graft-vs-host disease. Blood 1995; 86: 2429–38. Sykes M, Romick ML, Sachs DH. Interleukin 2 prevents graft-vs-host disease while preserving the graft-vs-leukemia effect of allogeneic T cells. Proc Natl Acad Sci U S A 1990; 87: 5633–7. Yang Y-G, Sergio JJ, Pearson DA, Szot GL, Shimizu A, Sykes M. Interleukin-12 preserves the graft-versus-leukemia effect of allogeneic CD8 T cells while inhibiting CD4-dependent graft-

230.

231.

232.

233.

234.

235.

236.

237. 238.

239.

240.

241. 242.

243.

vs-host disease in mice. Blood 1997; 90: 4651– 60. Yang YG, Sykes M. Graft-versus-host disease and graft-versus-leukemic effect in allogeneic bone marrow transplantation: role of interferongamma. Graft 2002; 5: 255. Bonini C, Ferrari G, Verzeletti S et al. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science 1997; 276: 1719–24. Chakraverty R, Cote D, Buchli J et al. An inflammatory checkpoint regulates recruitment of graftversus-host-reactive T cells to peripheral tissues. J Exp Med 2006; 203: 2021–31. Mapara MY, Leng C, Kim YM et al. Expression of chemokines in GVHD target organs is influenced by conditioning and genetic factors and amplified by GVHR. Biol Blood Marrow Transplant 2006; 12: 623–34. Xun CQ, Thompson JS, Jennings CD, Brown SA, Widmer MB. Effect of total body irradiation, busulfan-cyclophosphamide, or cyclophosphamide conditioning on inflammatory cytokine release and development of acute and chronic graft-versus-host disease in H-2-incompatible transplanted SCID mice. Blood 1994; 83: 2360– 7. Krivenko SI, Drik SI, Komarovskaya ME, Karkanitsa LV. Ionizing radiation increases TNF/ cachectin production by human peripheral blood mononuclear cells in vitro. Int J Hematol 1992; 55: 127. Schwaighofer H, Kernan NA, O’Reilly RJ et al. Serum levels of cytokines and secondary messages after T-cell-depleted and non-T-celldepleted bone marrow transplantation. Influence of conditioning and hematopietic reconstitution. Transplantation 1996; 62: 947–53. Carlos TM, Harlan JM. Leukocyte-endothelial adhesion molecules. Blood 1994; 84: 2068–101. Pober JS, LaPierre LA, Stolpen AH et al. Activation of cultured human endothelial cells by recombinant lymphotoxin: comparison with tumor necrosis factor and interleukin 1 species. J Immunol 1987; 138: 3319. Spitzer TR, MCafee S, Dey BR et al. Non-myeloablative haploidentical stem cell transplantation using anti-CD2 monoclonal antibody (MEDI507)-based conditioning for refractory hematologic malignancies. Transplantation 2003; 75: 1748–51. Kolb H, Holler E. Adoptive immunotherapy with donor lymphocyte transfusions. Curr Opin Oncol 1997; 9: 139–45. Antin JH. Graft-versus-leukemia: no longer an epiphenomenon. Blood 1993; 82: 2273–7. Johnson BD, Becker EE, LaBelle JL, Truitt RL. Role of immunoregulatory donor T cells in suppression of graft-versus-host disease following donor leukocyte infusion therapy. J Immunol 1999; 163: 6479–87. Blazar BR, Lees CJ, Martin PJ et al. Host T cells resist graft-versus-host disease mediated by donor leukocyte infusions. J Immunol 2000; 165: 4901– 9.

207

244. Johnson BD, Becker EE, Truitt RL. Graft-vs.-host and graft-vs.-leukemia reactions after delayed infusions of donor T-subsets. Biol Blood Marrow Transplant 1999; 5: 123–32. 245. Dey BR, MCafee S, Colby C et al. Impact of prophylactic donor leukocyte infusions on mixed chimerism, graft-vs-host disease and anti-tumor response in patients with advanced hematologic malignancies treated with nonmyeloablative conditioning and allogeneic bone marrow transplantation. Biol Blood Marrow Transplant 2003; 9: 320–9. 246. Shlomchik WD, Couzens MS, Tang CB et al. Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science 1999; 285: 412–15. 247. Gao E-K, Lo D, Sprent J. Strong T cell tolerance in parent → F1 bone marrow chimeras prepared with supralethal irradiation. J Exp Med 1990; 171: 1101–21. 248. Blackman M, Kappler J, Marrack P. The role of the T cell receptor in positive and negative selection of developing T cells. Science 1990; 248: 1335–41. 249. Sprent J, Gao E-K, Webb SR. T cell reactivity to MHC molecules: immunity versus tolerance. Science 1990; 248: 1357–63. 250. Speiser DE, Pircher H, Ohashi PS, Kyburz D, Hengartner H, Zinkernagel RM. Clonal deletion induced by either radioresistant thymic host cells or lymphohemopoietic donor cells at different stages of class I-restricted T cell ontogeny. J Exp Med 1992; 175: 1277–83. 251. Hoffman MW, Heath WR, Ruschmeyer D, Miller JFAP. Deletion of high-avidity T cells by thymic epithelium. Proc Natl Acad Sci U S A 1995; 92: 9851–5. 252. Laufer TM, DeKoning J, Markowitz JS, Lo D, Glimcher LH. Unopposed positive selection and autoreactivity in mice expressing class II MHC only on thymic cortex. Nature 1996; 383: 81–5. 253. Lapp WS, Ghayur T, Mendes M, Seddik M, Seemayer TA. The functional and histological basis for graft-versus-host-induced immunosuppression. Immunol Rev 1985; 88: 107–31. 254. Hauri-Hohl MM, Keller MP, Gill J et al. Donor T-cell alloreactivity against host thymic epithelium limits T-cell development after bone marrow transplantation. Blood 2007; 109: 4080–8. 255. Cheney RT, Sprent J. Capacity of cyclosporine to induce auto-graft-versus-host disease and impair intrathymic T cell differentiation. Transplant Proc 1985; 17: 528–30. 256. Gao E-K, Lo D, Cheney R, Kanagawa O, Sprent J. Abnormal differentiation of thymocytes in mice treated with cyclosporine A. Nature 1988; 336: 176–9. 257. Parkman R. Graft-versus-host disease: an alternative hypothesis. Immunol Today 1989; 10: 362– 4. 258. Rosenkrantz K, Keever C, Kirsch J et al. In vitro correlates of graft-host tolerance after HLAmatched and mismatched marrow transplants: suggestions from limiting dilution analysis. Transplant Proc 1987; 19(Suppl 7): 98–103.

16

James L.M. Ferrara & Joseph H. Antin

The Pathophysiology of Graft-Versus-Host Disease

Acute GVHD pathophysiology: a three-step model Acute graft-versus-host disease (GVHD) results from complex interactions between donor T cells and host tissues in an inflammatory milieu. The pathophysiology of acute GVHD can be considered as a three-step process involving both the innate and adaptive immune systems (Fig. 16.1). The three steps are: (1) tissue damage to the recipient by the radiation/chemotherapy pretransplant conditioning regimen; (2) donor T-cell activation and clonal expansion; and (3) cellular and inflammatory factors. This schema underscores the importance of mononuclear phagocytes and other accessory cells to the development of GVHD after complex interactions with cytokines secreted by activated donor T cells. In step 1, the conditioning regimen (irradiation and/or chemotherapy) leads to damage and activation of host tissues throughout the body and the secretion of the inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1). These cytokines enhance donor T-cell recognition of host alloantigens by increasing the expression of major histocompatibility complex (MHC) antigens and other molecules on host antigen-presenting cells (APCs). An important aspect of the mucosal injury in the gastrointestinal tract is the resultant leakage of endotoxins into the systemic circulation. In step 2, host APCs present alloantigen in the form of a peptide– human leukocyte antigen (HLA) complex to the resting T cells. Costimulatory signals are required for T-cell activation, and these signals further activate APCs and further enhance T-cell stimulation. Donor T-cell activation is characterized by cellular proliferation and the secretion of cytokines, including IL-2 and interferon-gamma (IFN-γ). IL-2 expands the T-cell clones and induces cytotoxic T lymphocyte (CTL) responses, whereas IFN-γ primes mononuclear phagocytes to produce TNF-α and IL-1. Regulatory T cells (Tregs) that can act as a powerful brake on the GVH reaction also expand at this time. In step 3, effector functions of mononuclear phagocytes and neutrophils are triggered through a secondary signal provided by mediators, such as lipopolysaccharide (LPS), that leak through the intestinal mucosa damaged during step 1. Release of inflammatory cytokines stimulates host tissues to produce inflammatory chemokines that direct activated T cells and other effector cells such as mononuclear cells and granulocytes into target organs where they adhere due to increased adhesion molecule expression. This mechanism results in the amplification of local tissue injury, other cellular effectors and further promotion of a

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

208

proinflammatory response which, together with CTLs, leads to target tissue destruction. It should be noted from the outset that all these steps do not carry equal weight in the pathogenesis of acute GVHD. The pivotal interaction occurs in step 2, where host APCs activate allogeneic donor T cells. The subsequent cytokine cascade is clearly important, but blockade of individual cytokines may not reverse established GVHD when other cellular effectors such as CTLs are present. GVHD can also occur when no conditioning of the host has occurred (e.g. transfusion-associated GVHD).

Step 1: Effects of conditioning in hematopoietic cell transplantation The first step of acute GVHD starts before donor cells are infused. Prior to hematopoietic cell transplantation (HCT), a patient’s tissues have been damaged, sometimes profoundly, by underlying disease and its treatment, infection, and transplant conditioning. High-intensity chemoradiotherapy characteristic of HCT conditioning regimens activates host APCs that are critical to the stimulation of donor T cells infused in the stem cell inoculum. These important effects help explain a number of unique and seemingly unrelated aspects of GVHD. For example, a number of clinical reports have noted increased risks of GVHD associated with advanced stage leukemia, certain intensive conditioning regimens, and histories of viral infections [1]. Total body irradiation is particularly important in this process because it activates host tissues to secrete inflammatory cytokines, such as TNF-α and IL-1 [2], and it induces endothelial apoptosis that leads to epithelial cell damage in the gastrointestinal tract [3]. Injury to the gut is transient and self-limited after autologous HCT. However, after allogeneic HCT, further damage by GVHD effectors amplifies gastrointestinal tract and systemic GVHD by allowing the translocation of microbial products such as LPS into the systemic circulation. This scenario helps to explain the increased risk of GVHD associated with intensive conditioning regimens. The relationship between conditioning intensity, inflammatory cytokines, and GVHD severity has been confirmed by animal models [4] and clinical observations [1].

Step 2: Donor T-cell activation and cytokine secretion Donor T-cell activation Donor T-cell activation occurs during the second step of acute GVHD. Murine studies have demonstrated that host APCs alone are both

The Pathophysiology of Graft-Versus-Host Disease

209

Conditioning: Tissue Damage

(I) Host APC activation

Host tissues

Small intestine

TNF-a IL-I LPS

LPS

Mf Host APC

TNF-a IL-I

TNF-g Donor T-cell

(II) Donor T-cell activation

Target cell apoptosis Thr

CD4 CTL

Treg

TNF-a IL-I CD8 CTL

CD8 CTL

(III) Cellular and inflammatory effectors

Fig. 16.1 The pathophysiology of acute graft-versus-host disease (GVHD). GVHD pathophysiology can be summarized in a three-step process. In step 1, the conditioning regimen (irradiation, chemotherapy or both) leads to the damage and activation of host tissues, especially the intestinal mucosa. This allows the translocation of lipopolysaccharide (LPS) and other inflammatory stimuli from the intestinal lumen to the circulation, stimulating the secretion of the inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) from host tissues. These mediators increase the expression of major histocompatibility complex (MHC) antigens and adhesion molecules on host antigen presenting cells (APCs), enhancing the recognition of MHC and minor histocompatibility antigens by mature donor T cells. In step 2, donor T-cell activation is characterized by a predominance of T helper type 1 (Th1) cells and the secretion of interferon-gamma (IFN-γ), which activates mononuclear phagocytes. Regulatory T cells (Tregs) limit the proliferation and clonal expansion of activated donor T cells. In step 3, effector functions of activated mononuclear phagocytes are triggered by the secondary signal provided by LPS and other stimulatory molecules that leak through the intestinal mucosa damaged during steps 1 and 2. Activated macrophages, along with cytotoxic T lymphocytes (CTL), secrete inflammatory cytokines that cause target cell apoptosis. CD8+ CTLs also lyse target cells directly. Damage to the gastrointestinal tract in this phase, principally by inflammatory cytokines, amplifies LPS release and leads to the “cytokine storm” characteristic of severe acute GVHD. This damage results in the amplification of local tissue injury and further promotes an inflammatory response.

necessary and sufficient to stimulate donor T cells [5,6]. Donor T cells migrate to secondary lymphoid organs, such as Peyer’s patches, lymph nodes, and the spleen, where they first encounter host APCs [7]. In murine models of GVHD to MHC differences between donor and host, robust donor T-cell proliferation is observed in the spleen as early as day 3 after HCT, preceding the engraftment of donor stem cells [5,7]. After allogeneic HCT, both host- and donor-derived APCs are present in secondary lymphoid organs. T-cell receptors (TCRs) of donor T cells can recognize alloantigens on either host APCs (direct presentation) or donor APCs (indirect presentation). During direct presentation, donor T cells recognize either the peptide bound to allogeneic MHC molecules or the foreign MHC molecules themselves [8]. During indirect presentation, T cells respond to the peptides generated by degradation of the allogeneic MHC molecules that are presented on self-MHCs [9]. In GVHD to minor histocompatibility antigens (mHAs), direct presentation is dominant because APCs derived from the host rather than from the donor are critical [6]. Several laboratories have identified that naïve (CD62L+) T cells cause experimental GVHD whereas memory (CD62L−) T cells do not, although memory stem cells may be involved [10,11]. In the majority of HLAidentical HCT, GVHD is induced by mHAs, which are peptides derived from polymorphic cellular proteins that are presented on the cell surface

by MHC molecules [12]. Because the genes for these proteins are located outside the MHC, two siblings often have many different peptides in the MHC groove. In this case, different peptides presented by the same MHCs are recognized by donor T cells and lead to GVHD. In mice, the actual number of so-called “major minor antigens” that can potentially induce GVHD is likely to be limited. In humans, of five previously characterized mHAs (HA-1, -2, -3, -4, and -5) recognized by T cells in association with HLA-A1 and -A2, mismatching of HA-1 alone was significantly correlated with acute grade II–IV GVHD. Theoretical models also predict substantial benefits if multiple minor loci could be typed [13]. Several mHAs are encoded on the male-specific Y chromosome and are associated with an increased risk of GVHD when male recipient cells are transplanted from female donors [14]. mHAs with tissue expression limited to the hematopoietic system are potential target antigens of graft-versus-leukemia (GVL) reactivity [15], and separation of GVHD and GVL by using CTLs specific for such antigens is an area of intense research [16]. Adhesion molecules mediate the initial binding of T cells to APCs. TCR signaling after antigen recognition induces a conformational change in adhesion molecules, resulting in higher-affinity binding to the APCs [17]. Full T-cell activation also requires co-stimulatory signals provided by APCs in addition to TCR signals. Two primary co-

210

Chapter 16

stimulatory pathways signal through either CD28 or TNF receptors. Currently, there are four known CD28 superfamily members expressed on T cells: CD28, cytotoxic T-lymphocyte antigen 4 (CTLA-4), inducible co-stimulator, and programmed death-1. In addition, there are four TNF receptor family members: CD40 ligand (CD154), 4–1BB (CD137), OX40, and herpes simplex virus glycoprotein D or herpesvirus entry mediator. The best-characterized co-stimulatory molecules, CD80 and CD86, deliver positive signals through CD28 that lower the threshold for T-cell activation and promote T-cell differentiation and survival, while signaling through CTLA-4 is inhibitory [18]. The most potent APCs are dendritic cells (DCs); however, the relative contribution of DCs and other semiprofessional APCs, such as monocytes/macrophages and B cells, to the development of GVHD remains to be elucidated. Donor APCs may also amplify GVHD, but in the skin, Langerhans cells of host origin appear to be essential for the activation of donor T cells [18,19]. Signaling through Toll-like receptors (TLRs) and through other innate immune receptors such as NOD may act as “danger signals” and activate host APCs, amplifying the donor T-cell response [20,22]. DCs can be matured and activated during HCT by: (1) inflammatory cytokines; (2) microbial products such as LPS and the dinucleotide CpG entering the systemic circulation from intestinal mucosa damaged by conditioning; and (3) necrotic cells that are damaged by recipient conditioning. All of these stimuli may be considered “danger signals” [20] and may make the difference between an immune response and tolerance [23]. When T cells are exposed to antigens in the presence of adjuvant such as LPS, the migration and survival of T cells are dramatically enhanced in vivo [24]. The effect of age in enhancing the allostimulatory activity of host APCs may also help explain the increased incidence of acute GVHD in older recipients [25]. The elimination of host APCs by activated natural killer (NK) cells can prevent GVHD [26]. This suppressive effect of NK cells on GVHD may have relevance in humans: HLA class I differences driving donor NK-mediated alloreactions in the GVH direction mediate potent GVL effects and produce higher engraftment rates without causing severe acute GVHD [26,27].

Cytokine secretion by donor T cells T-cell activation involves multiple, rapidly occurring intracellular biochemical changes, including the rise of cytoplasmic free calcium and activation of protein kinase C and tyrosine kinases [28]. These pathways in turn activate the transcription of genes for cytokines such as IL-2, IFN-γ, and their receptors. Cytokines secreted by activated T cells are often classified as T-helper type 1 subset (Th1) (secreting IL-2 and IFNγ) or T-helper type 2 subset (Th2) (secreting IL-4, IL-5, IL-10, and IL-13) [29]. The role of Th17 cells, a recently described functional Tcell subset, has not yet been clarified in GVHD [30]. Several factors influence the ability of DCs to instruct naïve CD4+ T cells to secrete Th1 or Th2 cytokines. These factors include the type and duration of DC activation along with the DC : T-cell ratio and the proportions of DC subsets present during T-cell interactions [31]. Differential activation of Th1 or Th2 cells has been evoked in the immunopathogenesis of GVHD and the development of infectious and autoimmune diseases. Although this dichotomy has many exceptions, in both settings activated Th1 cells: 1 amplify T-cell proliferation by secreting IL-2; 2 lyse target cells by Fas–Fas ligand (FasL) interactions; 3 induce macrophage differentiation in the bone marrow by secreting IL-3 and granulocyte macrophage colony-stimulating factor; 4 activate macrophages by secreting IFN-γ and by their CD40–CD40 ligand interactions; 5 activate endothelium to induce macrophage binding and extravasation;

6 recruit macrophages by secreting monocyte chemoattractant protein-1 [32]. During step 2 of acute GVHD pathophysiology, IL-2 has a pivotal role in both controlling and amplifying the immune response against alloantigens. IL-2 induces the expression of its own receptor (autocrine effect) and stimulates proliferation of other cells expressing the receptor (paracrine effect). IL-2 is secreted by donor CD4+ T cells in the first several days after GVHD induction [33]. In some studies, the addition of low doses of IL-2 during the first week after allogeneic bone marrow transplantation enhanced the severity and mortality of GVHD [34]. The precursor frequency of host-specific IL-2 producing cells predicts the occurrence of clinical acute GVHD [35,36]. Monoclonal antibodies (mAbs) against IL-2 or its receptor can prevent GVHD when administered shortly after the infusion of T cells [37,38], but this strategy was only moderately successful in reducing the incidence of severe GVHD [39]. Cyclosporine (CSP) and tacrolimus dramatically reduce IL-2 production and effectively prevent GVHD. IL-15 is another critical cytokine in initiating allogeneic T-cell division in vivo [40], and elevated serum levels of IL-15 are associated with acute GVHD in humans [41]. IL-15 may, therefore, also be important in the clonal expansion of donor T cells in step 2. IFN-γ is another crucial cytokine that can be implicated in the second step of the pathophysiology of acute GVHD. Increased levels of IFN-γ are associated with acute GVHD [42,43], and a large proportion of Tcell clones isolated from GVHD patients produce IFN-γ [44]. In animals with GVHD, IFN-γ levels peak between days 4 and 7 after transplantation before clinical manifestations are apparent. CTLs that are specific for mHAs and produce IFN-γ also correlate with the severity of GVH reaction in a skin-explant assay of human disease [45]. Experimental data demonstrate that IFN-γ modulates several aspects of the pathophysiology of acute GVHD. First, IFN-γ increases the expression of numerous molecules involved in GVHD, including adhesion molecules, chemokines, MHC antigens, and Fas, resulting in enhanced antigen presentation and the recruitment of effector cells into target organs [46]. Second, IFN-γ alters target cells in the gastrointestinal tract and skin so that they are more vulnerable to damage during GVHD; the administration of anti-IFN-γ mAbs prevents gastrointestinal tract GVHD [47], and high levels of both IFN-γ and TNF-α correlate with the most intense cellular damage in the skin [45]. Third, IFN-γ mediates GVHD-associated immunosuppression seen in several experimental HCT systems in part by the induction of nitric oxide (NO) [48–50]. Fourth, IFN-γ primes macrophages to produce proinflammatory cytokines and NO in response to LPS [51]. At early time points after HCT, IFN-γ may paradoxically reduce GVHD by enhancing Fas-mediated apoptosis of activated donor T cells [52,53]. Both cell-mediated and inflammatory cytokine GVHD effector mechanisms can sometimes be inhibited if donor T cells produce fewer Th1 cytokines [54]. Furthermore, cell mixtures of Th2 donor cells with an otherwise lethal inoculum of allogeneic bone marrow and T cells also protect recipient mice from LPS-induced lethality, demonstrating the ability of Th2 cells to modulate Th1 responses after allogeneic transplantation [55]. Polarization of donor T cells toward a Th2 phenotype by pretreating HCT donors with granulocyte colony-stimulating factor also results in less severe GVHD [56]. It should be noted, however, that systemic administration of Th2 cytokines IL-4 or IL-10 as experimental prophylaxis of GVHD was either ineffective or toxic [57,58]. On the other hand, administration of Th1 cytokines can also reduce GVHD. High doses of exogenous IL-2 early after bone marrow transplantation protect animals from GVHD mortality [59]. It has been suggested that IL-2 mediates its protective effect via inhibition of IFNγ [42]. However, injection of IFN-γ itself can prevent experimental GVHD [60], and neutralization of IFN-γ results in accelerated GVHD

The Pathophysiology of Graft-Versus-Host Disease

in lethally irradiated recipients [61]. These paradoxes may be explained by the complex dynamics of donor T-cell activation, expansion, and contraction. Activation-induced cell death is a chief mechanism of clonal deletion and is largely responsible for the rapid contraction of activated T cells following an initial massive expansion [62]. Thus, the complete absence of IFN-γ may result in an unrestrained expansion of activated donor T cells, leading to accelerated GVHD, which may be of particular importance in recipients of intensified conditioning [4]. Similarly, administration of IFN-γ-inducing cytokines, such as IL-12 or IL-18, protects mice from GVHD in a Fas-dependent fashion [53]. Thus, moderate amounts of Th1 cytokines production after donor T-cell expansion may amplify GVHD; extremes in production (either low or high), particularly during T-cell expansion, may hasten the death of activated donor T cells, aborting T-cell expansion and reducing GVHD. Regulatory T cells In both humans and mice, Treg deficiency results in immune dysregulation and autoimmunity, as characterized by the human IPEX syndrome resulting from loss of function mutations of FOXP3 [63]. A similar condition occurs in scurfy mice, a strain that lacks the transcription factor FOXP3 [64]. FOXP3 appears to function as a master control gene for the development and function of natural Tregs, which normally constitute approximately 5–10% of the circulating CD4+ T cell population. Tregs constitutively express markers associated with activated/ memory T cells: CD25 (IL-2 receptor), glucocorticoid-induced TNF-α receptor, CD152 (CLTA-4), CD103, CD62L, and neuropilin-1; and downregulate the expression of CD45RB and CD127 [65,66]. Tregs suppress both innate and the adaptive immune functions [67–69] by producing inhibitory cytokines (IL-10 and transforming growth factorbeta) as well as by cell contact-dependent inhibition of APC function and direct cytotoxicity against antigen-presenting B cells [68,70]. Additional purified CD4+CD25+ Treg populations can suppress the proliferation of conventional T cells and prevent GVHD [71,72]. Tregs do not themselves induce GVHD, and the small numbers of Treg cells present in a graft appear to be overwhelmed by the large number of conventional donor T cells present. The use of calcineurin inhibitors and thymic injury from GVHD may also interfere with the development of adequate numbers of Treg cells that can control GVHD [71,73–75]. NK1.1+ T cells may also possess regulatory function, and both peripheral blood and marrow NK1.1+ T cells can prevent GVHD by their IL-4 secretion [76].

Step 3: Cellular and inflammatory effectors The pathophysiology of acute GVHD culminates in the generation of multiple cytotoxic effectors that contribute to target tissue injury. At a minimum, step 3 includes: (1) several inflammatory cytokines; (2) specific antihost CTL activity using Fas and perforin pathways; (3) large granular lymphocytes or NK cells; and (4) NO. Significant experimental and clinical data suggest that soluble inflammatory mediators act in conjunction with direct cell-mediated cytolysis by CTL and NK cells to cause the full spectrum of deleterious effects seen during acute GVHD. As such, the effector phase of GVHD involves aspects of both the innate and adaptive immune response, and the synergistic interactions of components generated during steps 1 and 2.

Cellular effectors The Fas/FasL and perforin/granzyme (or granule exocytosis) pathways are the principal effector mechanisms used by CTLs and NK cells to lyse their target cells [77,78]. Following recognition of a target cell through TCR–MHC interaction, CTLs secrete perforin and insert it into

211

the target cell membrane, forming “perforin pores” that allow granzymes to enter the cell and induce apoptosis through various downstream effector pathways [79]. Ligation of Fas results in the formation of the deathinducing signaling complex and the subsequent activation of caspases [80]. A number of ligands on T cells also possess the capability to trimerize TNF-α receptor (TNFR)-like death receptors (DR) on their targets, such as TNF-related apoptosis-inducing ligand (TRAIL : DR4,5 ligand) and TNF-like weak inducer of apoptosis (TWEAK : DR3 ligand) [81,82]. The involvement of these pathways in GVHD has been tested by utilizing donor cells that are genetically deficient in each molecule. Lethal GVHD occurs even in the absence of perforin-dependent killing, demonstrating that the perforin/granzyme pathway plays a significant, but not exclusive, role in target organ damage. CD4+ CTLs preferentially use the Fas/FasL pathway during acute GVHD, while CD8+ CTLs primarily use the perforin/granzyme pathway, consistent with other conditions involving cell-mediated cytolysis [18]. Fas is a TNF-receptor family member that is expressed by many tissues, including GVHD target organs. Inflammatory cytokines such as IFN-γ and TNF-α can increase the expression of Fas during GVHD [83]. FasL expression on donor T cells is also increased during GVHD [84,85]. Elevated serum levels of soluble FasL and Fas have also been observed in some patients with acute GVHD [86,87]. FasLdefective donor T cells cause markedly reduced experimental GVHD in liver, skin, and lymphoid organs [88,89]. The Fas/FasL pathway is particularly important in hepatic GVHD, consistent with the marked sensitivity of hepatocytes to Fas-mediated cytotoxicity in models of murine hepatitis [90]. Fas-deficient recipients are protected from hepatic GVHD, but not from GVHD in other target organs [91]. Administration of anti-FasL (but not anti-TNF) mAbs significantly blocked hepatic GVHD damage occurring in one model [92], whereas the use of FasL-deficient donor T cells or the administration of neutralizing FasL mAbs had no effect on the development of intestinal GVHD in several studies [93,94].

Inflammatory effectors In the effector phase of acute GVHD, inflammatory cytokines synergize with CTLs, resulting in the amplification of local tissue injury and the development of target organ dysfunction in the transplant recipient. The cytokines TNF-α and IL-1 are produced by an abundance of cell types involved in innate and adoptive immune responses, and they have synergistic and redundant activities during several phases of acute GVHD. A central role for inflammatory cytokines in acute GVHD was confirmed by a recent murine study using bone marrow chimeras in which either MHC class I or MHC class II alloantigens were not expressed on target epithelium but on APCs alone [5]. GVHD target organ injury was induced in these chimeras even in the absence of epithelial alloantigens, and mortality and target organ injury were prevented by the neutralization of TNF-α and IL-1. These observations were particularly true for CD4-mediated acute GVHD but also applied, at least in part, for CD8mediated disease. A critical role for TNF-α in the pathophysiology of acute GVHD was first suggested almost 15 years ago because mice transplanted with mixtures of allogeneic bone marrow and T cells developed severe skin, gut, and lung lesions that were associated with high levels of TNF-α messenger RNA in these tissues [95]. Target organ damage could be inhibited by infusion of anti-TNF-α mAbs, and mortality could be reduced from 100% to 50% by the administration of the soluble form of the TNF-α receptor [2]. Extensive experimental data further suggest that TNF-α is involved in the multistep process of GVHD pathophysiology. TNF-α can:

212

Chapter 16

1 cause cachexia, a characteristic feature of GVHD; 2 induce activation of DCs, thus enhancing alloantigen presentation; 3 recruit effector T cells, neutrophils, and monocytes into target organs through the induction of inflammatory chemokines; 4 cause direct tissue damage by inducing apoptosis and necrosis [96]. TNF-α is also involved in donor T-cell activation directly through its signaling via TNFR1 and TNFR2 on T cells; TNF–TNFR1 interactions promote alloreactive T-cell responses [97], whereas TNF–TNFR2 interactions are critical for intestinal GVHD [98]. TNF-α plays a central role in intestinal GVHD in murine and human studies [92,95]. TNF-α also seems to be an important effector molecule in GVHD in skin and lymphoid tissue [94,95,99] and can contribute to hepatic GVHD, probably by enhancing effector cell migration to the liver via the induction of inflammatory chemokines. Studies in animals have demonstrated that neutralization of TNF-α alone or in combination with IL-1 resulted in a significant reduction of hepatic GVHD [5,100]. An important role for TNF-α in clinical acute GVHD has also been suggested by multiple studies. Elevations of TNF-α protein (serum) and messenger RNA (peripheral blood mononuclear cells) have been measured in patients with acute GVHD and other endothelial complications, such as hepatic veno-occlusive disease, now termed sinusoidal obstructive syndrome [101–103]. IL-1 is another proinflammatory cytokine that contributes to acute GVHD toxicity. Secretion of IL-1 appears to occur predominantly during the effector phase of GVHD in the spleen and skin, two major GVHD target organs [104]. Experimental models implicated IL-2 in GVHD pathophysiology, and direct inhibition of IL-1 activity with IL-1 receptor antagonist looked promising in mice and in phase II clinical trials. However, an attempt to use IL-1 receptor antagonist to prevent acute GVHD in a randomized trial was not successful [105]. It is likely that redundancy of inflammatory pathways bypasses the direct inhibition of IL-1, reducing the clinical benefit of this approach. Finally, macrophages can also produce significant amounts of NO as a result of activation during GVHD. Several studies have shown that NO contributes to the deleterious effects of GVHD on target tissues and specifically to GVHD-induced immunosuppression [50,106]. NO also inhibits repair mechanisms in target tissue by inhibiting the proliferation of epithelial stem cells in the gut and skin [107]. In humans and rats, development of GVHD is preceded by an increase of NO oxidation products in the serum [108].

Toll-like receptors and innate immunity As alluded to above, components of the HCT conditioning regimen (in particular total body irradiation) and the secretion of type 1 cytokines (specifically IFN-γ) prime mononuclear phagocytes to produce inflammatory cytokines. However, the actual secretion of soluble cytokines occurs after primed macrophages are triggered or stimulated by a second signal. This stimulus may be provided through TLRs by LPS and other microbial products that have leaked through an intestinal mucosa damaged initially by HCT conditioning regimens during step 1. Since the gastrointestinal tract is known to be particularly sensitive to the injurious effects of cytokines [95,109], damage to the gastrointestinal tract during the effector phase can lead to a positive feedback loop wherein increased translocation of LPS results in further cytokine production and progressive intestinal injury. Thus, the gastrointestinal tract may be critical to propagating the “cytokine storm” characteristic of acute GVHD; increasing experimental and clinical evidence suggests that damage to the gastrointestinal tract during acute GVHD plays a major role in the amplification of systemic disease [97]. This conceptual framework underscores the role of LPS in the development of acute GVHD as suggested by several groups [51,109,110].

LPS is a major structural component of Gram-negative bacteria and is a potent stimulator of cellular activation and cytokine release [111]. LPS shed from bacteria that comprise normal bowel flora can elicit a broad range of inflammatory responses from macrophages, monocytes, and neutrophils. In particular, LPS may stimulate gut-associated lymphocytes and macrophages [51]. Following allogeneic HCT, LPS accumulates in both the liver and spleen of animals with GVHD prior to its appearance in the systemic circulation [110]. Elevated serum levels of LPS have been shown to correlate directly with the degree of intestinal histopathology occurring after allogeneic HCT [109,112]. A genetic mutation in the TLR-4 (Tlr4) gene makes mice resistant to LPS, and transplantation with LPS-resistant donor cells results in a significant reduction of TNF-α levels, gastrointestinal tract histopathology, and systemic GVHD compared with animals receiving LPSsensitive HCT [109]. In addition, transplantation from deficient (CD14−/−) donors that are insensitive to LPS stimulation in vitro resulted in a major reduction in gastrointestinal tract GVHD and improved longterm survival compared with normal controls [113]. Early animal studies showed that death from GVHD could be prevented if transplanted mice were given antibiotics to decontaminate the gut [114]. After clinical HCT, endotoxemia is associated with intestinal injury, hepatic toxicity, acute GVHD, and fever in the absence of bacteremia [112,115]. Accordingly, Gram-negative gut decontamination during HCT has also been shown to reduce GVHD [116], and the intensity of this decontamination can predict severity of GVHD [117–119]. Naturally occurring antibody titres to a rough-mutant strain of Escherichia coli J5, which protect humans and animals from septic shock, are also associated with a decreased incidence of acute GVHD after allogeneic SCT [120]. Molecules that act as competitive inhibitors to LPS at the cell surface and block nuclear factor-kappa B activation and nuclear translocation block the biologic response to LPS and result in decreased inflammatory cytokine release and reduced GVHD severity while preserving potent GVL effects and improved leukemia-free survival [113]. Traffic of cellular effectors Regulation of effector cell migration into target tissues during the development of GVHD occurs in a complex milieu of chemotactic signals where several chemokine receptors may be triggered simultaneously or successively. Inflammatory chemokines expressed in inflamed tissues are specialized to recruit effector cells, such as T cells, neutrophils, and monocytes [121]. Chemokine receptors are differentially expressed on subsets of activated/effector T cells. Upon stimulation, T cells can rapidly switch chemokine receptor expression, acquiring new migratory capacity [24,122]. The involvement of inflammatory chemokines and their receptors in GVHD has been recently investigated in mouse models of GVHD. Macrophage inhibitory protein-1 alpha recruits CCR5+CD8+ T cells into the liver, lung, and spleen during GVHD [123,124], and levels of several chemokines are elevated in GVHD-associated lung injury [125]. DCs in the lymph node activate lymphocytes and induce a profile of adhesion molecules and chemokine receptors that direct the activated cells to migrate back to the organ of DC origin. For example, activated T cells that leave a lymph node in the skin express cutaneous lymphocyte antigen [126]. T cells expressing cutaneous lymphocyte antigen only home back to the skin where they may mediate cutaneous GVHD, but they do not traffic to other organs. Similarly, T cells exiting mesenteric lymph nodes express α4β7 (LPAM-1), which is necessary for homing to gastrointestinal tract and gut-associated lymphoid tissues [127], but they do not express cutaneous lymphocyte antigen. Lymphocytes homing to the small intestine bear chemokine receptors for the chemokine thymus-expressed chemokine (CCL25) that is produced in the small intestine, but not in the skin. Thus, cells bearing the appropriate cell

The Pathophysiology of Graft-Versus-Host Disease

surface adhesion receptors for the gastrointestinal tract (e.g. LPAM-1) and chemokine receptors (e.g. CCR25) undergo firm adhesion followed by transmigration from the vessel into the appropriate organ.

GVHD prevention: from animal models to clinical practice The development of strategies for GVHD prophylaxis and therapy has suffered from a limited understanding of the underlying pathophysiology of GVHD. This conceptual limitation led to approaches that were based on an “oncologic” model of GVHD, i.e. the notion that since GVHD is dangerous and life-threatening, management requires the utilization of high-dose immunosuppressant regimens. High doses of multiple immunosuppressive agents have been used much like we use combination chemotherapy to treat malignancies. While there is often reasonable control of the clinical manifestations of GVHD, this “shotgun” approach lacks elegance and is usually associated with opportunistic infections, lymphoproliferative disorders or relapse of the underlying malignancy. Indeed, it is often infection while GVHD is in remission that results in mortality. The objective should be to reduce the nonspecific tissue damage associated with GVHD, while allowing effective immunologic recovery. The three-phase model described above can be simply summarized. GVHD is an exaggerated and dysregulated response of a normal immune system (that of the donor) to tissue damage that is intrinsic to transplantation. The donor’s immune system reacts as if there is a massive and uncontrolled infection, and its efforts to deal with this injury result in the clinical manifestations of GVHD. It is quite possible that the tissue injury intrinsic to the administration of high-dose chemoradiotherapy initiates the breakdown of mucosal barriers, allowing endotoxin into the tissues. TLRs on DCs bind to endotoxin and activate signal transduction pathways that lead to DC maturation and induce inflammation [128]. The upregulation of co-stimulatory molecules, MHC molecules, adhesion molecules, cytokines, chemokines, prostanoids, and other inflammatory mediators prime and trigger the attack on target tissues, fueling the fire. Thus, it is likely that components of cytokine storm [129], the danger hypothesis [20], and more traditional notions of adaptive and innate immunity [130] all apply. Moreover, with our improved understanding of inhibitory pathways that regulate and suppress inflammation, it may be possible to enhance regulatory mechanisms rather than attempt to engender immunologic paralysis.

Step 1: Reduced-intensity conditioning regimens The three cardinal organs affected by GVHD are the skin, intestinal tract, and liver. The common thread would seem to be exposure to the environment. Skin and gut have very obvious barrier functions and a well-developed reticuloendothelial system. DCs resident in these organs present antigens to naïve T cells, which then have alterations in cell surface characteristics that cause them to return to the organ that originated the specific DC. Similarly, the liver is the first line of defense downstream of the gut. One would expect the lung to be a similar target; however, its less intense exposure to organisms, particularly Gramnegative rods, probably reduces its primacy as a target organ. Nevertheless, recent studies clearly demonstrate pulmonary injury that is likely to reflect GVHD. All of these organs are rich in DCs, a probable prerequisite for generating the injurious cytokines as well as directing the attack of the adaptive immune system. They are all subject to injury from conditioning, and breaches of the barrier will allow organisms or endotoxin into the circulation. One prediction of this model is that less-intense conditioning regimens would be associated with less GVHD. This effect was initially

213

observed after donor lymphocyte infusions. The T cells were administered without pharmacologic GVHD prophylaxis, and the resultant GVHD was less frequent and severe than would be expected in the absence of immunosuppressive drugs. It could be inferred that reducedintensity conditioning regimens might similarly be associated with less severe GVHD, both through less cellular injury per se and through a reduction in endotoxin exposure by virtue of less mucosal injury. Available data suggest that the rate of GVHD after reduced-intensity conditioning regimens is similar to the rate seen after conventional dose conditioning [131,132]. Interestingly, the onset of GVHD is delayed and an overlap syndrome of acute and chronic GVHD is more prevalent. This observation suggests that the initial tissue injury of high-dose regimens speeds the development of GVHD and reduced-intensity procedures seem to have little effect on chronic GVHD. Thus, while data on both reducing colonization with bacteria and reducing tissue injury with less-intense regimens are suggestive and support the basic concepts described above, the effect is insufficient to control GVHD adequately in human transplantation.

Step 2: Modulation of donor T cells Reduced T-cell numbers The advent of monoclonal technology in the 1980s led to the logical conclusion that if a pharmacologic reduction in T-cell numbers could partially control GVHD, albeit at the risk of drug-induced toxicity, then eliminating the cells with mAbs, immunotoxins, lectins, CD34 columns or physical techniques, might be more effective (reviewed by Ho and Soiffer in [133]). Furthermore, the ability to identify and target specific T-cell subsets opened the possibility that the graft could be engineered to control GVHD while allowing immunologic recovery as well as GVL. A typical unmanipulated marrow transplant entails the infusion of about 107 T cells/kg of recipient weight. Establishment of a clear dose–response relationship is difficult since the risk of acute GVHD depends on factors beyond simple dosing, for example minor histocompatibility disparities, virus exposure, disease and stage, donor age, recipient age, etc. Studies of counterflow elutriation showed that 106 T cells/kg plus CSP resulted in a GVHD rate similar to that of CSP alone, but 5 × 105 T cells/kg plus CSP resulted in an overall rate of 22%, and only limited skin involvement was observed [134]. After lectin depletion of T cells in the absence of supplemental immunosuppression, a T-cell dose of around 105/kg was associated with complete control of GVHD [135]. More recently, the combination of very high stem cell numbers and CD3 T-cell numbers of less than 3 × 104/kg allowed haploidentical transplantation without GVHD [136]. Global T-cell depletion (TCD) using a broad panel of mAbs has been largely abandoned because of the risk of graft failure [137]. However, it is clear that even depletion of T-cell subsets must be undertaken with caution. Depletion of CD4+ T cells or CD8+ T cells alone can be associated with a high graft failure rate [138]. However, depletion of CD5+ or CD6+ T cells seems to be associated with a low graft failure rate [139,140]. The presumption is that radioresistant T cells that survive the initial conditioning are responsible for graft rejection. When the stem cell source is rich in T cells, the GVH reaction will further reduce the residual population capable of alloreactivity, thus decreasing graft rejection. To some degree, the higher graft failure rates may be controlled by increasing the intensity of the conditioning regimen [141], adding back T cells [134], or with additional immunosuppressants [142]. Additional problems associated with T-cell depletion (TCD) include a higher incidence of Epstein–Barr virus-induced lymphoproliferative disorders, loss of the GVL effect with a consequent increase in relapses and slower immunologic recovery due to reduced passive transfer of

214

Chapter 16

immunocompetent donor T cells. These concerns also have been approached by increasing the conditioning intensity, delayed T-cell add-back [143], rituximab [144] or the infusion of specific T-cell lines [145]. However, in no case has there been an improvement in survival that can be definitively attributed to TCD. A large multicenter study of unrelated donor HCT similarly showed no improvement in outcomes after TCD [146]. Another interesting and novel approach to restore T-cell function without GVHD is to start by removing T cells from the initial graft using a technique such as CD34 selection. This step is followed by reacting donor and recipient lymphocytes in a mixed lymphocyte reaction in the presence of an anti-IL-2 receptor immunotoxin. This agent removes only activated T cells since it binds to CD25. The remaining resting cells are then infused to enhance immunologic recovery [147]. Alternatively, studies in mice have accomplished a similar effect, depleting donor cells activated in a mixed lymphocyte culture with antibodies to the activation antigen CD69 and immunomagnetic bead sorting [148]. Finally, both antithymocyte globulin and anti-CD52 therapy (alemtuzumab) deplete T cells in vivo. Antithymocyte globulin appears in some studies to reduce both acute and chronic GVHD [149]. Alemtuzumab also reduces acute and chronic GVHD but at the expense of high relapse rates and graft failure [150].

Reduced T-cell activation The introduction of CSP in the late 1970s was a significant advance in GVHD prevention. More recently a similar agent, tacrolimus, has been shown to provide similar control of GVHD. Both drugs inhibit T-lymphocyte activation, although the exact mechanism of action is not known. Both drugs bind to immunophilins: CSP binds cyclophilin, and tacrolimus binds FKBP12. The complex of drug and binding protein, calcium, calmodulin, and calcineurin is then formed, and the phosphatase activity of calcineurin is inhibited. This effect may prevent the dephosphorylation and translocation of nuclear factor of activated T cells, a nuclear component thought to initiate gene transcription for the formation of lymphokines (such as IL-2 and IFN-γ). The net result is the inhibition of T-lymphocyte activation. As a single agent, CSP was about as effective as methotrexate (MTX) [151]. However, in combination with MTX, there was a significant reduction in the incidence of GVHD and an improvement in survival [152]. Subsequent trials of tacrolimus and MTX compared with CSP and MTX showed no advantage for either combination [153,154]. The addition of prednisone to the conventional two-drug regimen resulted in similar rates of GVHD and no improvement in survival [155]. DCs appear to be critical to the development of acute GVHD [6]; therefore, interference with DC function would probably prevent GVHD. The goal would be to inhibit function selectively or temporarily until transplanted DC precursors had engrafted and become functional. Campath is an anti-CD52 mAb that has been extensively used for the prevention of GVHD, primarily in Europe. It is a promiscuous antibody, targeting B cells, T cells, monocytes, and other cells. Interestingly, it also appears to target DCs, and when the drug was administered prior to transplantation, host DCs were selectively depleted and the donor DCs repopulated the recipient [156], although the sensitivity of Langerhans cells to depletion is controversial [157]. These observations are difficult to separate from concomitant TCD, but they may be consistent with the theme that facilitation of DC turnover may ameliorate GVHD.

Reduced T-cell proliferation The first generally prescribed GVHD preventive regimen was the administration of intermittent low-dose MTX as developed in a dog

model by Thomas and colleagues [158]. Another regimen developed by Santos and based on cyclophosphamide [159] was never widely adopted. The principle of this approach is to administer a cell-cycle specific chemotherapeutic agent intermittently immediately after the transplant, when the T cells will have started to divide after exposure to allogeneic antigens. This was an effective approach compared with no prophylaxis [160]; however, GVHD control was incomplete. Subsequently, the addition of antithymocyte globulin or prednisone or both resulted in incremental improvement in the GVHD rate but no improvement in survival [161]. Long courses of MTX proved to be associated with a higher risk of interstitial pneumonitis and mucositis, and were abandoned with the advent of CSP. One concern regarding the use of MTX is that the enhanced mucositis might be associated with increased transfer of endotoxin across the mucosa. Ultimately, the course of MTX was abbreviated and combined with a T-cell activation inhibitor, such as CSP or tacrolimus. The use of cyclophosphamide immediately after HCT may also prevent both acute and chronic GVHD [162]. Mycophenolate mofetil (MMF) is another useful agent that may prevent GVHD with less mucositis and myelotoxicity than MTX. It is the prodrug of mycophenolic acid, a selective inhibitor of inosine monophosphate dehydrogenase, and inhibits the de novo pathway of guanosine nucleotide synthesis. Since T lymphocytes are dependent on the de novo synthesis of purines, mycophenolic acid inhibits the proliferative responses of T cells to both mitogenic and allogeneic stimulation. Myeloid and mucosal cells can utilize salvage pathways, so the drug is less toxic to mucosa and myeloid recovery. Mycophenolic acid also prevents the glycosylation of glycoproteins that are involved in adhesion to endothelium and may reduce recruitment of leukocytes into inflammatory sites. MMF does not inhibit the activation of T cells as such, but blocks the coupling of activation to DNA synthesis and proliferation [163]. Many trials of the combination of MMF with CSP or tacrolimus have shown reliable but incomplete control of GVHD when used in conjunction with a calcineurin inhibitor. Sirolimus is a macrocyclic lactone immunosuppressant that is similar in structure to tacrolimus and CSP. All three drugs bind to immunophilins; however, sirolimus complexed with FKBP12 inhibits T-cell proliferation by interfering with signal transduction and cell-cycle progression. Sirolimus (rapamycin) affects lymphocyte activation at a later stage than either CSP or tacrolimus, and activation stimuli that resist inhibition to the latter agents have been shown to be sensitive to sirolimus. Sirolimus has been shown to inhibit antibody-dependent cellular cytotoxicity and the cytolytic effects of NK cells and IL-2 lymphokine-activated killer cells. The sirolimus–FKBP12 complex binds to mammalian target of rapamycin (mTOR), which blocks IL-2-mediated signal transduction pathways that prevent G1/S phase transition. This effect is mediated through a complex pathway involving the inhibition of ribosomal protein synthesis at several levels, as well as effects on transcription and translation [164]. The drug has similar effects on proliferation of T cells induced by IL-2, IL-4, IL-7, IL-12, and IL-15. Interestingly, it may also interfere with co-stimulation [165] by inhibiting CD28-mediated blocking of inhibitor of nuclear factor kappa B and translocation of c-Rel to the nucleus [166]. Sirolimus contributes to massive T-cell apoptosis if signals 1 and 2 of T-cell activation are blocked, an effect that is not seen with CSP [167]. This effect may be related to its ability to inhibit the expression of bcl-2 and BAG-1 [168,169]. Sirolimus completely inhibits the kinase activity of the cdk4–cyclin B and cdk2–cyclin E complexes despite normal expression of these proteins and, as a consequence, blocks the hyperphosphorylation of the retinoblastoma gene product. Interestingly, sirolimus has inhibitory effects on integrin-mediated signal transduction [170], and dermal microvascular injury in a Hu-SCID transplantation model [171].

The Pathophysiology of Graft-Versus-Host Disease

Sirolimus has excellent antirejection activity in organ transplantation [170], and combination therapy with sirolimus and tacrolimus appears to be extremely effective in human organ allografting and murine models of marrow grafting. Since it acts through a separate mechanism from the tacrolimus–FKBP complex (and CSP–cyclophilin complex), which inhibits calcineurin, and thus T-cell activation, sirolimus is synergistic with both tacrolimus and CSP. While both tacrolimus and sirolimus bind to FKBP12, there appear to be adequate binding sites for both molecules. Therefore, in contrast to expectations, the drugs are not competitive, and in fact they appear to be synergistic. Since the sirolimus–mTOR complex does not bind to calcineurin, sirolimus also is free of nephrotoxicity and neurotoxicity, making combination therapy appealing. Clinical trials indicate that sirolimus is a promising addition to the GVHD armamentarium in both related and unrelated donor HCT [172].

Step 3: Blockade of inflammatory stimulation and effectors Reduced exposure to organisms Another hypothesis that flows from this model is that reduction of intestinal colonization with bacteria could prevent GVHD. The first indication that elimination of exposure to microorganisms could prevent GVHD was in germ-free experiments in mice, where GVHD was not observed until the mice were colonized with Gram-negative organisms [114]. Later, gut decontamination and use of a laminar airflow environment was associated with less GVHD and better survival in patients with severe aplastic anemia [116]. Similarly, studies of intestinal decontamination in patients with malignancies have shown less GVHD in some [118] but not all [173] studies. However, despite the power of a large number of patients in registry analyses [173], it is possible that the heterogeneity of care in the large number of participating centers obscured an important effect. Viral infections, particularly herpesviruses, may also be associated with GVHD [174]. Blockade of inflammatory effectors As alluded to above, an important role for TNF-α in clinical acute GVHD was suggested by studies demonstrating elevated levels of TNFα in the serum of patients with acute GVHD and other endothelial complications such as sinusoidal obstructive syndrome [101,102,175]. Importantly, the appearance of the increased levels was predictive of the severity of complications and overall survival. Patients with higher serum TNF-α levels during the conditioning regimen (pre-HCT) had a 90% incidence of grade II or greater acute GVHD and an overall mortality of 70%, compared with a mortality of 20% in patients without TNFα elevations [102]. Murine mAbs or F(ab)′2 fragments directed at TNF-α produced partial responses either as therapy for steroid-resistant GVHD [176] or as prophylaxis [177]. A phase II trial of etanercept, a chimeric fusion protein consisting of the extracellular ligand-binding portion of the human TNFR linked to the Fc portion of human immunoglobulin G1, has recently shown significant efficacy as primary treatment for acute GVHD when added to systemic steroids (Fig. 16.2). Almost 70% of patients had complete resolution of their GVHD within 1 month, compared with 33% of patients treated with steroids alone [178]. Blockade of chemokines may prevent the recruitment, localization, and activation of lymphocytes during GVHD. Prevention of lymphocyte entry into secondary lymphoid organs, acquisition of appropriate homing and adhesion molecules, or modification of their entry into target tissues could all reduce GVHD [179–181]. Such molecules as FTY720 [182,183] and alefacept [184–186] as well as new chemokine inhibitors may allow

215

selective targeting of organ-specific lymphocytes, although as yet little clinical experience has accumulated.

Chronic GVHD It is important to recognize that chronic GVHD was originally defined temporally rather than clinically or pathophysiologically. The initial clinical reports of chronic GVHD used descriptions of clinical problems occurring at least 150 days after HCT [187]. By convention, many transplant clinicians and scientists use day 100 after stem cell infusion as a convenient divider between acute and chronic GVHD. However, it should be recognized that acute manifestations of GVHD may occur after day 100, and problems typically associated with chronic GVHD may occur before day 100. Thus, it is preferable to consider the symptom and signs per se rather than the timing of the onset of clinical findings. The National Institutes of Health consensus conference on chronic GVHD recognizes four categories of GVHD [188] (see Chapter 87). The lack of animal models that reproduce the critical aspects of chronic GVHD has limited our ability to address the pathophysiology of this complication. Chronic GVHD is often considered an autoimmune disease because of distinctive similarities to various autoimmune disorders, especially collagen vascular disease. The relationship is difficult to prove clinically, but experimental studies have demonstrated an autoimmune aspect of chronic GVHD pathophysiology [189]. Indeed, autoantibody production is commonly observed after transplantation, and some of the clinical manifestations of chronic GVHD are similar to those of scleroderma, lichen planus, and other autoimmune diseases. T cells from animals with chronic GVHD produce unusual patterns of cytokines, such as IL-4, or IFN-γ in the absence of IL-2 [190–192]. These cytokines can stimulate collagen production by fibroblasts, which can be further amplified by IL-4. Interestingly, there is often an eosinophilia as an early manifestation of chronic GVHD, further supporting the notion that cells with a Th2 phenotype are important mediators of this problem [193]. One mechanism to induce autoimmune attack might be failure of normal regulatory mechanisms in the setting of thymic damage or immunosuppressants or both. Failure of Tregs results in autoimmune phenomena increasingly implicated in both acute and chronic GVHD [191,192,194–197]. Moreover, Tregs are implicated in preventing autoantibody production [198]. The notion that autoimmune attack can be induced by calcineurin inhibitors after autologous or syngeneic HCT has been observed in animals by preventing the development of Tregs [199]. In clinical trials, the manipulation of CSP to engender autoimmunity produced limited GVHD and limited GVL responses [200,201]. However, thymic injury and GVHD in the context of allogeneic HCT is more ominous, and one randomized trial suggested that calcineurin inhibitors might increase the risk of chronic GVHD [152]. Another explanation of autoimmunity may be that the massive apoptosis of T cells early in acute GVHD overwhelms the macrophage/DC system, resulting in autoantigen presentation [202]. The autoreactive cells of chronic GVHD are associated with a damaged thymus, which may be injured by acute GVHD, by the conditioning regimen, or subject to age-related involution and atrophy. Thus, the normal ability of the thymus to delete autoreactive T cells and to induce tolerance may be impaired. In addition, data are accumulating to support the concept that B cells may contribute to chronic GVHD. The anti-CD20 mAb rituximab has been used clinically to treat severe autoimmune disorders including autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura, and now several series indicate that anti-B-cell therapy is an interesting approach to treating chronic GVHD [203–205]. Alloantibodies can facilitate the cross-presentation of antigens to effector T cells, thereby amplifying T-cell responses to mHAs [206,207]. Donor B

Chapter 16

B. Related donor recipients

C. Unrelated donor recipients

100 90 80 70 60 50 40 30 20 10 0

100 90 80 70 60 50 40 30 20 10 0

p1,500 3.6 ×

109 107 106 104 104 108

Individual human genome SNP frequency Estimated number of CNVs per individual genome

239

1/300 base pairs >100

CNV, copy number variant; SNP, single nucleotide polymorphism. Adapted with permission from Mullaly and Ritz [194].

sequences that, in the setting of MHC-matched allogeneic HCT, could potentially stimulate and serve as targets for donor T- and B-cell responses that could mediate GVT activity. Dissection of GVT responses at the cellular and molecular level is, indeed, beginning to show that GVT in any one HCT recipient is likely to be mediated by a broadly focused immune response directed at both polymorphic alloantigens and nonpolymorphic, over- or aberrantly expressed self-antigens on recipient tumor cells. The diversity of this response is likely to be one of the keys to the remarkable potency of the GVT effect. Nonetheless, posttransplant persistence or recurrence of disease remains a major cause of treatment failure after allogeneic HCT, and developing a more comprehensive understanding of the GVT response is an urgent research priority. Recognition of the enormous diversity in the genome demonstrates that there is abundant polymorphism to be exploited, but also suggests that whole-genome approaches to understanding GVT will likely provide the key to its successful exploitation.

Strategies for exploiting the GVT response Early attempts to manipulate the GVT effect and decrease the relapse rate after allogeneic HCT were motivated by the recognition that GVT is mediated by cells contained in or derived from the hematopoietic cell graft, and that exogenous immune suppression, although it suppresses GVHD, also suppresses GVT. Complete omission of GVHD prophylaxis in the MHC-matched sibling BMT setting led to severe and hyperacute GVHD, and the increase in GVHD-related mortality precluded the identification of any decrease in the relapse rate [139]. The prophylactic administration of donor buffy-coat cells after MHC-identical sibling BMT, with a standard course of methotrexate alone as GVHD prophylaxis, was also associated with an increased rate of severe, and often fatal, acute GVHD, and also prevented the detection of any effect on the relapse rate [140]. Characterization of T- and B-cell immune responses associated with GVT activity after HCT or DLI, and identification of their antigenic targets, has demonstrated that the targets of GVT and GVHD responses are not completely overlapping. Many current efforts to manipulate GVT are consequently focused on the development of therapeutic strategies for enhancing donor immune responses against defined antigens that are selectively or exclusively expressed on recipient tumor cells, and not widely expressed on tissues that are targets for GVHD. In addition, characterization of the extensive sequence and

Identifying genomic loci associated with GVT activity The identification of extensive sequence and structural variation in the human genome suggests exciting possibilities for exploiting genomic diversity to enhance GVT, but also poses several challenging new questions. What fraction of the polymorphic loci in the genome can elicit T- or B-cell responses that can contribute to GVT? Are there specific autosomal loci at which donor–recipient disparity makes a measurable contribution to GVT activity? Are there genetic variants in the donor or the recipient that influence GVT activity in a manner that is independent of donor–recipient matching or mismatching? The advent of microarraybased genotyping technologies that permit simultaneous determination of SNP genotype and of copy number at hundreds of thousands of unique sites distributed throughout the genome has raised the prospect of using whole-genome association analysis to address these questions in a comprehensive fashion. A genome-wide, case-control approach to the identification of loci associated with GVT could potentially facilitate the identification of polymorphic loci at which donor–recipient mismatching is significantly associated with decreased risk for relapse, and could identify genetic variants in the donor or in the recipient that, independent of donor–recipient matching or mismatching, are also associated with decreased relapse risk. MHC genotypically identical sibling pairs represent a transplant population that is uniquely suited to the application of whole-genome association analysis for the identification of loci associated with GVT activity. Full siblings share 50% of their genomes and, on average, are expected to be genetically identical for both alleles at 25% of their genomes. If risk of relapse is in fact measurably influenced by donor– recipient disparity at specific autosomal loci, it is reasonable to hypothesize that HCT recipients who relapse post transplant will, on average, show more genetic identity with their donors for both chromosomes at such putative “GVT loci” than recipients who do not relapse. Thus, in the case of MHC-identical siblings, where donors and recipients are selected for genetic identity for both alleles within the MHC, this hypothesis predicts that HCT recipients who relapse post transplant will show more genetic identity for both alleles with their donors than the expected 25% at GVT loci, and patients who do not relapse post transplant will show less genetic identity for both alleles with their donors than the expected 25% (Fig. 18.5). This hypothesis is currently being tested in a population of 1000 MHC-identical sibling donor–recipient pairs – 500 “cases” in which the recipient relapsed, and 500 “controls” in which no relapse occurred – who underwent T-replete allogeneic HCT for the treatment of a myeloid malignancy at a single transplant center. Donor selection For patients for whom several potential hematopoietic cell donors can be identified, deliberate selection of a donor who is mismatched with the recipient at one or more “GVT loci” represents one potential strategy for enhancing GVT. In the T-cell-depleted haploidentical transplant setting, for example, where alloreactive donor NK cells are primary mediators of GVT responses, selection of a donor with a specific KIR genotype and appropriate MHC class I mismatch with the recipient can take advantage of predicted donor KIR–recipient MHC incompatibilities [141]. Since most HCT recipients will have several potential haploidentical donor candidates from which to choose – including parents, siblings, and children – donor selection based on donor–recipient KIR–MHC

240

Chapter 18

Relapse (cases) No relapse (controls)

Fraction identical at both alleles

1.00

GVT locus Δ/2 0.25 Δ/2

6p

Genome position

Fig. 18.5 Identification of a putative “graft-versus-tumor (GVT) locus” through assessment of donor–recipient genetic identity for both alleles at a large number of polymorphic loci distributed across the entire genome. Major histocompatibility complex (MHC)-identical sibling donor–recipient pairs are genotyped at hundreds of thousands of polymorphic loci distributed across the genome using high-density oligonucleotide arrays. Although the average fraction of donor–recipient pairs who have identical genotypes at any given polymorphic locus outside the MHC is expected to be 0.25, the observed fraction will fluctuate around this mean value. At a hypothetical “GVT locus,” the observed fraction of donor–recipient pairs with identical genotypes in a population in which the recipient relapsed (“cases”) is expected to be significantly higher than the observed fraction of pairs with identical genotypes in a population of pairs in which the recipient did not relapse (“cases”), with an effect size of Δ.

MHC-matched sibling donors of patients with multiple myeloma have been safely vaccinated with purified idiotype protein harvested from their respective siblings, and cellular and humoral idiotype-specific immune responses were subsequently detected in the donors as well as in the sibling recipients to whom they donated bone marrow [145–147]. CML patients with post-transplant relapse have been safely vaccinated with BCR–ABL fusion peptides, and CD4+ BCR–ABL fusion peptidespecific T-cell responses were subsequently detected in several patients [148]. A vaccine containing the nonameric PR1 peptide derived from the proteinase-3 and elastase proteins in incomplete Freund’s adjuvant has been administered to patients with AML, CML, and myelodysplastic syndrome in the allogeneic HCT as well as nontransplant settings, and has been associated with durable complete remissions in several patients [149], and WT1-derived peptide vaccines administered to patients with AML in the nontransplant setting have elicited clinical responses [150,151]. Leukemic cells from CML [152] and AML [153– 155] patients acquire characteristics of dendritic cells and stimulate CTL responses when cultured in vitro with selected cytokines, suggesting that such cells could be used for a cellular leukemia vaccine. A cellular vaccine consisting of autologous leukemic blasts admixed with skin fibroblasts transduced with adenoviral vectors encoding human IL-2 and CD40L was administered to patients in remission after HCT, and stimulated donor CD8+ and CD4+ T cell responses to the recipient leukemic blasts [156]. Preliminary results of other vaccination trials with wholecell vaccines, including a trial in AML patients undergoing reducedintensity HCT of vaccination with autologous, irradiated AML cells transduced with the granulocyte–macrophage colony-stimulating factor (GM-CSF) gene, have appeared in abstract form, with final results expected in the near future. Adoptive cellular therapy – donor lymphocytes

genotypes to optimize GVT activity will likely become an increasingly important consideration in haploidentical, T-cell-depleted HCT. Donor selection based on donor–recipient KIR–MHC genotypes could also conceivably prove to be useful in mismatched unrelated donor HCT, but retrospective studies have not consistently identified an association between donor–recipient KIR ligand mismatching and relapse rate in this setting [51,142–144]. For the minority of patients for whom multiple related or unrelated MHC-matched donor candidates are identified, donor selection based on donor–recipient disparity at non-MHC loci awaits the unambiguous identification of loci at which mismatching has a statistically significant effect on post-transplant relapse. The subject of donor selection is more extensively reviewed in Chapter 48. Donor or recipient vaccination Active immunotherapeutic approaches to enhancing GVT based on pretransplant vaccination of HCT donors or post-transplant vaccination of recipients are currently being investigated at many transplant centers. Vaccination strategies can be employed to augment donor T-cell responses to specific recipient mHAs, or alternatively to tumor-specific or tumor-associated antigens expressed on recipient tumor cells. The immunogens currently being evaluated in clinical trials include whole proteins, specific peptides or peptide mixtures, DNA, and whole cells, including tumor cells and dendritic cells, some of which are genetically or biochemically modified to enhance their immunogenicity. A variety of adjuvants are also being administered with the immunogens. The results of published trials demonstrate that vaccination is in general safe and can elicit frequent humoral or cellular vaccine-specific immune responses, and occasional clinical responses.

The remarkable potency of DLI for the treatment of recurrent malignancy after HCT, and its frequent serious toxicities – most notably GVHD and myelosuppression [14,16,17] – have generated intense interest in the development of more selective adoptive cellular therapies that have greater antitumor efficacy with less toxicity to normal, nonmalignant tissues. Strategies for exploiting GVT that are based on the adoptive transfer of donor-derived cells permit ex vivo selection and manipulation of the cellular product, and potentially of the donor from whom it is derived, prior to infusion, as well as independent manipulation of the recipient in order to increase the safety and the efficacy of adoptive therapy. Adoptive transfer approaches also allow the genetic characterization or marking of cells prior to infusion to facilitate precise monitoring of the in vivo persistence and trafficking of infused cells. Administration of DLI in escalating doses and selective depletion of CD8+ cells from donor lymphocyte products prior to infusion have been performed in an attempt to harness the antileukemic effect of DLI while minimizing the toxicity attributable to GVHD. Sequential administration of escalating doses of CD3+ donor lymphocytes has proven to be an effective approach to the treatment of recurrent CML post transplant [157,158]. Doses in the range of 1–2 × 107 CD3+ cells/kg recipient weight have been associated with high response rates and low rates of clinically significant GVHD. Patients who do not respond to an initial dose at this level often respond to subsequent higher doses, at which the risk of GVHD is greater. Based on the hypothesis that the therapeutic efficacy of DLI for CML is attributable to the activity of CD4+ T cells, and that the GVHD associated with DLI is attributable to the activity of infused CD8+ T cells, donor lymphocyte products selectively depleted of CD8+ T cells have been used to treat recurrent CML and multiple myeloma post transplant [56,57]. Complete responses were observed in a majority of the patients with CML and many myeloma patients, with

The Human Graft-versus-Tumor Response – and How to Exploit It

rates of GVHD that were lower than would be expected based on historical controls. Whether the lower rates of GVHD were attributable to the depletion of GVHD-causing CD8+ T cells from the DLI products administered in these studies, or simply due to a lower mean dose of cells administered, cannot be determined from the available data. Concomitant administration of cytokines with DLI and ex vivo activation of DLI products prior to infusion have been explored as means to enhance the antitumor efficacy of adoptive therapy with donor lymphocytes. IL-2 administration during or after DLI has dose-dependent effects that range from prolonging the in vivo survival and persistence of adoptively transferred cells at low doses to nonspecific activation of both CD3+ T cells and NK cells at higher doses [15]. A reproducibly significant enhancement of the antitumor efficacy of DLI by administration of IL-2 has yet to be demonstrated. A retrospective analysis suggested that administration of IFN-α did not significantly improve the efficacy of DLI for treatment of CML recurrence [14], but DLI and IFN-α combined with GM-CSF has produced clinical responses in patients who were refractory to DLI or to DLI and IFN-α [152]. IFN-α can, however, elicit durable tumor regression in patients with persistence of metastatic renal cell carcinoma after reduced-intensity allogeneic HCT and DLI [23,105]. Whether the effect of IFN-α in this setting is attributable to a primary effect on activation or expansion of donorderived effector cells in the host, or alternatively to a direct antitumor effect on recipient tumor cells, is unknown. Infusions of donor lymphocytes activated and expanded ex vivo via CD3–CD28 co-stimulation has been used in combination with chemotherapy and conventional DLI to treat patients with a variety of aggressive malignancies whose tumors recurred or persisted post transplant. Complete remissions were seen in eight of 18 patients treated in one phase I trial [159], suggesting that further studies of ex vivo activated DLIs are warranted. Adoptive cell therapy with antigen-specific effector cells The low frequency of T cells in unselected donor lymphocyte products that are reactive with recipient tumor cells, combined with the presence in such products of alloreactive cells that contribute to GVHD, has stimulated interest in adoptive therapy with products manipulated ex vivo to selectively expand the subsets of effector cells reactive with recipient tumor cells. Infusion of donor-derived CTL lines generated by repetitive in vitro stimulation of donor peripheral blood mononuclear cells with recipient CML cells was followed by a complete remission in a CML patient with a post-transplant recurrence of accelerated-phase disease who had not responded to unselected DLI [160]. Although the contribution of the ex vivo manipulated cells to elimination of residual leukemia in this case could not be unambiguously established, the results demonstrate that adoptive therapy with products that are enriched for tumor-reactive cells will likely be more effective than infusion of unselected donor lymphocytes. Techniques for large-scale ex vivo expansion of antigen-specific T cell clones [161] are now being used to support clinical trials evaluating the safety and efficacy of adoptive cell therapy with homogeneous populations of cloned CD8+ and CD4+ T cells specific for antigens selectively or exclusively expressed on recipient tumor cells. Three broad categories of target antigens expressed on tumor cells are being investigated: polymorphic mHAs encoded by genes that are expressed on normal and malignant hematopoietic cells but not widely expressed in nonhematopoietic tissues, tumor-specific antigens, and nonpolymorphic tumorassociated antigens that are encoded by normal cellular genes and over- or aberrantly expressed in tumor cells. There are advantages and limitations associated with adoptive cell therapy directed at each of these three categories of target antigen, and thus they will be discussed separately.

241

Defined mHAs that are expressed selectively in normal and malignant hematopoietic cells include those encoded by the HA-1 (KIAA0223) [89], MYO1G [73,104], HB-1 [79], UTY [162,163], BCL2A1 [82], CENPM [164], ECGF1 [165], P2RX5 [166], and HMSD [167] genes. It is likely that many more such mHAs with expression limited to hematopoietic cells remain to be identified [77]. The applicability of adoptive T-cell therapy with CD8+ T cells specific for MHC class I-restricted mHAs, however, is restricted to HCT recipients who (1) express the MHC class I allele that presents the mHA(s) to CD8+ T cells, and (2) are appropriately discordant with their donors for expression of the mHA (donor negative, recipient positive). Therefore, for mHAs that are presented by uncommon MHC class I alleles or are created by SNPs for which the minor allele frequency is low, these restrictions imply that eligibility for adoptive T-cell therapy targeting such antigens will be limited to a very small minority of HCT recipients. For any candidate hematopoietic-specific mHA, the proportion of HCT recipients who would be eligible for T-cell therapy targeting that antigen can be readily estimated from Internet resources which provide the allele frequencies in the four HapMap populations of most common (minor allele frequency ≥0.01) human SNPs (http://www.hapmap.org), as well as the frequency of various MHC alleles in several ethnic and racial groups (http://www.ashi-hla.org/publicationfiles/archives/prepr/motomi.htm). Tumor-specific antigens that could potentially be targeted with adoptive T-cell therapy to enhance GVT activity include fusion peptides such as BCR–ABL [168] and PML–RARα [169] that are created by tumorspecific chromosomal translocations, the Ig idiotype of B-lymphoid tumors [170], and novel peptide sequences created by the FLT3-internal tandem duplication mutation that is common in AML [171,172]. Many other potential tumor-specific antigens that could be targeted with adoptive cell therapy are likely to exist. Comprehensive genetic sequence analysis of breast and colorectal tumor cells, for example, has demonstrated that such tumors carry, on average, 90 mutations that generate amino acid substitutions and could therefore generate tumor-specific antigens [173]. The clinical utility of adoptive therapy targeting antigens created by tumor-specific mutations, however, will be highly dependent on the frequency with which those mutations recur in tumor cells from different patients. A recent comprehensive genetic analysis of pediatric ALL has shown that mutations causing nonsynonymous changes in the coding sequence of the PAX5 gene are common in tumors from different patients [174], suggesting that “shared” tumor-specific antigens that could be targeted in multiple different patients will, indeed, be identified. Antigens derived from nonpolymorphic proteins that are over- or aberrantly expressed in tumor cells and not widely expressed in nonhematopoietic tissues represent a third class of potential target for adoptive T-cell therapy to enhance GVT. The most extensively characterized antigens in this class include the PR1 peptide encoded by the proteinase3 and elastase genes and presented by HLA-A*0201 [120,121] and several different epitopes encoded by the WT1 gene [175,176]. Genes such as HOXA9 and BMI1 that are selectively expressed in normal hematopoietic and leukemic stem cells and whose products perform critical nonredundant functions in leukemic stem cells [177,178] also represent attractive candidates for T-cell therapy. A peptide encoded by BMI1 and recognized by CD8+ T cells has been identified [179], and is undergoing further evaluation as a potential target for GVT therapy. Adoptive cell therapy directed at nonpolymorphic antigens encoded by normal, unmutated genes that are overexpressed in tumor cells has potential limitations imposed by the expression of these genes, at some level, in normal tissues. Suppression of nonmalignant hematopoiesis might occur, for example, if normal hematopoietic progenitor cells also present the antigen to T cells, as has been suggested for an epitope encoded by WT1 [151]. Alternatively, expression of the antigen in

242

Chapter 18

normal tissues might lead to a deletion or functional inactivation of T cells with high avidity for the antigen, leaving only low-avidity cells with limited reactivity against tumor cells. Adoptive therapy with regulatory T cells Several subsets of human T cell with the capacity to suppress the in vitro activity or function of effector CD8+ or CD4+ T cells or of NK cells have been described, and there is evidence that these cells can regulate immune responses in vivo (reviewed in [180]) (see Chapter 15). The role of such cells in GVHD and GVT after allogeneic HCT in humans is poorly defined. Studies in murine systems, however, have suggested that adoptive transfer of CD4+CD25+ regulatory T cells can selectively inhibit GVHD but preserve GVT activity after MHC-mismatched HCT [181]. These studies have suggested that analogous strategies based on adoptive transfer of T cells with regulatory function to preserve GVT while inhibiting GVHD in human HCT could soon be developed. Adoptive therapy with genetically modified cells Genetic modification of donor lymphocytes to increase the safety and the efficacy of adoptive cell therapy is an active area of investigation in which significant progress has already been made. Donor lymphocytes transduced with multicomponent suicide genes combining a selectable marker with the Herpes simplex virus thymidine kinase gene, to permit in vivo ablation of adoptively transferred cells with acyclovir or ganciclovir, have been administered to HCT recipients in both the T-celldepleted, haploidentical [182,183] as well as T-replete, MHC-identical [184] transplant settings to treat post-transplant relapse or Epstein–Barr virus-related lymphoproliferative disease. The rationale for adoptive therapy with suicide gene-transduced cells is to permit administration of effector cells that are likely to have a potent antitumor effect, albeit with a risk for GVHD, and that can subsequently be ablated if clinically significant GVHD does, in fact, develop. The antitumor efficacy of suicide gene-transduced donor lymphocytes has been significantly greater in T-cell-depleted compared with T-replete settings due to the immunogenicity of the transgenes, which elicit CD8+ transgene-specific T-cell responses that dramatically limit the in vivo persistence of adoptively transferred, transgene-bearing cells in Treplete HCT recipients [182,184–186]. Although T-cell responses to transgene products have also been detected after transfer of transduced cells to recipients of T-cell-depleted HCT, GVT activity attributable to adoptively transferred cells has been observed in the majority of recipients [183,186]. Tumor regression was temporally correlated with the development of GVHD in most responding patients, and GVHD was largely controlled by the subsequent administration of ganciclovir. Novel suicide genes encoding human FAS or caspase death domains fused to a modified FK506-binding protein domain, which can be activated by a drug that dimerizes the FK506-binding protein domain, have been developed [187,188], and are currently being evaluated in clinical trials. Genetic modification of T cells is also being explored as a means to create GVT effector cells expressing high-affinity receptors for defined tumor-specific or tumor-associated antigens. Genes encoding chimeric antigen receptors comprising the antigen-binding domains of Igs fused to cytoplasmic signaling domains of the T-cell receptor (TCR)–CD3 complex, for example, have been constructed and introduced into polyclonal T cells from peripheral blood via retroviral transduction [189]. Transduced cells expressing the chimeric immunoreceptor demonstrate potent MHC-unrestricted cytotoxicity against cells expressing the target antigen, and can be expanded ex vivo with retention of chimeric receptor expression and antigenic specificity, for use in adoptive therapy.

Clinical trials of adoptive therapy with T cells transduced with a chimeric receptor specific for CD19 for the treatment of CD19-expressing B-lineage malignancies [190] are currently in progress. Effector T cells can also be rendered specific for tumor-specific or tumor-associated antigens through transfer of genes encoding the α- and β-chains of TCRs with defined specificity and affinity. T cells expressing transgenic TCRs retain the MHC restriction of the T cell from which the receptor was derived. The therapeutic potential of this approach was demonstrated in a recent clinical trial in patients with metastatic melanoma [191]. T cells expressing a transgenic αβ TCR specific for the melanoma-associated antigen encoded by the glycoprotein 100/PMEL17 (SILV) gene were adoptively transferred into patients with melanoma after the administration of lymphodepleting chemotherapy, followed by an infusion of IL-2. Durable engraftment of genetically engineered T cells at levels exceeding 10% of peripheral blood lymphocytes was seen in a majority of the patients for at least 2 months after T-cell transfer, and in two patients persistence of transduced cells was seen for 1 year after transfer, accompanied by objective regression of metastatic disease. TCR genes encoding a receptor specific for an HLA-B52-restricted H-Y antigen encoded by UTY have been transferred into peripheral blood T cells, and transduced cells exhibit HLA-B52-restricted UTY peptidespecific cytotoxicity [163]. Transduced cells can also be expanded ex vivo with retention of their antigenic specificity, and can potentially be used in adoptive therapy of male patients with leukemia or other tumors expressing UTY. In vivo persistence of adoptively transferred GVT effector cells will likely be required for optimal antitumor efficacy, and several different strategies for improving persistence and establishing durable antitumor immunity are currently being explored [192]. Genetic modification of human CD8+ T-cell clones to express chimeric cytokine receptors that deliver an IL-2 signal upon ligation of GM-CSF can endow them with GM-CSF-dependent growth, such that antigen recognition and consequent GM-CSF secretion will drive expansion of the T cells in an autocrine fashion [193]. Transfer into GVT effector cells of genes that enable resistance to immune suppressive agents commonly used in the allogeneic HCT setting, such as corticosteroids and mycophenolate mofetil, is also an active area of investigation, and such studies, if successful, will facilitate the use of adoptive cell therapy in patients suffering from or at risk for GVHD. Further development of T-cell-depleted transplant platforms in which alloreactive donor T cells are selectively removed from the hematopoietic cell graft prior to infusion will likewise facilitate the development of adoptive therapy in transplant settings where there is a high prevalence of GVHD with unmodified, T-replete grafts. It is anticipated that the development of T-cell-depletion strategies for selectively removing alloreactive donor cells that might potentially contribute to GVHD will alleviate, if not completely eliminate, the problem of poor immune reconstitution observed in recipients of nonselectively T-depleted grafts. Further characterization of the properties and function of the different subsets of human memory T cells, particularly the effector and central memory subsets, will likely provide valuable insight into the requirements for establishing sustained GVT activity via adoptive transfer.

Conclusion The GVT effect associated with allogeneic HCT is the clearest example of effective immunotherapy in humans. Genetic disparity between donor and recipient is the key to the therapeutic efficacy of HCT but is also the root of GVHD, its primary limitation. Cellular and molecular dissection of GVT responses has demonstrated that GVT after MHC-matched HCT is initiated by donor CD8+ and CD4+ T cells that recognize recipient mHAs encoded by polymorphic genes, and that this

The Human Graft-versus-Tumor Response – and How to Exploit It

alloresponse likely recruits additional effector cells recognizing tumorspecific or tumor-associated antigens encoded by nonpolymorphic genes that are over- or aberrantly expressed in recipient tumor cells. The GVT response in most HCT recipients is therefore likely to comprise a broadly focused immune response directed at a large number of polymorphic and nonpolymorphic target antigens. Selective expression of some GVT target antigens in recipient hematopoietic cells and recipient tumor cells,

243

with limited expression in nonhematopoietic tissues, may allow the development of therapeutic strategies for enhancing GVT activity without inducing or aggravating GVHD. Characterization of the extensive sequence and structural variation in the human genome suggests that there are likely to be a large number of polymorphic protein and peptide sequences at which immunotherapy to augment GVT could potentially be directed.

References 1. Barnes DW, Corp MJ, Loutit JF, Neal FE. Treatment of murine leukaemia with X rays and homologous bone marrow; preliminary communication. Br Med J 1956; 2: 626–7. 2. Barnes DW, Loutit JF. Treatment of murine leukaemia with x-rays and homologous bone marrow. II. Br J Haematol 1957; 3: 241–52. 3. Weiden PL, Flournoy N, Thomas ED et al. Antileukemic effect of graft-versus-host disease in human recipients of allogeneic-marrow grafts. N Engl J Med 1979; 300: 1068–73. 4. Weiden PL, Sullivan KM, Flournoy N, Storb R, Thomas ED. Antileukemic effect of chronic graftversus-host disease: contribution to improved survival after allogeneic marrow transplantation. N Engl J Med 1981; 304: 1529–33. 5. Odom LF, August CS, Githens JH et al. Remission of relapsed leukaemia during a graft-versushost reaction. A “graft-versus-leukaemia reaction” in man? Lancet 1978; 2: 537–40. 6. Sullivan KM, Shulman HM. Chronic graft-versushost disease, obliterative bronchiolitis, and graftversus-leukemia effect: case histories. Transplant Proc 1989; 21(3 Suppl 1): 51–62. 7. Higano CS, Brixey M, Bryant EM et al. Durable complete remission of acute nonlymphocytic leukemia associated with discontinuation of immunosuppression following relapse after allogeneic bone marrow transplantation. A case report of a probable graft-versus-leukemia effect. Transplantation 1990; 50: 175–7. 8. Collins RH Jr., Rogers ZR, Bennett M, Kumar V, Nikein A, Fay JW. Hematologic relapse of chronic myelogenous leukemia following allogeneic bone marrow transplantation: apparent graft-versusleukemia effect following abrupt discontinuation of immunosuppression. Bone Marrow Transplant 1992; 10: 391–5. 9. deMagalhaes-Silverman M, Donnenberg A, Hammert L et al. Induction of graft-versusleukemia effect in a patient with chronic lymphocytic leukemia. Bone Marrow Transplant 1997; 20: 175–7. 10. van Besien KW, de Lima M, Giralt SA et al. Management of lymphoma recurrence after allogeneic transplantation: the relevance of graftversus-lymphoma effect. Bone Marrow Transplant 1997; 19: 977–82. 11. Libura J, Hoffmann T, Passweg J et al. Graftversus-myeloma after withdrawal of immunosuppression following allogeneic peripheral stem cell transplantation. Bone Marrow Transplant 1999; 24: 925–7. 12. Moscardo F, Martinez JA, Sanz GF et al. Graftversus-tumour effect in non-small-cell lung cancer after allogeneic peripheral blood stem cell transplantation. Br J Haematol 2000; 111: 708–10. 13. Kolb HJ, Mittermuller J, Clemm C et al. Donor leukocyte transfusions for treatment of recurrent

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

chronic myelogenous leukemia in marrow transplant patients. Blood 1990; 76: 2462–5. Kolb HJ, Schattenberg A, Goldman JM et al. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. Blood 1995; 86: 2041–50. Slavin S, Naparstek E, Nagler A et al. Allogeneic cell therapy with donor peripheral blood cells and recombinant human interleuki