T Cell Subsets: Cellular Selection, Commitment and Identity

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ADVANCES IN

Immunology T Cell Subsets: Cellular Selection, Commitment and Identity VOLUME 83

This Page Intentionally Left Blank

ADVANCES IN

Immunology T Cell Subsets: Cellular Selection, Commitment and Identity EDITED BY HARVEY CANTOR Dana-Farber Cancer Institute Department of Cancer Immunology, Boston, Massachusetts Harvard Medical School Department of Pathology, Boston, Massachusetts

LAURIE GLIMCHER Harvard School of Public Health Department of Immunology and Infectious Disease, Boston, Massachusetts Harvard Medical School Department of Pathology, Boston, Massachusetts

SERIES EDITOR FREDERICK W. ALT Howard Hughes Medical Institute Children’s Hospital, Boston, Massachusetts

VOLUME 83

Elsevier Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK

This book is printed on acid-free paper. Copyright ß 2004, Elsevier Inc. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (www.copyright.com), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2004 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-2776/2004 $35.00 Permissions may be sought directly from Elsevier’s Science & Technology Right Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, E-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting ‘‘Customer Support’’ and then ‘‘Obtaining Permissions.’’ For all information on all Academic Press publications visit our Web site at www.books.elsevier.com ISBN: 0-12-022483-6 PRINTED IN THE UNITED STATES OF AMERICA 04 05 06 07 08 9 8 7 6 5 4 3 2

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CONTENTS

Contributors Introduction

ix xi

Lineage Commitment and Developmental Plasticity in Early Lymphoid Progenitor Subsets David Traver and Koichi Akashi I. Introduction II. Hematopoietic Stem Cells: Clonogenic Precursors of the Hematolymphoid System III. Cell Fate Determination: How do HSCs Commit to Each of the Hematolymphoid Lineages? IV. Overview of T and B Lymphoid Differentiation V. Defining the Earliest Stage in Lymphoid Commitment: Isolation of Common Lymphoid Progenitors (CLPs) VI. Is the CLP Stage Necessary for Adult Lymphoid Differentiation? VII. Common Myeloid Progenitors as the Counterpart of CLPs VIII. Lineage Priming by Promiscuous Gene Expression in Multipotent Stem and Progenitor Cells IX. Lymphoid and Myeloid Promiscuity Demonstrated by Single-Cell Analyses X. Fate Choices Made by Combinations of Instructive Signals at Lineage Promiscuous Stages XI. Lineage Plasticity in Lymphoid Progenitors: Fate Choices are Reversible XII. Comparison of Gene Expression Profiles among Early Hematopoietic Stem and Progenitor Cells XIII. Early Lymphoid Progenitors can Differentiate into Antigen-presenting Dendritic Cells v

1 2 3 6 7 13 17 18 19 22 28 32 33

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XIV. Fetal Hematopoietic Progenitors are Not Fully Committed to the Lymphoid and Myeloid Fates XV. Lymphoid- and Myeloid-restricted Progenitors in Human Bone Marrow XVI. Clinical Relevance XVII. Conclusion References

35 38 39 39 40

The CD4/CD8 Lineage Choice: New Insights into Epigenetic Regulation during T Cell Development Ichiro Taniuchi, Wilfried Ellmeier, and Dan R. Littman I. II. III. IV. V.

Introduction Role of Coreceptors in T Cell Development Regulation of CD4 Gene Expression Regulation of CD8 Gene Expression Conclusion References

55 56 59 77 86 86

CD4/CD8 Coreceptors in Thymocyte Development, Selection, and Lineage Commitment: Analysis of the CD4/CD8 Lineage Decision Alfred Singer and Remy Bosselut I. II. III. IV. V.

Introduction Early Thymocyte Development CD4 and CD8 Coreceptor Molecules on DP Thymocytes Selection and Commitment: Classical Models Kinetic Signaling as an Alternative to Classical Models of Lineage Commitment VI. Conclusions References

91 91 95 99 110 121 121

Development and Function of T Helper 1 Cells Anne O’Garra and Douglas Robinson I. Introduction II. Factors Inducing the Development of Th1 Cells And Their Production of IFN-g III. Role of Th1 Signaling Components in In Vivo Immune Responses

133 135 144

CONTENTS

IV. Transcriptional Regulation for Th1 Cells Producing IFN-g V. New Cytokines: What is Their Role in Th1 Cell Responses? VI. Inherited Disorders of IL-12 and IFN-g-mediated Immunity: Clinical Outcomes of Defects in Th1 Development References

vii 146 149 151 153

Th2 Cells: Orchestrating Barrier Immunity Daniel B. Stetson, David Voehringer, Jane L. Grogan, Min Xu, R. Lee Reinhardt, Stefanie Scheu, Ben L. Kelly, and Richard M. Locksley I. II. III. IV. V. VI. VII. VIII.

Introduction: History and Definitions Activation of IL-4 Expression in Naive CD4 T Cells Stabilization of IL-4 Expression in T Cells Phenotype and Genotype Analysis of Th2 Cells Mutations Impacting IL-4 Expression in T Cells Expression of IL-4 in Non-Th2 Cells Where Does Type 2 Immunity Operate? Concluding Remarks References

163 164 167 170 171 171 177 179 180

Generation, Maintenance, and Function of Memory T Cells Patrick R. Burkett, Rima Koka, Marcia Chien, David L. Boone, and Averil Ma I. II. III. IV. V.

Introduction What Is a Memory Cell? Where Do Memory CD8þ T Cells Come From? How Are Memory Cells Maintained? Conclusions References

191 192 198 212 220 221

CD8þ Effector Cells Pierre A. Henkart and Marta Catalfamo I. II. III. IV.

Effector Cells Defined Secretion as the Mechanism of Effector Function Cytotoxicity In Vivo Cytotoxicity In Vitro

233 235 236 238

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V. VI. VII. VIII. IX.

Perforin-dependent Granule Exocytosis Pathway FasL/Fas Death Pathway Cytokine Secretory Effector Cells CD8þ T Cell Differentiation Conclusions References

238 242 243 245 249 250

An Integrated Model of Immunoregulation Mediated by Regulatory T Cell Subsets Hong Jiang and Leonard Chess I. Introduction and Objectives II. Historical Considerations: Clonal Selection Theory, Immunoregulation, and Regulatory T Cell Subsets III. The T Cell Subsets which Mediate Suppression of the Immune Response IV. An Integrated Model of Immunoregulation by NKT, CD4þCD25þ, and Qa-1 Restricted CD8þ T Cell Subsets References Index Contents of Recent Volumes

253 254 261 277 281 289 301

CONTRIBUTORS

Numbers in parenthesis indicated the pages on which the authors’ contributions begin.

Koichi Akashi (1), Dana-Farber Cancer Institute, Boston, Massachusetts 02115 David L. Boone (191), Department of Medicine and The Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois 60637 Remy Bosselut (91), Laboratory of Immune Cell Biology, National Cancer Institute, Bethesda, Maryland 20892 Patrick R. Burkett (191), Department of Medicine and The Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois 60637 Marta Catalfamo (233), National Institutes of Health, Bethesda, Maryland 20892-1360 Leonard Chess (253), Department of Medicine and Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10032 Marcia Chien (191), Department of Medicine and The Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois 60637 Wilfried Ellmeier (55), University of Vienna, 1235 Vienna, Austria Jane L. Grogan (163), Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California 94143 Pierre A. Henkart (233), National Institutes of Health, Bethesda, Maryland 20892-1360 Hong Jiang (253), Department of Medicine and Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10032 Ben L. Kelly (163), Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California 94143 Rima Koka (191), Department of Medicine and The Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois 60637 Dan R. Littman (55), New York University School of Medicine, New York, New York 10016 Richard M. Locksley (163), Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California 94143 ix

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CONTRIBUTORS

Averil Ma (191), Department of Medicine and The Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois 60637 Anne O’Garra (133), National Institute for Medical Research, London NW7 1AA, United Kingdom R. Lee Reinhardt (163), Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California 94143 Douglas Robinson (133), Imperial College London, London SW7 2AZ, United Kingdom Stefanie Scheu (163), Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California 94143 Alfred Singer (91), Experimental Immunology Branch, National Cancer Institute, Bethesda, Maryland 20892 Daniel B. Stetson (163), Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California 94143 Ichiro Taniuchi (55), Kyushu University, Fukuoka 812-8582, Japan David Traver (1), Dana-Farber Cancer Institute, Boston, Massachusetts 02115 David Voehringer (163), Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California 94143 Min Xu (163), Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California 94143

INTRODUCTION

Until fairly recently, immunologists were most concerned with understanding genetic mechanisms that generated very large numbers of antibodies and T cell receptors from a relatively small set of genes. The discovery of recombination-based genetic mechanisms in T and B cells shifted attention to the obverse problem: how does each cell lineage and sublineage select and use a tiny fraction of the genome to establish its differentiated state? This question has resolved itself into understanding the epigenetic mechanisms that regulate lineage decisions and irreversible lineage commitment. T cell differentiation has, arguably, become the most tractable experimental system for addressing these questions in mammalian cells. The process begins with migration of precursor cells into the thymus, and ends with the formation of two major differentiated subsets. One is equipped to detect and eliminate virally infected cells; a second is programmed to interact with B cells and dendritic cells to induce antibody and/or inflammatory reactions. The surface antigens of cells at each differentiative step in this program have been extensively characterized, beginning with hematopoietic stem cells and ending with mature CD8 cells and sublineages of CD4 helper cells. Isolation and characterization of the hematopoietic stem cell (HSC) has been the foundation for analysis of the early events in thymocyte development. Traver and Akashi describe isolation of HSC from bone marrow cells according to reactivity with antibodies to sca-1, c-kit, Thy1, and CD34. To appreciate this remarkable feat, it is only necessary to imagine a baseball game, say between the Red Sox and the NY Yankees at Fenway Park. The 20,000 or so fans represent the differentiated lymphoid, myeloid and erythroid elements within the hematopoietic system. The position players on the field represent the committed progenitors for each of these lineages; the pitcher represents the hematopoietic stem cell. Genetic profiling of HSC suggests that transcriptional access can predict both their immediate and full differentiative potentials. Traver and Akashi discuss evidence of significant plasticity within HSC and progenitor xi

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populations, including findings that some non-hematopoietic genes are transcriptionally accessible and/or active. Although chromatin accessibility and low level transcriptional expression do not normally lead HSC down non-hematopoietic paths, recognition that these precursor cells have alternative genetic potentials is likely to promote a search for inducing agents and receptors that might stimulate HSC to give rise to non-hematopoietic progeny. Mechanisms that underlie CD4/CD8 initial lineage choices and stabilization of these genetic decisions are the subject of reviews by Taniuchi et al. and Singer and Bosselut. Taniuchi et al. delineate the remarkably detailed and complex molecular interactions that regulate CD4 and CD8 expression at discrete stages of T cell development. Expression of the CD8 co-receptor depends mainly on interaction between silencer elements and members of the Runx transcription factor family (and other proteins) to repress CD4 gene expression at early stages of development and to facilitate epigenetic changes that stably quench CD4 gene expression in mature CD8 cells. CD8 gene expression is regulated through a quite different mechanism that depends on tandemly-arranged clusters of cis-acting enhancer elements that sequentially act on CD8 gene expression. Why does regulation of CD4 and CD8 expression depend on many layers of silencing and enhancer elements that are brought to bear at discrete stages in ontogeny? One answer comes from the need for expression of both genes followed by selection-dependent extinction of one of the pair. Additional layers of regulation might be required if additional revisions of co-receptor expression were imposed by positive selection. Singer and Bosselut suggest the possibility that DP thymocytes test TCR/coreceptor compatibility and fix mismatched cells through co-receptor revision. According to this view, TCR signaling of DP cells by MHC (class I or class II) uniformly down regulates CD8 but not CD4 expression. If unabated TCRdependent signaling in this selection intermediate validates the cell’s TCR/ CD8 pairing, the cell continues on to become a full-fledged SP CD4 cell, possibly quenching IL-7R dependent signals along the way. On the other hand, disruption of TCR signaling may direct the cell to reverse its developmental direction, through re-expression of CD8 and repression of CD4 gene expression, to yield a ‘revised’ CD8 cell. Early plasticity followed by irreversible commitment is also a major feature of Th1 and Th2 sublineage development. In the case of Th1 cells, O’Garra and Robinson discuss some of the factors that can impinge upon the STAT1-mediated signaling pathway initiated from the IFN-g receptor that induce expression of the master regulator of Th1 cells, T-bet. Like all good instructors, T-bet-dependent guidance leads to fully independent progeny

INTRODUCTION

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through remodeling of target genes such as IFN-g. This is certainly not the whole story, and new cytokines that enhance the overall expression of IFN-g acting at both early and late stages of Th1 development, mainly through IFN-g, can modify this process. Stetson et al. delineate the central role of GATA-3 as the master regulator of Th2 gene transcription through integration of signals from the T cell synapse and remodeling of chromatin during Th2 development. Additional features of this differentiative path include coordinate regulation of a group of Th2 cytokine genes by inter-genic sequences, designated CNS1 and CNS2 (conserved, non-coding sequences) that facilitate locus-remodeling and epigenetic memory. Terminal cellular commitment that is independent of pioneer transcription factors also involves DNA methylation and heterochromatin patches that stably inhibit non-expressed cytokine loci. This developmental path is placed within the broader biological and clinical context of Th2 immune responses that normally discourage pathogen invasion at epithelial surfaces at the risk of allergic and hypersensitivity reactions. Henkart and Catalfamo and Burkett et al. discuss the generation of memory and cytotoxic effector cells within the CD8 lineage in the context of lineage modeling. Will a single lineage do as CD8 cells pass from effector cells to memory cells? Or do multiple lineages account for memory development, despite the fact that they add complexity to this area. We are also reminded that cytotoxic effector cells are one of the more straightforward elements of the immune response, particularly since much is know about the classic perforin dependent granule exocytosis pathway. Nonetheless, the generation and maintenance of CD8 memory cells has many fascinating biological features. The unexpected interaction in trans between IL-15 and its receptor in the support of CD8 memory, as delineated by Burkett et al., is an excellent case in point. These reviews highlight a series of remarkable new insights into the mechanisms that govern development and function of T cell lineages. As Jiang and Chess point out ‘‘in contrast, the precise biological definition of immunologic suppressive activity has largely remained an enigma’’. Current approaches to this relatively neglected problem will undoubtedly be sharpened by recent molecular insights into T cell development and potential inhibitory effects of certain cytokines. Jiang and Chess contribute a scholarly review of experimental evidence supporting the view that CD4 and CD8 development may include cells that are genetically programmed to mediate suppressive or effector activity. They cite findings that the recently-cloned transcription factor Foxp3 is expressed in a subpopulation of regulatory CD4 cells, as a step in this direction. They also summarize the increasingly impressive experimental data that defines suppressive activity mediated by a sublineage of CD8 cells that

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depend on the MHC class Ib molecule Qa-1. It is fitting that this volume end with an overview of an area of immunology that is just being revived after many years of neglect. Jiang and Chess serve to remind us that the remarkable progress that has been made in understanding epigenetic regulation of T cell development should not be viewed as the last word in this field. It may also provide a foundation for future analysis of the development of effector and regulatory sublineages of CD4 and CD8 cells. Harvey Cantor

advances in immunology, vol. 83

Lineage Commitment and Developmental Plasticity in Early Lymphoid Progenitor Subsets DAVID TRAVER AND KOICHI AKASHI Dana-Farber Cancer Institute, Boston Massachusetts 02115

I. Introduction

All blood cell types including lymphocytes are derived from HSCs that both self-renew and maintain multilineage hematopoiesis over the lifetime of the host. Differentiation is defined as the sequence of events through which immature precursors become mature, effector cells. During this stepwise commitment process, it has long been assumed that oligopotent progenitors exist that are daughters of HSCs. Whereas many findings have retrospectively suggested the existence of such cell types, prospective isolation of clonal progenitors is essential for the precise understanding of both normal and aberrant hematopoiesis. An important issue in the commitment sequence from HSCs to lymphoid cells was whether lymphocytes are directly derived from certain HSC subsets, from bipotent (T-cell/myeloid or B-cell/myeloid) progenitors, or from progenitors that exclusively give rise to all lymphoid cells, including T and B lymphocytes, and natural killer (NK) cells. Since both B and T lymphocytes display similar mechanisms for antigen receptor rearrangement and selection, it was postulated that T and B cells arise from common progenitors that have lost myeloid differentiation potential. Support for the existence of lymphoidrestricted progenitors came from studies using chromosomally marked HSCs (Abramson et al., 1977), from the results of cultured bone marrow cells transplanted into immunodeficient mice (Fulop and Phillips, 1989), and from the loss of both T and B cell subsets in patients with severe combined immunodeficiency (SCID) (Fischer, 1992; Hirschhorn, 1990). More recently, a rare population within whole mouse bone marrow was shown to generate all lymphoid subsets but that completely lacked myeloerythroid differentiation potential. These were termed common lymphoid progenitors (CLPs) (Kondo et al., 1997). Subsequently, counterpart myeloerythroid-restricted progenitors (common myeloid progenitors: CMPs) were identified (Akashi et al., 2000). Continuing studies support the concept that the lymphoid and myeloerythroid pathways diverge downstream of HSCs within the bone marrow of adult mice (Gounari et al., 2002; Igarashi et al., 2002). Using these prospectively isolated populations, lineage commitment can now be investigated directly at each of the major hematopoietic branchpoints (Fig. 1). 1 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2776/04 $35.00

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DAVID TRAVER AND KOICHI AKASHI

Fig 1 Hematopoietic commitment model based on prospective isolation of lineage-restricted progenitors. HSC, hematopoietic stem cells, CMP, common myeloid progenitors, CLP, common lymphoid progenitors; GMP, granulocyte/monocyte progenitors; MEP, megakaryocyte/erythrocyte progenitors. (See Color Insert.)

II. Hematopoietic Stem Cells: Clonogenic Precursors of the Hematolymphoid System

The search for hematopoietic stem cells began with the observation by Till and McCulloch that bone marrow transplants into lethally irradiated mice generated clonal spleen colonies, and that some of these colonies could generate multilineage hematopoiesis in serially transplanted animals (Becker, 1963; Till and McCulloch, 1961; Wu et al., 1968). Many subsequent retrospective transplantation experiments suggested that rare populations of HSCs existed within total bone marrow (Mulder and Visser, 1987; Visser et al., 1984). The modern era of HSC biology began with the first rigorous, prospective isolation of murine HSCs by cell surface phenotype (Spangrude et al., 1988; Uchida and Weissman, 1992; Uchida et al., 1994). These investigators showed that long-term, multilineage reconstitution activity was present only within a population of Lin/loThy-1.1loSca-1þ bone marrow cells. A subset of these cells displayed long-term self-renewing potential (Spangrude et al., 1991) that, at the single-cell level, could give rise to both myeloid and lymphoid outcomes (Smith et al., 1991). Subsequent studies, however, showed that the Lin/loThy-1.1loSca-1þ bone marrow fraction was heterogeneous in terms of self-renewal activity; HSCs with long-term self-renewal activity could be further subfractionated by isolating cells expressing c-Kit, a receptor for Steel

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factor (Slf) (Morrison and Weissman, 1994). Another group showed that within the LinSca-1þc-Kitþ population, only CD34/lo cells are long-term HSCs, whereas the remaining CD34þ cell can display only short-term, multilineage reconstitution (Osawa et al., 1996). Both Thy-1.1loLinSca-1þc-Kitþ and CD34/loLinSca-1þc-Kitþ populations constitute 0.01% of total bone marrow cells, and we have confirmed that >60% of these populations phenotypically overlap. Multilineage, long-term reconstitution from single cells was observed in 20% and 35% of Thy-1.1loLinSca-1þc-Kitþ and CD34/loLinSca-1þc-Kitþ populations, respectively, after competitive reconstitution assays (Osawa et al., 1996; Wagers et al., 2002). Another marker for HSCs, as well as for stem cell subsets in other tissues (Goodell et al., 1997, 2001; Jackson et al., 2001; Storms et al., 2000) is the differential efflux of the intracellular dye Hoechst 33342. Hoechstlow cells, termed the side population (SP), almost exclusively contain the long-term HSC subset (Goodell et al., 1997). Purified SP cells were LinSca-1þc-Kitþ, and contained >30% of CD34/lo cells (Okuno et al., 2002). In a study in 2001, the Thy1.1loLinSca-1þc-Kitþ population was further divided by expression of the fms-like tyrosine kinase-3 (Flt-3 or Flk-2). (Adolfsson et al., 2001; Christensen and Weissman, 2001). Around 60% of Thy-1.1loLinSca-1þc-Kitþ cells express Flk-2, and long-term HSC activity was only found within Flk-2Thy-1.1 lo LinSca-1þc-Kitþ cells, enabling the further enrichment of long-term HSCs. Considering the noted inefficiency of intravenously injected cells to seed the bone marrow (Morrison et al., 1996), isolation of long-term HSCs by the above phenotypes has likely reached purity. III. Cell Fate Determination: How do HSCs Commit to Each of the Hematolymphoid Lineages?

A fundamental question is how multipotent cells select one cell fate from a choice of several. Lineage commitment and subsequent differentiation of multipotent cells likely occurs due to the selective activation and silencing of particular gene expression programs. This process likely involves the formation of transcriptional complexes at the regulatory regions of lineage-specific gene loci (Wadman et al., 1997). Changes in chromatin structure permitting or denying access to transcriptional machinery is probably critical for this process (Berger and Felsenfeld, 2001; Felsenfeld et al., 1996). The development of in vitro clonogenic assays has defined retrospective subsets of myeloerythroid progenitors that appear to have restricted differentiation capacity (Bradley and Metcalf, 1966; Pluznik and Sachs, 1965). Unfractionated bone marrow was found to contain oligopotent colony-forming units (CFU) for all myeloid lineage cells (CFU-GEMMeg or CFU-Mix) (Johnson and Metcalf, 1977; Metcalf et al., 1979), for granulocytes and

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macrophages (CFU-GM), and for megakaryocytes and erythrocytes (CFUMegE). Monopotent CFU for granulocytes (CFU-G), macrophages (CFUM), erythrocytes (CFU-E), or megakaryocytes (CFU-Meg) were also found. The combinations of cell types present within these colonies supported the notion of clonal progenitor subsets with progressive loss of lineage potentials, and suggested close relationships between the GM and MegE branches of the hematopoietic tree. If multipotent HSCs indeed give rise to progenitors with progressive lineage restriction in vitro, it is logical to place these progenitors in a hierarchical order in the hematopoietic lineage map (Dexter and Testa, 1980) (Fig. 2A), and the myeloerythroid differentiation pathway might initiate from clonogenic progenitors of all mature cell types, such as CFU-GEMMeg. More precise lineal studies using in vitro blast colony formation, however, did not necessarily support this hierarchical model. Cells harvested from multipotent blast colonies (Nakahata and Ogawa, 1982) were able to efficiently form secondary colonies, and paired daughter cells derived from single blast cells frequently gave rise to different combinations of myeloid progeny when split into identical conditions (Suda et al., 1984a,b). Based on this phenomenon, ‘‘stochastic’’ commitment of HSCs to the myeloerythroid fates has been proposed (Ogawa, 1993) (Fig. 2B). This model states that the commitment decision of multipotent cells is essentially random and cell autonomous, and that differentiation is subsequently determined by the availability of survival or growth signals. Another model of hematopoietic commitment was proposed largely based on the analysis of in vitro differentiation potentials of immortalized cell lines. The ‘‘sequential determination model’’ proposed by Brown and colleagues states that there is a predetermined order of developmental choices (Brown et al., 1985, 1987) (Fig. 2C). This model proposes that HSCs undergo an intrinsic decision program to generate cells that can differentiate along one discrete pathway. While all of these commitment models accommodate intermediate progenitor cells, all have the critical caveat that retrospective in vitro lineage outcomes may not reflect the full commitment potential of each assayed cell type. First, there exists no single in vitro assay system that is permissive for each blood cell lineage. Whereas retrospective identification of a CFU-GEMM colony is often attributed to an early myeloerythroid progenitor, this colony could have equally resulted from a plated HSC that could not produce lymphocytes due to culture limitations. Second, if in vitro colony conditions are fully permissive for all myeloerythroid fates, it may be logically assumed that HSCs should always give rise to CFU-GEMMeg. It is known, however, that single Thy1.1loLinSca-1þc-Kitþ HSCs give rise to many different colony types, including burst-forming unit-erythroid (BFU-E), CFU-E, CFU-GM, CFU-Meg, and multilineage CFU-GEMMeg colonies at high frequencies (Akashi et al., 2000; Heimfeld et al., 1991; Morrison et al., 1996). It is thus still unclear

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Fig 2 Historical hematopoietic commitment models. (A) The hierarchical commitment model based on cell component contained in clonogenic myeloid colonies. (B) The stochastic commitment model based on the analysis of colonies from paired daughter progenitors. (C) The sequential determination model based on the analysis of cell line phenotypes.

whether highly purified HSCs do indeed commit randomly to the various myeloerythroid fates at least in vitro, or whether these phenomena simply represent the unstable nature of in vitro assay systems. These models have been established mainly on the in vitro behavior of stem and progenitor cells to read-out myeloerythroid, but not lymphoid, differentiation. This is largely because lymphoid assay systems are inefficient compared to myeloerythroid assays, as discussed in the following section. Nonetheless, in each model, there should be a variety of lineage-restricted progenitors in early hematopoiesis that are intermediates between HSCs and mature blood cells. An important approach to understanding hematopoietic commitment was to purify lineage-restricted progenitor subsets and to test their lineal relationships.

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IV. Overview of T and B Lymphoid Differentiation

Lymphoid development requires interactions between lymphoid precursor cells and stromal cells; this relationship is responsible for the expansion and selection of immature lymphocytes. The requirement of complex conditions for lymphoid development has thus hindered the precise analysis of lymphoid development and commitment processes. For T lymphocytes, the thymic stromal microenvironment is critical in the selection of developing thymocytes and in the elimination of cells expressing inappropriate T cell receptor (TCR) genes. The thymic microenvironment consists of thymic epithelial cells, macrophages, B cells, and dendritic cells. Lymphoid progenitors from the bone marrow need to seed the thymus for T cell production. It is unclear what cell population homes to thymus in normal T lymphopoiesis, which will be discussed later in this chapter. Within the thymus, the earliest thymic precursor has been identified within the fraction of CD4loCD8CD3 (triple negative; TN) cells. The earliest thymic progenitors (or proT1 cells) are CD25 þCD44þc-Kitþ, and as a population, they are capable of NK, B, and dendritic cell differentiation, but have lost most myeloid potential (Matsuzaki et al., 1993; Wu et al., 1991a,b). This supports the hypothesis that lymphoid commitment may occur in the bone marrow before cells home to the thymus. In the next proT (proT2 cells) stage, CD25þCD44þc-Kitþ proT cells begin to rearrange TCR genes. The molecules that initiate the recombination of VDJ for TCRb or of VJ for TCRg genes remain largely unclear. This rearrangement requires the two tightly regulated lymphoid-specific proteins’ recombination activation gene (RAG)-1 and RAG-2, which form a complex resulting in cleavage of DNA (McBlane et al., 1995; van Gent et al., 1996). Mice that lack RAG-1 or RAG-2 genes have a complete block of early T (and early B) cell development (Mombaerts et al., 1992; Shinkai et al., 1992). Ectopic expression of transfected RAG-1 and RAG-2 genes in nonlymphoid tissue does not result in antigen receptor rearrangement (Schatz et al., 1992), suggesting that the RAG genes do not directly instruct lymphoid commitment. Developing preT cells (CD25þCD44c-Kit) are first selected based on the appropriateness of rearranged TCRb chains coupled with the invariant preT cell receptor a chain (pTa). Cells that successfully pass through this b-selection stage are further selected by low- and high-affinity major histocompatibility (MHC) molecule interactions that positively select self-restrictive T cells and negatively select autoreactive T cells, respectively. T cell development is thus critically regulated by these two checkpoints that exist specifically in the thymus. Importantly, to estimate the T cell potential of candidate lymphoid progenitor populations, one should ensure that the assayed cell subset reaches the thymic microenvironment. For example, by intravenous transplantation, high numbers of T cell progenitors are required to obtain T cell reconstitution, whereas injection of

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progenitors directly into the thymus significantly increases T cell differentiation efficiency (Kondo et al., 1997). This requirement limits the detection of T cell potential by intravenous injection alone, especially when assayed cell types possess limited expansion potential. B cell development also involves selective outgrowth of randomly generated clonal cell populations. Proper rearrangement of B cell antigen receptor (BCR) genes is required for B lymphocyte development. Despite the lack of a requirement for BCR interaction with MHC molecules, B cells need to pass through a number of developmental checkpoints; failure results in apoptotic death. Progressive commitment to the B cell lineage occurs through a phenotypically defined differentiation pathway based on the surface markers AA4.1 and CD43 (Hardy et al., 1991; Li et al., 1996). An early AA4.1þCD43þ preproB cell subset can be further subdivided into A0, A1, and A2 fractions (Allman et al., 1999) based on the additional B220 and CD4 markers. The CD4loB220 A0 fraction expresses heterogeneous levels of Sca-1, c-Kit, and Mac-1, and contains cells that can give rise to myeloid and T cell as well as B cell progeny. This population may contain Thy-1loLinSca-1þCD4lo multipotent progenitors (Morrison and Weissman, 1994). The CD4hiB220þ A1 fraction expresses low levels of Sca-1 and Mac-1, but does not express c-Kit. The A1 fraction lacks myeloid differentiation activity, and possesses minimal T cell differentiation potential as assessed by intrathymic injection. It is unknown whether the A1 fraction contains B and T biopotent precursors. The majority of CD4B220þ A2 cells are B cell-committed. Pro-B cells rearrange DH-JH genes and express B220, CD43, and HSA on their surface (Fraction B), and subsequently express 6C3/BP-1 and undergo V-DJ recombination (Fraction C). Acquisition of CD19 represents an important marker for B cell commitment and corresponds to the ability of late proB cells to proliferate in IL-7 without other stromal cell-derived factors (Hardy et al., 1991; Hayashi et al., 1990). These CD19þ precursors should be categorized in fraction B in Hardy’s classification (Tudor et al., 2000). ProB cells in fractions B and C express the surrogate light chain genes, l5 and VpreB, to form pre-BCR. Following rearrangement of the immunoglobulin light chain gene, the heavy and light chains form a complex and are expressed on the surface together with Iga and Igb to become mature B cells. V. Defining the Earliest Stage in Lymphoid Commitment: Isolation of Common Lymphoid Progenitors (CLPs)

To prospectively isolate early lymphoid-commited progenitors, one could either employ the random, biochemical approach that led to HSC isolation or a candidate approach based on lymphoid-specific cell surface markers. Both Thy-1 and Sca-1, critical markers for HSC fractionation, are also expressed

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on the majority of mature T cells. Based on targeted gene disruptions, Thy-1 does not play a critical role in myeloid or lymphoid development (NostenBertrand et al., 1996), and Sca-1 functions in HSC self-renewal and granulocyte development but is not required for lymphoid development (Ito et al., 2003). As has been discussed, much is known regarding the molecular determinants of both T and B lymphocytes. Markers specific for lymphoid functions could therefore be systematically analyzed to phenotypically delineate early steps in lymphoid differentiation. The first such key molecule used to enrich for CLPs was the receptor for interleukin 7 (IL-7), an essential cytokine for both T and B cell development (Peschon et al., 1994; von Freeden-Jeffry et al., 1995) (Fig. 3). As will be discussed, mice carrying reporter genes for other functional lymphoid markers, such as the lymphoid-specific RAG-1 gene (Mombaerts et al., 1992) and the early T lymphoid preT cell receptor a chain

Fig 3 Identification of IL-7Raþ CLPs and myeloid progenitors in mouse bone marrow. (a) Cells negative for lineage (Lin) markers including B220, CD4, CD8, CD3, Gr-1, Mac-1, and TER119 were subdivided into IL-7Ra positive and negative fractions. (b) Sca-1/c-Kit profiles of LinIL-7Raþ cells. The LinIL-7RaþSca-1loc-Kitlo cells are CLPs. (c) Sca-1/c-Kit profiles of LinIL-7Ra cells. The LinIL-7RaSca-1hic-Kithi subset represents the HSC population, whereas the LinIL-7RaSca-1c-Kithi subset contains all myeloid progenitor populations. (d) The LinIL-7RaSca-1c-Kithi population is subdivided into 3 distinct myeloid progenitor subsets such as CMPs, GMPs, and MEPs, according to their FcgRII/III and CD34 profiles.

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Fig 4 Phenotypic differences in primitive lymphoid progenitor subsets possessing T and B lymphoid potential. The early lymphocyte progenitor (ELP) population is defined in the bone marrow as a population that initiates RAG-1 transcription (Igarashi et al., 2002). This population corresponds to a few percent of cells within the conventional short-term HSC subset (LinSca1þc-Kitþ). Although ELPs have ‘‘lymphoid’’ RAG-1 mRNA, they still possess a weak GM potential. ELPs do not express IL-7Ra. Common lymphoid progenitors (CLPs) are cells that begin to express IL-7Ra (Kondo et al., 1997). CLPs are all transcribing the RAG-1 gene, and a small fraction of them also initiate pTa transcription, which is upregulated in thymic proT cells (Miyamoto et al., 2002). In the B lymphoid pathway in the bone marrow, PTa transcription is maintained in a minority of B220þ (c-Kit) cells, which maintain T/B bipotentiality (Gounari et al., 2002). During this sequential lymphoid development in the bone marrow, both Sca-1 and c-Kit are gradually downregulated during the transition of ELPs, CLPs, to B220þ cells. In contrast, the earliest thymic progenitors, called early T lineage progenitors (ETPs) express Sca-1 and c-Kit at a high level, and still possess weak B and GM potentials (Allman et al., 2003). It is unclear whether ETPs originate from ELPs, CLPs, or B220þpTaþ cells. Although the high expression pattern of Sca-1 and c-Kit in ELPs and ETPs may represent their direct precursor/progeny relationship, direct evidence is lacking (see text).

(pTa) (Groettrup et al., 1993), have also been used to search for the earliest lymphoid progenitors that can give rise to all lymphoid components. Lineal relationships and phenotypic overlaps among these lymphoid progenitors defined by each criterion are illustrated in Fig. 4. A. The Role of Flk-2 in Early Stem and Progenitor Cells The Flk-2/Flt-3 receptor, like c-Kit, is a cytokine tyrosine kinase receptor expressed primarily on early hematopoietic precursors (Matthews et al., 1991). Flk-2 was originally isolated based on its expression in HSCs (Mackarehtschian et al., 1995). Mice deficient in Flk-2 or Flt-3 ligand (FL) display loss of early B, NK, and dendritic cell development, and reduced numbers of T cell progenitors (Mackarehtschian et al., 1995; McKenna et al., 2000; Sitnicka et al., 2002). These phenotypes could result due to a requirement for FL signals in common precursors for each lymphoid lineage. As has been discussed, Adolfsson et al. showed that Flk-2 is not expressed in long-term reconstituting HSCs, but is upregulated in non-self-renewing cells within the LinSca-1þc-Kitþ fraction (Adolfsson et al., 2001). Importantly, these authors presented clonal data

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indicating that Flk-2þSca-1þc-Kitþ cells contain cells with T, B, and myeloid (GM) potentials, supporting the existence of multipotent progenitors, although MegE potentials were not tested. Christensen et al. further subfractionated the Flk-2þSca-1þc-Kitþ population by using Thy1.1 expression (Christensen and Weissman, 2001). They showed that the Thy1.1loSca-1þc-Kitþ population can be divided into Flk-2 long-term HSC and Flk-2þ short-term HSC populations, and all Thy1.1Sca-1þc-Kitþ multipotent progenitors are Flk-2þ. In an independent study, the Thy1.1Sca-1þc-Kitþ population displayed rapid B cell reconstitution after intravenous transplantation with minimal contributions to the T and myeloid lineages (Searles et al., 2000). Compatible with these data, Flk-2 is expressed in IL-7Raþ CLPs (Kondo et al., 1997), but not in common myeloid progenitors (CMPs) (Akashi et al., 2000) (see following text). Furthermore, FL-deficient mice lack CLPs, but possess normal numbers of CMPs (Adolfsson et al., 2001). These data strongly suggest that Flt-2/FL interactions might play an important role in early lymphoid development. B. CLPs are Defined as the Most Primitive IL-7Ra-Expressing Cells A more definitive marker for lymphoid commitment might be the expression of the receptor for IL-7, an essential cytokine for both T and B cell development. The IL-7 receptor (IL-7R) is composed of the IL-7Ra chain and the common cytokine receptor g chain (g c) (Kondo et al., 1994; Noguchi et al., 1993). Mice genetically deficient for IL-7 or the IL-7 receptor (IL-7R) lack both T and B cells (Peschon et al., 1994; von Freeden-Jeffry et al., 1995), whereas mice deficient for gc lack NK cells as well as T and B lymphocytes (Cao et al., 1995; Ohbo et al., 1996). NK cell deficiency in the latter animals is due to the lack of the IL-15 receptor, which is composed of both the IL-15Ra and gc (Giri et al., 1994). The IL-7R is expressed in early pro-T and pro-B cells (Akashi et al., 1998). The critical role of IL-7 in abT cell differentiation is to maintain survival of developing T cells through the upregulation of a survivalpromoting protein, Bcl-2 (Akashi et al., 1997). Signaling through the IL-7R is necessary for the rearrangement of immunoglobulin heavy chain V segments through the activation of the pax-5 gene (Corcoran et al., 1998), and for V-J recombination of gd TCR genes through the activation of STAT5 (Maki et al., 1996). Administration of neutralizing anti-IL-7 antibodies resulted in severe inhibition of T and B lymphopoiesis, but not myelopoiesis (Bhatia et al., 1995). Taken together, these data suggested that IL-7Ra expression is one of the most reliable functional markers for lymphoid commitment. The isolation of CLPs was performed according to the following steps. First, IL-7Ra-expressing cells were devoid of myeloerythroid potential in vitro and in vivo. Second, within IL-7Raþ bone marrow cells, robust

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differentiation potential to the T, B, and NK cell lineages existed within the LinSca-1loc-Kitlo population in vivo. In mice transplanted with CLPs, donorderived T and B cells began to decline after 4 to 6 weeks, indicating that this population has no significant self-renewal activity. Third, by a two-step clonogenic assay, approximately 20% of single IL-7RaþLinSca-1loc-Kitlo cells could give rise to both T and B cells. Thus, IL-7RaþLinSca-1loc-Kitlo cells contain clonogenic T and B cell progenitors and completely lack myeloerythroid differentiation potential. In a subsequent study, more than 40% of single IL-7RaþLinSca-1loc-Kitlo cells could differentiate to NK cells. The vast majority of CLPs express Flk-2 (Sitnicka et al., 2002). The clonogenic T/B bipotentiality of IL-7Ra-expressing CLPs has also been demonstrated by another group (Izon et al., 2001), using a modified fetal thymic organ culture (FTOC) system (Kawamoto et al., 1997). C. Initiation of Lymphoid Commitment May Occur Prior to the CLP Stage: Early Lymphoid Progenitors Defined by RAG-1 Transcriptional Activation The RAG genes are indispensable for the rearrangement of TCR and Ig genes, and RAG-1 or RAG-2 deficient mice display a complete loss of T and B cells (Mombaerts et al., 1992; Shinkai et al., 1992). Mice carrying GFP knocked into the RAG-1 locus were generated (Igarashi et al., 2001; Kuwata et al., 1999) and similarly assayed for early lymphoid progenitors initiating transcription of the RAG-1 gene (Igarashi et al., 2002). Over 90% of IL-7RaþLinSca-1lo c-Kitlo CLPs expressed GFP in these animals (Igarashi et al., 2002). Interestingly, RAG-1 expression was also observed in 5% of the LinSca1þc-Kitþ HSC fraction. Like CLPs (Sitnicka et al., 2002), GFPþLinSca-1þcKitþ cells also expressed Flk-2, indicating that they are not long-term HSCs (Adolfsson et al., 2001; Christensen and Weissman, 2001) but likely are committed progenitors (Igarashi et al., 2002) and included within the (Flt-2þ)Thy1.1LinSca-1þc-Kitþ population (Christensen and Weissman, 2001; Searles et al., 2000). A vast majority of LinSca-1þc-Kitþ cells and GFPþLinSca-1þcKitþ cells express CD27 (Wiesmann et al., 2000), a member of the TNF receptor family previously shown to play a role in lymphoid proliferation, differentiation, and apoptosis. RAG-1/GFPþLinSca-1þc-KitþCD27þ cells exhibited potent T, B, and NK differentiation potential after transplantation into congenic hosts. The timing of thymic T cell reconstitution of RAG-1/ GFPþLinSca-1þc-KitþCD27þ cells preceded that of CLPs by 7 days, suggesting that these cells were more immature than IL-7RaþLinSca-1locKitlo CLPs. A fraction of RAG-1/GFPþLinSca-1þc-KitþCD27þ cells, however, formed CFU-GM in vitro, indicating that this population is not entirely committed to the lymphoid fates. Furthermore, this study did not demonstrate that T and B cell progeny can originate from single

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RAG-1/GFPþLinSca-1þc-KitþCD27þ cells, and therefore the homogeneity of this population is still in question. Nonetheless, these data indicate that the majority of RAG-1/GFPþLinSca-1þc-KitþCD27þ cells are committed to the lymphoid lineages, and that they likely contain cells similar to, or slightly upstream of CLPs. The authors termed this population ‘‘early lymphocyte progenitors (ELPs)’’ (Igarashi et al., 2002). D. Use of pTa Reporter Constructs to Define Early Lymphoid Progenitors The pTa protein pairs with the T cell receptor (TCR) b chain to form the pre-TCR (Groettrup et al., 1993) that plays a critical role in the efficient generation of mature T cells (Fehling et al., 1995). Since pTa is used for preTCR formation after TCRb gene rearrangement, pTa transcription should be a relatively late marker for lymphoid development as compared to RAG-1 transcription. A transgene containing a 9 kb fragment spanning all characterized pTa promoter and enhancer sequences was used to drive expression of a hCD25 minigene (Miyamoto et al., 2002; Reizis and Leder, 2001). In pTa/hCD25 transgenic mice, the expression of hCD25 was highly correlated with that of the endogenous pTa, as quantified by real-time RT-PCR analyses (Gounari et al., 2002). However, a small fraction of B220þ and CD4þ cells in the bone marrow were hCD25þ, but did not express pTa mRNA, presumably because hCD25 transcripts persist for a while after pTa downregulation. In the thymus, the expression of pTa reported by hCD25 was detected in 30% of mCD25c-KitþCD3CD4lo cells, termed the earliest thymic precursors (Wu et al., 1991b), and in the majority of mCD25þc-Kitþ proT cells, consistent with the normal expression of pTa. In the bone marrow, only 6% of Lin cells were hCD25þ, all of which displayed the IL-7RaþSca-1lo-c-Kitlo CLP phenotype, whereas only 7% of CLPs were positive for hCD25 expression (Miyamoto et al., 2002). This result indicates that CLPs are the earliest population to initiate pTa transcription, but that the majority of CLPs have not initiated pTa transcription. The transcription of pTa does not mark T cell commitment at the CLP stage, since pTa/hCD25þ CLPs differentiated into B cells at the same efficiency as pTa/hCD25þ CLPs differentiated into B cells at the same efficiency as pTa/hCD25þ CLPs. In two-step clonogenic assays similar to those used in the original CLP report (Kondo et al., 1997), single pTa/hCD25þ CLPs were demonstrated to give rise to both T and B cells (Gounari et al., 2002). This study also showed that CLP activity persists in a small cell fraction of cells phenotypically downstream from CLPs: pTa/ hCD25þCD19B220þc-Kit cells express IL-7Ra, and possess some T and B cell potential, suggesting that CLP potential is maintained in at least a fraction of B220þ cells that have downregulated c-Kit.

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E. Which Markers are Most Useful to Separate the Earliest Lymphoid Progenitors? The phenotype and biological activities of each progenitor subset are summarized in Fig. 4 and Table I. From the already cited studies, it is highly likely that lymphoid commitment occurs in adult mouse bone marrow, and that CLPs are an important intermediate in steady-state hematopoiesis. Although the majority of CLP activity resides in cells within the originally described IL-7RaþLinSca-1loc-Kitlo fraction, similar activity can also be found within a population of LinSca-1þc-Kitþ HSCs (as evidenced by RAG-1/GFP mice) and within B220þCD19c-Kit cells (as evidenced by hCD25-pTa mice). The differences in the distribution of CLP activity described in these studies demonstrates the difficulty in finding markers that precisely correlate with cell functions, although IL-7Ra-expression is still the only available marker for CLPs in normal mice. In separating cells according to positive or negative marker expression, the sensitivity of each marker is critical. The threshold for positive detection will be decided by the sensitivity of the detectors and by the amount and/or longevity of antigens or reporters. For example, although IL7Raþ cells are undetectable in the LinSca-1þc-Kitþ population by FACS, IL-7Raþ cells are detectable by highly sensitive nested RT-PCR analyses. However, such low level of receptor expression may not be sufficient to transmit functional IL-7 signals. In addition, as will be discussed, the expression of lineage-related genes at low levels precedes lineage commitment in multi- or oligopotent cells. Therefore, if the marker is too sensitive, this may result in the isolation of uncommitted cells. In this context, it is likely that the CFU-GM potential detected within the RAG-1/GFPþ LinSca-1þc-Kitþ fraction is a reflection of multipotent cells with low level ‘‘priming’’ of lymphoid genes (see following text). Conversely, when a late lymphoid marker is used, such as pTa, a failure to efficiently isolate CLPs may result, since 50% of hGM-CSFRþ CLPs and >20% hGM-CSFRþ proT cells gave rise to granulocytes, monocytes, and/or dendritic cells, but not MegE lineage cells in the presence of hGM-CSF (Iwasaki-Arai et al., 2003). These data collectively suggest that conversion to GM fates by ectopic GM-CSF signals can occur in CLPs and their downstream lymphoid progeny, but that this potential progressively disappears as cells become more mature. During the conversion of CLPs into the myelomonocytic lineage, a number of GM-related cytokine receptors (e.g., G-CSFR, M-CSFR) and transcription factors (e.g., C/EBPa, PU.1) were reactivated, whereas MegE-related genes such as EpoR and GATA-1 were not (Iwasaki-Arai et al., 2003). Thus, GM-CSF signals can activate GM but not MegE differentiation programs, suggesting their GM-specific ‘‘instructive’’ role in lineage commitment. Furthermore, this effect is specifically found with the GM-CSFR, since retrovirally transduced G-CSFR and M-CSFR could not induce GM conversion from CLPs. It should be interesting to test whether the transcription factors downstream of the GM-CSFR can similarly induce GM conversion from CLPs. B. Megakaryocyte/Erythrocyte Conversion from Lymphoid-commited Progenitors The loss of GM and MegE potentials in lymphoid cells might occur simultaneously as multipotent stem cells generate CLPs (Kondo et al., 1997) and common myeloid progenitors (CMPs) (Akashi et al., 2000) since each subset displayed mutually exclusive differentiation potentials. If the inducible GM potential in CLPs reflects residual multipotentiality from an upstream progenitor, it is reasonable to expect that MegE conversion could also be induced in CLPs or their lymphoid progeny.

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GATA-1, a critical transcription factor for MegE development, has been reported to instruct MegE and eosinophil commitment. GATA-1 is expressed in erythroblasts, megakaryocytes, eosinophils and, mast cells. The lineage instructive effect of GATA-1 was first demonstrated in a multipotential chicken cell line transformed by the Myb-Ets-encoding E26 leukemia virus (Kulessa et al., 1995). This cell line differentiates to the GM lineages following enforced expression of PU.1 whereas enforced expression of GATA-1 caused erythroid or eosinophilic differentiation (Kulessa et al., 1995). Within the myelomonocytic lineages, overexpression of GATA transcription factors can induce MegE phenotypes (for review, see Graf, 2002). Introduction of GATA-1 into myeloid cell lines induces megakaryocyte differentiation (Visvader et al., 1992) with upregulation of MegE-affiliated genes such as the erythropoietin receptor (EpoR) and a-globin (Seshasayee et al., 1998; Yamaguchi et al., 1998). Furthermore, both GM-restricted colony-forming cells, which are selectively generated in culture, and purified GMPs can give rise to erythroblasts, megakaryocytes, and eosinophils by GATA-1 transduction (Heyworth et al., 2002; Iwasaki and Akashi, 2001). These studies suggest that GATA-1 is sufficient to reactivate the MegE and/or eosinophil differentiation programs in immature myelomonocytic cells. In a recent report, GATA-1 was retrovirally introduced into CLPs (Iwasaki et al., 2002). Strikingly, GATA-1 converted CLPs into the MegE lineages, inducing differentiation of hemoglobinized erythroblasts and mature megakaryocytes even in the absence of Tpo or Epo. Furthermore, GATA-1transduced CLPs could not differentiate into T or B cells in vivo, indicating that ectopic GATA-1 inhibited normal lymphoid differentiation from CLPs. GATA-1 altered the expression profiles of lineage-affiliated genes in CLPs into those observed in MegE-committed MEPs, inducing the upregulation of genes essential for MegE development such as FOG-1, and concomitantly downregulated genes related to the GM and lymphoid lineages including PU.1, Pax5, and IL-7Ra. The reactivation of GATA-1 appears to be sufficient for, and a minimum requirement for, MegE conversion from CLPs (Iwasaki et al., 2002). Together, these data suggest that CLPs are normally lymphoid-restricted because they have downregulated myeloerythroid genes such as GM-CSFR and GATA-1. In turn, latent GM and MegE potentials of CLPs and early T and B lymphoid precursors indicate that the GM and MegE programs are still accessible after physiological lymphoid commitment, presumably by chromatin remodeling. It is of interest to test whether CMPs, the myeloid counterpart of CLPs, possess similar ‘‘plasticity’’ for lymphoid differentiation. Observed plasticity of lineage commitment at the early stages of lymphoid development likely reflects residual multipotency from upstream priming stages. This residual multipotency appears to be latent in normal commitment progenitors, presumably by epigenetic programs that control transcriptional accessibility, as

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evidenced by gene expression profiling in microarray studies (see following text). Stabilization of lineage commitment could be achieved by downregulation of master ‘‘instructive’’ genes that ectopically induce lineage conversion as well as the upregulation of gene sets to directly ‘‘exclude’’ other fate choices. XII. Comparison of Gene Expression Profiles among Early Hematopoietic Stem and Progenitor Cells

That promiscuous gene expression exists at each of the major hematopoietic branchpoints suggests that priming of multiple lineage-affiliated programs allows fate choice flexibility at each progenitor stage. The PCR studies that generated these findings used single cells and assayed only partially representative myeloid and lymphoid genes. Thus, a more global view of gene expression, including hematopoietic and nonhematopoietic genes is essential to unravel the complexity of genetic programs within early hematopoietic subsets. Oligonucleotide microarray analyses were thus performed using highly purified long-term HSCs, short-term HSCs, CMPs, and CLPs (Akashi et al., 2003). HSCs expressed a variety of myeloid (GM and MegE-affiliated) but not lymphoid genes. CMPs coexpressed many GM and MegE-affiliated genes, and CLPs coexpressed many T, B, and NK lymphoid-related genes on Affymetrix chips. Thus, genome-wide gene profiling revealed that HSCs predominantly exhibit myeloid promiscuity, and that CLPs and CMPs exclusively possess lymphoid and myeloid priming programs, respectively (Akashi et al., 2003). In short-term HSCs (or multipotential progenitors), both lymphoid and myeloid genes were primed. However, as has been mentioned, myeloid and lymphoid gene coexpression was not assayed at the single-cell level in these cell types. Therefore, these findings in ‘‘short-term HSCs’’ could be due to a combination of heterogeneous expression profiles. Nonetheless, this study strongly suggests that lineage promiscuous priming might be a common transcriptional feature in uncommitted stem and progenitor cells, and that primed genes may represent their full and immediate differentiation potentials (Miyamoto et al., 2002). This genome-wide approach discloses another important ‘‘priming’’ event at the level of HSCs. Primitive HSCs expressing CD45, a hematopoietic cellspecific marker, express approximately 70% of all nonhematopoietic genes, including genes characteristic of neuronal, endothelial, pancreatic, kidney, liver, heart, hair, epithelial, and muscle cell types (Akashi et al., 2003). These nonhematopoietic genes were detectable by nested RT-PCR in CD45þ HCS at the single- to 10-cell levels. This broad transcriptional usage, however, is lost as HSCs generate CMPs and CLPs; these cell types displayed only myeloid and lymphoid expression profiles, respectively.

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These data also demonstrate that HSCs possess transcriptional accessibility for nonhematopoietic genes associated with multiple organ systems (Akashi et al., 2003). Recent reports demonstrate that murine bone marrow contains cells capable of differentiation into multiple organs, including endothelial cells, skeletal and cardiac muscle (Orlic et al., 2001), neurons and glia (Priller et al., 2001), parenchymal liver cells (Lagasse et al., 2000), and/or epithelial cells (Krause et al., 2001), as well as hematopoietic cells. While these reports suggest that the bone marrow may be special in harboring precursors of nonhematopoietic fates, the notion of HSC plasticity was challenged by a study in which mice reconstituted with single, GFPþ HSCs were extensively analyzed for GFP expression in nonhematopoietic tissues (Wagers et al., 2002). These clonal experiments demonstrated that HSCs very rarely contributed to nonhematopoietic fates, suggesting that ‘‘transdifferentiation’’ of HSCs is unlikely in this setting (Wagers et al., 2002). It was also reported that cell fusion can occur during co-culture of embryonic stem (ES) cells with HSCs or neural stem cells (NSCs). Although cell fusion was observed at an extremely rare incidence (104 to 105), ‘‘conversion’’ from NSCs (or HSCs) to other cell types could be obtained through spontaneous generation of hybrid cells rather than epigenetic reprogramming of the somatic stem cells (Terada et al., 2002; Ying et al., 2002). Thus, it is likely that ‘‘transdifferentiation’’ is a rare event under physiological conditions (Lemischka, 2002; McKay, 2002). Transdifferentiation in the settings of increased tissue renewal or tissue damage remains to be precisely addressed using prospectively isolated HSCs. Based on the findings that HSCs normally express many nonhematopoietic genes, it will be similarly interesting to search among these genes for candidate molecules that may instruct nonhematopoietic fate outcomes from bona fide HSCs. XIII. Early Lymphoid Progenitors can Differentiate into Antigen-presenting Dendritic Cells

Dendritic cells (DCs) are bone marrow-derived leukocytes that were initially defined by their high antigen presentation capacities and their ability to prime antigen responsiveness in naive T cells (Banchereau and Steinman, 1998; Hart, 1997). Since monocytes can give rise to DCs in vitro (Inaba et al., 1993), it was initially believed that DCs were of myeloid origin. It is now recognized that DCs develop from both lymphoid and myeloid precursors (Manz et al., 2001). A lymphoid origin for DCs was first reported using early thymocyte progenitors (TPs) (CD4loCD44þCD25c-Kitþ) and thymic proT cells (CD44þCD25þcKitþ). Each population is capable of generating CD8aþ DCs in vivo (Ardavin et al., 1993; Wu et al., 1996). Because the majority of thymic DCs

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express CD8aþ, it was proposed that CD8a expression is a marker for DCs of lymphoid derivation, whereas conventional CD8a DCs were of ‘‘myeloid’’ origin. This model appeared to be supported by the fact that mice deficient for the myeloid transcription factors, RelB (Wu et al., 1998) or PU.1 (Guerriero et al., 2000) lack CD8a DCs, while ‘‘lymphoid’’ transcription factors, Ikaros (Wu et al., 1997), or Id2 (Hacker et al., 2003) lack only CD8aþ DC subsets. On the other hand, developmental dissociation of CD8aþ DC and T cells was reported in T cell-deficient c-Kit/ gc/ (Rodewald et al., 1999) or Notch1/ (Radtke et al., 2000) mice, suggesting that CD8a is not necessarily a marker of lymphoid derivation. Each myeloid and lymphoid progenitor subset was thus examined for DC differentiation potential. Interestingly, CMPs could generate both CD8aþ and CD8a DCs (Traver et al., 2000). Both CD8aþ and CD8a DCs were produced from CLPs and CMPs with similar efficiency on a per-cell basis (Manz et al., 2001; Wu et al., 2001). Thus, expression of CD8a is not indicative of a lymphoid origin, and the presence or absence of CD8a expression among DC subsets is likely to reflect maturation status rather than ontogeny. The precursor/progeny relationship between CD8aþ and CD8a DCs, however, is still controversial. Splenic CD8aCD11cþ DCs were reportedly able to give rise to CD8aþCD11cþ DCs (Martinez del Hoyo et al., 2002), but this finding was not supported by another report (Naik et al., 2003). It has also been suggested that the acquisition of CD8a expression is likely to initiate in splenic DC precursors that are CD11c, since CD8aþCD11c spleen cells gave rise to mature CD8aþCD11cþ DCs (Wang et al., 2002). On the other hand, both CD8aþ and CD8a DCs are reported to originate from CD11cþMHCII DC progenitors which were devoid of other myeloid or lymphoid differentiation potential (Martinez del Hoyo et al., 2002). This population was also shown to give rise to interferon (IFN)-g-producing plasmacytoid DCs (PDCs; see following text) (Martinez del Hoyo et al., 2002). These data suggest that the CD11cþMHCII DC progenitors may represent a DC stage independent of DC pathways from CLPs or CMP. It is more likely, however, that CLPs and/or CMPs generate CD11cþMHCII DC precursors that are upstream of mature CD8aþ and CD8a DCs. Interestingly, DC potential is maintained downstream of CLPs in proT cells and downstream of CMPs in GMPs, whereas DC potential is lost once B cell or megakaryocyte/erythrocyte commitment occurs (Manz et al., 2001). Although murine DCs and their precursors are usually isolated from lymphoid organs or bone marrow, human DCs are usually isolated from peripheral blood (for review, see Shortman and Liu, 2002). Human DCs can be derived from CD34þ progenitors, lymphoid restricted progenitors, and from peripheral blood monocytes, suggesting that human DCs similarly derive from both myeloid and lymphoid pathways (Caux et al., 1996; Hao et al., 2001;

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Randolph et al., 1998; Romani et al., 1994; Sallusto and Lanzavecchia, 1994; Young et al., 1995). In both mice and humans, there appears to be no functional difference between lymphoid- and myeloid-derived DCs as evaluated by mixed leukocyte reactions, immunophenotyping, and cytokine production (Manz et al., 2001; Traver et al., 2000). A precursor population that immediately generates DCs capable of producing interferon a was identified in humans (Cella et al., 1999; Grouard et al., 1997; Jarrossay et al., 2001; Kadowaki et al., 2000, 2001) and subsequently in mice (Asselin-Paturel et al., 2001; Bjorck, 2001; Nakano et al., 2001). In addition to the conventional DC markers such as CD11c and MHC class II, these cells express CD45RA. This population has been termed plasmacytoid dendritic cells (PDCs) or DC2 (Liu, 2001). In humans, DCs of this phenotype were reportedly generated from at least M-CSFR-expressing myeloid progenitors (Olweus et al., 1996). On the other hand, human CD34þ cells with enforced Id2 or Id3 expression reportedly differentiated into conventional DCs, but not T, B, or PDCs (Heemskerk et al., 1997; Jaleco et al., 1999; Spits et al., 2000), suggesting that PDCs may have a lymphoid origin. Accordingly, PDCs express genes usually found in the lymphoid lineage such as preTa, 14.1, and Spi-B (Bendriss-Vermare et al., 2001; Spits et al., 2000). In our hands, murine PDC equivalents could be induced from both CLPs and CMPs (unpublished data). Thus, it is likely that PDCs as well as conventional DCs originate from both lymphoid and myeloid pathways. CLPs and proT cells, but not preT or proB cells, can differentiate into DCs (Manz et al., 2001; Traver et al., 2000; Wu et al., 1996). It is interesting to note that DC potential is contained within the same lymphoid precursor subsets that can be converted to myeloid fates by ectopic cytokine signals (Iwasaki-Arai et al., 2003) (see previous text). This correlation may suggest that ‘‘lymphoid’’ DC potential within CLPs and proT is a residual function of latent myeloid potential. It is therefore important to characterize the signals that induce DC commitment from lymphoid progenitors. XIV. Fetal Hematopoietic Progenitors are Not Fully Committed to the Lymphoid and Myeloid Fates

Although the hematopoietic hierarchy and effector cell types produced in fetal life are similar to those in adult blood development, some phenotypic and functional differences exist (Holyoake et al., 1999; Jordan et al., 1995; Morrison et al., 1995; Pawliuk et al., 1996). For example, fetal liver HSCs can generate Vg3þ and Vg4þ T cells (Ikuta et al., 1990) and B-1a lymphocytes (Hayakawa and Hardy, 2000) while adult HSCs cannot. Using surface markers similar to those used to isolate adult progenitor populations, fetal liver counterparts have been prospectively purified. These include the fetal CLPs

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(LinIL-7RaþB220/loSca-1loc-Kitlo) (Mebius et al., 2001), CMPs (LinIL7RaSca-1c-KitþAA4.1FcgRII/IIIloCD34þ), GMPs (LinIL-7RaSca1c-KitþAA4.1FcgRII/IIIhiCD34þ), and MEPs (LinIL-7RaSca-1cKitþAA4.1FcgRII/IIIloCD34) (Traver et al., 2001). Each fetal population displays similar differentiation potentials, but, as will be discussed, appear to be somewhat less restricted than their adult counterparts. Clonogenic progenitors for T cells, B cells, and macrophages were demonstrated to exist within the AA4.1þFcgRII/IIIþ fraction in mouse fetal liver (Lacaud et al., 1998). Cumano et al. demonstrated clonogenic B cell and macrophage progenitors within a fetal liver AA4.1þB220Mac-1Sca-1þ fraction, although T cell differentiation potential was not tested (Cumano et al., 1992). B220þc-Kitþ fetal liver cells (also positive for the IL-7Ra chain) differentiated into T cells, B cells, and macrophages at a high frequency (Sagara et al., 1997). Interestingly, IL-7Ra expression is not a definitive marker to exclude cells with macrophage potential in fetal liver hematopoiesis. A fetal counterpart to adult CLPs, IL-7RaþB220/loSca-1loc-Kitlo cells, comprises 0.5 to 1.2% of E12.5 to E14.5 fetal liver cells. These cells are positive for AA4.1 but negative for FcgRII/III, indicating that there is no overlap with the cells reported by Lacaud et al. (1998). The populations reported by Cumano et al. (1992) and Sagara et al. (1997) should include IL-7RaþB220/loSca-1locKitlo cells. In vitro culture of fetal CLPs on S17 stromal layers showed that 5% of single cells differentiated into macrophages and B cells (Mebius et al., 2001). This population never gave rise to myeloerythroid cells other than macrophages. The burst-size of macrophage differentiation appeared minimal (Mebius et al., 2001). Accordingly, injection of fetal CLPs into the livers (Domen et al., 1998) of sublethally irradiated newborn mice showed, T, B, and NK cell-restricted differentiation without detectable macrophage progeny (Mebius et al., 2001). Taken together, these findings suggest that macrophage potential is maintained following commitment into the T/B lymphoid lineages during fetal life. In early myelopoiesis, fetal liver CMPs, which form all myeloerythroid colony types, show a relatively high propensity to differentiate into B cells (Traver et al., 2001). In limiting dilution assays, B cell frequency was 0.8% from fetal CMPs. The B cell potential in fetal CMPs has not been tested at the single-cell level and, therefore, this still could be from a minor contaminant of B cell progenitors in the phenotypic CMP fraction. T cell developmental potential, however, was completely absent as no donor-derived progeny were found following intrathymic injection of 10,000 fetal CMPs. Thus, in the model established by prospective isolation of fetal counterparts of adult progenitors, fetal CMPs and CLPs are placed downstream of HSCs as in adult hematopoiesis, allowing them to maintain a minor potential for B cell and macrophage potentials, respectively (Mebius et al., 2001; Traver et al., 2001) (Fig. 7A).

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Fig 7 A developmental model in fetal liver hematopoiesis. (A) A developmental model based on purification of phenotypic counterparts of adult CMPs and CLPs in the fetal liver (FL). In this model, B cell potential in fetal CMPs and macrophage potential in fetal CLPs represent their relatively incomplete lineage restriction.

The differentiation potentials of single fetal liver LinSca-1þc-Kitþ HSCs and IL-7RaþLinSca-1þc-Kitþ lymphoid progenitors were carefully evaluated by high-oxygen FTOC in the presence of IL-3, IL-7, and SLF (Kawamoto et al., 1997, 2000). This has been termed the multilineage progenitor (MLP) assay and is powerful since it can support myelomonocytic and lymphoid fates from fetal liver progenitors. Unfortunately, this assay is not suitable to evaluate megakaryocyte/erythrocyte potential. Throughout these experiments, single progenitors giving rise only to T and B cells were never found, although T/GM, B/GM, or T/B/GM outcomes were detectable (Kawamoto et al., 1997). Myeloid progeny in this culture system mainly consisted of macrophages, with minor populations of dendritic cells and granulocytes produced. These data led to the proposal of a unique hematopoietic differentiation model that does not include the CLP stage (Katsura, 2002) (Fig. 7B). In this model, T and B lymphoid development is always associated with myelomonocytic development, and originates from the common myelolymphoid progenitor (CMMP) (Fig. 7B). How can we reconcile this model with the conventional developmental scheme based on prospective purification studies? Retrospective studies such as these base upstream developmental sequences on functional endpoints such as colony formation. The ensuing developmental models can only be justified if the assay system is fully permissive for all fate potentials of plated stem and progenitor cells. In the MLP assay system, only 5% of single fetal liver HSCs exhibited multilineage (T/B/GM) readouts. Nonetheless, the fact that T/B only differentiation was never found from single cells raises the possibility that CLPs are not a requisite step in the generation of fetal lymphocytes. As has been discussed, it is highly likely that macrophage potential is well preserved along the early lymphoid pathway in

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the fetal liver (Mebius et al., 2001). This may account for the relatively high incidence of lymphoid/myeloid differentiation from fetal progenitors in the MLP assay. Interestingly, the high oxygen concentration used in the MLP assay dramatically promotes macrophage differentiation (Anderson et al., 2002). Therefore, the macrophage potentials of fetal liver cells could also be overestimated by this assay. Another group has subsequently reported that single, adult CLPs frequently gave rise to both T and B but not myeloid cells in the MLP assay (Izon et al., 2001), highlighting an apparent difference between fetal and adult CLP subsets. It will be interesting to determine the developmental boundaries at which the relatively incomplete lineage restriction observed in fetal progenitors become restricted to the lymphoid and myeloerythroid fates. XV. Lymphoid- and Myeloid-restricted Progenitors in Human Bone Marrow

In human hematopoiesis, similar progenitor populations have been isolated by cell surface phenotypes. A population having significant CLP activity has been reported to exist within the LinCD34þCD10þ subset of human bone marrow cells (Galy et al., 1995). Lymphoid potential was determined by reconstitution of human bone and thymus fragments implanted into SCID mice as well as by in vitro culture systems. LinCD34þCD10þThy-1þ multipotent HSC are CD45RA, whereas CD45RAþ LinCD34þCD10þThy-1 CLPs differentiated only into T, B, NK, and dendritic cells (Galy et al., 1995). Similar to mouse CLPs, this population expresses IL-7Ra. Many studies support the existence of human oligopotent myeloid progenitors (de Wynter et al., 2001; Fritsch et al., 1993; Huang et al., 1999; Lansdorp et al., 1990; Olweus et al., 1996, 1997; Rappold et al., 1997). Human counterparts of murine CMPs, GMPs, and MEPs were isolated from human bone marrow and cord blood cells (Manz et al., 2002). All are negative for multiple mature lineage markers, including the early lymphoid markers CD7, CD10, and IL-7Ra, and all are CD34þCD38þ. This CD34þCD38þ fraction was divided by the expression of CD45RA, an isoform of CD45 that can negatively regulate at least some classes of cytokine receptor signaling (Irie-Sasaki et al., 2001), and IL-3Ra, a receptor that, upon activation, supports proliferation and differentiation of primitive progenitors (Kimura et al., 1997). CD45RAIL3Ralo (CMPs), CD45RAþIL-3Ralo (GMPs), and CD45RAIL-3Ra (MEPs) formed distinct myeloid colony types according to their definitions at a high frequency, but possess little or no LTC-IC activity. The FcgRII/III (CD16/ CD32) that distinguishes mouse CMPs and GMPs was not detectable on human myeloid progenitor populations. CMPs give rise to MEPs and GMPs in vitro and a significant proportion of CMPs were demonstrated to possess clonal granulocyte/macrophage and megakaryocyte/erythrocyte potential

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(Manz et al., 2002). Thus, the hierarchical progenitor relationships demonstrated in the mouse also exist in human hematopoiesis and can be prospectively isolated by cell surface phenotype. XVI. Clinical Relevance

Bone marrow transplantation (BMT) into myeloablated individuals often gives rise to graft versus host disease (GVHD) when using total bone marrow or mobilized peripheral blood cells. This is due to the transfer of allogeneic lymphocytes within the transplanted cells. It has been shown that transplantation of purified CD34þ HSCs alleviates the vast majority of GVHD, presumably because donor-derived lymphocytes can be educated in the host thymus to prevent alloreactivity (Link et al., 1996). Pure HSC transplants, however, are relatively slow to generate sufficient numbers of mature cell types. Using a mouse progenitor transplantation model, it has been shown that high-dose transplantation of either CMPs or MEPs was sufficient for radioprotection over a 1-month interval (Na Nakorn et al., 2002). After this time, rare surviving host HSCs recovered to produce all blood cell subsets (Na Nakorn et al., 2002). Another important complication following BMT is infection. Myeloablative irradiation and/or cytoreductive drugs cause a rapid disappearance of myelomonocytes as well as lymphocytes. This leaves a window following transplantation for opportunistic pathogens such as Aspergillus fumigatus, Pseudomonas aeruginos, and cytomegaloviruses to flourish. Using another mouse model, it was shown that transplantation of CMPs and/or GMPs in conjunction with HSC transplants could protect against otherwise lethal challenges of either pathogen due to increased numbers of myelomonocytic cells (BitMansour et al., 2002). Transplantation of CLPs in conjunction with HSCs also protected mice against murine CMV infection (Arber et al., 2003). Based on these preclinical findings, it may be of clinical interest to purify human hematopoietic progenitor subsets to augment purified HSC transplants. XVII. Conclusion

In this chapter, we have provided a current understanding of hematolymphoid development from rare HSCs that give rise to progenitor subsets that progressively lose fate potential as they differentiate down their respective lineages. The ability to prospectively isolate the major branchpoints along the hematopoietic tree allows a molecular profiling of each, both at the population and single-cell level. Transcriptional profiling at the population level has shown that the ‘‘master regulator’’ genes identified from mouse knockout studies are expressed in the progenitor subsets upstream of the noted defective lineages. Single-cell profiling has supported the hypothesis of ‘‘priming stages,’’ whereby

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promiscuous, low-level expression of many genes appears to maintain flexibility in cell fate choices. Interestingly, enforced expression of single, inappropriate genes within progenitors normally exclusively commited to the lymphoid fates can reprogram myeloerythroid outcomes. This demonstrates that the nucleus maintains developmental flexibility and that the progressive loss of fate potential upon differentiation is likely controlled initially by strict regulation of growth factor receptor expression. This finding may explain the clinical observations of lineage infidelity and mixed-lineage leukemias. Future work with hematopoietic progenitor populations should allow a more precise understanding of the molecular mechanisms of lineage commitment and may help elucidate how stem cells choose between self-renewal and differentiation. References Abramson, S., Miller, R. G., and Phillips, R. A. (1977). The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems. J. Exp. Med. 145, 1567–1579. Adolfsson, J., Borge, O. J., Bryder, D., Theilgaard-Monch, K., Astrand-Grundstrom, I., Sitnicka, E., Sasaki, Y., and Jacobsen, S. E. (2001). Upregulation of Flt3 expression within the bone marrow Lin()Sca1(þ)c-kit(þ) stem cell compartment is accompanied by loss of self-renewal capacity. Immunity 15, 659–669. Aifantis, I., Azogui, O., Feinberg, J., Saint-Ruf, C., Buer, J., and von Boehmer, H. (1998). On the role of the pre-T cell receptor in alphabeta versus gammadelta T lineage commitment. Immunity 9, 649–655. Akashi, K., Harada, M., Shibuya, T., Fukagawa, K., Kimura, N., Sagawa, K., Yoshikai, Y., Teshima, T., Kikuchi, M., and Niho, Y. (1991). Simultaneous occurrence of myelomonocytic leukemia and multiple myeloma: Involvement of common leukemic progenitors and their developmental abnormality of ‘‘lineage infidelity.’’ J. Cell Phys. 148, 446–456. Akashi, K., He, X., Chen, J., Iwasaki, H., Niu, C., Steenhard, B., Zhang, J., Haug, J., and Li, L. (2003). Transcriptional accessibility for genes of multiple tissues and hematopoietic lineages is hierarchically controlled during early hematopoiesis. Blood 101, 383–389. Akashi, K., Kondo, M., and Weissman, I. L. (1998). Role of interleukin-7 in T-cell development from hematopoietic stem cells. Immunol. Rev. 165, 13–28. Akashi, K., Kondo, M., von Freeden-Jeffry, U., Murray, R., and Weissman, I. L. (1997). Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor-deficient mice. Cell 89, 1033–1041. Akashi, K., Traver, D., Miyamoto, T., and Weissman, I. L. (2000). A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 404, 193–197. Allman, D., Li, J., and Hardy, R. R. (1999). Commitment to the B lymphoid lineage occurs before DH–JH recombination. J. Exp. Med. 189, 735–740. Allman, D., Sambandam, A., Kim, S., Miller, J. P., Pagan, A., Well, D., Meraz, A., and Bhandoola, A. (2003). Thymopoiesis independent of common lymphoid progenitors. Nat. Immunol. 4, 168–174. Anderson, A. C., Robey, E. A., and Huang, Y. H. (2001). Notch signaling in lymphocyte development. Cur. Op. Gen. 11, 554–560. Anderson, M. K., Weiss, A. H., Hernandez-Hoyos, G., Dionne, C. J., and Rothenberg, E. V. (2002). Constitutive expression of PU.1 in fetal hematopoietic progenitors blocks T cell development at the pro-T cell stage. Immunity 16, 285–296.

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Arber, C., BitMansour, A., Sparer, T. E., Higgins, J. P., Mocarski, E. S., Weissman, I. L., Shizuru, J. A., and Brown, J. M. Y. (2003). Common lymphoid progenitors rapidly engraft and protect against lethal murine cytomegalovirus infection after hematopoietic stem cell transplantation. Blood 2002, 2012–3834. Ardavin, C., Wu, L., Li, C.-L., and Shortman, K. (1993). Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population. Nature 362, 761–763. Asselin-Paturel, C., Boonstra, A., Dalod, M., Durand, I., Yessaad, N., Dezutter-Dambuyant, C., Vicari, A., O’Garra, A., Biron, C., Briere, F., and Trinchieri, G. (2001). Mouse type I IFNproducing cells are immature APCs with plasmacytoid morphology. Nat. Immunol. 2, 1144–1150. Banchereau, J., and Steinman, R. M. (1998). Dendritic cells and the control of immunity. Nature 392, 245–252. Becker, A., McCulloch, E., and Till, J. (1963). Cytological demonstration of the clonal nature of spleen colonies dervived from transplanted mouse marrow cells. Nature 197, 452–454. Bendriss-Vermare, N., Barthelemy, C., Durand, I., Bruand, C., Dezutter-Dambuyant, C., Moulian, N., Berrih-Aknin, S., Caux, C., Trinchieri, G., and Briere, F. (2001). Human thymus contains IFN-alpha-producing CD11c(), myeloid CD11c(þ), and mature interdigitating dendritic cells. J. Clin. Inv. 107, 835–844. Berger, S. L., and Felsenfeld, G. (2001). Chromatin goes global. Mol. Cell 8, 263–268. Bhatia, S. K., Tygrett, L. T., Grabstein, K. H., and Waldschmidt, T. J. (1995). The effect of in vivo IL-7 deprivation on T cell maturation. J. Exp. Med. 181, 1399–1409. BitMansour, A., Burns, S. M., Traver, D., Akashi, K., Contag, C. H., Weissman, I. L., and Brown, J. M. Y. (2002). Myeloid progenitors protect against invasive aspergillosis and Pseudomonas aeruginosa infection following hematopoietic stem cell transplantation. Blood 100, 4660–4667. Bjorck, P. (2001). Isolation and characterization of plasmacytoid dendritic cells from Flt3 ligand and granulocyte-macrophage colony-stimulating factor-treated mice. Blood 98, 3520–3526. Boyd, A. W., and Schrader, J. W. (1982). Derivation of macrophage-like lines from the pre-B lymphoma ABLS 8.1 using 5-azacytidine. Nature 297, 691–693. Bradley, T. R., and Metcalf, D. (1966). The growth of mouse bone marrow cells in vitro. Aust. J. Exp. Biol. Med. Sci. 44, 287–299. Brown, G., Bunce, C. M., and Guy, G. R. (1985). Sequential determination of lineage potentials during haemopoiesis. Br. J. Cancer 52, 681–686. Brown, G., Bunce, C. M., Howie, A. J., and Lord, J. M. (1987). Stochastic or ordered lineage commitment during hemopoiesis? Leukemia 1, 150–153. Busslinger, M., Nutt, S. L., and Rolink, A. G. (2000). Lineage commitment in lymphopoiesis. Curr. Op. Im. 12, 151–158. Cao, X., Shores, E. W., Hu-Li, J., Anver, M. R., Kelsall, B. L., Russell, S. M., Drago, J., Noguchi, M., Grinberg, A., Bloom, E. T., et al. (1995). Defective lymphoid development in mice lacking expression of the common cytokine receptor gamma chain. Immunity 2, 223–238. Caux, C., Vanbervliet, B., Massacrier, C., Dezutter-Dambuyant, C., de Saint-Vis, B., Jacquet, C., Yoneda, K., Imamura, S., Schmitt, D., and Banchereau, J. (1996). CD34þ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSFþTNF alpha. J. Exp. Med. 184, 695–706. Cella, M., Jarrossay, D., Facchetti, F., Alebardi, O., Nakajima, H., Lanzavecchia, A., and Colonna, M. (1999). Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon. Nat. Med. 5, 919–923. Chen, H., Ray-Gallet, D., Zhang, P., Hetherington, C. J., Gonzalez, D. A., Zhang, D. E., MoreauGachelin, F., and Tenen, D. G. (1995). PU.1 (Spi-1) autoregulates its expression in myeloid cells. Oncogene 11, 1549–1560.

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Cheng, G. Y., Minden, M. D., Toyonaga, B., Mak, T. W., and McCulloch, E. A. (1986). T cell receptor and immunoglobulin gene rearrangements in acute myeloblastic leukemia. J. Exp. Med. 163, 414–424. Chiang, M. Y., and Monroe, J. G. (1999). BSAP/Pax5A expression blocks survival and expansion of early myeloid cells implicating its involvement in maintaining commitment to the B-lymphocyte lineage. Blood 94, 3621–3632. Christensen, J. L., and Weissman, I. L. (2001). Flk-2 is a marker in hematopoietic stem cell differentiation: A simple method to isolate long-term stem cells. Proc. Natl. Acad. Sci. USA 98, 14541–14546. Colucci, F., Samson, S. I., DeKoter, R. P., Lantz, O., Singh, H., and Di Santo, J. P. (2001). Differential requirement for the transcription factor PU.1 in the generation of natural killer cells versus B and T cells. Blood 97, 2625–2632. Corcoran, A. E., Riddell, A., Krooshoop, D., and Venkitaraman, A. R. (1998). Impaired immunoglobulin gene rearrangement in mice lacking the IL-7 receptor. Nature 391, 904–907. Cotta, C. V., Zhang, Z., Kim, H. G., and Klug, C. A. (2003). Pax5 determines B versus T cell fate and does not block early myeloid-lineage development. Blood 101, 4342–4346. Cross, M. A., and Enver, T. (1997). The lineage commitment of haemopoietic progenitor cells. Cur. Op. Gen. 7, 609–613. Cumano, A., Paige, C. J., Iscove, N. N., and Brady, G. (1992). Bipotential precursors of B cells and macrophages in murine fetal liver. Nature 356, 612–615. de Wynter, E. A., Heyworth, C. M., Mukaida, N., Jaworska, E., Weffort-Santos, A., Matushima, K., and Testa, N. G. (2001). CCR1 chemokine receptor expression isolates erythroid from granulocyte-macrophage progenitors. J. Leukoc. Biol. 70, 455–460. Deftos, M. L., Huang, E., Ojala, E. W., Forbush, K. A., and Bevan, M. J. (2000). Notch1 signaling promotes the maturation of CD4 and CD8 SP thymocytes. Immunity 13, 73–84. DeKoter, R. P., and Singh, H. (2000). Regulation of B lymphocyte and macrophage development by graded expression of PU.1. Science 288, 1439–1441. Delassus, S., Titley, I., and Enver, T. (1999). Functional and molecular analysis of hematopoietic progenitors derived from the aorta–gonad–mesonephros region of the mouse embryo. Blood 94, 1495–1503. Dexter, T. M., and Testa, N. G. (1980). In vitro methods in haemopoiesis and lymphopoiesis. J. Immunol. M. 38, 177–190. Doi, H., Inaba, M., Yamamoto, Y., Taketani, S., Mori, S. I., Sugihara, A., Ogata, H., Toki, J., Hisha, H., Inaba, K., et al. (1997). Pluripotent hemopoietic stem cells are c-kit2.5% of the repertoire, was not observed in EAE-recovered mice with intact CD8þ T cells. In contrast, the TCR

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HONG JIANG AND LEONARD CHESS

Fig 3 Regulatory CD8þ T cells control self-reactive TCR Vb repertoire in EAE mice. The figure shows the outline of the experimental design and a schematic illustration of representative results demonstrating that regulatory CD8þ T cells control self-reactive TCR Vb repertoire in EAE mice. The MBP-reactive TCR repertoire, following MBP immunization, in naive mice (control), mice recovered from EAE and CD8þ T cell-depleted, EAE-recovered mice (CD8-/EAE) (Jiang et al., 2003) were compared by TCR Vb surface staining, PCR-based CDR3 length distribution analysis of the TCR repertoire, and direct sequence analysis of the CDR3 regions. The studies demonstrated that regulatory CD8þ T cells selectively downregulate certain but not all MBP-reactive T cells within the TCR Vb8.2 family in EAE-recovered mice.

repertoire of MBP-reactive CD4þ T cells in the EAE-recovered mice was composed predominantly of a highly diverse set of self-reactive clones with limited outgrowth. Because the CD8þ T cell depleted EAE-recovered mice are highly susceptible to clinical EAE, whereas CD8þ T cell intact EAErecovered mice are not, it is likely that the dominant clones which emerge following secondary MBP immunization in CD8þ T cell depleted EAErecovered mice contain the potentially encephalitogenic CD4þ T cells. This idea was further supported by our observation that MBP-reactive CD4þ Vb8.2þ T cell clones derived from CD8þ T cell depleted EAErecovered mice were more likely to induce EAE following adoptive transfer into naive mice than clones derived from EAE-recovered mice with intact CD8þ T cells. Furthermore, adoptive transfer of CD8þ T cells isolated from EAE-recovered mice but not naive mice modified MBP-reactive TCR Vb8.2 but not Vb6 repertoire in recipient mice (Jiang et al., 2003). Taken together, these in vivo and in vitro studies provide evidence that in addition to their TCR Vb specificity, CD8þ T cells only selectively downregulate certain

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but not all self-reactive T cells within the TCR Vb8.2 family. These regulatory CD8þ T cells play a key role in controlling self-reactive TCR repertoire by selectively downregulating the clones enriched in the potentially pathogenic self-reactive T cells in the periphery. The residual highly diverse nonpathogenic self-reactive TCR repertoire is preserved by this selective downregulation. Taken together, it thus appears that the regulatory CD8þ T cells described are Qa-1 restricted and that they regulate immune response in a TCR Vb and TH type specific manner. Since regulatory CD8þ T cells control peripheral immune responses to both self and foreign antigens by selectively downregulating certain but not all antigen-activated T cells thus regulating peripheral TCR repertoire, it is of interest to consider why only some but not all antigenactivated clones are downregulated. As has been noted, it is known that Qa-1 is only minimally expressed on resting lymphoid cells and that, unlike classical MHC class Ia molecules, its expression is dependent on activation. Although the precise Qa-1 binding peptide/s in this system have not been identified yet, it is likely that the major determinant of whether or not a CD4þ T cell will process and present Qa-1/self-peptide will be the consequence of initial cognitive encounter between the particular ab TCR expressed by antigen-reactive CD4þ T cells and the MHC class II/antigen peptide complex presented on antigen-presenting cells. One possible explanation for the specific recognition of certain clones that could be envisioned is an idiotype model in which the TCR expressed by regulatory CD8þ T cells will preferentially recognize Qa-1 complexed with only certain TCR Vb peptides. Alternatively, one can envision an affinity model in which the precise threshold for antigen-activated CD4þ T cells to process and present Qa-1/self-peptides may be a function of the affinity/avidity of the interaction between antigen-specific CD4þ T cells and MHC class II/antigen peptides expressed on antigen-presenting cells. We have previously proposed an ‘‘affinity model’’ that hypothesizes that CD4þ T cells will process and present their own Qa-1/self-peptide as a function of the affinity/avidity of their TCRs for the MHC/antigen peptide on APC. Thus, TCRs with either low or high affinity interaction with MHC/ antigen peptide will not express Qa-1/peptide complexes whereas TCRs with intermediate affinity will express Qa-1/peptide. As a consequence, only T cells with ‘‘intermediate’’ affinity, above and under certain thresholds, will be regulated by the Qa-1 restricted CD8þ regulatory cells (Jiang and Chess, 2000). Evidence in support of this hypothesis came from our observation in EAE that MBP-reactive CD4þ clones which are under the control of CD8þ T cells are the clones with higher growth potential responding to MBP (Jiang et al., 2003). Because high affinity self-reactive clones are deleted intrathymically and the low affinity clones are not properly triggered by MBP, these higher growth potential clones presumably possess intermediate affinity to MBP. The precise

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threshold for these CD4þ T cells to process and present self-peptide coupled to Qa-1 molecules will not only be a function of the quality, intensity, and duration of the initial tri-molecular complex interaction between antigenspecific CD4þ T cells and antigen-presenting cells but may also be influenced by signaling via co-stimulatory molecules including possible CD28/B7, CTLA-4/B7, and CD40/CD40L interactions. Moreover, these same factors may determine whether the surface Qa-1 molecules on CD4þ T cells are predominately composed of Qa-1/Qdm or Qa-1/self-peptide or both. The expression of Qa-1/self-peptide will induce specific regulatory CD8þ T cells, which may be further regulated by the Qa-1/Qdm expression through their CD94/NKG2 receptor. Thus, either the particular Qa-1 binding peptide/s (idiotype model) or the affinity/avidity of antigen-reactive T cells (affinity model), or both, determines the susceptibility of antigen-activated CD4þ T cells to the selective downregulation by the CD8þ T cells. We initially proposed that the source of Qa-1 binding peptide/s would be derived from the TCR Vb proteins. This could clearly explain the Vb specificity of the regulation by the CD8þ T cells. Based on the newly developed experimental evidence in analyzing the MBP-reactive TCR Vb repertoire that CD8þ T cells selectively downregulate certain but not all MBP-reactive Vb8.2 and Vb13 T cells which have higher growth potential responding to MBP in vivo in EAE mice (Jiang et al., 2003), we have modified our theory. The modified theory suggests that whether or not a particular CD4þ T cell is subject to CD8þ T cell downregulation depends on whether the CD4þ T cell is capable of presenting self-peptide/s coupled to Qa-1 rather than which Qa-1 binding self-peptide the CD4þ T cell presents. The selective expression of Qa-1/self-peptide/s on certain activated CD4þ T cells is determined by the affinity/avidity of T cells as a consequence of activation. This modified theory suggests that a non-TCR Vb peptide, which could be common in all activated T cells, could also, like Vb peptide/s, bind to Qa-1 and be recognized by the Qa-1 restricted regulatory CD8þ T cells. For example, heat shock proteins are induced by T cell activation and heat shock protein peptide/s are known to bind to Qa-1 (Imani and Soloski, 1991). In either case, the expression of Qa-1/self-peptide complex by activated CD4þ T cells could be governed by the affinity/avidity of the TCR on CD4þ T cells and the co-stimulatory molecules involved during the initial encounter of TCR on CD4þ T cells and MHC class II/antigen peptide on antigen-presenting cells. In either case, certain TCR Vb specific regulation may be observed due to the preferential usage of certain TCR Vb chains by the responding T cell population to a particular antigen. For example, we have shown that CD8þ T cells selectively downregulate certain but not all MBP-reactive T cell clones in EAE-recovered mice only in TCR Vb8.2 and Vb13 but not in other Vb families (Jiang et al., 2003). It is of interest that in B10PL mice, the Vb8.2


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and Vb13 families of CD4þ T cells represent the two major T cell populations that are activated by 1-9Nac MBP and are largely responsible for clinical EAE (Acha-Orbea et al., 1988; Zamvil et al., 1985). In this case, either ‘‘Vb peptide’’ or ‘‘non-Vb peptide’’ or both could account for the ‘‘Vb specific’’ phenomenon. It is important to emphasize that CD8þ T cells require priming during the first episode of EAE to regulate the outgrowth of potentially pathogenic CD4þ T cells triggered by secondary MBP challenge in vivo. The evidence that regulatory CD8þ T cells require priming during the first episode of EAE is simply that B10PL mice depleted of CD8þ T cells during the initial induction of EAE recover from EAE normally but are not resistant to rechallenge with 1–9Nac MBP. In contrast, EAE-recovered mice with CD8þ T cells primed during the first episode are resistant to reinduction of EAE unless they are depleted of CD8þ cells prior to reinduction of EAE (Jiang et al., 1992, 2001, 2003). Based on these observations, we envision that the cellular events during the evolution of natural EAE are as follows: MBP-reactive CD4þ T cells are activated by the first encounter with MBP to induce both clinical EAE and regulatory CD8þ T cells. The regulatory CD8þ T cells are not induced in time to downregulate MBP-reactive CD4þ T cells during the first episode of EAE but are primed to downregulate MBP-reactive CD4þ T cells activated by secondary MBP immunization. In this study, CD8þ T cells were depleted during the induction of EAE (first episode). These mice developed EAE and clinically recovered. Newly generated CD8þ T cells reappear during the recovery and are present at the time of secondary MBP immunization in vivo. When the MBP-reactive TCR Vb repertoire of these mice was compared to that of EAE-recovered mice with primed CD8þ T cells, the profound effect of the primed CD8þ T cells on the CD4þ MBP-reactive TCR Vb repertoire was observed. This sequence of events is reminiscent of the general biology of CD8þ T cells involved in the response to viruses. During the initial infection with most viruses, CD8þ T cells are not involved in the recovery that may occur within the first week or so (this initial recovery is mediated, in part, by the innate immune response, including macrophages, NK and NKT cells, and gd T cells) but they are primed during the initial infection period and are clearly involved in resistance to reinfection or persistent virus infection. In summary, a general model of peripheral regulation by Qa-1 restricted regulatory CD8þ T cells is illustrated in Fig. 4. There are two unique features of Qa-1 restricted regulatory CD8þ T cells, which distinguish them from other regulatory NKT or CD4þ T cells. First, the Qa-1 restricted regulatory CD8þ T cells selectively downregulate certain but not all antigen-activated T cells. The molecular interaction between the regulatory CD8þ T cells and target CD4þ T cells is through the recognition of Qa-1/self-peptide complex, which may be only preferentially expressed by T cells with intermediate affinity/avidity to the antigens, by ab TCR on regulatory CD8þ T cells

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Fig 4 Model of cognate interactions in the induction and function of Qa-1 restricted regulatory CD8þ T cells. The Qa-1 restricted regulatory CD8þ T cell selectively downregulate certain but not all antigen-activated CD4þ T cells based on the specific recognition of Qa-1/self-peptide/s expressed on certain CD4þ T cells by the ab TCR on the CD8þ T cells. In this regard, we have demonstrated in the EAE model that self-reactive CD4þ T cells which are selectively downregulated by the CD8þ T cells are enriched in potentially pathogenic self-reactive T cell clones (Jiang et al., 2003). (See Color Insert.)

(Jiang et al., 1998, 2001). Second, the Qa-1 restricted regulatory CD8þ T cells require priming in vivo. The regulatory CD8þ T cells are induced by antigenactivated T cells expressing TCRs of intermediate affinity/avidity during the primary immune response, perhaps with help from professional antigenpresenting cells, and then, in turn, selectively downregulate those T cells in the later stage of immunity. Ultimately, the regulatory CD8þ T cells, in concert with other regulatory mechanisms, control the peripheral TCR repertoire during the course of immune responses to both self and foreign antigens. With respect to the self-reactive clones, the peripheral repertoire is composed of mainly low and intermediate affinity/avidity CD4þ T cells. The intermediate affinity T cells are enriched in the potentially pathogenic self-reactive T cell clones and are controlled by CD8þ regulatory cells. The very high affinity selfreactive clones have already been eliminated in the thymus so that the biological consequence of selective regulation of these intermediate clones will be the control of autoimmunity. However, with respect to foreign antigens,


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the repertoire is primarily composed of intermediate and high affinity clones. The biological consequence of the selective downregulation of the intermediate affinity but not the high affinity foreign-reactive T cell population will be to promote affinity maturation. Thus, the biological consequences of the selective downregulation of T cells with intermediate affinity/avidity TCRs to antigens by the Qa-1 restricted CD8þ T cells is to shape the peripheral T cell repertoire in order to ensure peripheral self-tolerance and facilitate affinity maturation to foreign antigens. IV. An Integrated Model of Immunoregulation by NKT, CD41CD251, and Qa-1 Restricted CD81 T Cell Subsets

In this chapter, we have emphasized the fact that the immune system has evolved a variety of regulatory mechanisms mediated by distinct T cell subsets to control peripheral immunity. In particular, we have discussed various pathways of immunoregulation mediated by suppressor subsets of CD4þ and CD8þ T cells as well as by NKT cells. The regulation of immune responses mediated by these regulatory T cell subsets is superimposed on the regulatory mechanisms induced by antigen activation of the immunocompetent cells which are independent of the regulatory T cells and include antigen-induced apoptosis, anergy as well as antigen-induced differentiation of precursor T cells into Th subsets secreting regulatory cytokines (see Fig. 1). Each of the regulatory T cell subsets expresses distinct receptors, employs different effector mechanisms, and functions predominately at different stages during the evolution of the peripheral immunity (see Table I). Thus, the NKT cells and CD4þCD25þ regulatory cells exist in the early neonatal period as ‘‘natural suppressor cells’’ prior to antigen activation and function primarily during the early ‘‘innate’’ and/or primary immune responses (see Table I and Fig. 5). The NKT cells are endowed with ab TCRs which specifically recognize glycolipid molecules often expressed by various pathogens and are presumably expressed also by tumor cells, activated blasts, and injured ‘‘apoptotic’’ cells. These NKT cells are poised to secrete IL-4 and IL-10, which are known to influence the balance of Th1 or Th2 cells that emerge during the primary immune response. It is of interest that in addition to NKT cells, the CD4þCD25þ regulatory subset also exists in the periphery in the early neonatal period and persist in the peripheral lymphoid system as cells capable of potently suppressing the outgrowth of immunocompetent cells. These cells, like the NKT cells, can function during the primary immune response and do not require specific induction. In vitro, the suppressor function of these cells can be shown to dependent of cell-cell contact but these cells can also secrete immunoregulatory cytokines including TGFb which may be involved in the their suppressor function in vivo. The precise specificity of these cells for their targets remain

TABLE I Characteristics of NKT, CD4þCD25þ and Qa-1 restricted CD8þ Regulatory T Cell Subsets Subsets of regulatory T cells NKT cells

CD4þCD25þ regulatory T cells CD8þ regulatory cells

Target cells for suppression

Restriction element/antigen

Stage of immunity affected Innate

Regulatory mechanisms

Tumor cells, pathogenactivated T cells, and/or APCs expressing CD1d/glycolipid T cells, probably APCs

CD1d/glycolipid

IL-4, IL-10 TGFb, IFN-g

Unknown; not MHC restricteda

Primary, earlyb

Requires cell–cell contact, cytokines

Certain activated T cells expressing Qa-1/self-peptide/s

Qa-1/hydrophobic self-peptides

Secondary, lateb

Cytotoxicity; requires cell–cell contact; cytokines

In vivo function Destruction of tumors and infectious pathogens; regulation of Th1-mediated autoimmune diseases Prevention of a variety of autoimmune diseases, regulation of allo-graft rejection Fine-tune peripheral TCR repertoire; maintain self-tolerance and control autoimmune disease

The specific interaction between CD4þCD25þ regulatory T cells and the target T cells is currently unknown. CD4þCD25þ regulatory T cells isolated from naive unprimed mice protect recipient animals from autoimmune diseases when adoptively transferred. In contrast, Qa-1 restricted CD8þ regulatory T cells require priming during primary immune response in order to regulate the secondary immune response in vivo. a

b


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Fig 5 Regulatory T cells control the peripheral induction and clonal outgrowth of self-reactive T cells. This figure illustrates various pathways of immunoregulation mediated by suppressor subsets of NKT, CD4þ, and CD8þ T subsets. Each of the regulatory T cell subsets expresses distinct receptors, employs different effector mechanisms, and functions predominately at different stages during the course of the peripheral immune response. The NKT and CD4þCD25þ regulatory cells are ‘‘natural suppressor cells’’ and are present prior to antigen activation and function primarily during the early ‘‘innate’’ and/or primary adaptive immune responses. In contrast, the CD8þ regulatory cells are induced to differentiate into suppressor effector cells during the primary immune response and they function as effector suppressor cells predominately during the secondary and memory phases of immunity. (See Color Insert.)

unknown and it is not clear whether antigen presenting cells and/or T cells are the targets of CD4þCD25þ mediated suppression. Moreover, although the CD4þCD25þ suppressor cells express conventional ab TCRs, the evidence suggests that these TCRs are not involved in the recognition of the targets of suppression. In contrast to both the NKT cells and the CD4þCD25þ regulatory cells, the CD8þ regulatory T cells are not observed in naive animals prior to antigen encounter. As a consequence, adoptive transfer of CD8þ T cells from naive animals has no effect on the outcome of autoimmune responses and depletion of CD8þ T cells prior to the first induction of EAE has no effect on the first episode of disease. However, the CD8þ regulatory cells function like classical

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immunocompetent cells activated during adaptive immune responses. Thus, the CD8þ regulatory cells are induced to differentiate into suppressor effector cells during the primary immune response and they function as effector suppressor cells predominately during the secondary and memory phases of immunity. Thus, adoptive transfer of CD8þ T cells from self-antigen primed mice will retard the outgrowth of the potentially pathogenic self-reactive T cells. In this regard, it is of interest that CD8þ regulatory T cells are known to mediate resistance to autoimmunity following initial recovery from disease and to decrease the incidence and severity of relapse of the disease. In contrast to the CD4þCD25þ T cells, the CD8þ regulatory T cells utilize their ab TCRs to recognize target cells in a MHC-restricted fashion. Thus, the CD8þ regulatory cells are Qa-1 restricted and selectively downregulate Qa-1/self-peptide complexes preferentially expressed on certain but not all activated T cells. We propose a general synthesized model of peripheral regulation of immunity incorporating pathways mediated by the three different T cell subsets we have discussed. Here, we use regulation in control of autoimmunity as an example. As shown in Fig. 5, we envision that during the initiation of autoimmune disease, cell injury or death leads to the release of self-peptide/s which can be processed and presented by antigen-presenting cells to selfreactive CD4þ T cells, which subsequently induce and effect immune responses to self. During these early stages of the primary autoimmunity, the NKT and CD4þCD25þ regulatory cells will directly influence the emergence of self-reactive clones by controlling the balance of Th1 to Th2 differentiation and cytokine release. In addition, CD4þCD25þ T cells by cell–cell contact will directly suppress certain self-reactive cells by mechanisms that are currently unknown. We envision that the majority of the activated self-reactive T cells are controlled by these regulatory T cells working in concert with the conventional Th1 and Th2 cells as well as activated CD4þCD25- T cells which uniquely express immunosuppressive cytokines (i.e., the Tr1 and Th3 cells). This level of control prevents the majority of potentially pathogenic self-reactive T cells from functioning and therefore generally maintains self-tolerance in the periphery. However, by analogy to thymic negative selection which deletes the majority but not all potentially pathogenic self-reactive T cells, the very existence of autoimmune disease in mammals implies that this first level of peripheral regulatory defense mechanisms may not control all potentially pathogenic self-reactive T cell clones. This leak in the first level of control may be due to subtle defects in the mechanisms that normally control CD4þCD25þ T cell differentiation, such as defects in Foxp3 or subtle defects in the differentiation of the CD1-restricted NKT cells. Thus, we believe that the immune system developed a second level of control mechanisms, including the pathway


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mediated by Qa-1 restricted CD8þ T cells, to fine-tune the TCR repertoire of self-reactive T cells that have escaped both thymic selection and peripheral regulation of the primary response to self-reactive T cells. Presumably, this was necessary because self-reactive T cells that have escaped both thymic deletion and primary peripheral regulation may contain the potentially pathogenic autoreactive T cells. The Qa-1 restricted regulatory CD8þ T cellmediated pathway represents the only regulatory pathway capable of finetuning the TCR repertoire. Thus, we envision that self-reactive T cells, which escape the first level of regulatory defense mechanisms, are activated by further self-antigen triggering, in vivo, and undergo clonal growth. Some of these outgrowth clones are potentially pathogenic and probably possess intermediate affinity/avidity to self. We have proposed that the intermediate affinity/avidity T cells, which are enriched in potentially pathogenic clones, express Qa-1/self-peptide complexes on their surface. The activated CD4þ T cells expressing Qa-1/self-peptide/s are capable of inducing Qa-1 restricted regulatory CD8þ T cell and also are subject to the specific downregulation by the CD8þ regulatory T cells. On the other hand, the low affinity self-reactive clones are not pathogenic and they persist in the periphery by escaping downregulation by the CD8þ T cells. In summary, the peripheral immunity is controlled by a well-designed program consisting of a variety of regulatory mechanisms. Thus, despite the abundance of self-reactive clones in the periphery, clinical autoimmunity is usually well controlled. Acknowledgments The research was supported by NIH grants AI39630, AI39675, and National Multiple Sclerosis Society grant RG2938A to HJ and NIH grant U19 AI/46132 to LC.

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INDEX

A

Attrition, affect of memory T cells, 220 Autoantibodies, 256 Autoimmune diseases cause of, 255 cytokines and, 256 IL-18 and, 146 initiation of, 279f, 280 NK T cell function on, 265–267 types of, 255 Autoimmune pathologies, 133 Autoimmune responses, CD8þ T cell controlled, 270 Autoimmunity, 255 CD8þ T cells and, 268–269 characteristics of, 256 control of, 256 NK T cells and, 266 Autoreactive cells, 253 controlling outgrowth of, 255

Activated T cells Bcl-3 and survival of, 211 death of, 208 ROS and, 208–209 Alloantigens, 257–258 Alloantisera, Qa-1, 259–260 Anergy, 256 Antibodies, 145–146 anti-CD3, 103–104 anti-TCR, 103 memory B cells producing, 191 monoclonal, 258 Antibody-dependent cellular type cytotoxicity (ADCC), 134 Antigens activation of immunocompetent cells, 277 doses of, 269 effector function and, 249 epithelial, 177 foreign, 194, 257, 276–277 HEL, 269 self, 255 type 2 immune responses and, 177, 178f in vivo loss of, 237 Antigen-specific CD8þ T cells expansion/decline of, 199f, 204 gene expression pattern of, 207 Apoptosis antigen induced, 277 of DP thymocytes, 94–95 granzyme A triggered, 241 granzyme B induced, 240 target cell, 238 TNF-a induced, 242 Apoptotic mechanisms, 255 regulation of self-reactive cells and, 256

B B cells CLPs as progenitors for, 16 differentiation, 15, 257, 261 IL-4 expression by, 176 lymophocyte precursors differentiation into, 92 Pax-5 and commitment of, 27–28 Basophiles, IL-4 expression in, 176 Bcl-3, 211 Bi-potential model, origin of memory CD8þ T cells and, 205, 205f

C

CD4þ cells, B cell differentiation inducer function of, 261 CD4 genes c-Myb/SAF binding in silencing of, 66f, 69 289

290

INDEX

CD4 genes (continued) enhancer regulated expression of, 67–68 HEB/E2A heterodimeric complex in activation of, 68 trans-acting proteins and regulation of, 67–71 transcription factors for negative regulation of, 69–71 CD4 intronic enhancer, 63 CD4 promoter (P4), 59–60 expression in DP thymocytes and, 62 CD4 proximal enhancer nuclear protein-binding sites of, 60 in transgenic reporter genes expression, 61, 61f CD4þ regulatory cells, 55 CD4 silencer, 62–63 in CD4 silencing, 73 functional sites in, 64–65, 66f mammalian SWI/SNF chromatin remodeling complex in, 71 mechanisms of, 71–73, 75–76, a98f mutations in, 65, 66f, 67 temporal activation of, 64 trans-acting proteins acting on, 68–71 CD4 SP cells, 56 process of, 57–58 CD4þ suppressor cells biological foundation of, 262 expressing CD25/CTLA-4, 262 Foxp3 and, 264 CD4þ T cells, 193 antigen-reactive, 273 as autoreactive clones, 255 CD8þ T cells downregulation of, 274 CD40 direct ligation on, 210–211 CD40/CD40L signals increase survival of, 210 cytokine secretion of, 244 encephalitogenic, 272 inducing Qa-1 restricted regulatory CD8þ T cells, 281 LAG-3 expressed on, 59 Lck-deficient thymocytes differentiation into, 102 MHC class II restricted, 258 outgrowth of, 255 Qa-1 restricted regulatory CD8þ T cells interaction with target, 270, 271f Qa-1/self-peptide and, 273–274

regulatory, 253–254 response patterns of, 199f, 212 response patterns of antigen-specific, 212 SEB activated, 270 self-reactive, 280 suppressed by CTLA-4, 263 suppression by, 261 surface Qa-1 molecules on, 274 TCR employed by, 264 Th1 development in, 137 TH1/TH2 subsets of, 256 CD4þ Th cells, 55 subsets of, 133 CD4þ8 intermediate thymocytes, 113–115, 114f coreceptor reversal as function of, 116 CD4/CD8 coreceptor molecules, 99 CD4þCD8 T cells. See CD4 SP cells CD4CD8þ T cells. See CD8 SP cells CD4þCD25þ cells Foxp3 expressed in, 264 self/nonself distinguished by, 264–265 TCRs expressed by, 279 CD4þCD25þ regulatory cells characteristics of, 278t immunocompetent cells suppressed by, 277 self-reactive clones influenced by, 280 CD4þCD25þ T cells, immunosuppresive cytokines expressed by, 280 CD8þ cytotoxic cells, 55 CD8þ effector cells, 259 CD8þ effector T cells acquiring gene expression profiles, 207 cell division into, 200 as defined, 234 identifying, 234 memory CD8þ T cells v., 194–195 CD8 genes cis-regulatory element and regulation of, 77, 78f, 79–84 DP thymocytes terminate, 117f expression regulation of, 77, 78f, 79–86 genomic fractions in regulation of, 78f, 79 regulation of, 83–84 trans-acting factors in, 83–84 CD8þ regulatory cells, 253–254 recognition of Qa-1/peptide complex, 261 T cells regulated by, 273

INDEX

CD8þ regulatory T cells acting as effector suppressor cells, 279–280 autoimmunity resistance mediated by, 280 biological function of, 270 characteristics of Qa-1, 278t features of Qa-1 restricted, 275–277, 276f importance of, 268–269 peripheral regulation by Qa-1 restricted, 275, 276f prior to antigen encounter, 279 CD8 SP cells, 56 CD4 silencing in, 71–72 enhancer directed expression in, 80 process of, 57–58 CD8þ suppressor T cells autoimmunity and, 268–269 EAE model and, 268 as induced during immune response, 265 role in regulating immune responses, 268 CD8þ T cell phenotypic subsets central memory, 247 effector, 248–249 effector memory, 248 naive, 245 CD8þ T cells APC initiated response of, 201 autoimmune responses controlled by, 270 bcl-2 and survival of, 211 CD4 silencing in, 73 CD4þ T cells downregulation by, 274 cell death during response of, 209 costimulatory regulation of responses of, 202 cytokine secretion of, 244 DC and expansion of, 201–202 differential receptor expression, 218 differentiation of, 198–99, 199f, 245, 246f, 247–249 EAE mice and, 271–272, 272f enhancer expression in, 81–82 expansion of, 199, 199f expansion/contraction/memory pattern of, 200 generating memory CD8þ T cells, 206–207 IL-2 and expansion differentiation of, 204 IL-7 role in differentiation of, 117–118 IL-15 and function survival of, 204 ILs supporting responses of, 203 immune exhaustion and, 209 immune response to foreign/self antigens governed by, 269

291

LAG-3 expressed on, 59 memory development of, 208 MHC class I restricted, 258 naive, 217–218 peripheral MBP-reactive CD4þ TCR repertoire regulated by, 270–272, 272f priming during EAE of, 275 response kinetics of, 200–201 Runx 3 and TCR-mediated response of, 76 selective downregulation of self-reactive T cells by, 272–273 suppression by, 260–261 surface markers of, 192t, 198, 199f viral clearance and, 197 CD8a expression of, 58 induction of, 84 CD28, 256 CD40 CD40L reaction with, 256 direct ligation of CD48þ T cell, 210–211 survival of activated CD8þ T cells regulated by, 210 CD45 molecule, TCR signaling on T cells regulated by, 263 CD48, 256 CD80, 256 CDL. See Cytotoxic lymphocytes Cells. See also specific cells activation-induced death of, 255 central memory, 196 death/injury of, 279f, 280 distinguishing self from nonself, 264–265 division of, 200 experiments of transitional, 106–108, 107f multiple lineage differentiation of, 21 Qa-1 restricted CD8þ T cells, 277 regulatory, 261 self-reactive, 256 suppressor, 260 suppressor T, 257 Cellular immunity, 191 Central thymic selection, 253 CFU. See Colony-forming units Chromatin GATA-4 in, 164 remodeling of, 18–19, 83 Clinical autoimmunity, 253

292

INDEX

Clonal selection, 254–255 Clones, 254–255 autoreactive, 255 downregulation of antigen-activated, 273 possessing intermediate affinity/avidity to self, 281 recognition of, 273 self-reactive, 255–257, 271, 272f, 276, 280 T cell, 270 CLPs. See Common lymphoid progenitors CMPs. See Common myeloid progenitors Colony-forming units (CFU), 3–4 Common lymphoid progenitors (CLPs), 1, 2f, 9f activity of, 13 adult lymphoid differentiation and, 13, 15–17 CMPs as counterpart of, 17–18 fetal, 35–37, 37f IL-7R and, 8, 8f as IL-7Ra-expressing cells, 10–11 isolation of, 7, 8f, 13 lymphoid lineage priming in, 20f, 28 Notch 1 distribution in, 26 Pax-5 in, 28 pTa transcription initiated by, 12 Common myeloid progenitors (CMPs), 10 as CLPs counterpart, 17–18 myeloerythroid gene priming in, 19, 20f, 21 Coreceptor genes, 58 Coreceptor molecules, 91. See also CD4/CD8 coreceptor molecules auto-immune diseases and, 146 CD4þ T cell/CD8þ T cell differentiation and, 103 on DP thymocytes, 95–99 IL-12 synergy between, 143 intracellular Lck binding to, 96–97 IRAK pathway signaling of, 142 lineage choice of, 100–101 protective role of, 145 regulation/expression of genes encoding, 55–56 structural difference between, 95–96 T cell development role of, 56–58 T cell differentiation development stages and, 92, 93f as TCR coreceptors, 95 in Th1 responses, 144–145

tissue specificity of, 142–143 Coreceptor reversal model. See Kinetic signaling model; Strength of signal instructional model Costimulatory molecules, regulating amplitude of CD8þ T cell response by, 202 CPS, 264–265 CTL. See Cytotoxic T lymphocytes CTLA-4 molecule, 256 CD4þ T cell suppression regulated by, 263 Cytokine secretory effector cells, 243–245 Cytokine secretory effector function memory cells with, 233 Cytokines, 256, 260 autoimmune diseases and, 256–257 autoimmunity and, 266 immunosuppresive, 280 memory CD8þ T cells production of, 194 memory T cell maintenance and, 214–215 new, 149, 151 NK T cells producing, 265–266 production of proinflammatory, 140–141 regulation of CD8þ T response, 202 role in suppression by, 263 secretion of, 235–236, 243–244 signals, 29–30 Th cell produced, 134 Th2-specific, 143 Cytotoxic effector cells degranulation of, 241–242 differentiation of, 245, 246f Cytotoxic effector function CD45RA expression and, 234 FasL/Fas death pathway and, 235–236, 242 measuring, 238 memory cells with, 233 perforin-dependent granule exocytes pathway and, 235–236, 238–239 Cytotoxic lymphocytes (CDL) CD8 molecules and, 258–259 function of, 259 Rab 27a required by, 239 Cytotoxic T cells CD4 silencing in, 72–73 TCRab, 77 Cytotoxic T lymphocytes (CTL), 236, 257. See also Effector CTLs extrinsic signals and, 209–210 memory, 58, 84

293

INDEX

memory CD8þ T cells and, 206 recognize/kill infected cells function of, 209 TcR-triggered granule exocytosis in, 239 Cytotoxicity Antibody-dependent cellular type, 134 measuring, 236 NK T cells and, 265 perforin-dependent, 237 in vitro, 238 in vivo, 236–238

D DCs. See Dendritic cells Delayed type hypersensitivity (DTH), 257 Dendritic cells (DCs) CD8þ T cells expansion and, 201 CD8a expression on, 82 CD40/CD40L and, 210 cytokine production by, 148f early lymphoid progenitors differentiate into, 33–35 IL-12 production by, 139–140 initiating T cell responses, 201–202 markers of, 35 maturation of, 140 T cell polarization and, 172t–174t, 177 Th cells development and, 140 Th1 cells development and, 136, 138 DH. See DNase hypersensitivity DN cells, 56 DN thymocytes. See Double negative thymocytes DNase hypersensitivity (DH) cluster II deletion of, 81 clusters of, 79, 85 sites of, 77, 78f, 79–80 Double negative (DN) thymocytes CD4 repressed in, 72, 74f CD4 silencing in, 71–72 development of, 92–93, 93f negative regulatory element in, 64 proliferative phases of, 93–94 selection of, 56–57 subsets of, 92, 93f Double positive (DP) thymocytes, 92 Bcl-2/IL-7R upregulated by, 117 CD4 expression in, 62 CD4/CD8 coreceptor molecules expressed by, 99

CD4-dependent TCR/ligand interactions signal to, 113–114, 114f CD4SP/CD8SP T cells differentiation from, 104, 111–112, 111f, 115 CD8-dependent TCR/ligand interactions signal to, 112, 113f CD8-negative, 81 cell fate determination of, 76 coreceptor function in, 97 development of, 94–95 differentiation into CD4þ T cells of, 102–103 enhancer directed expression in, 80–82 intracellular kinase activity in, 102 intracellular pools of Lck in, 96 Lck depletion and, 97–98 Lck signaling and signaled by, 120 as lineage-committed cells, 100 reporter transgene expression in, 61, 61f SP T cell conversion of, 99–100 stochastic/selection model and, 105–108 TCR complexes on, 98 TCR signaling of, 106 TCR signals generated in, 101 DP thymocytes. See Double positive thymocytes DTH. See Delayed type hypersensitivity

E EAE, 263–264, 268 cellular events during, 275 IFN-y production in, 146 induction of, 145 NK T cells and, 266 TCV induced protection from, 270 Early lymphoid progenitors differentiation into DCs of, 33–35 pTa proteins defining, 12 Early T lineage progenitors (ETPs) myeloid potential of, 16 T cell production by, 15–16 Early thymic progenitors, c-Kitþ, 15–16 Effector cells, 253. See also Effector function as defined, 233–235, 236, 249 Effector CTLs memory CD8þ T cells by, 207–208, 211–212 molecular mechanism change of CD8þ T cells to, 208 Effector function. See also Cytokine secretory effector function; Cytotoxic effector function

294

INDEX

Effector function (continued) secretion as mechanism of, 235–236 as subpopulation, 234 Effector memory CD4þ T cells, Listeria and, 135 Effector memory CD8þ T cells clear pathogens upon reinfection, 197–198 differentiate into central memory CD8þ T cells, 197 Listeria and, 135 protective immunity and, 197 Effector molecules, 191 Eosinophils, IL-4 storage/expression by, 176 ETPs. See Early T lineage progenitors Extrinsic signals, 209 CD40, 210

F Fas ligand, 255 FasL/Fas death pathway, 235, 242 Fetal hematopoietic progenitors, as not fully committed to lymphoid/myeloidfates, 35–38, 37f Flk-2, in early stem and progenitor cells, 9–10 Foreign antigens, 257 clones and, 276–277 immune tolerance to, 257 memory T cell recognition/elimination of, 194 Foxp3 CD4þCD25þ regulation and, 264 defects in, 280 mutational defects in, 264

G GATA-1 in hematolyphoid commitment, 22, 24–25 lineage instructive effect of, 31 GATA-3, 146 binding sites of, 164–165 T cell activation synapse signals and, 165 Th1 development down-regulated by, 148 Th2 cells expression of, 147–148 Th2 differentiation role of, 164 GATA-4, in decompacting condensed chromatin, 164 Genes activation/silencing of batteries of, 55 priming of, 19 GMPs, GM-related genes expressed by, 19

Granzymes, 239 A, 241 B, 240–241

H Helminth immunity, Th2 cells and, 133 Hematopoietic stem cells (HSCs), 2–3 commitment models of, 4–5, 5f commitment sequence to lymphoid cells from, 1, 2f fetal, 35 gene expression profiles and, 32–33 hematolymphoid lineages commitment of, 3–5, 5f Hetero-trimeric receptors, 203–204 Homeostatic regulation, 256–257 HSCs. See Hematopoietic stem cells Human bone marrow, lymphoid- and myeloid-restricted progenitors in, 38–39 Humoral immunity reinfection protection and, 191

I IEL. See Intestinal intraepithelial lymophocytes IFN. See Interferons IFN-g IL-12 signaling and production of, 138 IL-18/IL-12 induced production of, 142 macrophage effector function stimulated by, 144 regulating elements in, 146 responses of, 134 role of, 163 secretion of, 237 T cell produced, 141 TCR induced production of, 143 Th1 cell produced, 134, 256 IFN-g receptor (IFN-gR), 152 IFN-g-inducing factor (IGIF), 141 IFN-gR. See IFN-g receptor IgG, antibody synthesis, 257 IGIF. See IFN-g-inducing factor IL. See Interleukins IL-2 controlling lymphocyte homeostasis, 204 as effector cytokine, 244 IL-4, 256 in coreceptor reversal, 117–118 expression activation of, 166

INDEX

mutations affecting expression of, 171 non-Th2 cell expression of, 171, 175–176 T cell expression of, 167–170 T cell production of, 168 IL-7 ab T cell differentiation role of, 10 memory CD8þ T cells support by, 216, 216f naive T cells and, 203 as survival factor for memory CD8þ T cells, 215 IL-7 receptor (IL-7R) DP thymocyte upregulation of, 117 PU.1 regulated, 23 role of signals of, 118 T/B cell development and, 8, 8f T/B cell expression of, 10 IL-7R. See IL-7 receptor IL-10, 256 cell production of, 140–141 IL-12 DC stimulation by, 139–140 G protein coupled signaling via CCR5 and, 139 IL-18 synergy between, 143 p40, 144–145 as secreted, 136 signaling, 138 Th1 cells development and, 136, 141–142 IL-15 maintaining memory of memory CD8þ T cells by, 217 memory CD8þ T cells support by, 216–217, 216f protective immunity to intracellular pathogens and, 219 regulating CD8þ T cells function/ survival, 204 STATS and memory CD8þ T cells response to, 218 as survival/proliferation factor for antigen-specific memory CD8þ T cells, 215–216 as survival/proliferation factor for memory CD8þ T cells, 215 IL-18 Autoimmune diseases and, 146 IL-12 synergy between, 143 in Th1 cells responses of, 144–145 IL-23, 145, 151

295

Immune memory, 220–221 Immune responses, 208 cytotixicity pathways role in, 237 to foreign pathogens, 253 NK T cells and tumor rejection, 265 NK T cells primary, 277 regulation of, 254 to SEB, 269 suppressor cells regulating, 261 suppressor T cells control of, 257 of T cells, 199f triggers of, 256 Immune system, 133, 255 suppressor mechanisms of, 255 Immunity cellular, 191 helminth, 133 humoral, 191 peripheral regulation of, 280 protective, 197–198 suppressor T cells regulation of, 257 Immunocompetent cells, 268 antigen activation of, 277 autoimmune disease and, 255 NK T cells as, 267 Immunodominance, 206 Immuno-dysregulation, polyendocrinopathy, enteropathy, X linked syndrome (IPEX), 264 Immunological memory, 212 Immunopathology development of, 143 prevention of, 148f Th subsets contribution to, 133 Immunoregulation, 269–270 helper/suppressor functions of, 258 NK T cells and, 267 paradigms of, 253–254 by regulatory T cell subsets, 277 Immunosuppresive cytokines, Th3 secreting, 256 Interferons (IFN), 202–203 type I, 137–138 Interleukins (IL), 203. See also IL-2; IL-4; IL-7; IL-10; IL-12; IL-15; IL-18; IL-23 Intestinal intraepithelial lymophocytes (IEL), 82 CD8þ, 55 Intracellular pathogens, 144 Th1 caused death of, 134

296

INDEX

IPEX. See Immuno-dysregulation, polyendocrinopathy, enteropathy, X linked syndrome

Lymphokines, structural characterization of, 261 Lyt2, 257 SRBC and, 258

K Kinetic signaling model, 111f. See also Strength of signal instructional model analysis of, 112–115, 113f, 114f coreceptor reversal in, 115–119 lineage commitment of DP thymocytes in, 110–111 MHC-I-signaled thymocytes differentiation into CD8SPT cells in, 117f signal intensity/duration distinctions of, 119–121

L Ligand, engagement, 204 Lineage commitment models classical, 100–101 Linear differentiation model origin of memory CD8þ T cells and, 205, 205f predictions of, 206–207 Listeria monocytogenes (Lysteria), controlling primary immune responses to, 135 LKLF, 221 Lymphadenopathy, 256 Lymphocytes development of, 6 differentiation of T/B, 6–8 IL-4 expression of, 166, 166f manipulating, 235 markers of, 13 memory, 192 precursors, 91 promiscuity of, 19, 20f, 21–22 regulatory T, 253 secretory granules of, 235 Lymphoid progenitors as differentiate into T cells, 56 lineage elasticity in, 28–32 lineage promiscuity in, 20f, 21 Lymphoid-committed progenitors instructive cytokine signaling myelomonocytic conversion of, 29–30 megakaryocyte/erythrocyte conversion from, 30–32

M Macrophages cytokine production by, 148f differentiation of, 15 effector functions of, 144 fetal CLPs and, 36–38, 38f TNF induction by, 145 Major Histocompatibility Complex (MHC), 99, 258–259, 264 CD4/CD8 coreceptor binding to, 95 class I, 91 class II, 91 class specificity of, 101 positive/negative selection by, 6 role in memory T cell homeostasis, 213–214 TCR signals in DP thymocytes stimulated by, 94 Mast cells, IL-4 expression in, 176 MBP, immunization with, 270 MeCP1 complexes, 164 Megakaryocyte-restricted progenitors (MKPs), 18 Memory B cells, antibodies produced by, 191 Memory CD4þ T cells functional plasticity and, 212 maintenance of, 219 Memory CD8þ T cells, 191 in absence of IL-15, 216 acquisition kinetics of, 207 attrition of, 220 CD8þ effector T cells v., 194–195 CD8þ T cells generating, 206–207 cytokine production of, 194 differentiation of, 197 effector CTLs create, 207–208, 211–212 effector CTLs relation to, 205, 205f expansion of, 194 frequency of, 193 heterologous stimuli and, 195–196 HKLM-induced, 197–198 homeostasis of, 217 IL-7 as survival factor for, 215 IL-15 and maintaining memory of, 217 IL-15 survival/proliferation factor for, 215 immunodominance and, 206

297

INDEX

LCMV infection of, 196 LKLF and survival of, 221 magnitude of pool/size of CTL response of, 206 maintenance of, 218 memory pool of, 198 naive CD8þ T cells/effector CTLs relationship to, 198 protective immunity to intracellular pathogens of, 219 recruited to peripheral tissues, 195–196 response to IL-15 and STAT5, 218 role of subsets of, 196–197 secondary response mediated by, 198 support by IL-7/IL-15, 216–217, 216f Memory T cells antigen specific, 196 cellular immunity of, 191 foreign antigen recognition/elimination by, 194 induced by organism/antigen, 193 induced/maintained, 191 negative regulation of, 219–220 persistent antigen and function of, 214 protective immunity mediated by, 219 surface markers on, 192, 192t, 194 tonic MHC/TCR signals and survival of, 213 Memory-phenotype CD8þ T cells, 193 Mendelian susceptibility to mycobacterial disease (MSMD) gene mutations and, 151–152 MHC. See Major Histocompatibility Complex MHC I CD8 SP cells with cytotoxic function and, 57 TCR/ligand interactions specific to, 115 MHC II CD4 SP cells with helper functions and, 57 TCR/ligand interactions specific to, 115 MKPs. See Megakaryocyte-restricted progenitors Molecular immunology, 259–260 Molecules, 262 antigen activation-induced cell surface, 256 antigen-specific suppressor, 260 CD45, 263 costimulatory, 202 CTLA-4, 263 cytoplasmic tails of, 258 effector, 191 glycolipid, 277

MHC, 258–259, 264 types of, 255 Monoclonal antibodies, 258 MSMD. See Mendelian susceptibility to mycobacterial disease

N Naive CD4 T cells IL-4 expression activation in, 164–167 IL-4 genes in, 165 Natural killer (NK) cells, 1 NK cells. See Natural killer cells NK T cells activators for, 265–266 autoimmunity controlled by, 266 characteristics of, 278t diabetes and, 266–267 function on autoimmune disease, 265–267 IL-4 expression in, 175 immunoregulation and, 267 as primary immune response, 277 with restricted TCRs, 55, 253–254 self-reactive clones influence by, 280 Notch 1, in T cell commitment, 25–26 Notch intracellular domain (Notch-IC), gene transcription activated by, 92 Notch molecule cell fate determined by, 91 lymphocyte precursors differentiation into T cells by, 92 Notch-IC. See Notch intracellular domain

P P4. See CD4 promoter Pathogens, 141 foreign, 253 immunity to, 191 intracellular, 134, 152 Pax-5, in B cell commitment, 27–28 Perforin, 239–240 Perforin-dependent granule exocytosis pathway cytotoxic activity via, 244 perforin/granzymes and, 239 process of, 238 secretion of, 235 Peripheral immunity regulation of, 253, 254f regulatory mechanisms controlling, 281

298

INDEX

Peripheral regulation, 253 of immune responses, 261 Physiologic immune regulation, 253 Progenitor cells, differentiation of, 55 PU.1, in hematolyphoid commitment, 22–25

Q Qa-1, 273 Qa-1 restricted CD8þ T cells biological consequences of down regulation of, 277 CD4þ T cells inducing, 281 Qa-1/peptide complex, 273–274 CD8þ regulatory cells recognition of, 261 TCRs expressing, 273

R Rab 27a molecule, mutations in, 239 RAG. See Recombination Activating Genes RAG proteins DNA cleavage by, 6 thymocyte proliferative phase expression of, 93 RAG-1, ELP defined by transcriptional activation of, 11–12 Reactive oxygen species (ROS), 208 Receptor ligand interactions, 256 Recombination Activating Genes (RAG), 92. See also RAG-1 DNA cleavage by, 6 function of, 11 Regulatory mechanisms MHC/peptide triggering CD4þ cells cause of, 256 sets of, 256–257 TCR/MHC/peptide interaction initiating, 256 Regulatory T cells as component of immune system, 265 subsets of, 277 ROS. See Reactive oxygen species Runx genes CD4 silencing and, 70–71 Runx 3, 76 silencing roles of, 75–76 TCR signaling in, 76–77

S SEB, immune response to, 269 Self peptide/MHC complexes, T cell reaction to, 253

Self-reactive cells regulation of, 256 suppression of, 273 Self-reactive clones, 255 CD4þ T cells and, 276 death of, 256 limiting outgrowth of, 271–272, 272f outgrowth/functions of, 256–257 Self-reactive T cells CD8þ T cells selective downregulation of, 272–273 clonal growth of, 281 express ligands, 265 Signal instructional model DP thymocytes and, 102 strength of, 101–104 Single positive (SP) cells. See CD4 SP cells; CD8 SP cells SP cells. See Single positive cells Splenomegaly, 256 Stat6, Th2 effector development role of, 168 Stochastic/selection model contradictions to, 107f, 108–110 support for, 105–108 Strength of signal instructional model, 58. See also Kinetic signaling model contradictions to, 104–105 signal intensity/duration distinction of, 119–121 Superantigens, 269 Suppressor cells, 260 immune responses regulated by, 261 Suppressor T cells, control of immune responses and, 257

T T cell receptors (TCR), 253, 264 activated transgenic, 136 antigen activity of, 260 CD4þ8 signaling of, 113, 113f CD8 gene expression and, 111–112 cell death and, 255 downregulation and, 272–273 DP thymocytes and, 94, 97–98 DP thymocytes expressing, 99 expressed by CD4þCD25þ, 279 expressing Qa-1/peptide, 273 heterogeneity to pathogens of, 133 IL-7R desensitized by signaling of, 118 of intermediate affinity, 56–57

INDEX

intermediate thymocytes signaling of, 112 memory CD8þ T cells and, 194 role in memory phenotype T cell maintenance, 214 signal intensity/duration of, 120–121 stochastic/selection model and, 106–108 in Th2 development, 167 transgenic cells and, 200 T cell responses DCs initiating, 201–202 increase of T cells due to, 219–220 T cell vaccination (TCV), CD8þ T cell hybrids and, 270 T cells. See Tumor cells T helper cells (Th cells) DC subsets on development of, 140–141 disease outcome and, 134–135 IL-12 and, 141 protein antigens and induction of, 135–136 specific cytokines, 134 subsets of, 133 T-bet, 147 Tc1 cells, 244–245 Tc2 cells, 244–245 expression of IL-4 in, 171, 175 TCR. See T cell Receptors TCV. See T cell vaccination TGFb, 256 Th cells. See T helper cells Th1 cells, 256. See also T helper cells autoimmune pathologies and, 133 cell-mediated immunity role of, 133 commitment, 137 DC and development of, 136, 138 development of, 148f, 150f downregulatory influences on, 256–257 IFN-y secreted by, 134, 256 IL-4 location in, 169 IL-10 production by, 140 IL-12 driven development, 136–137 IL-18 in responses of, 144–145 impaired responses of, 149, 151 intracellular pathogen protection by, 152–153 proinflammatory cytokines produced by, 134 as subset of CD4þ Th cells, 133 systematic invasion response of, 163 T-bet and, 147 Th1 clones, DTH responses induced by, 134 Th2 cells, 256. See also T helper cells

299

development molecular mechanisms of, 136 development of, 167 differentiation and function of, 256 GATA-3 in differentiation of, 164 helminth immunity and, 133–134 IL-4 expression plasticity in differentiated, 169 IL-4-expressing, 163 IL-4/IL-4R/Stat6-independent differentiation of, 167 IL-12 signaling and, 138 interleukins produced by, 133 mutations affecting IL-4 expression from, 171 naive T cell polarization by, 165, 166f phenotype/genotype analysis of, 170 production of Ige/IgG1 antibodies, 134 responses of, 144 as subset of CD4þ Th cells, 133 terminal differentiation of, 177–178 type 2 immunity orchestrated by, 163, 179 as unresponsive to IL-12, 136 Thymic negative selection, T cell elimination through, 253, 255 Thymocytes apoptosis of, 253 CD4-CD8-DN, 63–64, 72 CD8 lineage, 80 CD8 single positive, 63–64 into CD8þ T cell lineage, 103 CD8SP T cells differentiation from, 117f differentiation of, 55, 92 kinetic signaling model and, 110–115, 111f, 113f, 114f lineage commitment development in, 101–110 Runx sites and, 70 TCR transgenic, 101–102 TLR. See Toll like receptors TLR9 expression of, 139 TLRLs. See Toll like receptor ligands TNF, Listeria clearance by, 135 TNF-a apoptosis induced by, 242 secretion of, 237 Toll like receptor ligands (TLRLs), 195, 201 cytokines induced by, 215

300 Toll like receptors (TLR), pathogen-derived products recognition by, 139 Transcription factors Notch1 as, 25–26 Pax-5, 27–28 PU.1/GATA/1 as, 22–25 Transgenes CD4, 63 lineage-specific expression of, 63 Tumor cells (T cells) activation regulation of, 264 autoimmune disease and, 253 autoreactive, 281 CD4þ/CD8þ interaction, 260 CD8abþ, 84 DC interaction with, 201–202 DC40L expressed by, 256 death of, 199, 208 differentiation, 15–17, 258 division of, 204 enhancer directed expression in, 80 functional fate of, 256 growth and proliferation of, 262–263 Hybrids, 270 IL-4 production by, 168 IL-7 and, 203 immune response of, 199f Lyt2, 257–258 mediated immunity, 191

INDEX

memory, 191 memory-phenotype, 192–193 mutations impacting IL-4 expression in, 171 polyclonal stimulation of, 136 pTa gene as Notch target in, 26 recruitment of antigen-specific, 200 as regulated by CD8þ regulatory cells, 273 response to foreign antigens of, 254 Runx function in, 76–77 scurfy mice/IPEX patients defects in activation of, 264 self-reactive, 261, 265, 272–273, 281 stabilization of IL-4 expression in, 167–170 suppressor, 257 survival of, 202–203 thymic negative selection elimination of, 253 Type 2 immunity, 163 initiation/expression of, 177, 178f, 179 mutations impacting, 172t–174t Type I IFNs STAT4 and, 137 in Th1 development, 137–138

V Vaccines, 260 humoral immunity and, 191 subsets of memory CD8þ T cells and, 198 T cell based, 199

CONTENTS OF RECENT VOLUMES

Volume 74

Ju¨rgen Hess, Ulrich Schaible, Ba¨rbel Raupach, and Stefan H. E. Kaufmann

Biochemical Basis of Antigen-Specific Suppressor T Cell Factors: Controversies and Possible Answers Kimishice Isihzaka, Yasuyuki Ishii, Tatsumi Nakano, and Katsuji Sugik

The Cytoskeleton in Lymphocyte Signaling A. Bauch, F. W. Alt, G. R. Crabtree, and S. B. Snapper

The Role of Complement in B Cell Activation and Tolerance Michael C. Carroll

TGF- Signaling by Smad Proteins Kohei Miyazono, Peter ten Dijke, and Carl-Henrik Heldin

Receptor Editing in B Cells David Nemazee

MHC Class II-Restricted Antigen Processing and Presentation Jean Pieters

Chemokines and Their Receptors in Lymphocyte Traffic and HIV Infection Pius Loetscher, Bernhard Moser, and Marco Bacciolini

T-Cell Receptor Crossreactivity and Autoimmune Disease Harvey Cantor

Escape of Human Solid Tumors from T-Cell Recognition: Molecular Mechanisms and Functional Significance Francesco M. Marincola, Elizabeth M. Jaffee, Daniel J. Hicklin, and Soldano Ferrone

Strategies for Immunotherapy of Cancer Cornelis J. M. Meliey, Rene E. M. Toes, Jan Paul Medema, Sjoerd H. van der Burg, Ferry Ossendorp, and Rienk Offringa

The Host Response to Leishmania Infection Werner Solbacii and Tamas Laskay

Tyrosine Kinase Activation in the Decision between Growth, Differentiation, and Death Responses Initiated from the B Cell Antigen Receptor Robert C. Hsueh and Richard H. Scheuermann

Index

Volume 75

The 30 IgH Regulatory Region: A Complex Structure in a Search for a Function

Exploiting the Immune System: Toward New Vaccines against Intracellular Bacteria 301

302


Ahmed Amine Khamlichi, Eric Pinaud, Catherine Decourt, Christine Chauveau, and Michel Cogne´

Human Basophils: Mediator Release and Cytokine Production John T. Schroeder, Donald W. MacGlashan, Jr., and Lawrence M. Lichtenstein

Index

Volume 76 MIC Genes: From Genetics tok Biology Seiamak Bahram CD40-Mediated Regulation of Immune Responses by TRAF-Dependent and TRAF-Independent Signaling Mechanisms Amrif C. Grammer and Peter E. Lipsky Cell Death Control in Lymphocytes Kim Newton and Andreas Strassen Systemic Lupus Erythematosus, Complement Deficiency, and Apoptosis M. C. Pickering, M. Botto, P. R. Taylor, P. J. Lachmann, and M. J. Walport Signal Transduction by the High-Affinity Immunoglobulin E Receptor FceRI: Coupling Form to Function Monica J. S. Nadler, Sharon A. Matthews, Helen Tuhner, and Jean-Pierre Kinet Index

Volume 77 The Actin Cytoskeleton, Membrane Lipid Microdomains, and T Cell Signal Transduction S. Celeste Posey Morley and Barbara E. Bierer Raft Membrane Domains and Immunoreceptor Functions Thomas Harder

Btk and BLNK in B Cell Development Satoshi Tsukada, Yoshihiro Baba, and Dai Watanabe Diversity and Regulatory Functions of Mammalian Secretory Phospholipase A2s Makoto Murakami and Ichiro Kudo The Antiviral Activity of Antibodies in Vitro and in Vivo Paul W. H. I. Parren and Dennis R. Burton Mouse Models of Allergic Airway Disease Clare M. Lloyd, Jose-Angel Gonzalo, Anthony J. Coyle, and Jose-Carlos Gutierrez-Ramos Selected Comparison of Immune and Nervous System Development Jerold Chun Index

Volume 78 Toll-like Receptors and Innate Immunity Shizuo Akira Chemokines in Immunity Osamu Yoshie, Toshio Imai, and Hisayuki Nomiyama Attractions and Migrations of Lymphoid Cells in the Organization of Humoral Immune Responses Christoph Schaniel, Antonius G. Rolink, and Fritz Melchers Factors and Forces Controlling V(D)J Recombination


David G. T. Hesslein and David G. Schatz T Cell Effector Subsets: Extending the Th1/Th2 Paradigm Tatyana Chtanova and Charles R. Mackay MHC-Restricted T Cell Responses against Posttranslationally Modified Peptide Antigens Ingelise Bjerring Kastrup, Mads Hald Andersen, Tim Elliot, and John S. Haurum Gastrointestinal Eosinophils in Health and Disease Marc E. Rothenberg, Anil Mishra, Eric B. Brandt, and Simon P. Hogan Index

Volume 79 Neutralizing Antiviral Antibody Responses Rolf M. Zinkernagel, Alain Lamarre, Adrian Ciurea, Lukas Hunziker, Adrian F. Ochsenbein, Kathy D. McCoy, Thomas Fehr, Martin F. Bachmann, Ulrich Kalinke, and Hans Hengartner Regulation of Interleukin-12 Production in Antigen-Presenting Cells Xiaojing Ma and Giorgio Trinchieri Mechanisms of Signaling by the Hematopoietic-Specific Adaptor Proteins, SLP-76 and LAT and Their B Cell Counterpart, BLNK/SLP-65 Deborah Yablonski and Arthur Weiss Xenotransplantation David H. Sachs, Megan Sykes, Simon C. Robson, and David K. C. Cooper Regulation of Antibacterial and Antifungal Innate Immunity in Fruitflies and Humans Michael J. Williams

303

Functional Heavy-Chain Antibodies in Camelidae Viet Khong Nguyen, Aline Desmyter, and Serge Muyldermans Uterine Natural Killer Cells in the Pregnant Uterus Chau-Ching Liu and John Ding-E Young Index

Volume 80 Protein Degradation and the Generation of MHC Class I-Presented Peptides Kenneth L. Rock, Ian A. York, Tomo Saric, and Alfred L. Goldberg Proteoanalysis and Antigen Presentation by MHC Class II Molecules Paula Wolf Bryant, Ana-Maria Lennon-Dumeńil, Edda Fiebiger, Cećile Lagaudrie´re-Gesbert, and Hidde L. Ploegh Cytokine Memore of T Helper Lymphocytes Max Lo¨hning, Anne Richter, and Andreas Radbruch Ig Gene Hypermutation: A Mechanism is Due Jean-Claude Weill, Barbara Bertocci, Ahmad Faili, Said Aoufouchi, Ste´phane Frey, Annie De Smet, Se´bastian Storck, Auriel Dahan, Fre´de´ric Delbos, Sandra Weller, Eric Flatter, and Claude-Agne´s Reynaud Generalization of Single Immunological Experiences by Idiotypically Mediated Clonal Connections Hilmar Lemke and Hans Lange The Aging of the Immune System B. Grubeck-Loebenstein and G. Wick Index

304


Volume 81 Regulation of the Immune Response by the Interaction of Chemokines and Proteases Jo Van Damme and Sofie Struyf Molecular Mechanisms of Host-Pathogen Interaction: Entry and Survival of Mycobacteria in Macrophages Jean Pieters and John Gatfield

Tumor Vaccines Freda K. Stevenson, Jason Rice, and Delin Zhu Immunotherapy of Allergic Disease R. Valenta, T. Ball, M. Focke, B. Linhart, N. Mothes, V. Niederberger, S. Spitzauer, I. Swoboda, S.Vrtala, K. Westritschnic, and D. Kraft

B Lymphoid Neoplasms of Mice: Characteristics of Naturally Occurring and Engineered Diseasse and Relationships to Human disorders Herbert Morse et al.

Interactions of Immunoglobulins Outside the Antigen-Combining Site Roald Nezlin and Victor Ghetie

Prions and the Immune System: A Journey Through Gut Spleen, and Nerves Adriano Aguzzi

The Roles of Antibodies in Mouse Models of Rheumatoid Arthritis, and Relevance to Human Disease Paul A. Monach, Christophe Benoist, and Diane Mathis

Roles of the Semaphorin Family in Immune Regulation H. Kikutani and A. Kumanogoh HLA-G Molecules: from Maternal-Fetal Tolerance to Tissue Acceptance Edgardo Carosella et al. The Zebrafish as a Model Organism to Study Development of the Immune System Nick Trede et al. Control of Autoimmunity by Naturally Arising Regulatory CD4þ T Cells S. Sakaguchi Index

Volume 82 Transcriptional Regulation in Neutrophils: Teaching Old Cells New Tricks Patrick P. McDonald

MUC1 Immunology: From Discovery to Clinical Applications Anda M. Vlad, Jessica C. Kettel, Nehad M. Alajez, Casey A. Carlos, and Olivera J. Finn Human Models of Inherited Immunoglobulin Class Switch Recombination and Somatic Hypermutation Defects (Hyper-IgM Syndromes) Anne Durandy, Patrick Revy, and Alain Fischer The Biological Role of the C1 Inhibitor in Regulation of Vascular Permeability and Modulation of Inflammation Alvin E. Davis, III, Shenghe Cai, and Dongxu Liu Index

T Cell Subsets: Cellular Selection, Commitment and Identity

Advances in Immunology Volume 83 T Cell Subsets: Cellular Selection, Commitment and Identity

Antibodies and T-Cell Receptors,

T Cell Protocols

T Cell Protocols

T cell protocols

T Cell Protocols

Receptors of Cell Adhesion and Cellular Recognition

T Cell Protocols: Development and Activation

Factoring groups into subsets

Commitment

Commitment

T-Cell Receptor FactsBook (FACTSBOOK SERIES)

Gamma-Herpesvirus Latency Requires T Cell Evasion

Gamma-Herpesvirus Latency Requires T Cell Evasion

Reason and Commitment

T-64 and T-80

Natural Killer Cell Protocols. Cellular and Molecular Methods

Natural killer cell protocols: cellular and molecular methods (volume 612)

Presentism and Ontological Commitment

T-Cell Autoimmunity and Multiple Sclerosis (Neuroscience Intelligence Unit 5)

Rationality and Commitment

Paula's Commitment

Mr. Commitment

Paula's Commitment

Mr. Commitment

Commitment Issues

Cellular

Commitment Hour

T Cell Subsets: Cellular Selection, Commitment and Identity

Advances in Immunology Volume 83 T Cell Subsets: Cellular Selection, Commitment and Identity

Antibodies and T-Cell Receptors,

T Cell Protocols

T Cell Protocols

T cell protocols

T Cell Protocols

Receptors of Cell Adhesion and Cellular Recognition

T Cell Protocols: Development and Activation

Factoring groups into subsets

Commitment

Commitment

T-Cell Receptor FactsBook (FACTSBOOK SERIES)

Gamma-Herpesvirus Latency Requires T Cell Evasion

Gamma-Herpesvirus Latency Requires T Cell Evasion

Reason and Commitment

T-64 and T-80

Natural Killer Cell Protocols. Cellular and Molecular Methods

Natural killer cell protocols: cellular and molecular methods (volume 612)

Presentism and Ontological Commitment

T-Cell Autoimmunity and Multiple Sclerosis (Neuroscience Intelligence Unit 5)

Rationality and Commitment

Paula's Commitment

Mr. Commitment

Paula's Commitment

Mr. Commitment

Commitment Issues

Cellular

Commitment Hour

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