Advances in Immunology Volume 17

ADVANCES IN Immunology VOLUME 17 CONTRIBUTORS TO THIS VOLUME ELMERL. BECKEX SAM M. BEISER VINCENTP. BUTLER,JR. KEJ...

Author: Frank J. Dixon | Henry G. Kunkel

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ADVANCES IN

Immunology

VOLUME 17

CONTRIBUTORS TO THIS VOLUME ELMERL. BECKEX SAM M. BEISER VINCENTP. BUTLER,JR.

KEJTH M. COWAN PETERM. HENSON EUGENEM. LANCE

P. B. MEDAWAR

ROBERTN. TAUB

ADVANCES IN

Immunology E D I T E D BY

F. J. DIXON

HENRY G. KUNKEL

Division of Experimentof Pothology Scrippr Clink ond Reseorch Foundation lo Jollo, Colifornio

The Rockefeller Univerrity New York, New York

VOLUME 17 1973

ACADEMIC PRESS

New York and London

A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT 0 1973, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART O F 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.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York. New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NW1

LIBRARY OF CONGRESS CATALOO CARDNUMBER:61-17057

PRINTED IN THE UNITED STATES OF AMERICA

CONTENTS

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LIST OF CONTRIBUTORS PREFACE

CONTENTS OF PREVIOUS VOLUMES.

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Vii

ix

xi

Antilymphocyte Serum EUGENEM LANCE.P B MEDAWAR. AND ROBERT N TAUB

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. . . I . Introduction . . . . . . . . . . . . . I1. History . . . . . . . . . . . . . . I11. Preparation of Antilymphocytic Antisera . . . . . . . IV . Purification of Antilymphocytic Serum . . . . . . . V . Assays of Potency . . . . . . . . . . . . VI . Effect on Lymphoid Cells or Tissue . . . . . . . . . . . . . VII . Scope of Antilymphocytic Serum Action in Vioo VIII . Inimunogenicity of Antilyniphocytic Serum Immunoglobulin G . . IX . Discriminate Action of Antilymphocytic Serum on Cell-Mediated . . . . . . . . . . . . . Immunity X . Chronic Administration of Antilymphocytic Serum . . . . . . . . . . . . . . XI . Synergism with Other Agents XI1. Antilymphocytic Serum and the Induction of Immunological Tolerance . . . . . . XI11. Mode of Action of Antilymphocytic Serum XIV. Effects in Man . . . . . . . . . . . . XV. Projections for the Future . . . . . . . . . . References . . . . . . . . . . . . .

2 3 4 15 24 27 36 46 47 51 54 55 57 62 73 73

In Vitro Studies of Immunologically Induced Secretion of Mediators from Cells and Related Phenomena ELMERL BECXERAND PETER M HENSON

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I . Introduction . . . . . . . . . I1. General Characteristics of Secretory Process . . I11. Mediator Secretion from Isolated Tissues and Organs . . . . IV . Mediator Secretion from Mast Cells V. Mediator Secretion from Basophiles . . . . VI. Mediator Secretion from Platelets . . . . VII . Mediator Secretion from Neutrophiles . . . VIII . Phagocytosis by Neutrophiles . . . . . IX . Mediator Secretion from Monocytes and Macrophages X . Chemotaxis . . . . . . . . . XI . Lymphocyte Transformation . . . . . . XI1. General Summary . . . . . . . . References . . . . . . . . . V

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94 96 99 106 122 126 143 152 158 159 162 166 178

vi

CONTENTS

Antibody Response to Viral Antigens

KEITH M . COWAN

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I. Introduction I1 Virus Structure and Viral Antigens . . I11. Measurement of Antibody to Viral Antigens IV. The Antibody Response . . . . V . Concluding Reinarks . . . . . References . . . . . . .

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195 197 208 223 244 245

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255 256 261 297

311

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Antibodies to Small Molecules: Biological and Clinical Applications

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. . . . . . . . . I . Introduction . I1. General Principles . . . . . . . . I11. Specific Applications . . . . . . . References . . . . . . . . . VINCENT P BUTLER. JR.,

AND

SAMM BEISER

AUTHORINDEX.

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SUBJECTINDEX.

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343

LIST OF CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

ELMERL. BECKER, Department of Pathology, University of Connecticut Health Center, Farmington, Connecticut ( 93) SAM M . BEISER,Departments of Medicine and Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York (255)

VINCENTP. BUTLER,JR., Departments of Medicine and Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York (255) KEITH M. COWAN,Plum Island Animal Disease Laboratory, Agricultural Research Service, U . S . Department of Agriculture, Greenport, New York (195)

PETERM . HENSON, Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California ( 9 3 ) EUGENEM . LANCE,Division of Surgical Sciences, Clinical Research Centre, Harrow, Middlesex, England ( 1 ) P. B. MEDAWAR, Division of Surgical Sciences, Clinical Research Centre, Harrow, Middlesex, England (1) ROBERT N . TAUB,Transplantation Immunology Laboratory, Mount Sinai Hospital, New York, New York ( 1 )

vii

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PREFACE The extent to which inmmnology continues to permeate new frontiers of biology and medicine is a constant source of amazement. It is certainly in part a consequence of the more diversified usages of immunological methodology which is clearly exemplified by the last article, by Drs. Butler and Beiser, on the extraordinary array of small molecules to which antibodies can now be obtained. More significantly, perhaps, it stems from the broad significance of the science of immunology itself to mammalian systems and their derangements. The scope of relevant subjects to be covered by reviews of this type is necessarily broadened, and this is clearly apparent in the diversification in Volume 17. In the first contribution, Drs. Lance, Medawar, and Taub present a very complete analysis of the question of antilymphocyte serum. These workers were pioneers in the experimental work with this material and this wide experience is very apparent in the exhaustive and thorough treatment afforded the subject. There is no doubt that the use of antilymphocyte serum, particularly preparations with defined specificities for single types of lymphocytes, will have broad usage in experimental immunology. Its place in human therapy remains unanswered, although impressive results have been obtained in certain centers. The authors review this work critically and completely. In the second article, Drs. Becker and Henson have attempted a very difficult task in trying to review the intricate and in most instances illdefined topic of mediators, not only those involved in immunological reactions but many others as well. Of necessity, it has to represent a progress report, but it will undoubtedly prove of considerable value to a wide audience and the bibliography is particularly complete. Certain common denominators become apparent in this review, such as the role of Ca and Mg ions and the cyclic AMP system, and these topics are well covered by the authors. The contribution by Dr. Cowan contains many examples of the use of immunological techniques in the dissection of the components of both the simple and the more complex viruses. Emphasis is placed on the picornavirus-type foot-and-mouth disease virus, where very advanced methodology has been utilized in the definition of a spectrum of different antigens. A number of practical and theoretical developments in radial immunodiffusion have evolved from the studies on this virus by Dr. Cowan and his associates at Brookhaven. A variety of other viral antigens including those of the influenza group are also reviewed. ix

X

PREFACE

The final article deals primarily with the biological and clinical applications of antibodies to various low molecular weight, biologically active molecules. There does not appear to be any limit to the type of molecule to which antibodies may be produced; the only limitation is the ingenuity of the chemist to conjugate a specific type to a suitable carrier, Important new examples are cyclic AMP and angiotensin. The diagnostic value of such antisera when combined with the newer methods of radioimmunoassay is abundantly clear. The therapeutic possibilities of antisera to such a substance as angiotensin in ameliorating disease manifestations are certainly intriguing but not as yet realized. Work on this review was one of the last activities of Dr. Beiser prior to his untimely death; his outstanding contributions to the field add greatly to this section. The complete cooperation of the publishers in the production of Volume 17 is gratefully acknowledged.

HENRYG. KUNKEL FRANK J. DIXON

Contents of Previous Volumes Volume 1 Transplantation Immunity and Tolerance AND T. HRABA M. HA~EK, A. LENGEROVL,

Immunological Tolerance of Nonliving Antigens

RICHARDT. SMITH Functions of the Complement System

ABRAHAM G. OSLER In Vifro Studies of the Antibody Response

ABRAMB. STAVITSKY Duration of Immunity in Virus Diseases

J. H. HALE Fate and Biological Action of Antigen-Antibody Complexes

WILLIAM0. WEICLE Delayed Hypersensitivity to Simple Protein Antigens

P. G. H. CELLAND B. BENACERRAF The Antigenic Structure o f Tumors

P. A. GORER AUTHORINDEX-SUB JECX INDEX Volume 2 Immunologic Specificity and Molecular Structure

FREDKARUSH Heterogeneity of y-Globulins JOHN

L. FAHEY

The Immunological Significance o f the Thymus

J. F. A. P. MILLER,A. H. E. MARSHALL, AND R. G. WHITE Cellular Genetics of Immune Responses

G. J. V. NOSSAL Antibody Production by Transferred Cells

CHARLESG. COCHRANE AND FRANK J. DIXON Phagocytosis

DERRICK ROWLEY xi

xii

CONTENTS OF PREVIOUS VOLUMES

Antigen-Antibody Reactions in Helminth Infections

E. J. L. SOUL~BY Embryalogical Development of Antigens

REED A. FLICKINCER AUTHORINDEX-SUB JECT INDEX Volume 3 In Vifro Studies of the Mechanism of Anaphylaxis

K. FRANKAUSTENAND JOHN H. HUMPHREY The Role of Humoral Antibody in the Homograft Reaction

CHANDLER A. STETSON Immune Adherence

D. S. NELSON Reaginic Antibodies

D. R. STANWORTH Nature of Retained Antigen and Its Role in Immune Mechanisms

DANH. CAMPBELL AND JUSTINE S. GARVEY Blood Groups in Animals Other Than Man

W.H. STONEAND M. R. IRWIN Heterophile Antigens and Their Significance in the Host-Parasite Relationship

C. R. JENKIN

AUTHORINDEX-SUB JECT INDEX Volume 4 Ontogeny and Phylogeny of Adaptive Immunity ROBERTA. GOODAND BENW. PAPERMASTER Cellular Reactions in Infection

EMANUEL SUTERAND HANSRUEDY RAMSEIER Ultrastructure of Immunologic Processes JOSEPH

D. FELDMAN

Cell Wall Antigens of Gram-Positive Bacteria

MACLYNMCCARTYAND STEPHENI. MORSE Structure and Biological Activity of Immunoglobulins

SYDNEYCOHENAND RODNEYR. PORTER


Autoantibodies and Disease

H. G. KUNKELAND E. M. TAN Effect of Bacteria and Bacterial Products on Antibody Response

J. Mmoz AUTHOR INDEX-SUB JECT INDEX Volume 5

Natural Antibodies and the Immune Response

STEPHEN V. BOYDEN Immunological Studies with Synthetic Polypeptides

MICHAELSELA Experimental Allergic Encephalomyelitis and Autoimmune Disease

P ~ I Y. P PATERSON The Immunology of Insulin

c. G. POPE

Tissue-Specific Antigens

D. C. DUMONDE

AUTHOR INDEX-SUB JECT INDEX Volume 6

Experimental Glomerulonephritis: Immunological Events and Pathogenetic Mechanisms

EMILR.UNANUE AND FRANK J. DIXON Chemical Suppression of Adaptive Immunity

ANN E. GABRIELSON AND ROBERT A. GOOD Nucleic Acids as Antigens

OTTOJ. PLESCU AND WERNER BRAUN In Vifro Studies of Immunological Responses of Lymphoid Cells

RICHARDW. DUITON Developmental Aspects of Immunity

JAROSLAV STERZLAND ARTHURM. SILVERSTEIN Anti-antibodies

PHILIPG. H. GELLAND ANDREWS. KELUS Conglutinin and lmmunoconglutinins

P. J. LACHMANN AUTHORINDEX-SUB JECT INDEX

xiii

xiv


Volume 7 Structure and Biological Properties of Immunoglobulins SYDNEY C O ~ AND N CESAR MrrsTEJN Genetics of Immunoglobulins in the Mouse

MICHAELPOTI-ERAND ROSELIEBERMAN Mimetic Relationships between Group A Streptococci and Mammalian Tissues JOHN

B. ZABRISKIE

lymphocytes and Transplantation Immunity

DARCY B. WILSON AND R. E. BILLINGHAM Human Tissue Transplantation

JOHNP. MERRILL AUTHORINDEX-SUBJECT INDEX Volume 8 Chemistry and Reaction Mechanisms of Complement

HANSJ. M~~LLER-EBERHARD Regulatory Effect of Antibody on the Immune Response JONATHAN

W. Urn AND GORANMOLLER

The Mechanism of Immunological Paralysis

D. W. DRESSER AND N. A. MITCHISON In Vifro Studies of Human Reaginic Allergy

ABRAHAMG. OSLER, LAWRENCE M. LICHTENSTEIN, AND DAVID A. LEVY AUTHORINDEX-SUBJECTINDEX Volume 9 Secretory Immunoglobulins

THOMAS B. TOMASI, JR.,

AND JOHN

BIENENSTOCK

Immunologic Tissue Injury Mediated by Neutrophilic Leukocytes

CHARLES G. COCHRANE The Structure and Function of Monocytes and Macrophages

ZANVIL A. COHN The Immunology and Pathology of NZB Mice

J. B. HOWIE AND B. J. HELYER

AUTHORINDEX-SUB JECT INDEX


Volume 10 Cell Selection by Antigen in the Immune Response

GREGORY W. SISKINDAND BARUJ BENACERRAF Phylogeny of Immunoglobulins

HOWARD M. GREY Slow Reacting Substance of Anaphylaxis

ROBERTP. ORANGE AND K. FRANKAUSTEN Some Relationships among Hemostasis, Fibrinolytic Phenomena, Immunity, and the Inflammatory Response

OSCARD. RATNOFF Antigens o f Virus-Induced Tumors

KARL HABEL Genetic and Antigenetic Aspects of Human Histocompatibility Systems

D. BERNARDAMOS AUTHOR INDEX-SUB j ~ c INDEX r Volume 11 Electron Microscopy o f the Immunoglobulins

N. MICHAELGREEN Genetic Control of Specific Immune Responses

HUGH0. MCDEVITTAND BARUJBENACERRAF The lesions in Cell Membranes Caused b y Complement JOHN

H. HUMPHREY AND ROBERTR. DOURMASHKIN

Cytotoxic Effects of lymphoid Cells In Vifro

PETERF’ERLMANNAND GORANHOLM Transfer Factor

H. S. LAWRENCE Immunological Aspects of Malaria Infection

IVOR N. BROWN AUTHOR INDEX-SUB j ~ c INDEX r Volume 12 The Search for Antibodies with Molecular Uniformity

h x ~ m M.KRAUSE Structure and Function o f y M Macroglobulins

HENRYMETZGER

xv

xvi


Transplantation Antigens

R. A. REISFELD AND B. D. KAHAN The Role of Bone Marrow in the Immune Response

NABIH I. ABWU

AND

MAXWELLRICHTER

Cell Interaction in Antibody Synthesis

D. W. TALMAGE, J. RADOVICH, AND H. HEMMINGSEN The Role of Lysosomes i n Immune Responses

GERALD WEISSMANN AND PETERDUKOR Molecular Size and Conformation of Immunoglobulins

KEITH J. DORRINGTON AND CHARLES TANFORD AUTHORINDEX-SUB JECT INDEX Volume 13

E HANSBENNICHAND S. GUNNAR 0. JOHANSSON

Structure and Function o f Human Immunoglobulin

Individual Antigenic Specificity of Immunoglobulins

JOHNE. HOPPERAND ALFREDNISONOFF In Vitro Approaches to the Mechanism of Cell-Mediated Immune Reactions

BARRYR. BLOOM Immunological Phenomena in Leprosy and Related Diseases

J. L. TURKAND A. D. M. BRYCESON Nature and Classification of Immediate-Type Allergic Reactions

ELMERL. BECKER AUTHORINDEX-SUB JECT INDEX Volume 14 lmmunobiology of Mammalian Reproduction

ALANE. BEERAND R. E. BILLINGHAM Thyroid Antigens and Autoimmunity

SIDNEY SHULMAN lmmunological Aspects of Burkitt’s Lymphoma

GEORGE KLEIN Genetic Aspects o f the Complement System

CHESTER A. ALPER AND FREDS. ROSEN The Immune System: A Model for Differentiation in Higher Organisms

L. HOODAND J. PRAHL AUTHORINDEX-SUB j ~ c INDEX r


Volume 15 The Regulatory Influence of Activated T Cells on B Cell Responses t o Antigen

DAVDH. KATZ AND BARUJBENACERRAF The Regulatory Role of Macrophages in Antigenic Stimulation

E. R. UNANUE Immunological Enhancement: A Study of Blocking Antibodies

JOSEPHD. FELDMAN Genetics and Immunology of Sex-linked Antigens

DAVIDL. GASSER AND WILLYS K. SILVERS Current Concepts of Amyloid

EDWARD C. FRANKLIN AND DOROTHEA ZUCKER-FRANKLIN AUTHOR INDEX-SUB JECT INDEX Volume 16 Human Immunoglobulins: Classes, Subclasses, Genetic Variants, and ldiotypes

J. B. NATVICAND H. G. KUNKEL Immunological Unresponsiveness

WILLIAM0. WEIGLE Participation of Lymphocytes i n Viral Infections

E. FREDERICK WHEELOCK AND STEPHENT. TOY Immune Complex Disease in Experimental Animals and Man

C. G. COCHRANE AND D. KOFFLER The lmmunopathology of Joint Inflammation in Rheumatoid Arthritis

NATHAN J. ZVAIFLER AUTHORINDEX-SUB j ~ c INDEX r

xvii

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An ti lymphocyte Serum

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EUGENE M LANCE. P B MEDAWAR. AND ROBERT N TAUB Division o f Surgicol Sciences. Clinical Reseorch Cenfre. Harrow. Middlesex. England. and Transplanfation Immunology Laborotory. Mount Sinai Hospifal. New York. N e w York

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I. Introduction I1. History . . . . . . . . . . . . . . I11. Preparation of Antilymphocytic Antisera . . . . . . . A. Source of Antigen . . . . . . . . . . . B Choice of Species . . . . . . . . . . . C Schedule of Immunization . . . . . . . . . D . Comment on the Preparation of Antilymphocytic Sera . . . . . . . . . . IV. Purification of Antilymphocytic Serum . A Absorption . . . . . . . . . . . . . B . Antilymphocytic Serum Fractions and Antibody Fragments . . C . Antibody Eluates . . . . . . . . . . . V Assays of Potency . . . . . . . . . . . . A . In Vivo . . . . . . . . . . . . . B . In Vitro . . . . . . . . . . . . . VI . Effect on Lymphoid Cells or Tissue . . . . . . . . A. In Vitro . . . . . . . . . . . . . B In Vioo . . . . . . . . . . . . . . . . . VII . Scope of Antilymphocytic Serum Action in Viuo . A Effect on Inflammation . . . . . . . . . . B. Effect on Cell-Mediated Immunity . . . . . . . . C. Humoral Immunity . . . . . . . . . . . D. Erasure of Memory . . . . . . . . . . . VIII. Imniunogenicity of Antilymphocytic Serum Immunoglobulin G . . IX . Discriminate Action of Antilymphocytic Serum on Cell-Mediated . . . . . . . . . . . . . Immunity . A. Suppression of Responses in Virgin Animals . . . . . . B . Effect in Sensitized Animals . . . . . . . . . . . . C . Effect of Antilymphocytic Serum on Viral Systems . D . Morphological Evidence . . . . . . . . . . X . Chronic Administration of Antilymphocytic Serum . . . . . XI. Synergism with Other Agents . . . . . . . . . . XI1. Antilymphocytic Serum and the Induction of Immunological Tolerance . . . . . . XI11. Mode of Action of Antilymphocytic Serum . A . Selective Action on Recirculating Lymphocytes . . . . . B Alternative Possibilities . . . . . . . . . . XIV Effects in Man . . . . . . . . . . . . . A . Parallelism between Clinical and Experimental Evidence . . . B . Special Aspects of Antilymphocytic Serum Production for Use in Man C. Administration and Side Effects . . . . . . . . D . Clinical Use of Antilymphocytic Globulin . . . . . . 1

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4 9 12 14 15 15 16 23 24 24 26 27 27 29 36 36 37 44 45 46

47 48 49 51 51 51 54 55 57 57 60 62 62 64 65 69

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E. M. LANCE, P. B. MEDAWAR, AND R . N. TAUB

XV. Projections for the Future References . . .

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73 73

I. Introduction

It is always di5cult to be sure of perspective when viewing a subject at close range, yet, judging from the number of published papers and even reviews (James, 1968, 1969; Medawar, 1968; Lance and Medawar, 1970c; Lance et al., 1971; Denman, 1969; Taub, 1970a; Sell, 1969; Gum, 1969; Caron, 1968; Woodruff, 1969, 1971; van Bekkuni, 1969; Renoux and Mikol, 1967; Russell, 1968, 1969; Shanfield and McLean, 1969; Brendel, 1969), antilymphocytic serum ( ALS) has captured the imagination of the scientific community. There seem to be clear and adequate reasons for this enthusiasm, although the ultimate role of this agent both with respect to therapeutics and research remains to be clarified. Impelled by the success and growing demands of clinical transplantation, a wide variety of agents has been used to suppress the rejection response. The great majority of these “conventional” immunosuppressive agents share several features in common: ( a ) often they have been the side product of the search for drugs to control malignancy (immunosuppression is an indirect result of some very general antimetabolic effects, and a wide variety of tissues and organs are affected); ( b ) the dosage range for immunosuppression is close to or coextensive with the toxic range, and finally ( c ) these agents do not generally discriminate between the two arms of the immune response-cellular and humoral. In so far as they do discriminate they appear to be more effective in opposing humoral rather than cell-mediated immunity. For reasons which will be fully developed in this review, ALS promises to be a considerable advance. Although it is too early for a final judgment, yet in some ways this is a good time for a reappraisal of research in this field. For it may well be asked whether the early promise heralded in the publications of the 1960s has been fulfilled. For some time it appeared as if the biological usefulness of ALS ended with the evolution of Homo sapiens. Although remarkable and reproducible effects could be achieved in a wide variety of animal species, there was little convincing evidence that ALS was useful in man. Surgeons of considerable seniority and experience in the field of transplantation became sceptical, remarking when asked their opinion of ALS, “Oh! Yes . . . I have heard of it . . . Jolly useful in mice.” We believe that this is a good time to inquire carefully into the cause of this apparent discrepancy and examine the more recent clinical evidence which, to some extent, suggests that man is, indeed, not exceptional in his response to ALS. Antilymphocytic serum, in the context of this review, refers to the product obtained when lymphoid cells or cell fractions from animals of

ANTILYMPHOCYTE SERUM

3

one species are injected into animals of another species. Strictly speaking, this definition excludes sera produced by the injection of cells other than lymphoid cells. However, as will be pointed out below, this exclusion is an operational one, and in some cases homologous sera or sera raised against tissue other than lymphoid tissue may share the properties of heterologous ALS. Reference is made throughout this review to the potency or activity of ALS. Unless otheiwise stated we are referring to the power to prolong the survival of tissue allografts in vivo. This stipulation and restriction are necessary because many of the in vivo and in oitro properties of ALS appear to have little or nothing to do with the defining property: immunosuppression. II. History

The current intense interest in ALS stems from its remarkable properties as an immunosuppressive agent. Inderbitzin (1956) is credited with the first demonstration of this effect. While working in the laboratory of John Humphrey, he showed that such antisera could strongly inhibit the skin reactions of delayed hypersensitivity in the guinea pig. In 1961, Waksman et al. confirmed these findings and extended the range of effects to inhibition of autoimmune disease and the prolongation of skin homograft survival. The abrogation of the homograft response recorded in his experiments, although feeble, was undoubtedly significant, and it was not until the demonstration by Woodruff and Anderson (1963, 1964) of striking prolongation of the life of skin homografts in rats under the aegis of ALS that the present frenetic phase of investigation began. Although knowledge of the immunosuppressive action of ALS is barely 15 years old, similar sera have been used for almost 70 years as a tool in biological research. The first recorded application was by the great Russian zoologist Metchnikoff ( l899), who used ALS to investigate the cellular basis of inflammation. Over the ensuing decades, sporadic reports anticipated much of our current knowledge of the properties of these antisera. Besredka, in 1900, showed that ALS agglutinated and killed lymphocytes in vitro, that cytotoxicity was destroyed by heating the serum to 55°C. for 30 minutes, that these agents showed species specificity, and caused a leukopenia in vivo. The specificity of ALS for lymphocytes was first shown by Bunting in 1903. He reported relatively specific and immediate fall in blood lymphocytes after intraperitoneal ALS injection. Some of the sera used by early workers failed to discriminate among leukocytes ( Flexner, 1902; Christian and Leen, 1905; Besredka, 1900). However, Chew and Lawrence (1937) were able to establish that sera that discriminated rather specifically between lymphocytes and polymorphonuclear leukocytes could be raised. Cruickshank (1941) investigated the role of the spleen in the media-

4


tion of ALS-induced lymphopenia. In contrast with the immune hemolysis of erythrocytes, splenectoniy did not alter the rapid and profound lymphopenia induced by ALS. Moorhead (1905) was apparently the first to attempt to raise antithymocyte sera to investigate thymic function; however, his efforts were not rewarded with success. Ritchie (1907) and later Pappenheimer (1917) were able to raise antithymic sera that did not discriminate between lymphoid cells from various sources. The studies of Pappenheimer are of additional significance, since he showed that at least two populations of antibodies were raised by the injection of thymocytes-one directed toward lymphoid cells and another directed toward erythrocytes. Absorption with erythrocytes did not affect the titer of lymph agglutinins; however, these could be completely absorbed with thymocytes. Woodruff and Forman were among the first to recognize the immunosuppressive potential of ALS but were discouraged in their initial efforts (1960). They found that repeated injection of rabbit antirat lymph node antiserum into rats produced an initial but short-lived lymphopenia which was followed by a lymphocytosis despite continued administration of antiserum. Antilymphocytic serum-induced lymphopenia was a direct effect, since it was elicited in adrenalectomized rats. Initial attempts to prolong the survival of allografts in Wistar rats were unsuccessful. Woodruff and Anderson (1963) reopened the question by combining ALS treatment with thoracic duct drainage and were able to show striking skin graft prolongation. This demonstration was quickly followed by the work of Gray et al. (1964), of Monaco et al. (1967a), and of Levey and Medawar (1966a,b) establishing ALS as the most potent known inhibitor of the transplant-rejection reaction. Clinical application of ALS was pioneered by Starzl and his collaborators ( 1966, 1967a,b,c, 1968, 1969b) . After extensive experience in dogs, they used ALS as adjunctive immunosuppression to the usual programs of azathioprine and corticosteroids in humans. Although these clinical studies were not strictly controlled, there seemed to be a decrease in morbidity, a reduced requirement for corticosteroids, and a reduction in the loss of kidneys from rejection. I l l . Preparation of Antilymphocytic Antiserd

A. SOURCEOF ANTIGEN Potent antisera have been raised by using a wide variety of lymphoid cells. Indeed, it seems a general principle that any source of lymphoid tissue may be used. The types of lymphoid tissue that have been reported to yield potent antisera are summarized in Table I. A point of some theo-

ANTILY MPHOCY TE SERUM

5

TABLE I Souiiciss OF ANTICICN USICDTO RAISE ANTILYMPHOCYTE SERA Normal viable cells Thymus Lymph nodes Spleen Thoracic duct Peripheral blood Tonsil Preserved lymphoid cells Epidermal cells Embryonic fibroblasts Lymphoid cells from antilymphocytic serum-lreated animals Viable malignant cells Cells from patients with lymphomns L-cells Lymphoblastic rell lines Myeloma cells Nonviable cells or cell components Heat-killed lymphoid cells Lymphoid cells from cadavers Chemical extracts from lymphoid cells Subcellular component8 from lymphoid cells

retical interest is that potent antisera can be raised by using the residual lymph node population from ALS-treated animals ( Miller et d.,1970). At various times, claims for the superiority of one lymphocyte type as antigen over others have been made. Thymocytes have been especially favored, and there is some evidence that antithymocyte antisera may be better than, for instance, anti-lymph node antisera. The possibility that antithymocyte antisera may have special advantages because of a postulated antibody toward thymic humoral factors ( thymosin ) was raised by Nagaya and Sieker (1965, 1967, 1969a,b). Not only have others failed to confirm the superiority of antithymocyte antisera, but Wood and Vriesendorp (1969) found the exact opposite. The critical experiment, which would take the form of a comparison of the effects of antithymic and anti-lymph node antisera in both thymectomized and intact animals, has not yet been reported. The choice of antigen source is strongly conditioned by expedience and availability, especially in clincal settings. Lymphocytes from most sources are contaminated to a greater or lesser extent by other tissue components. Upon injection into animals, such crude preparations give rise to a number of antibodies that are not only irrelevant to the immuno-

6


suppressive effect of ALS but also are potentially toxic ( Woiwood et al., 1970). There are in general two possible approaches to this problem: purification of the antigen so that irrelevant antibodies do not arise or absorption of unwanted antibodies from a multivalent antiserum. The latter approach will be dealt with below. The first stage in antigen preparation is the production of a single cell suspension. When the starting material is blood or lymph this occasions no problem, but solid lymphoid organs must be disrupted either in mechanical homogenizers or preferably by passage through fine mesh screens. Great care must be taken to remove stromal elements, as these seem particularly prone to give rise to antibodies that react with basement membrane. The cells must, then, be thoroughly washed to eliminate unwanted serum proteins after which some attempt to remove contaminating erythrocytes is warranted. To this end perfusion of vascularized organs prior to disruption may be helpful (Iwasaki et al., 1967). Flash osmotic shock which will preferentially lyse erythrocytes can be applied to the cell suspension. At this point the cell suspension is more or less heterogeneous, depending upon the antigen source. Filtration through cotton or glass wool columns enriches such suspensions in small lymphocytes by preferentially retaining polymorphonuclear leukocytes and monocytes. When whole blood is used as the starting material, an additional processing step is required to remove platelets. From the point of view of ease of processing and homogeneity sources of lymphocytes would probably rank in the order: thoracic duct, thymus, lymph nodes, peripheral blood, and spleen. Accessibility and availability are two practical considerations when selecting an antigen source. In this respect it seems possible to raise suitable antisera by using neoplastic lymphoid tissue (Witz et al., 1968; Pichlmayr, 1970). The potential use of cultured lymphoblasts seems particularly attractive ( Najarian et al., 1969a,b,c; Perper et al., 1970a,b; Moore, 1969a,b) for a relatively unlimited supply of pure lymphocytes. A further stage in antigen purification involves the isolation of subcellular lymphocyte components. Levey and Medawar ( 1966b) were the first to approach this subject. They prepared by relatively simple manipulations a crude membrane fraction, a nucleoprotein fraction, and soluble extract. The crude membrane fraction was by far the most effective but shared several disadvantages with whole cells-namely, potency tended to fall off rather than increase with repeated immunization, and high titers of potentially toxic antibodies were also produced. Lance et al. (1968, 197Oa) and Zola et al. (1970) took this approach a step further (see also Grabar, 1970; Aschkenasy et al., 1970; Traeger et al., 1970a,b; Nagaya et al., 1970; Hayes et al., 1970; Knight et al.,


7

1970). Through the use of differential centrifugation and sucrose gradients, they isolated various subcellular fractions from mouse thymocytes that had been disrupted by intracellular cavitation in a nitrogen decompression apparatus. The fractions were identified by enzyme markers and by electron microscopy and consisted of supernatant, microsomal, plasma membrane, niitochondrial, and nuclear fractions. Rabbits were repeatedly immunized by intravenous inoculations of aliquots of these fractions, and the sera were collected at intervals. The resulting antisera were assayed for i n vivo potency and toxicity as well as for i n vitro lymphocytoxins and hemagglutinins. All subcellular fractions could raise active antisera, but the supernatant fraction was extremely feeble. The nuclear fraction (which also contained some undisrupted whole cells) gave rise to active antisera initially, but repeated immunization led to decline in potency. Antisera raised with mitochondria contained very high titers of hemagglutins. The membrane fraction led most consistently to formation of potent and nontoxic antisera and, moreover, could be combined with adjuvant. Antiserum raised in this way was much more potent than that resulting from membrane alone (Fig. l), remained nontoxic, and could be used without prior absorption. There was no tendency for decline in potency with repeated immunization. The membrane ingredient responsible for potency could be solubilized by a variety of treatments, including sonication, high salt concentration, 8 M urea, and deoxycholate. Treatment of solubilized membrane with a variety of proteolytic enzymes or by butanol extraction decreased or eliminated the efficacy of membrane antigen, whereas ribonuclease and deoxyribonuclease were without effect. These results were consistent with the possibility that the relevant antigens were lipoproteins. Further characterization of the relevant antigens or antigen must await more detailed fractionation and analysis. The facts that the antigens are widely distributed throughout all lymphoid tissue and that strain differences, if they exist, are minimal suggest an analogy with the MSLA antigens of the mouse, i.e., a differentiation antigen characteristic of the species lymphocyte. Boyse et al. (1968) studied the activity of ALS in relation to alloantigens of murine lymphocytes. They suggested that heterologous AES was directed against an antigenic configuration which included but which was larger than the LY series of antigens. Shigeno et al. (1968) concluded that ALS was not directed toward the 0, TL, or LY specificities, whereas Asakumah and Reif (1968) thought that a portion of ALS activity might, in fact, be directed against a species variety of 0 antigen ( a marker for thymus-derived lymphocytes in mice). This possibility is fully compatible with the observations that thymocytes carry cell surface antigens not shared by other lymphocytes (Grabar

8

E. M. LANCE, P. B. MEDAWAR, AND R. N. TAUB

A:Mmbrane + Freund's alone t antigen pulse iserum sample

0 B:hmkane

'1 0

t

ANTIGEN DOSE IN

250

750

250 20

30

t

ti

tl

10

400 MICROGRAMSOF PROTEIN

40

11

50

60

DAY OF EXPERIMNT

CYTOTOXIC TITERS

Group A all 4 bleeds 50% lysls >> 1120.480 Group B 50% lysls bleed 1 < 112560 2 115120 3 115120 4 < 115120

FIG. 1. Potency assays of group A and B antisera. New Zealand rabbits were immunized with aliquots of thymocyte membrane at the indicated times and dosages. The only difference between groups A and B was the incorporation of complete Freund's adjuvant with the first dose of membrane in group A which was injected into the footpads. All injections in group B and all subsequent injections in group A were intravenous. Note the consistently greater potency both in uioo and in oitro of the group A serum. In oivo potency was assayed by the ability to prolong the survival of A-strain skin homografts on CBA mice.

et al., 1965; Potworowski and Nairn, 1967; Grabar, 1970; Colley et al., 1970). The relevant antigens may not be possessed exclusively by lymphocytes. Levey and Medawar (1967b) reported that active antisera could be raised by using epidermal cells, L cells, and mouse embryo fibroblasts; these findings were confirmed in part by Barth et al. ( 1968). These observations suggest that the differentiation antigens of mouse lymphoid tissue may be to some extent represented in other tissues. The fact that the 6' antigen is found on brain (Boyse and Old, 1969) and the more


9

recent discovery of B on epidermal cells (M. Scheid, personal communication) make this a likely possibility. Neither fresh nor live cells are essential to the production of ALS. Cells taken from animals 24 hours after death have been demonstrated effective by Brent et al. (1968). Lymphoid cells killed by heating to 48.5"C. for 20 minutes were every bit as effective as live cells, thereby discounting the possibility that the ability to home to lymphoid organs played a role in immunization (Jooste et al., 1968). Of some importance is the fact that lymphoid cells may be stored, accumulated, and used at a later date (Nossa et al., 1969). Jooste et al. (1968) reported that mouse thymocytes equilibrated with dimethyl sulfoxide (DMSO) at a final concentration of between 10 to 15%and stored frozen at -79°C. were still effective although noticeably inferior to live cells. It may well be that thet best conditions for storage have not been achieved. Symes and Riddell ( 1966) have described the controlled cooling of lymphoid cells suspensions in DMSO with liquid nitrogen. Human spleen cells stored under these conditions ( -196°C.) for 50 days could still transform after thawing and stimulation with phytohemagglutinin (PHA) (Symes et al., 1966; Meek et al., 1967). More recently, thoracic duct cells stored in a similar fashion have been useful in raising ALS (Carraz et al., 1967; Traeger et nl., 1968c, 1969). Almost all the available evidence suggests that ALS does not possess strain specificity, so that within a species any source of lymphoid cells may be used. For example, Jooste et al. (1968) found that there were no systematic differences of potency among antisera raised in rabbits to the thymocytes of CBA, A, VS, Parkes, Albino, or miscellaneous outbred mice when tested in CBA mice. Sera that are active in CBA mice are also active in mice of other strains, e.g., C57/B16, A, Balb/c, C3H. Gowland et al. (1969) found that ALS raised against C3H cells was more effective when tested in CBA than in C3H mice. However, slight differences may exist: Brent et al. (1967) reported that rabbit anti-CBA antisera seemed somewhat more effective in CBA than in C57B1 mice, and Chard (1968) found that the F( a b ) ? fragment of anti-C57B1 antiserum would block the cytotoxicity of anti-C57B1 but not that of anti-CBA antisera, suggesting that some portion of the rabbit antiserum is directed toward strain-specific antigen. To what extent this is important with respect to immunosuppression remains unclear.

B. CHOICEOF SPECIES Heterologous ALS has been raised in a wide variety of species, but, surprisingly, the question of whether there are distinct advantages or disadvantages in the choice of species has not been investigated to any

10


great extent. It seems clear that within the mammalian order any recipient can give potent antisera. The range of recorded experience is summarized in Table 11. The species that have been most thoroughly studied have been the rabbit and the horse. More recently, the use of calves or pigs has been advocated by Binns et al. (1970). The degree of genetic disparity between antigen donor and antiserum producer may be of some importance. Balner and Dersjant (1967) reported that a monkey antimonkey ALS gave relatively feeble results in comparison to rabbit antimonkey TABLE I1 RANGEOF SPECIES CHOICEIN RAISING ANTILYMPHOCYTIC SERUM ~ _ _ _ _ _ _

Source of serum

Source of antigen

References.

Rabbit

Mouse Rat Guinea pig Hamster Dog Monkey Man Mouse Rat Rabbit Dog Pig Goat Human Monkey Mouse Dog Mouse Monkey Man Dog Mouse Dog Mouse Man Mouse Man

Levey and Medawar (1966a,b) Woodruff and Anderson (1963) Waksman et al. (1961) Wallace et al. (1971) Lance et aE. (1971) Balner and Dersjant (1967) Monaco et al. (1967a) James and Medawar (1967) James and Anderson (1967) Burde et al. (1971) Iwasaki et al. (1967) Lucke el al. (1968) Gunnarson et al. (1969) Iwasaki et al. (1967) Balner and Dersjant (1967) Jeejeebhoy (1967) Pichlmayr et al. (1967a) Binns et al. (1970) Lance and Medawar (1970a,b) Shorter et al. (1967) Halpern et al. (1969) Southworth et al. (1970) Pichlmayr el al. (1967s) Binns et al. (1970) Southworth et al. (1970) Binns et al. (1970) E. Simpson and R. M. Binns (personal communication) Binns et al. (1970) Binns et al. (1970) Binns rt al. (1970) Jooste et al. (1968)

Horse

Monkey Dog Sheep Goat

cow Pig

Duck

Dog Rat Chicken Rabbit

a A single reference only is given ahhough in most categories mult,iple references exist.


11

ALS. Moreover, with the exception of the report by Taub (1969), antisera raised within a species are regarded as impotent. Taub raised ALS in C57/ Ks mice against CBA thyniocytes which significantly prolonged the survival of A-strain grafts on CBA mice (mean survival time 17.5 clays), In general, intraspecific antisera ( alloantisera) are likely to exert their effects through enhancement, It has been suggested that alloantisera may be directed against the hypothetical receptor sites of a recipient’s lymphocytes ( Ramseier and Lindenmann, 1969). In contrast to heterologous ALS, the activity can be completely absorbed with nonlymphoid tissues (Taub, 1970a). Reports of a naturally occurring syndrome with cell-mediated, complement-dependent lymphopenia mimicking the functional deficient in ALS-treated animals ( Kretschmer et aZ., 1969) and the induction of “autoimmune” lymphopenia in dogs (Chiba et aZ., 1965) suggest lymphotoxic action of autologous antibodies ( Schwartz, 1969). Two theoretical factors bearing on ALS potency have received little attention. First, the half-life of r-globulin in the circulation varies considerably depending on the choice of donor and recipient ( Spiegelberg and Weigle, 1965). Since the duration of effect may depend on the continued availability of antibody in the immunosuppressed host, it would seem that pairs selected for this property might improve the efficacy of antisera. Second, if, as suggested by the work of R. N. Taub and M. Ruskiewicz (personal communication), different antigens on lymphocytes are “seen” by different species ( a result suggested by the ability of lymphocytes saturated with ALS antibodies of one species to absorb antilymphocytic antibodies from ALS produced by another species), then pooled antisera raised in different species might prove more effective than the use of an antiserum of single provenance. Although the choice of species within mammals seems fairly unrestricted, avians are a poor choice for the production of antisera intended for mammalian use (Jooste et al., 1968; Lance, 1968d; Riethmuller, 1967a,b; Riethmuller et al., 1968); the converse may not be true (Jankovic et al., 1970). Antisera raised in chickens and ducks to mouse thymocytes were totally inactive in uiuo. Duck anti-rabbit ALS could prolong the survival of skin allografts, although this activity was feeble. The lack of potency could not be attributed to a failure on the part of antigeninjected animals to produce antilymphocytic antibodies, for such antisera were strongly cytotoxic in vitro in the presence of avian sources of complement (Fig. 2 ) . Some activity could be demonstrated in the presence of rabbit complement, but guinea pig and mouse complement were virtually ineffective. Therefore, one could surmise that failure resulted from the known inefficiency with which antibodies produced

12

E. M.LANCE, P. B. MEDAWAR, AND R. N. TAUB I Duck ALS absorbed

I

Duck ALS unabsorbed

Chicken ALS absorbed c r D u c k 1:4 c. Rabbit .1:5 MG-pig 1:2 CI Mouse 1:l

15

160

10,240

1 5

160

lq240

15

160

14240

Reciprocalof serum dilution

FIG.2. Influence of complement source on in uitro cytotoxicity of avian antilymphocytic serum ( ALS ) on mouse lymphocytes. High titers are found when avian complement is used. Rabbit and guinea pig complement are less effective, and there is no demonstrable cytotoxicity with mouse complement.

in one species may interact with the complement of different species (Cushing, 1945; Rice, 1950). This inefficiency is not limited to mammalian-avian combinations and could, therefore, be of some importance in guiding the choice of species for the production of ALS. Apart from the preliminary report by Alexander et al. (1968, 1969) that goat ALS may not activate human complement effectively, little cognizance has been taken of this factor. Because patients may be immunized to heterologous proteins either before or during treatment, in clinical practice it may be advisable to have two different species of ALS available. Studies of Amemiya et al. (1970) suggest that cross-reactivity between inimunoglobulin G ( IgG) of different species might guide this choice.

C. SCHEDULEOF IMMUNIZATION The pattern of injection of serum donors falls into three general categories: a short course of antigen injection (two- or three-pulse sera) ; the repeated administration of antigen over relatively long periods of time (hyperimmune sera), and finally those schedules that incorporate the use of adjuvants. Levey and Medawar introduced the two-pulse method for raising rabbit antimouse thymocyte serum ( 1966a). Rabbits received two injections of lon murine thymocytes 2 weeks apart and were exsanguinated a week after the last injection. Such antisera were not toxic in a clinical sense and could be administered without absorption. Moreover, they were reliably potent, augmenting the survival of skin allografts across an H-2


13

barrier two- to threcfold. The distribution of median survival times of such allografts in a large number of such sera, reproduced from Jooste et al. ( 1968), is as follows:

15-19 20-24 25-29 3034 34-39 40-44

+ ++++ +++++++++ ++++ ++ +

Levey and Medawar found that a single pulse of antigen produced generally weaker antisera, whereas the addition of a third pulse did not produce antisera significantly better than two. Indeed, the repeated injection of antigen pulses 4-6 actually resulted in the formation of less effective antisera. The short course schedule has been adopted by a number of workers ( Lance, 1968a,c,d; Berenbaum, 1967; Berenbaum et al., 1971; Prince, 1970; Levey et al., 1970; Shorter et al., 1967, 1968) whose experience generally confirms that of Levey and Medawar. Schedules of raising hyperimmune antisera have been employed by a number of workers (Woodruff et al., 1967a,b; Iwasaki et al., 1967; Pichlmayr et al., 1967a,b,c,d;Traeger et al., 1967; Carraz et al., 1967), but there is no evidence that such antisera are more potent than those raised by the short course (Thomas et al., 1970). There is, of course, the practical advantage that by repeated bleeding a greater yield of antiserum can be achieved per animal. However, this must be counterbalanced by the disadvantages: a rise in potentially toxic antibodies increasing the need for absorption (Edwards et al., 1970) and the progressive decline in potency often observed with such hyperimmune sera. This decay phenomenon has been widely reported and includes antisera raised in horses and rabbits (Woodruff et al., 1967a,b; Lance et al., 1968; B. Fisher et al., 1969; E. R. Fisher et al., 1969a,b; Jooste et al., 1968; Lee et al., 1964; Dormoiit et al., 1970; James et al., 1970; Levey and Medawar, 1966b). Moreover, the phenomenon is observed with antigens from a variety of sources including thymocytes, lymphocytes, splenic cells, epidermal cells, and crude membrane preparations. The phenomenon is not limited to the production of ALS but has been documented for the analogous situation in which the repeated immunization of rabbits with mouse erythrocytes produced a progressive fall in 19 S and 7 S hemolysins (Lee et al., 1964). The reason for decay is unknown, but it seems reasonable that the recipient may become preoccupied with lesser and irrelevant constituents of the antigenic mix that interfere with production of “active” antibodies. Alternatively, antilymphocytic antibodies may become local-

14

E. M.

LANCE,

P. B.

MEDAWAR,

AND R. N. TAUB

ized to a nonimniuiiosuppressive fraction of the immunoglobulins ( James et al., 1969a,b). An encouraging finding is that relatively pure membrane preparations may be repeatedly injected without decline in potency (Lance et al., 1968). The use of adjuvants in the production of ALS has been widely advocated. Most investigators have recommended emulsification of antigen in Freunds complete adjuvant (Gray et al., 1966; Nagaya and Sieker, 1965; Denman et al., 1!367a,b; Guttman et al., 1967a,b,c,d; Monaco et al., 1966a,b,c; Wood and Vriesendorp, 1969; Traeger et al., 1 9 6 8 ~ ) . There is no question that highly potent antisera can be raised in this way in rabbits and horses. Nevertheless, a note of caution comes from the findings of Jooste et al. (1968) which were subsequently confirmed (Koumans et al., 1971). They reported that the inclusion of adjuvants into the immunization procedure produced antisera that, although undoubtedly potent, were at the same time highly toxic. Such antisera injected into mice ( even after extensive absorption with erythrocyte, serum proteins, and suspensions of kidney and lung) caused interference with wound healing and pathological changes in the liver and kidney sometimes followed by wasting, paralysis, and death. They concluded that such antisera might be rendered useful but only after a very extensive program of purification. Wood and Vriesendorp (1969) found higher titers of hemagglutinins in sera raised with adjuvants than without; Pichlmayr ( 1970) found higher antiplatelet titers with adjuvant. There are few comparative studies of antigen dosage or route of administration. Jooste et al. (1968) investigated the dosage range of los10'O mouse thymocytes administered intravenously to rabbits, and it appeared that the intermediate dose lou was most effective. Gozzo et al. (1971) studied a wider range of antigen doses, again in the rabbit, and found that with the use of adjuvant a much lower dose of antigen was effective (see also Pichlmayr, 1970). Iwasaki et al. (1967) injected horses with large numbers of lymphoid cells to raise antisera; they favored 2 X 1O'O cells per inoculum. In general, choices of dose and route have been made empirically, and there is too little information on this score for further comment.

D. COMMENT ON

THE

PREPARATION OF ANTIL,YMPHOCYTIC SERA

Considering the obvious importance of this subject for prospective clinical use, the relevant literature reveals little that is systematic and comparative. It is unlikely that antihuman lymphocytic serum can be produced in the absence of accurate, predictive, in uitro assays considering the difficulty in evaluating the results from clinical practice. Therefore guide lines will have to be drawn from the results of animal experi-


15

mentation. On the basis of currcnt information the antigen sourcc of choice lies between thymocytes and thoracic duct lymphocytes ( cultured lymphoblasts remain a distinct possibility). The rabbit must represent the species of choice, regardless of the practical and logistic implications, because most evidence now establishes them as the best producers of predictably potent and nontoxic antisera. The horsc remains a second choice, whereas the possibilitics inherent in the pig, calf, goat, and sheep require further study. IV. Purification of Antilymphocytic Serum

A. ABSORPTION Whole unabsorbed ALS contains a mixture of antibodies some of which are antilymphocytic, whereas others are directed at various constituents of donor tissue, e.g., erythrocytes, serum proteins, platelets, and stromal antigens. The extent to which ALS is contaminated by this latter class of antibodies depends on the heterogeneity of the antigenic preparation and the schedule of immunization. Nonetheless, no known method of raising ALS produces a product free from these irrelevant antibodies which are potentially toxic to the recipient when present in moderate amounts. Most antisera, therefore, require absorption before use. The most frequent need seems to be the removal of hemagglutinins which can be accomplished by absorption with whole erythrocytes or with eiythrocyte stroma (Eyquem et al., 1970). An ingenious approach, recommended by Pichlmayr ( 1970), was injection of antierythrocyte antibodies simultaneously with antigen which lowered hemagglutinin titers, Induction of tolerance to erythrocytes in serum donors is another possible approach (Seiler et aZ., 1970). Monaco has suggested that erythrocyte stroma may be coupled to diethylaminoethyl ( DEAE )-Sepharose columns so that hemagglutinins are removed concurrently with protein fractionation (Latham et al., 1970). The advantage of this approach is that such columns may be regenerated and used repeatedly. Absorption procedures may sometimes take advantage of cross-reacting antigens of related species; for example, antimouse ALS may be absorbed with rat erythrocytes as an initial step prior to a final absorption with mouse erythrocytes. In man the use of outdated, stored, AB erythrocytes seems an ideal source of absorbing material. Absorption with serum proteins is recommended by Iwasaki et 01. ( 1967). Insoluble antigen-antibody complexes may be removed by relntively low-speed centrifugation, but it may also be desirable to remove the soluble complexes by high-speed ultracentrifugation as these are potentially damaging to the recipient.

16


The need for removal of antiplatelet or antibasement membrane antibodies is best avoided by selection of antigen, and preparations containing high titers of these antibodies might best be discarded. However, if for some reason it is considered desirable to use such antisera, techniques for their removal have been described (Starzl et al., 1970a,b). The suggestion that antithymocyte serum might be improved by absorption with splenic cells (Leuker and Tribble, 1969) cannot be taken seriously, unless tests of potency can be produced to substantiate these claims. B. ANTILYMPHOCYTIC SERUM FRACTIONS AND ANTIBODYFRAGMENTS After removal or reduction of contaminating antibodies the serum still contains antilymphocytic antibodies belonging to a variety of antibody classes and serum proteins of nonantibody character. Waksman et al. (1961) were the first to show that the immunosuppressive activity of ALS resided in the crude y-globulin fraction by using the technique of ammonium sulfate precipitation. This finding was soon confirmed by many others (Monaco et al., 1965a,b; Currey and Ziff, 1966). Formal studies of antisera fractionated by DEAE or Sephadex chromatography (James and Medawar, 1967; Lance, 1967, 1968d) of both horse and rabbit antimouse ALS have shown that the bulk if not all the activity resided within the 7 s IgG class of antibody. In these studies, careful comparison was made in a standard skin allograft assay' of the various fractions, and the activity of the native serum could be equalled by amounts of IgG equal to that contained in the test dose of whole serum. Moreover, treatment of whole serum or y-globulin fractions with 2-mercaptoethanol (2-ME) under conditions that destroyed the activity of immunoglobulin ( IgM ) antibodies did not appreciably alter potency. The restriction of ALS activity to the IgG fraction has been confirmed by a number of studies (Woodruff et al., 1967b); James and Anderson, 1967; Iwasaki et al., 1967; Monaco et al., 1967a,b; Betel et al., 1970). Some of the experimental evidence on this point is reproduced in Table 111. Aliquots of three rabbit antimouse pools were fractionated by column chromatography with Sephadex G-150, and the 1 9 s and 7 s components were restored to original serum concentration. Immunoelectrophoresis against specific antisera showed the 7 S fraction to be free of IgM and the 19 S fraction to be free of IgG. The relative abilities of these fractions to protect skin homografts in the standard assay procedure were compared to a simultaneous assay of the whole native serum pool from which they were prepared. The 1 9 s fraction did not demonstrably alter the fate of first-set A-strain skin grafts on CBA mice, whereas the 7 S fraction was in one

17


TABLE I11 ASSAYSOF POTENCY: COMPARISON OF 19 S A N D 7 S FRACTIONS O F RABBIT ANTILYMPHOCYTIC SERUM (SURVIVAL OF A-STRAINS K I N ON CBA MALEMICE) Rabbit serum pool

Fraction or fragment

Protein concentration (mg./ml.)

No. Animals tested

MEL'

S.D.

(100) 10 10

(11.6) 21.0 20.6

(1.3) 3.1 3.8

Whole serum Whole serum +2mercaptoethanol 7 s 19 s

10.5 5.8

10 17

22.8 11.8

2.7 1.6

B

Whole serum 7 s 19 S

13.0 5.4

10 10 10

28.0 22.9 11.2

5.6 3.2 1.8

C

Whole serum 19 s 19 S b

-

14.0' 14.0

20 8 7

26.4 12.4 16.0

3.5 2.6 2.1

A

' MEL

= Mean expectation of life. Animals received 0.5 ml. on days +2, $3, +4, +5, +6. Represents 3-5 times concentration over original serum content.

instance as potent as the whole serum (pool A ) and in the other only slightly less effective (pool B ) . Reduction of whole serum with 2-ME under conditions that degraded macroglobulins did not affect potency. Concentration of the 19s fraction to 3 to 5 times the original serum concentration (pool C ) proved ineffective. However, repeated administration at closely spaced intervals of this concentrated fraction did produce a feeble but definite effect. We shall return to the significance of this last observation. Since the 7 S fraction could be shown to contain IgA as well as IgG, the relative contributions of these two components were studied by preparing pure IgG fractions after chromatography on DEAE-cellulose columns. Immunoglobulin G, eluted from two pools of rabbit ALS and one batch of horse ALS, was restored to 10 mg./ml. and by immunoelectrophoresis was shown to be free of immunoglobulin A ( IgA) and in the case of horse, immunoglobulin T (IgT), a protein fraction of horse serum. The results of assays employing these preparations (Table IV) establish that the graft-protecting ability is largely if not wholly within the IgG fraction. If the relevant molecules are of IgG specificity, is the intact molecule necessary for the expression of activity or would molecular fragments

18


TABLE IV ASSAYS

O F POTENCY: IMMUNOGLOIIULIN

Serum pool

Prot,ein concentration Fraction or fragment (mg./ml.)

D horse

E rabbit adjuvant

5.6 7.2

-

10

43.0

4.7

7

40.2

3.6

10

27.0 25.3

6.1 5.4

10 10

15.8 11.5 10.7

2.7 1.3 0.4

10

23,s

9.4

10

22.3

6.7

6 4

29.2 32.5

5.6 4.4

(3

5

19.0

3.3

6 4.6d

5

16.5

2.1

50%*

Whole serum Whole serum F(ab)z

50% 50% 5

I rabbit

IgG IgG

+ F(abh

5 12

J rabbit

IgG IgG

+ Fab’

+

=

24.8 25.1 10,s

Whole serum

Whole serum F(ab)z Fab’

MEL

S.D.

6

10

H rabbit

MEL.

16 6 7

Whole serum IgG

G rabbit

No. of animals

10

10

F rabbit

G FKZCMICNTS

Whole serum I& Fab’

IgG

a

AND IMMUNOCLOIIULIN

6

6

10 10

1.4

Mean expectation of life.

* I n this assay, 0.25 ml. was given on days + 2

and +5 instead of t,he usual 0.5-ml. dose. c Given as a single injection on day + 4 in the afternoon. Dose of 2.3 mg. Fab’ given A.M. and P.M. on day + 3 and twice in A.M. on day $4.

bearing the antigen-combining sites do as well? This question has been investigated by preparing the F( a b ) z or Fab’ fragments from ALS-IgG through enzymatic digestion and comparing the ability of these products to protect skin allografts with equimolar amounts of the parent IgG (Table IV). None of the fragments prepared from either horse or rabbit ALS-IgG prolonged the survival of allografts over control levels. An additional check on this point was made when either F( ab)? or Fab‘ was given in combination with ALS-IgG to see whether synergy might occur or whether the fragments by binding with potential antigens on the lymphocyte surface would block the action of subsequently administered


19

intact molecules. Neither synergism nor iiiterfcrence was found with the possible exception of experiment J, where massive amounts of Fab’ given prior to ALS-IgG did seem to curtail graft survival. Since the IgM fraction contained antilymphocytic antibodies and the IgG antibody fragments contained the antigen-binding sites, why were these fractions and fragments ineffective in vivo? In the hope of identifying those properties essential to its action, an extensive comparison of the in vitro and in vivo properties of ALS fractions and fragments was undertaken (Lance, 1968d) to learn in what ways the intact ALS-IgG molecule differed from either intact ALS-IgM or -1gG fragments. These results are summarized in Table V. Alterations in morphology and function which have been attributed to the use of whole ALS are duplicated by the use of the isolated IgG fraction. In all test systems in which the interaction of antibody and cells is made to occur in vitru, the 1 9 s fraction is as effective as the 7s or IgG fraction. In the presence of complement, equal cytotoxic titers are developed. Exposure in the absence of complement induces equal alteration of the ability of lymphoid cells to migrate to lymphoid organs or to carry out immunological transactions. This is clearly a function of immune sera, as the corresponding fractions of normal rabbit sera do not lead to these changes. These findings leave no doubt that within the 1 9 s fraction of ALS, antibodies directed against lymphocytes exist in high titer. However, when the interaction between cells and antibody occurs in vivo, there are widely divergent results. The implication of these findings is that in vivo the access of 1 9 s antibody to these cells is restricted. A relatively simple explanation which may account for this discrepancy takes cognizance of the rapid elimination of 19 S antibodies from the body when contrasted with those of 7s size (half-life in the circulation for 19 S is 2-2.5 days, whereas that for 7 S is 5.6-7.0days). Thus at any given time after administration, 1 9 s antibodies are present in the circulation in lower concentration than the corresponding 7 S antibodies and are, therefore, at a relative disadvantage. Implicit in this argument is the assumption that apart from differences in clearance and tissue penetration the effect of the two fractions is similar. In vitro and some in vivo findings support this line of reasoning. The differences between 7 S and 19 S fractions are predominantly in magnitude and duration, i.e., quantitative rather than qualitative, which could be explained by the shorter biological half-life and, therefore, the shorter interval during which biologically effective concentrations of 19 S antibodics would be maintained in the recipient. If this explanation is true then it should be possible to duplicate the effects of ALS by repeated and frequent injections of 19 S antibodies, an

20


TABLE SURVEYOF BIOLOGICAL ACTIVITIESOF Effect on lymphocyte migration: treat,ment Material"

Immunosuppression

Cytotoxicity

Normal rabbit

Nil

Trace

serum WholeALS ALS 7 S

++++ ++++

1/10,000 1/5,000

ALS 19 S

Trace

1/5,000

ALS-IgG

++++

1/10,000

Nil Nil

1/500 Nil

ALS F(ab)*

ALS Fab'

In vitro

Cell recipients

Cell donor

Lymphopenia

Nil

Nil

Nil

Nil

++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ Sf++ 4Trace ++

++++ ++++ ++++ ++++ Nil ++ + Trace

Trace

Nil

Nil

Nil

~~

ALS, antilymphocytic serum; IgG, immunoglobulin G.

* GVH, graft versus host.

assumption confirmed by the results shown in Table 111, serum pool C. To insure the absence of 7 S contaminants this particular preparation had been passed through Sephadex G-150 twice. The total amount of protein given to each animal represented the 1 9 s content of approximately 10 ml. of whole ALS, i.e., 10 times as much as is given in the ordinary assay procedure. These results in Table I11 received support from the observations of Mandel and Asofsky (1968a,b) that synergism could be demonstrated between IgM and IgG fractions and also from the finding of Anderson et al. (1967, 1968) of a slight ability of 19 S ALS antibody to prolong skin allografts in rats. The biological properties of the enzymatically derived F( ab) and Fab' fragments of ALS IgG have been extensively studied with agreement that these fragments have no effect on the rejection of skin allografts (Anderson et aZ., 1967, 1968; Woodruff et al., 1967a; Riethmuller, 1.967a; Lance, 1967, 1968d). Loss of the Fc portion of the molecule is believed responsible for this lack of effect. The Fc portion of the niolecule mediates many diverse functions which include complement fixation and ability to cross the placental barrier and also determines the rate of

21


V ANTILYMPHOCYTIC SERUM FRACTIONS AND GVH reaction treatmentb

Zn vitro Nil

Cell donor Nil

FR.4GMlcNTS

Lymphocyte depletion histopathology Lymph node Nil

Spleen Nil

++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++ ++ ++++ ++

++++ ++++ ++++ ++++ Nil Nil ++++ Nil

Nil

Nil

Nil

Nil

Half-life in blood NRS-IgG 6-7 days -

Dist,ribution in vivo Parallele the blood distribution curve

-

ParalleLs the blood 6-7Daysin distribution curve tolerant recipient By 24 hours the percent2-2.5 Days age found in lymphoid organs was 36 that of the 7 S fraction. By 48 hours the distribution approximates background. 6-7Daysin tolerant recipient Less t,han 6 hours Less t'han 6 hours

excretion of the intact molecule (Porter, 1963). It has been assumed that the loss of ability to fix complement accounts for the failure of the fragments to duplicate the activities of intact ALS-IgG (James, 1967a,b). Woodruff and his colleagues have studied the fragments derived from horse and rabbit ALS (Woodruff et al., 1967a; James, 1967a,b) and have concluded that these fragments could not fix complement. The findings of Lance did not entirely support this contention, since rabbit F ( a b ) , was strongly cytotoxic in the presence of both guinea pig and rabbit complement although notably less efficient than the intact molecule. Schur and Becker ( 1963) also reported that immune rabbit F( ab), was 40% as efficient as the intact IgG molecule in complement fixation. A parallel exists concerning F( ab), and the 19 S fractions; namely, the discrepancy in results depends on whether the interaction with cells occurs in uiuo or in uitro. The clearance studies of the F( ab), fragment establish that the half-life of this material is extremely short (circa 6 hours). Spiegelberg and Weigle ( 1965) have shown the extreme rapidity with which fragments devoid of the Fc piece are eliminated from the body. Therefore, the F( ab), fragment is less effective than the parent

22


molecule for two reasons. First because it has a reduced ability to bind complement and, second, because it is eliminated from recipients SO rapidly, Both these deficiencies are attributable to destruction of the Fc portion of the molecule. The Fab’ fragment is totally devoid of activity and is absolutely incapable of causing complement fixation. In light of this interpretation, the seemingly contradictory findings of Guttman and his colleagues ( 1 9 6 7 ~ )become understandable. They have shown in a model system of organ transplantation that the F( ab)? fragment of ALS-IgG was able to protect renal allografts to some extent. The F( a b ) r fragment was however much less efficient than the parent IgG molecule and accordingly much larger quantities of the fragment were needed to achieve an effect ( Guttman et al., 1968). Our studies shed no light on their claim that the Fab’ fragment was as effective as the F( ab)? derivative, and, indeed, we remain dubious on this point. The administration of large quantities of Fab‘ seemed to interfere with rather than augment the action of ALS-IgG. This may have occurred because antigenic determinants were covered by an inactive fragment thereby protecting such cells from the action of the intact molecule. From the above the logical conclusion seems to be that raw ALS should be purified by the extraction of the IgG fraction, retaining the whole molecule for use. Advances in techniques for extracting IgG from raw serum should facilitate acceptance of this principle (Moberg et al., 1969; Perper et al., 1!367; Najarian et al., 1970a,b,c; Brummelhuis and Krijnen, 1970). The small amount of activity lost by discarding the IgM fraction is compensated for by being rid of immunogenic foreign proteins to which it would be difficult to induce tolerance (for significance, see below). Moreover there is the chance that, by discarding the IgM fraction, a fair amount of undesirable antibody, e.g., hemagglutinin is removed as well. Woodruff (1967a,b) noted that a large proportion of the antierythrocyte antibodies were contained in the 19 S fraction, a finding not entirely unquestioned (Iwasaki et al., 1967). Horse serum contains a protein fraction, IgT which is excluded in the preparation of the IgG. Although N. Klinman and E. M. Lance (unpublished data) could find no immunosuppressive activity in highly purified horse IgT prepared from two-pulse horse antimouse ALS, others have found antilymphocytic antibodies in this fraction (Funck, 1900; Starzl et al., 1970a,b). Kashiwagi et al. (1970) reported that ALS activity remained confined to the IgG fractions of rabbit and goat antisera but that many of the leukoagglutinins were found in the IgT fraction of hyperimmune horse serum. The possibility cannot be discounted, especially in hyperimmune horse ALS, that activity would be lost if the IgT fraction were discarded. More recent work suggests that the relevant IgG antibodies may be


23

restricted to an IgG subclass, and Perper et al. (1971) have found that IgG subclass antibodies may be actually antagonistic with respect to potency. Therefore, it may become desirnblc to recommcnd a further step in the purification of ALS.

c. ANTIBODY

ELUATES

Although the antibodies responsible in vivo for the in~munosuippressive action of ALS are of IgG specificity, only a small fraction of the total IgG fraction is effectively antilymphocytic. Lance ( 1967) and Woodruff et al. (196%) have estimated, after absorption studies of radioactive ALS-IgG, that the relevant antibodies comprise 15%of the total serum IgG. Woodruff (1968) purified ALS-IgG by acid elution from human lymphoid cell membranes. He tested the resulting preparation in vitro and found that the ability to bind with, agglutinate, and stimulate lymphocytes had been retained but that the cytotoxic properties were greatly reduced. Horse ALS-IgG was used and the acid elution step was carried out at 37°C. Lance (1969) purified rabbit 7 S ALS by absorption onto mouse thymocyte membranes and elution at pH 3.0 at 4°C. Although some denaturation was noted, the eluate retained in large part its biological activity and consisted of a relatively homogeneous solution of IgG molecules. Approximately 2.0% of the original IgG was recovered which could recombine with lymphocytes to the extent of about 75%. Gram for gram the eluate was 10 times as effective in killing lymphocytes in vitro and 50 times as effective in prolonging the survival of skin allografts as the parent IgG preparation. Moreover, the biological halflife of acid-treated molecules did not differ substantially from that of native IgG. This eluate, which accurately reflected the potency of whole ALS, was extremely valuable for studying the fates of relevant molecules in vivo but was not considered to have any practical application for clinical use. The tremendous effort required to obtain such eluates, especially in quantity, as well as the unavoidable denaturation more than offset the potential advantage of discarding the majority of irrelevant IgG molecules. This view may have to be modified since Wilson et al. (1971) have recently described rapid and efficient purification of antilymphocytic antibodies on an immunoadsorbent column. The methods available to extract and purify the IgG fraction from crude ALS have been summarized in detail by James (1968) and will not be elaborated here. Suffice it to say that by a variety of differential precipitations, by chromatographic techniques, or by selective electrophoresis, pure preparations of IgG may be obtained. The advantages of this purification step include discarding the great bulk of irrelevant and immunogenic serum proteins, eliminating some potentially noxious

24

E. M. LANCE, P. B. MEDAWAR, AND

n.

N. TAUB

antibodies with reduction in the requirement for absorption, and using an IgG fraction to which it is relatively easy to induce a state of immunological tolerance. These at the moment would seem to outweigh the disadvantages, namely, the time and cost of such procedures, the inevitable though small amount of denaturation, the possibility of introducing contaminants, and the possibility of discarding some immunoglobulins that may be relevant to potency. V. Assays of Potency

One of the most pressing problems for those who would apply ALS clinically is the search for a suitable and reliable assay of potency.

A. InVivo Medawar and his colleagues routinely assay mouse ALS by its ability to prolong the survival of skin allografts in a standardized system (Jooste et al., 1968). The advantage of this assay is that it measures directly the property in which we are most interested. Monaco et al. (1967b) have shown that antihuman ALS can prolong the survival of skin allografts in man, and Najarian and co-workers (1970a,b,c) have carried out careful dose-response evaluations in a similar system. However, it is difficult if not impossible to see how such tests could come to be applied as a matter of routine. In the search for a substitute, the potency of antihuman ALS has been appraised in surrogate hosts. Standardized doses have been administered to subhuman primates bearing skin allografts (Lance, 1968c; Balner et al., 1968b, 1969a,b,c; Lance and Medawar, 1970b; Bonneau et al., 1970; Darrow et al., 1971; Barnes et al., 1971). Although this procedure conflicts with the basic principle that ALS is species-specific, a good deal of cross-reactivity has been noted between the leukocyte antigens of man and other primates, especially the chimpanzee (Balner et al., 1967). Balner and colleagues (1969b) have shown that the chiinpanzee offers a more sensitive test system than do lower primates. Sera that are without demonstrable effect in the latter may prove to be effective in the former. However, the expense and limited availability of chimpanzees exclude the widespread use of this species for ALS assay. Lance and Medawar (1970b) suggested that the relative insensitivity of the monkey assay might not necessarily be a disadvantage, since in this way only the more potent antisera might be selected. This assumption has been widely accepted, and Balner (1969, 1970a,b) has adopted the monkey assay as a routine screening procedure for antihuman ALS. They have tested many antisera produced in different laboratories and


25

have shown that this assay system can give important information with respect to potential toxicity (Balner et al., 1970) and can also be used to rank antisera in order of potency. Yet no confirmation exists at present to allow direct reliable cxtrapolation. Indeed, it is slightly disquieting that antisera tested and found effective in a hunlan skin allograft system by Simmons et a/. (1971) were not considered very potent when screened by Balner in the monkey assay. Although it is agreed that intraspecies allograft assays constitute the most direct and, indeed, the definitive assay of potency, it is also true that the advent of a reliable replacement would be of great value. A variety of alternatives have been proposed and these will be considered briefly. The lymphopenia produced by injection of ALS has been used to assay potency (Jeejeebhoy, 1965a; Denman and Frenkel, 1968a). However, most reports have tended to emphasize the lack of correlation with allograft protection ( Jeejeebhoy and Vela-Martinez, 1968; Balner et al., 1968a,b; Starzl et al., 1967b). Treatment of lymphocytes in vitro or pretreatment of lymphocyte donors with ALS has been shown to reduce drastically the subsequent performance of these cells in a graftversus-host ( GVH) test (Levey and Medawar, 1967a; Ledney and van Bekkum, 1968; van der Werf et al., 1968; Mandel and Asofsky, 1968a,b; Brent et al., 1967, 1968),On this basis a potential assay can be formulated (Saleh et al., 1969a,b). Levey and Medawar (1966a) found that a relatively small dose of ALS inoculum could suppress the lymphocyte transfer response (Brent and Medawar, 1966) in guinea pigs and, therefore, could be used to assay anti-guinea-pig ALS. However this system takes advantage of the fact that inbred lines of guinea pigs are available and the intended recipient can be heavily irradiated. These requirements cannot be met or easily circumvented in outbred large mammals or in man. In mice, ALS given just prior to an injection of syngeneic lymph node lymphocytes that were labeled with radioactive chromium caused a marked alteration in the migration of these cells (Taub and Lance, 1968a). They were prevented from homing to lymphoid tissue, and the bulk of the radioactivity was recovered in the liver. It has been proposed that this test could be adapted in man (Lance, 1968c), and Martin (1969a,b,c,d) reported that human lymphocytes could be assayed by a variation of this test in mice. He showed that such cells exposed to ALS in vitro did not home to the spleens of recipients in normal numbers but were diverted to the liver. He proposed that this assay measured opsonizing antibody and found a correlation with the protection of skin allografts in mice (see also Lanioreux et al., 1970). If the problems inherent in what is essentially a xenogeneic migration assay such as occurs in the

26


presence of preformed heteroantibodies can be overcome, this might well be a promising test system. B. In Vitro There is no disagreement that a reliable in uitro test which could be rapidly and inexpensively performed with no risk to life or health would provide the best solution to the problem. Lymphocyte agglutination is used by some as the definitive assay (Iwasaki et al., 1967) but is not entirely satisfactory. For instance, avian ALS as well as F( ab) and 19 S antibodies agglutinate lymphocytes but are ineffective in uiuo ( Riethmuller, 1967a,b; Woodruff et al., 1967a,b,c; Jooste et ah, 1968; Mandel and Asofsky, 1968a,b; Lance 1968c; Naysmith and James, 1968). Tests based upon the measurement of lymphocyte activation are subject to the same criticism (Holt et al., 1966; Humphrey et al., 1967). There is no confirmation that this property is related to the in uiuo immunosuppression achieved by ALS, and, furthermore, the test is nonspecific in the sense that antiallotypic antibody (Gell and Sell, 1965; Sell and Gell, 1965) and ALS F( ab)z can activate lymphocytes strongly but do not produce immunosuppression (Monaco et al., 1966b; Levey and Medawar, 1966a; Anderson et al., 1967, 1968). Recently inhibition by ALS of antigen-induced blast transformation has been used ( Eijvoogel et al., 1970) and found to correlate with in uiuo measures of potency. The cytotoxic test has thus far not been an accurate predictive tool (Bach et al., 1967). Whereas all sera tested that are active in uiuo have been cytotoxic in uitro [see Govallo and Kosmiadi (1968) for a striking exception] the converse is not always true (Jooste et al., 1968; M. Ruszkiewicz, personal communication). Some of the possible sources of error may be enumerated. Tests on whole sera are misleading because the 19 S antibodies contribute to the in uitro titer out of proportion to their action in uiuo (James and Medawar, 1967; Lance, 1967, 1968c; Mandel and Asofsky, 1968a,b). Moreover, the IgG fraction may contain antibodies directed against irrelevant antigens. Antibodies against H-2 antigens are cytotoxic in uitro (Reif, 1963; Wigzell, 1965) but, when injected into the whole animal, are ineffective presumably because the great bulk of tissue, other than lymphocytes, which possesses these antigens absorbs them (Garver and Cole, 1961). Another source of error may be introduced when complement other than that of the intended recipient species is used in the titration (Cushing, 1945; Rice, 1950; Lance, 1968c; Jooste et al., 1968). It remains to be seen whether only IgG that has been fully absorbed and tested against the lymphocytes and complement of the intended recipient would enhance the value of the cytotoxic test. Studies specifically devoted to a search for a reliable in vitro assay have failed


27

to correlate significantly the various tests enumerated above with the promotion of allograft survival (Bach et al., 1967; Antoine et d.,1968; Wood and Vriesdorp, 1969; Jeejeebhoy and Vela-Martinez, 1968). Two relatively new test systems appear promising. The rosette inhibition test introduced by Bach and Antoine (1968) appears to measure a function that correlates reasonably well with skin allograft assays. Bach (1970) has introduced evidence to show that rosette-forming cells are thymus-derived lymphocytes. The correlation, although not exact, has been well worked out in murine systems (Bach et al., 1969a,b; Mosedale et al., 1970). Tentatively, results of surrogate skin allograft assays in monkeys and the rosette inhibition test appear to correspond in evaluations of antihuman ALS (Bach, 1970; J. F. Bach et al., 1970; Levey et al., 1970; Bach and Dormont, 1971). As currently performed the test employs whole unabsorbed antiserum and is subject to many of the same criticisms that were directed against the cytotoxic test. Perhaps the reliability of this assay could be enhanced by attention to these points. The opsonization tests performed in uitro by Greaves et al. (1969) or in uiuo by Martin (1969a,b; Martin and Miller, 1969) are appealing because they measure a function that is believed to be important in the mechanism of action of ALS (see below). The information at present available regarding its accuracy as a predictor of in uiuo potency (Clayman et al., 1969; Roitt et al., 1970; M. K. Bach et al., 1970a,b; Svehag and Manhem, 1970) suggests a relationship to graft survival. For the sake of completeness, we shall mention inhibition of macrophage migration ( Southworth et al., 1970), inhibition of blast transformation induced by contact of sensitized cells with antigen in uitro (Greaves et al., 1967), inhibition of the mixed lymphocyte reaction ( Brochier and Revillard, 1971), and promotion of allogeneic tumor growth (Deodhar et al., 1968) among test for potency of ALS. None of these possess obvious advantages over methods already available. In summation, no present in uitro test can replace the intraspecies allograft assay to predict in uiuo potency of ALS. The best alternatives appear to be a combination of the surrogate host assay with either the rosette inhibition or opsinization test or both. VI. Effect on Lymphoid Cells or Tissue

A. In Vitro Components of ALS coat lymphocytes ( Denman and Frenkel, 1968a,b) as demonstrated by immunofluorescence ( Levey and Medawar, 1966a; Woodruff et al., 1967d; Russell and Monaco, 1967), radioautography (Lance, 1969), and uptake of radiolabeled antibody (Woodruff

28


et al., 1 9 6 7 ~ ).A consequence of this binding is strong agglutination (Gray et al., 1964, 1966; Abaza et al., 1966; Abaza and Woodruff, 1966). Although high concentrations of antibody alone may prove cytotoxic to lymphocytes (Humphrey et al., 1967; Mosedale et al., 1968), this effect is usually not manifest until complement is added (Pappenheimer, 1917; Reif, 1963; Abaza and Woodruff, 1966; Gray et al., 1966; Amiel, 1969). Bitensky (1963) has shown that cytotoxic antibodies do not penetrate the cell in the absence of complement but react with cell surface antigens. Induced distortions of the cell surfaces have been studied by Clarke et al. (1970). Addition of complement causes blebs to form on the cell surface followed by cytoplasmic swelling and nuclear changes. There is no reason to think that ALS achieves its cytotoxic action on lymphocytes in a way different from that of other cytotoxic cell systems (Humphrey and Dourmashkin, 1965; Dumonde et al., 1965). Under suitable conditions, ALS can bring about blast transformation of lymphocytes (Grasbeck et al., 1963, 1964; Sell et al., 1965; Holt et al., 1966; Bach and Bach, 1970) or thymocytes (La Via et al., 1968; Claman and Brunstetter, 1968).This property is presumably related to the ability to bind to cell surface receptors and, perhaps, inflict microinjury, but the whole mechanism of cell transformation is at present poorly understood. The magnitude of this effect compares well with mitogens such as phytohemagglutinin ( PHA) . Antilymphocytic serum may either interfere with or enhance the mitogenic effect achieved in mixed lymphocyte reactions (Greaves et al., 1967; Mosedale et al., 1968; Revillard and Brochier, 1970) or by PHA (Ling et al., 1967). Moreover activation by ALS is sometimes more effective in the presence of small amounts of complement ( Ling et al., 1967). Treatment with ALS in vitro interferes with the subsequent immunological performance of lymphoid cells. This is true both when the function is assayed in vivo, i.e., the ability to cause GVH disease (van der Werf et al., 1967, 1968; Brent et al., 1968; Field and Gibbs, 1968; Ledney and van Bekkum, lW), to transfer immunity adoptively (James, 1968) or to home to the lymphoid tissues of syngeneic hosts (Martin and Miller, 1967; Taub and Lance, 1968a) and in vitro, i.e., mixed lymphocyte reactions (Greaves et al., 1967) or response to antigen by ribonucleic acid ( RNA) and deoxyribonucleic acid (DNA) synthesis (Greaves et al., 1967). Antilymphocytic serum can stimulate or interfere with the inhibitory effect that lymphoid cells exert on target cells in culture, possibly by coating recognition sites (Holm and Perlmann, 1969a,b, 1970; Lundgren and Moller, 1969, Lundgren, 1969a,b; Lundgren et al., 1968). Changes in electrical charge and cell metabolism after ALS treatment in vitro have been reported (Bert et al., 1970; Averdunk and Kirstaedter,


29

1969; Phondke et al., 1970). Brent and colleagues (1967, 1968) have provided powerful support for the coating notion and its reversibility by showing that competence can be restored to ALS-treated cells by removal of the protein coat with trypsin prior to testing. Studies with ALS serum fractions showed that the major portion of the lymphocyte-transforming ability was located within the IgG class of antibodies (James et al., 1969a,b; James, 1968). The F( ab)z but not the Fab’ derivative was also active in this system. The species specificity of ALS has been repeatedly documented (Cruickshank, 1941; Abaza and Woodruff, 1966; Gray et al., 1964, 1966), yet there is no question that some cross-reactivity, generally of a low order of magnitude, may occur ( Anigstein et al., 1965;Carraz et al., 1967; Iwasaki et al., 1967; Caspary et al., 1971). Cross-reactivity may be greater for leukemic lymphocytes than for normal cells (Schrek et al., 1969; Schrek and Preston, 1967). Crude unabsorbed ALS shows considerable cross-reactivity with tissues of nonlymphoid origin. Reactivity in vitro has been documented for erythrocytes and other lymphoid cells (Pichlmayr, 1967; Pichlmayr et al., 1967a,d), platelets ( Starzl et al., 1967b), polymorphonuclear leukocytes (Thorsby, 1967; Schroder and Schroder, 1969), mast cells (Guttman et al., 1967a,c), fibroblasts (Thorsby and Lie, 1968), hematopoetic stem cells (Field and Gibbs, 1968), and macrophages ( Hughes et al., 1971; Huber et al., 1969). These cross-reactions are probably to a large extent a reflection of antibodies directed toward histocompatability antigens and may be removed by absorption with nonlymphoid tissue without altering the immunosuppressive or lymphocyte cytotoxic properties of such antisera (Gray et al., 1966; Levey and Medawar, 1966a; McKenzie et al., 1969). Unabsorbed ALS forms precipitin lines with the saline extracts of many different nonlymphoid tissues (Sachs et al., 1964; Gray et al., 1966; Lawson et al., 1967). Some of this cross-reactivity may reflect the presence of serum proteins in these extracts, and absorption with serum proteins has been recommended on a routine basis (Lawson et al., 1967; Iwasaki et al., 1967). Perper et aZ. ( 1970a,b) have studied the tissue specificity of an antiserum raised to cultured lymphoblasts and found that there was a reduction in lymphocyte-specificantibodies and increased cross-reactivity with other tissues. These results raise serious questions about the use of cultured cells that may undergo dedifferentiation.

B. In Vivo Injection of ALS into intact animals causes prompt and profound lymphopenia (Gray et al., 1964, 1966; Iwasaki et al., 1967; Denman et al., 1968a,b; Twb, 1968a; Taub and Lance, 1968b). This is associated with

30

E. M.

LANCE, P. B. MEDAWAR,

AND R. N. TAUB

a drop in the lymphocyte content of the thoracic duct lymph (Agnew, 1968; Tyler et al., 1969; Lance, 1970c), although Martin and Miller (1967) could not demonstrate this. The kinetics of this response are to some extent conditioned by the route of injection (Besredka, 1900; Sachs et al., 1964); the effect develops more rapidly after intravenous inoculation than after injection through other parenteral routes. The rate of recovery of lymphocytes in the peripheral blood has been variously reported as relatively rapid (Cruikshank, 1941; Nagaya and Sieker, 1966; Levey and Medawar, 1966a; Iwasaki et al., 1967) or rather prolonged with full recovery requiring 3 4 weeks (Mandel and Asofsky, 1968a, Taub, 1968a; Taub and Lance, 1968b). There is also some conflict of opinion on the effect of repeated doses of ALS on the peripheral lymphocyte count. Some reports emphasize the persistence of low levels of lymphocytes with closely spaced injections (Pichlmayr, 1966; Monaco et al., 1966a,b,c; Pichlmayr et al., 1967a), whereas others have found a pharmacological tolerance with progressive rise of blood lymphocytes despite continued treatment ( Besredka, 1900; Nagaya and Sieker, 1966; Carraz et al., 1967; Starzl et al., 1967~).To a large extent these opposing reports may be reconciled by taking into account the host immunological response to the heterologous antibodies ( Lance, 1968a). There is general agreement that the level of lymphopenia does not correlate well with the alteration of immunological responsiveness ( Russe et al., 1965; Abaza et al., 1966; Starzl et al., 196713; Balner et al., 1968a,b; Jeejeebhoy and Vela-Martinez, 1968; Jeejeebhoy, 1965a,b; Levey and Medawar, 1966b; Woodruff and Anderson, 1963) raising the question whether or not the regenerant population of lymphocytes found in the blood after ALS treatment is qualitatively different from that found in untreated animals (Woodruff, 1967a; Lance, 1968a; Boak et al., 1968a,b, 1969). Denman et al. (1968b) hsave shown that when the number of blood lymphocytes has returned to normal or near normal levels the capacity to transact immune responses is still reduced. Tursi et al. (1969) have shown that lymphocytes from ALS-treated mice responded poorly to PHA, and furthermore recovery of immune competence could be monitored by following responsiveness to PHA of peripheral blood cells. The regenerant cell populations found after ALS treatment synthesize DNA to a far greater extent than do normal cells (Nagaya and Sieker, 1967; Denman and Frenkel, 1968a,b; Taub, 1968,a,b, 1970a,b; Denman et al., 1968a,b), implying either that they belong to a rapidly turning over pool of cells or are being formed in large numbers and, hence, are very young cells. The studies of lymphocyte kinetics in ALS-treated animals can only be understood in light of knowledge of the heterogeneity of lymphocyte populations and their kinetics. It has been estimated that the tissue


31

reservoir of small lymphocytes is 40 times that ordinarily found in blood (Cronkite et al., 1!264), and, therefore, the number of cells in the circulation at any given point in time may not reflect accurately the total availability of these cells in the entire animal. Moreover, there are at least two types of lymphoid cells in the peripheral blood, and these have markedly different life-spans (Everett et al., 1964). The kinetics and function of the short-lived peripheral blood lymphocyte has not as yet been fully characterized, but there is good evidcnce that after ALS treatment this population expands ( Denman et al., 1968a,b) at the expense of the long-lived lymphocytes ( Denman and Frenkel, 1968a,b; Denman et aZ., 1968a,b; Taub, 1969).

I . Alterations in Lymphoid Tissue The recorded alterations in lymphoid tissue cover the entire range of possibilities from gross hypertrophy and hyperplasia (Chew and Lawrence, 1937; Levey and Medawar, 1966a) to virtually complete destruction (Monaco et al., 1965a,b, 1966a,c; Gray et al., 1966; Lawson et al., 1967). The variation is due in large measure to differences in experimental conditions, i.e., the time of observation (Bunting, 1903), the use of antisera prepared by a variety of measures (Taub and Lance, 1968b), and the lack of systematic observation. However, difficulty also arises because whole serum contains many components that produce characteristic but varying effects. Taub and Lance have proposed a classification of morphological changes (Taub and Lance, 196Sb) and within this framework the lesions that arise may be attributed to the following factors. a. Serum proteins, which are irrelevant to the action of ALS but which are immunogenic, produce lesions after administration of both immune and nonimmune serum falling under the general heading of serum sickness, and include germinal center formation, medullary hyperplasia of the lymph nodes, analogous changes in the spleen, and the late development of arteritis and glomerulonephritis ( Iwasaki et al., 1967; Guttman et al., 1967b,c; Lance, 1968a; Balner et al., 1968a). b. “Irrelevant” antibodies may produce effects by direct action, e.g., immune hemolysis of erythrocytes leading, in turn, to marrow hyperplasia and foci of extramedullary hematopoiesis ( Flexner, 1902; Lance, ,1968a) or by a secondary effect, e.g., thymic atrophy as a consequence of stress (Taub and Lance, 1968b). c. The fraction of ALS responsible for immunosuppression causes a triad of findings, which seem to be an invariable and necessary concomitant to the injection of potent antisera. Within lymph nodes the characteristic sign is selective depletion of small lymphocytes from the

32


thymus-dependent or paracortical area (Parrott, 1967; Turk and Willoughby, 1967; Lance, 1968a; Simpson and Nehlsen, 1971a,b). The medulla, true cortex, and germinal centers are initially unaffected. In the spleen the characteristic lesion is a depletion of small lymphocytes from the peiiarteriolar area of the Malphigian bodies (Lance, 1968a; Taub and Lance, 196813). The third significant finding is a negative one, namely, the absence of significant alteration in the thymus even after prolonged treatment (Russe and Crowle, 1965; Iwasaki et al., 1967; Parrott, 1967; Lance, 196th). It should be pointed out that the existence of this triad of characteristic effects has not been universally accepted. Some workers have stressed the apparently gross and indiscriminate destruction of lymph node architecture after ALS injection (Monaco et al., 1965a,b, 1966a,c; Lawson et al., 1967; Denman and Frenkel, 1967, 196813). This lesion is associated with sera raised with adjuvants and may be mediated through the stress provoked. Shrinkage of the thymus cortex and atrophy of the thymus have also been reported (Nagaya and Sieker, 1965; Monaco et al., 1965a,b; Denman et al., 1967a). That some reduction in cortical thymocytes may occur cannot be categorically refuted, but the bulk of the evidence is against the necessary occui-rence of any major gross or microscopic change in the thymus. The lymphoid hyperplasia reported even in the earliest studies of ALS, and subsequently documented, may be ascribed to the immune response evoked by the immunogenicity of ALS-IgG (see Everett et al., 1970). These changes are found after “pathognomonic” lesions have developed, and their irrelevance is attested to by the fact that ALS-IgG given to animals preparalyzed to IgG either does not provoke these changes or elicits them to a greatly reduced degree without impairing the immunosuppressive action (Lance, 1967; Taub and Lance, 1968b; RodriguezParadisi et al., 1971). 2. In Vivo Fate of Antilymphocytic Antibodies Nava et al. (1969) found no significant difference in the distribution of lZ5I-labeledALS and normal rabbit globulin in mice. Moore (1959) found no difference in the affinity for lymphoid cells between antibody eluted from the kidney or from lymph nodes. Hintz and Webber (1965) reported that ALS-radiolabeled antibody showed the greatest affinity for the thymus. Significant localization was found in lymph nodes, bone marrow, muscle, and the gastrointestinal ( GI ) tract, whereas uptake in the spleen was paradoxically low. Denman and Frenkel (1968a) found heavy localization of fluorescein-labeled ALS-IgG in lymphoid organs but exclusion from the thymus. To a large extent the significance of these findings is obscured and interpretation hampered by the presence of


33

large amounts of nonantilyinphocytic antibody globulin. To overcome these difficulties, Lance ( 1969) prepared *251-labeled,specific, rabbit, antimouse thymus antibody which had been absorbed onto and subsequently eluted from mouse thymus membrane. This material is rapidly cleared from the bloodstream and eliminated from the body. The bulk of injected material is excreted within 24 hours and over 90%is gone in 48 hours. The plot of disappearance curves from the whole body is closely paralleled by the elimination from the various lymphoid and nonlymphoid organs as well. This finding strongly suggests that the direct or primary action of R single pulse of ALS must be quite evanescent however long its secondary biological effects may last. Both Pichlmayr et al. (19674 and R. N. Taub and M. Ruszkiewicz (personal communication) have followed the disappearance of cytotoxic antibody from the bloodstream after a single dose of ALS and have found that, within a matter of hours, cytotoxic antibody can no longer be detected. Traeger et al. (1970a) have proposed monitoring cytotoxic antibody in recipient serum as a means of judging adequate dose administration. This rapid elimination of antibody is compatible with the potency of such preparations, for when animals are intentionally preimmunized to ALSIgG, the elimination of a subsequent dose of IgG is extremely rapid, often being complete within 48 hours (Lance and Dresser, 1967; Clark et al., 1967). Nonetheless, even under these circumstances, ALS can exert a powerful immunosuppressive action (Lance, 1967; Levey and Medawar, 1966a). The mechanism for the very rapid clearance of active antibody eluate is unlikely to be due to an active immune clearance mechanism on the part of the host because it develops far too rapidly. The period of rapid clearance after injection of ALS-IgG into virgin animals does not begin for about 6 days (Lance and Dresser, 1967). Denaturation was also ruled out by internal controls of the elution procedure. The most likely explanation was felt to be that lymphocytes became coated with antibody in the circulation and these antigen-antibody complexes are rapidly cleared, degraded, and excreted. Humphrey has shown that antigen and antibody are simultaneously degraded after immune clearance of complexes (1965). If the clearance of eluted antibody is due to a passive mechanism, i.e., piggy back on circulating lymphocytes, then depletion of these cells should slow down clearance. E. M. Lance and S. V. Jooste ( unpublished observations) showed that the clearance of labeled eluate became slower (half-life 36 hours) in mice that had been previously treated with ALS. The interpretation is that the smaller number of lymphocytes resulted in a diminished number of binding sites for passive clearance of antibody.

34


The distribution of eluted ALS antibody was considerably different from that of nonantibody IgG. The uptake of eluate at 24 hours in lymphoid tissues exceeded that of normal IgG, whereas the converse was true for nonlymphoid tissue. Over the first 48 hours the distribution of normal IgG in the tissues was increasing, while the rate of disappearance of eluate was at a maximum. The elimination of eluate from lymphoid organs appeared to proceed at a more rapid rate and to a more complete degree than that from nonlymphoid organs. Finally the uptake by lymphoid organs of eluate was at all times only a minute fraction of the total injected. Radioautography showed labeled cells in the peripheral blood shortly after injection, In the lymph nodes the heaviest labeling was confined to the paracortex with sparing of the true cortex, germinal centers, and the medulla. The label in the spleen was initially seen around the periphery of the Malphigian follicles but later became diffuse over the white pulp. The thymus was lightly labeled at all times with more label in the cortex than the medulla. [For confirmation of thymic exclusion of label, see also Ranl@vet al. ( 1970) and Denman and Frenkel (1968a,b).] In the kidney, a dense accumulation of label was found in relation to the proximal tubules suggesting the route of excretion, whereas in the liver the label was clearly localized to the cytoplasm of the Kupffer cells and was often associated with phagocytosis of labeled leukocytes. This distribution of label within lymphoid tissues was felt to reflect the pathway of migration of cells in the recirculating pool (Gowans and Knight, 1964). In the thymus, lymph nodes, and spleen, some cells that were coated with antibody were not destroyed. Why this happened remains a matter of speculation. A number of factors may come into play. J. H. Humphrey and R. R. Dourmashkin (personal communication, 1968) have shown that for lysis of erythrocytes by IgG antibody, several thousand molecules must attach to the same cell. Woodruff et al. (1967b) have calculated that each lymphocyte can take up as many as 5.0 X lo6 molecules on its surface, but the exact number required for lysis is not known. Therefore, although lymphocytes in lymphoid organs are exposed to IgG antibody, the concentration may be insufficient for irreversible damage. Another limiting factor may be the local availability of complement. Waksman et al. (1961) documented the fall in circulating complement after ALS. If complement is necessary for the direct destruction of cells (Bitensky, 1963; Dumonde et al., 1965) then a limited availability of complement within lymphoid tissue might explain the relative protection that these cells receive. There is also the possibility that cells in the circulation that are opsonized by antibody are rapidly cleared and destroyed by cells of the reticuloendothelial system (Moller and MBller, 1966; Cajano,


35

1960) but that within lymphoid tissue these cclls are not exposed to phagocytes to the same extent. Finally there is the analogy to the markedly different effects antibody is known to exert against dissociated tumor cells in comparison to solid tissue growths (Gorer and Amos, 1956; Garver and Cole, 1961; Kaliss, 1 9 6 ) .

3. Fates of ALS Afected Lymphoid Cells With the experimental model developed by Bainbridge et al. (1966; Bainbridge and Gowland, 1966), the fates of lymphocytes labeled with chromiumdl, exposed to ALS in vitro, and reintroduced into normal syngeneic recipients were studied by Martin and Miller (1967) and by Taub and Lance (1968a). Instead of the expected distribution of such cells to the lymphoid compartment of the host, ALS-treated cells were rapidly and almost completely taken up by the liver. Their fates were identical to those of cells that had been killed by intentional exposure to heat. The high liver uptake reflects either the outright killing of ALScoated cells after interaction with host complement or opsonization or, most probably, a combination of these two mechanisms. Since exposure in vitro of the heterogeneous population of lymph node cells resulted in a uniform diversion, ALS antibody per se does not discriminate between populations. The fates of labeled cells given to ALS-treated recipients depended on the relative timing between administration of ALS and cells. Antilymphocytic serum, given at the time of or just before the cells, duplicated the effects of in vitro treatment (Taub and Lance, 1968a; Seifert and Brendel, 1969). As the interval between ALS and cell introduction was prolonged the effect became progressively less marked: ALS given to recipients after labeled cell injection was virtually ineffective. In view of the known short half-life of antilymphocytic antibody (see Section VI,B,2), these data suggest that the ALS-mediated cell damage in vivo is largely exerted on circulating cells at a time when they are external to lymphoid compartments. Treatment of cell donors with a short but intensive course of ALS resulted in a residual cell population that migrated differently from normal cells when introduced into untreated syngeneic recipients. This residual population was deficient in cells localizing in recipient lymph ngdes, whereas the spleen-seeking proportion was unaffected. This pattern of altered migration was comparable to that found in neonatally thyinectomized animals, animals thymectomized as adults and subjected to lethal whole-body irradiation, and animals that had been drained chronically through a thoracic duct fistula (Taub and Lance, 1971). These procedures are known to deplete the animal of the population of

36

E. M.LANCE, P. B. MEDAWAR, AND R. N. TAUB

recirculating small lymphocytes, and it is this population of cells that localizes preferentially in recipient lymph nodes (Lance and Taub, 1969; Zatz and Lance, 1971). Alternative possibilities to explain the altered distribution of cells from ALS-treated donors include poor viability of the cells or coating of the cells with antilymphocytic antibody. These are rendered unlikely because such cell suspensions did not show increased staining with trypan blue (Lance, 1968d), migration to the spleen was largely unaffected unlike the behavior of cells coated with ALS in uitro or killed by exposure to heat, no cytotoxicity could be developed by incubation of these cells with rabbit complement, and finally the disturbance in proportional migration, which extended in some cases for more than a month after the last serum injection, long outlasted the metabolic lifetime of ALS antibody in the host (Lance and Cooper, 1970). VII. Scope of Antilymphocytic Serum Action in Vivo

Antilymphocytic serum has been demonstrated to exert powerful immunosuppression in a wide variety of species, and there is no reason to believe that this effect is not an expression of a broad biological phenomenon. The list which is ever growing now extends to mice (Gray et al., 1964), rats (Woodruff and Anderson, 1963), guinea pigs (Waksman et al., 1961)) rabbits (Lance, 1968c), dogs (Abaza et al., 1966), pigs (Lucke et al., 1968), several species of monkeys (Balner and Dersjant, 1967), chimpanzees (Balner et al., 1968a), ,and man (Starzl et al., 1 9 6 7 ~ )There . is, however, one important exception. R. M. Binns and E. Simpson (personal communication) have raised bovine antipig ALS by using a regimen that has been successful against other species (Binns et al., 1970, 1971), but they were unable to demonstrate prolongation of skin allografts in the pig. The peculiar lymphoid anatomy of the pig and the unique response to allografts of liver suggest that lymphoid kinetics may be quite different in this species.

A. EFFECTON INFLAMMATION Some aspects of nonspecific inflammation may be depressed by ALS including those incited by nonspecific irritants (Turk and Polak, 1969; Turk et al., 1968; Waksman et al., 1961) and those that depend on the active production of antigen-antibody complexes ( Turk et al., 1968; Turk and Polak, 1969). On the other hand, in models of passive cutaneous anaphylaxis ( Waksman et al., 1961) and immune complex nephritis (Denman et d.,1966; Taub and Lance, 1968b), no suppression was noted. The extent to which these effects depend on the presence of antilymphocytic antibody or antibody to other cellular mediators of in-


37

flammation or, in the latter, case on the initiation of serum sickness, has not been determined, However, Perper et al. (1969) showed that the anti-inflammatory effect was not mediated by the adrenals and was separable from the immunosuppressive effect.

B. EFFECTON CELL-MEDIATED IMMUNITY 1. Delayed Hypersensitivity Since the time of the report of Inderbitzin (1956), there has been abundant confirmation of the ability of ALS to suppress the dermal manifestations of delayed hypersensitivity ( Waksman et al., 1961; Wilhelm et d., 1958; Russe and Crowle, 1965; Nagaya and Sieker, 1965, 1966; Lance, 1967, 1968; Turk and Willoughby, 1967; Brunstetter and Claman, 1968; Turk et al., 1968). This finding applies as well to primates ( Balner and Dersjant, 1967) including man (Iwasaki et al., 1967; Monaco et al., 1967a). Antilymphocytic serum administered at or just prior to challenge causes a marked reduction in responsiveness. Inderbitzin showed that the release of histamine was reduced and reported reduction in the mononuclear infiltrate at the site of challenge, a finding confirmed by Waksman et al. (1961). Turk and Willoughby (1967) have shown that ALS can block the central lymphoid changes characteristic of these responses. They as well as Waksman et al. pointed out that nonspecific inhibition of inflammation may play a role, but the most likely explanation for these effects is the reduction of potentially reactive cells at the challenge site. Despite this dramatic effect when ALS and challenge are concomitant, animals recover reactivity very rapidly after cessation of ALS treatment. This finding is in many ways analogous to the observations of Levey and Medawar (1966a, 1967a) in their studies of the lymphocyte transfer reaction. The response to challenge corresponds to a peripheral reaction susceptible to the action of ALS. Once treatment ceases, reactivity is restored by the release of competent cells from central lymphocyte stores into the periphery. Abrogation of central reactivity ( immunological memory) in presensitized animals has been extremely difficult to achieve, possibly because persistent antigen (retained for long periods when given in conjunction with Freunds adjuvant) served to reimmunize animals after the lapse of treatment (Russe and Crowle, 1965; Lance, 1967). Antilymphocytic serum can, however, prevent sensitization during the induction of immunity to contact sensitivity (Table VI). Guinea pigs were immunized to dinitrochlorobenzene ( DNCB ) . Two animals received 5 ml. of ALS on the day prior to sensitization, and 2.5 ml. on the first, second, and third days thereafter. A second group received ALS as above, and on the fourth day the sensitization site was surgically removed. The

38

E. M.

LANCE,

P. B.

MEDAWAR,

AND R. N. TAUB

TABLE VI EFFECTOF ANTILYMPHOCYTIC SERUMON INDUCTION OF IMMUNITY TO CONTACT SENSITIVITY I N THE GUINEAPIG

Group

Immunizationa

Serumb

Removal of sensitization site

A B C

DNCB DNCB DNCB

ALS ALS None

No Yes Yes

(I

Response to challenge

+++, ++++ 0, 0 +++, ++

DNCB, dinitrochlorobenxene. ALS, antilymphocytic serum.

third group received no serum but the sensitization site was removed. All animals were challenged 18 days after immunization. The animals receiving ALS in combination with removal of the sensitization site gave no reaction, i.e., they behaved as if unimmunized. The remaining groups showed vigorous reactivity. 2. Transplantation Immunity Antilymphocytic serum is the most powerful experimental agent for overcoming the allograft response. The rejection of both first and second set skin allografts can be prevented (Waksman et al., 1961; Monaco et al., 196!5a,b; Woodruff and Anderson, 1963, 1964; Levey and Medawar, 1966a,b; Brent et al., 1967; Gray et al., 1966; Balner et al., 1968a,b; Balner and Dersjant, 1969; Grabar and Chouroulinkov, 1970). The extent and duration of this effect seems to be limited only by the willingness of the experimenter to continue treatment and the ability of the animal to support the untoward sequelae (Lance, 1968a; Nehlsen, l970,1971a,b). Monaco et al. ( 1 9 6 6 ~ )felt that ALS was more effective if given prior to grafting, but Levey and Medawar (1966a) did not find this critical with respect to skin allografts in as much as ALS could reverse a rejection response which had already been allowed to start (see also Waltman et aZ., 1969, for similar findings with corneal allografts). The effect of ALS in overcoming rejection in presensitized animals (Levey and Medawar,. 1966a) as well as its ability to ablate memory of prior exposure distinguish it from all other immunosuppressive agents (Levey and Medawar, 1966b; Lance, 1968b). The results achieved with skin allografts extend to organ grafts as well, e.g., canine renal and hepatic transplants (Abaza et al., 1966; Monaco et al., 1966c; Starzl et al., 1966, 1967a;b,c; Pichlmayr et al., 1966, 1967a,b, 1968b,c; Atai and Kelly, 1967; Ellis et al., 1967; Lawson et al., 1967; Abbott et al., 1966, 1969; Braf et al., 1969; Shanfield et al., 1968;


39

Clunie et al., 1968), cardiac allografts (Halpern et al., 1969), lung allografts (Iwasaki et al., 1970), transplants of whole joints (Reeves, 1969) or whole limbs (Lance et al., 1971), and renal transplants in rats (Guttman et al., 1967a,b,c,d, 1968). In these systems it seems clear that the best results were achieved when ALS administration began prior to transplantation. A review of reports on organ transplants in large mammals leaves the distinct impression that the effects have not been as dramatic as those achieved with skin grafts in rodents. At present there is no need to invoke any basic biological differences to explain this discrepancy, which may be due to difficulties in working with outbred stock and the proportionally smaller doses of ALS usually employed. Nonetheless, the possibility that other factors are involved, e.g., damage inflicted upon organ grafts by humoral antibodies ( Porter, 1967), cannot be discounted. The degree of genetic disparity between donor and host often critical for other immunosuppressive regimens makes little difference to the effects of ALS. “Easy” allograft combinations are prolonged to about the same extent as difficult transplants across the major histocompatibility loci (Levey and Medawar, 1966b). Nowhere is this principle more obvious than in the survival of xenografts across the widest possible barriers. Xenografts of skin, considered before the advent of ALS an impossible transplant, survive for prolonged periods making the xenograft response amenable to study. Monaco et al. (1966a) were the first to show the acceptance of rat skin on mice treated with ALS. Lance and Medawar (1968) reported prolonged survival of both first-set and secondset xenografts of rat and guinea pig skin on ALS-treated mice, and human skin grafts survived for over 2 months without histological evidence of cellular reaction. Monaco (1970) confirmed the long survival of human adrenal tissue in ALS-treated thymectomized mice. Others have used human fetal transplants (Phillips and Gazet, 1969). In parallel with its effects upon normal tissues, ALS can promote the acceptance and survival of tumor allo- and xenografts ( Anigstein et al., 1965; Phillips and Gazet, 1967; Deodhar et al., 1968; Lewis et al., 1969; Stanbridge and Perkins, 1969; Beverley and Simpson, 1970; Wallace et al., 1971). 3. Graft-versus-Host Response

Numerous reports attest that ALS inhibits this reaction when the cell inoculum is exposed to ALS in vitro (van der Wed et al., 1967, 1968; Ledney and van Bekkum, 1968; Brent et al., 1968; James and Naysmith, 1968; Field and Gibbs, 1968). In mice and rats, pretreatment of cell donors with ALS reduces or ablates the GVH response (Levey and Medawar, 1967a; Ledney and van Bekkum, 1968; Brent et al., 1967, 1968;

40


Boak et al., 1967, 1968a,b; Boak and Wilson, 1968; Monaco et al., 196713; Schwartz et al., 1968; von Thierfelder et al., 1970; Thierfelder et al., 1971). These results have their parallel in the finding by Levey and Medawar (1967a) that splenic cells from ALS-treated donors were less well able to bring about the rejection of skin allografts when injected into lethally irradiated, syngeneic recipients than were cells from normal donors. Treatment of recipients with ALS is also effective (Nouza et al., 1971; Boak et al., 1968a,b; Ledney, 1969). The efficacy of donor pretreatment in abolishing GVH reactions does not apply to all species. Balner et al. (1968a,b, Balner, 1969) found this an ineffective approach in monkeys. In his model the best results were obtained by ALS treatment of recipients, but far greater doses were required than those necessary, for instance, to prolong allograft survival (van Bekkum et al., 1967). Difficulty in suppressing GVH reactions in chickens was reported by Tucker ( 1968). Levey and Medawar studied a model of the GVH reaction in guinea pigs ( 1966a, 1967a). Antilymphocytic serum completely abolished all components of the normal and immune lymphocyte transfer reaction. This could be achieved either by brief pretreatment of recipients or by more prolonged and intense treatment of cell donors. Conventional immunosuppressive agents were ineffective in opposing the first phase of this reaction.

4. Infectious Disease One of the most remarkable “clinical” (if that word can be applied to experimental animals) observations is the freedom from infection by common bacterial pathogens in ALS-treated animals. Susceptibility to infections of this genre is not enhanced by ALS treatment, although the acute inflammatory response to deliberate infection with staphylococci and Pseudomonus aeruginosa may be diminished (Morris and Burke, 1967; Grogan, 1969a,b). However, Grogan ( 1969a,b) reported increased mortality in P . ueruginosa-infected ALS-treated rats and resistance to mycobacterial infection ( Gaugas and Rees, 1968), mycoplasma (Allison, 1970), Protozoa (Spira et al., 1970; Barker and Powers, 1971), nematodes, and helminthic parasites is reduced (Doming0 and Warren, 1968; Kassai et al., 1968). Although relatively little attention has been focused on the modification of bacterial infections by ALS, a great deal is known about the effects of ALS on viral disease (Hirsch and Muiphy, 1968a,b). In general the potentiation of viral infection by ALS has been attributed to blocking of cell-mediated immunity, but titers of antiviral antibodies have not been affected. Reduction in interferon production has been reported


41

( Barth et al., 1969a,b). Antilymphocytic serum depresses the primary cellular response to a variety of viral agents: vaccinia (Hirsch et al., 1968; Hirsch et al., 1968), herpes simplex (Nahmias et al., 1968), yellow fever ( Hirsch and Murphy, 1967), mousepox ( Blandin, 1970), lymphocytic choriomeningitis (Gledhill, 1967; Hirsch et al., 1967, 1968; Lundstedt and Volkert, 1967), Rauscher leukemia (Hirsch and Murphy, 1968a,b), Moloney leukemia (Allison and Law, 1968), adenovirus-12 (Allison et al., 1967), and polyoma (Allison and Law, 1968; Gaugas et al., 1969; Nehlsen, 1971a,b). In vitro treatment with ALS of human lymphocytes causing blast transformation increased susceptibility to infection with vesicular stomatitis virus (Edelnian and Wheelock, 1968) and distemper virus (Poste, 1970). A warning that ALS may interfere with preexisting immunity to virus or potentiate latent disease is contained in the findings of Abaza et al. (1966) and van Bekkum et al. (1967). The former report distemper infections in ALS-treated dogs previously immunized with attenuated distemper vaccine, and the latter noted a number of deaths in ALStreated monkeys from presumably latent virus infections. Volkert and Lundstedt ( 1968) found that protracted ALS treatment could provoke latent lymphocytic choriomeningitis virus infection. On the other hand, Hirsch et al. (1968) found no difference between normal rabbit serum and ALS-treated mice which had been previously vaccinated and then challenged with intracerebral vaccinia. The reduction in cellular response to virus induced by ALS may have paradoxical effects. Thus, ALS protected against infection by low doses of yellow fever virus, although this effect became inapparent at high doses of virus (Hirsch and Murphy, 1967). No changes in clinical or morphological effects of influenza infection in mice were noted (Hirsch and Kaye, 1968). However, ALS treatment prevented the lethal effects of lymphocytic choriomeningitis viral infection ( Gledhill, 1967; Hirsch et al., 1967, 1968; Lundstedt and Volkert, 1967)-persisting viremia and high titers of circulating antibody were found, but encephalitis was obtunded or ablated. Clearly the immune response to this virus is of more potential damage than viral infection per se. In view of the above evidence, it is surprising that no one has reported the occurrence in man of viral disease potentiated by ALS therapy.

5. Autoimmune Disease The effect of ALS has been evaluated in a number of experimental models of autoimmunity, and the subject has been thoroughly reviewed by Dennian ( 1969). Allergic encephalomyelitis can be suppressed by

42

E. M. LANCE, P. B. MEDAWAR, AND R. N. T A D

ALS given concurrently with immunization or after several weeks (Waksman et al., 1961; Leibowitz et al., 1968a,b). Even when first treated, advanced neurological findings showed some amelioration, suggesting that established disease could be affected, a result reminiscent of the reversal of skin allograft rejection recorded by Levey and Medawar (1966a). Kalden et al. (1968) reported that ALS given prior to immunization completely inhibited the histological manifestations of experimental thyroiditis with suppression of antithyroglobulin antibodies. The manifestations of adjuvant arthritis were completely suppressed by ALS provided that treatment followed shortly after adjuvant administration. Late treatment was ineffective (Currey and Ziff, 1966, 1968). Similar observations pertain to autologous immune complex nephritis in rats (Barabas et al., 1969). The effects of ALS on the spontaneous autoimmune disease of NZB mice have been extensively studied by Denman and co-workers (1965, 1966, 1967b, 1968b; Denman and Denman, 1970; Holborow and Denman, 1968; Denman and Ziff, 1964). Antilymphocytic serum treatment could significantly retard the development of Coomb's positive hemolytic anemia which could be reinstated by the transfer of splenic cells from older affected mice. Once hemolytic anemia developed, ALS was powerless to oppose it. By contrast, ALS could not prevent the renal disease and appeared to accelerate its development. Immune complexes formed in response to ALS administration itself seemed an unlikely cause for this acceleration, because the same results were noted in animals rendered tolerant to ALS 7 S IgG. On the other hand, enhancement of renal disease in ALS-treated mice could be forestalled if they were reconstituted with allogeneic lymphoid cells. In Denman's view ( 1969), renal disease occurs when a delicate balance between virus and host lymphocytes is upset. Excessive depletion of small lymphocytes by ALS favors renal disease in mice, just as neonatal thymectomy (East et al., 1967). 6. Tumor Immunity

We have already mentioned that ALS treatment favors the take and growth of allogeneic and xenogeneic tumors and enhances the effect of oncogenic viruses. Antilymphocytic serum treatment alone increases susceptibility to tumor induction by adenovirus, polyoma, and Maloney virus (Allison et al., 1967; Allison and Law, 1968; Gaugus and Rees, 1968; Vredevoe and Hays, 1969; Nehlsen, 1971a,b), duplicating in many ways the effects of neonatal thymectomy. Thymectomy prevents the development of Moloney leukemia ( the thymus participates and is required) ;


43

however, thymectomized ALS-treated mice developed reticulum cell sarcoma at the site of ALS inoculation (Allison and Law, 1968). In the case of syngeneic transplantable tumors, B. Fisher et al. (1969) and E. R. Fisher et al. (1969a) have shown that ALS treatment increases take, growth rate, and the incidence of metastasis. An example of this effect is shown in Fig. 3 where a transplantable Balb/c myeloma grew faster and to a larger size in ALS-treated animals (E. M. Lance, previously unpublished observations; Mandel and DeCrosse, 1969). Tumor induction by chemical carcinogens is affected by ALS. Balner

TINT IN DAYS

FIG. 3. Growth curves of a syngencic myeloma (RPC 23) in Balb/c mice. Mice were treated with repeated injections of either antilyinphocyte sernni ( ALS ), antiplasma cell serum ( APCS), or nomial rabbit sernm (control), beginning day - 1 and continued on alternate days nntil day 17. On clay 0, 15 x lo* inyeloma cells were introduced subcntaneously in all animals. The APCS my have produced slight but insignificant inhibition of growth, whereas the growth curve of ALS-treated mice is significantly enhanced.

44

E. M. LANCE, P. R.

MEDAWAR,

AND R. N. T A D

and Dersjant (1969) found a more rapid rate of induction and growth of methylcholanthrene ( MCA) tumors in ALS-treated mice, although the final incidence of tumor was comparable to controls (see also Wagner and Haughton, 1971; Grant and Roe, 1969). Others have reported an increased incidence of MCA tumor induction in ALS-treated animals ( Cerilli and Treat, 1969; Rabbat and Jeejeebhoy, 1970). Nevertheless, Balner (1971) found the incidence of X-ray-induced leukemia to be reduced in animals treated with rabbit but not horse ALS. Nagaya and Sieker (1969a,b) found that ALS treatment did not influence the development of leukemia in AKR mice. C. HUMORAL IMMUNITY Antilymphocytic serum inhibits the primary response to a wide variety of antigens, both soluble and particulate (Gray et al., 1964; Denman et al., 1966; Berenbaum, 1967; Atai and Kelley, 1967; James and Anderson, 1967, 1968; James and Jubb, 1967; Pichlmayr et al., 1967c; Guttman et al., 1968; Barth and Southworth, 1968; Moller and Zukoski, 1968; Reithmuller et al., 1968; Muschel et al., 1968; Lance, 1967, 1970a). Those antigens most closely studied have been Salmonella antigens, bovine serum albumin (BSA), and sheep erythrocytes, and the effect includes not only a reduction in the quantity of antibody but also a delay in the kinetics of the response. In summary, the principal findings of these studies indicate that, although the primary response may be suppressed, the degree of inhibition depends on several factors, i.e., the amounts of antigen and ALS used and the timing between ALS and antigen exposure. The relative timing between ALS and antigen is critical, for ALS given prior to antigen is effective, whereas ALS given with or after antigen is largely ineffective. Increasing doses of ALS exert continuously greater inhibition upon the primary response, but at least for “strong” immunogens, even massive doses of ALS do not completely abort this response. The effect of ALS can be countered to some extent by increasing the dose of antigen. Not all antigens are equally susceptible to ALS. The response to keyhole limpet hemocyanin ( KLH ) or pneumococcal polysacchai-ide may, in fact, be augmented (Bauni et al., 19f39; Taub et al., 1969; Baker et al., 1970; Barth et al., 1971). The IgG component of the primary response is suppressed to a greater extent than is the IgM response (Lance, 1968d; Bauni et al., 1969), a finding reminiscent of the defect in thymectomized, irradiated animals (Taylor et al., 1967). The possibility that some of the effects of ALS are due to interference with macrophage function must be considered (Barth et al., 1969a,b; Marshall and Knight, 1969). Chare and Boak (1970) considered these effects to be a secondary feature of ALS administration.


45

The effect of ALS on the secondary response was far less than that on the primary response. In some cases slight immunosuppression was observed, but in others no suppression was found. It would appear that ALS affects primarily the antigen-sensitive cell. The crucial importance of timing of ALS injection on the primary response and the insensitivity of the secondary response provide the chief evidence for believing that this must be so, for once the chain of events launched by antigen recognition is under way, ALS exerts little or a greatly reduced effect. The studies of Martin and Miller (1968) and Barth ( 1969) strongly support this conclusion. The relative insensitivity to suppression by ALS of the primary response to so-called thymusindependent antigens and the ability to restore competence to thymusdependent antigens by the infusion of thymocytes (Miller and Mitchell, 1968; Leuchars et al., 1968; Martin and Miller, 1968) suggest that the thymus-derived antigen-sensitive cell is the chief target of action, whereas the antibody-producing cell is unaffected. Jeejeebhoy ( 1971) found both cell types to be affected. That the immunosuppressive action of ALS can be overcome by increasing the dose of antigen is siniilar to the effect observed by Sinclair and Elliot (1968) after neonatal thymectomy. If, as Mitchison (1970) proposes, the thymus-derived, antigen-sensitive cell cooperates with the antibody-producing cell by effecting a local concentration of antigen, then a rational explanation of the effect of antigen dosage in these two situations is readily available. Finally, ALS may bc used to abet the induction of tolerance to BSA ( Lance, 1970a).

D. ERASURE OF MEhfORY Levey and Medawar (1966b) reported that mice previously sensitized by skin allografts could be restored to “virgin” reactivity by treatment with ALS, but Russell and Monaco were unable to duplicate these results (1967). Lance studied this phenomenon in animals which had been sensitized to BSA and in others previously grafted with skin across the H-2 barrier (1968b). The dosage and schedule of administration of ALS after immunization were identical in both systems, With respect to BSA, all animals responded to a subsequent challenge dose with titers above those characteristic of a primary response, i.e., in no case had erasure of memory occurred. Although some of the animals treated with ALS gave secondary responses lower than those of normal rabbit serum ( NRS )-treated controls, sensitization was evident since the challenge dose (100 pg.) would not be expected to provoke a detectable response by the technique employed when given to mice de nouo (Mitchison, 1964).

46


Animals sensitized to transplantation antigens and treated with ALS thereafter rejected second skin allografts at a tempo characteristic of firstset rejection, i.e., as if they had not seen these antigens before. Memory in this system did not return even when long periods of time were allowed to elapse between ALS treatment and challenge. In a repeat of this experiment with a less potent ALS second-set rejection was modified but not ablated-these animals rejected the challenge allograft at a pace intermediate between first- and second-set rates. These results are important not only because they document an unique immunosuppressive attribute of ALS but also because they hold out hope that in certain clinical situations where immunosuppression is desired, already immune patients may be amenable to treatment. The way in which ALS achieves these effects is speculative, but the following explanation was offered. In skin allograft rejection, the effector cell is a circulating lymphocyte and is highly susceptible, therefore, to the action of ALS. The pace of rejection reflects the number of cells in the recirculating population capable of responding to graft antigens. Immunization increases the proportion of specifically reactive cells; ALS destroys this population, and the regenerant population reflects the original reactivity of the host. In humoral immune systems, the effector cell (or at least a considerable portion) remains sequestered in host lymph node or spleen where it enjoys a measure of protection from ALS treatment and, consequently, a second dose of antigen confronts a still enriched population of cells capable of specific response. VIII. lmmunogenicity of Antilymphocytic Serum Immunoglobulin G

The potential immunogenicity of ALS-IgG has both practical and theoretical implications. The subject has been most thoroughly studied in mice where the lack of immunogenicity of proteins of the IgG class is well-established (Dresser, 1962, 1965). By contrast the highly immunogenic character of ALS-IgG has been repeatedly documented (Lance and Dresser, 1967; Clark et al., 1967; Denman and Frenkel, 1967; Guttman et al., 1967b; James and Anderson, 1968; Iwasaki et al., 1967; Pichlmayr et al., 1968c; Lance and Medawar, 1968; Jasin et al., 1968; Kashiwagi et al., 1968, 1969) after administration to a wide variety of animal species including man. Recent experiments by Amemiya et al. (1972) suggest that immunogenicity may be reduced by treatment with Takaprotease; unfortunately potency was also severely reduced. However, a state of nonreactivity to ALS-IgG can be induced either by prior paralysis with NRS-IgG or by the%administrationover a long time of closely spaced doses of ALS (high zone paralysis) (Lance and Dresser, 1967; Lance and Medawar, 1968; Pichlmayr et al., 1968c;


47

Howard et al., 1968, 1969; Gewurz et al., 1971; Mergciihagen and Howard, 1970; Wood, 1970). The possibility that ALS might prevent reactivity against itself (Monaco et al., 1966c) has not been confirmed by the experience of others (Lance and Dresser, 1967; Currey and Ziff, 1966; Clark et al., 1967). The inimunogenicity of ALS-IgG might be explained in one of two ways: it could act as a nonspecific adjuvant by virtue of the “normal” 7-globulins or the adherence of IgG molecules to lymphocytes drawing the attention of iiiacrophagcs could be the decisive factor favoring immunity in contrast to the paralysis induced by normal (nonadherent) IgG (Howard et nl., 1968). The first possibility can be discounted, because doses of ALS-IgG that induced immunity to normal IgG still caused immunosuppression with respect to other antigens. The immunogenicity of ALS raises the question whether its in viva effects might not be due to antigenic competition (Adler, 1964). However, this possibility is eliminated by the finding that the prior induction of paralysis to IgG did not diminish but rather enhanced the therapeutic effect of a subsequent dose of ALS (Lance, 1967; Denman and Frenkel, 1967; Gaugas and Rees, 1968; Raju and Grogan, 1969). In contrast, either active or passive immunization to IgG curtailed the expression of potency of subsequently administered ALS (Lance, 1967; Judd et al,, 1969; Hardy et al., 1970). Under these circumstances ALS-IgG is rapidly cleared from the circulation ( Lance and Dresser, 1967), suggesting that the maintenance of a titer of circulating antibody is important for the expression of ALS activity and emphasizing the importance of preventing immunization to IgG in ALS recipients. Considering the very rapid rate of elimination of IgG in immunized animals, it is remarkable that under these circumstances ALS should be effective at all (Lance, 1967; Levey and Medawar, 1966a; Raju and Grogan, 1969). The clear implication of this result is that the critical reaction between lymphocyte and antibody can take place extremely rapidly and that prolonged persistence of ALS antibodies is not necessary. This review correlates well with the short biological half-life of the relevant, antilyinphocytic IgG ( ALg ) antibody molecules even in iionimmunized animals (see above). In practice the immunogenicity of ALS-IgG has two undesirable consequences: it creates the hazard of serum sickness and reduces effective immunosuppression. The clinical correlates will be fully developed below. IX. Discriminate Action of Antilymphocytic Serum an Cell-Mediated Immunity

In this section we shall summarize the evidence that ALS has a greater inimunosuppressive effect when directed toward cell-mediated

48


immunological reactions (rejection of transplants, delayed skin hypersensitivities, GVH reactions, etc. ) than those mediated by circulating antibodies. In as much as some of the evidence collated here has been presented elsewhere, detailed reference will be made only to new data. In fairness it must be mentioned that not everyone will accept the conclusions in this section (James and Anderson, 1968), although the evidence presented contra is not incompatible with these conclusions. From the outset it was apparent that ALS administration to experimental animals was remarkably free from infectious complications, suggesting that ALS did not interfere with defense mechanisms against common bacterial pathogens (presumably largely mediated by humoral antibodies and nonlymphoid cells) (see Medawar, 1968). However, because of the difficulty inherent in comparing directly two such different processes, it was hard to establish objective criteria of relative effects. Levey and Medawar (1967a) pointed out that the effects of ALS on cell-mediated and humoral mechanisms could be contrasted by reference to an external standard. They stated that a 0.25-ml. dose of ALS led to prolongation of skin allograft survival in mice equivalent to that obtained after 6OOR of whole-body irradiation. This same dose of ALS was virtually without effect on humoral immunity, whereas the suppression of humoral immunity by 600 R was profound. A. SUPPRESSION OF RESPONSES IN VIRGIN ANIMALS Although ALS can inhibit the primary response in both cell-mediated and humoral reactions it is interesting to contrast the requirements in these two types of immune response. Timing is critical for humoral immunities-for ALS to be effective it must be administered prior to or simultaneously with antigen. If ALS is given after the antigen, its suppressive effect is largely lost. On the other hand, one of the distinctive properties of ALS in transplantation systems is that it is capable of inhibiting rejection when first given after the reaction has already begun. A second contrast is that increasing the dose of antigen in humoral antibody systems appears to mitigate the effects of ALS. An analogous situation has not been observed in the suppression of cell-mediated immunities by ALS. Lance and Medawar (1969) have found that skin allografts of sizes over a tenfold range do not influence the tempo of rejection in ALS-treated mice. Perhaps the most striking evidence bearing on this point stems from the observations of responsiveness of mice chronically treated with low doses of ALS (Lance, 1968a). In this experiment, virtually indefinite survival of skin allografts across an H-2 barrier was achieved, while animals maintained normal responsiveness to heterologous serum proteins


49

and to primary immunization with Salmonella typhosa H antigen. Lance and Batchelor (1968) reported a very interesting dissociation of responsiveness in these animals which elaborated circulating antibodies to skin graft isoantigens but, at the same time, had neither gross nor histological evidence of a cellular response to the very same allograft. Beverley and Simpson (1970) found a similar dissociation in ALS-treated mice bearing hamster tumor xenografts. However, Monaco and Franco (1969) did not observe dissociation in their studies, Finally, the humoral response to some antigens (those completely or relatively thymusindependent) may not be suppressed at all, but enhanced; no parallel exists for cell-mediated immune reactions where transplantation reactions over the widest possible ranges are susceptible to suppression.

B. EFFECT m SENSITIZEDANIMALS The effect of ALS on the response to antigen in sensitized animals is even more strikingly divergent for the two types of immunity. Resistance of the secondary humoral response is heavily documented, and the susceptibility of the second-set reaction manifested most vividly in the abrogation of immunological memory is one of the outstanding characteristics of ALS. Lance (1969) studied the discriminative action of ALS by parallel assays of cell-mediated and humoral immune processes in the same animals. The results of one of these experiments is reproduced in Table VII. Two groups of CBA mice were immunized by S . typhosa H antigen and were also allowed to reject bilateral C57BL/ 6 skin grafts. After immunization, one group was treated with ALS and the other received comparable amounts of NRS. Large doses of cells (roughly 1O* cells/recipient ) prepared from the pooled lymph nodes and spleens of these animals were injected intravenously into either (CBA X C57BL/6) F, adult hybrids or CBA female mice which had been exposed to 600 R of whole-body irradiation the day before. Cells from the ALS-treated animals were markedly inferior in causing a GVH response when compared with cells from untreated or NRS-treated donors, On the other hand, no reduction was evident in the capacity to transfer adoptively the secondary response to H antigen. A second experiment was slightly different in design. Bovine serum albumin was substituted for H antigen and the IgG fraction of either ALS or NRS was used. The experimental animals were normal mice or mice primed to BSA or C57BL antigens. The humoral antibody response was measured in both intact animals and an adoptive cell transfer system. Cells from animals treated with NRS-IgG mounted a vigorous GVH response in hybrid recipients, and splenic enlargement was greater when the cell donors had been presensitized. The ALS-IgG treatment caused a drastic reduction

TABLE VII ADOPTIVETRANSFER OF CELL-MEDIATED AND HUMORAL ANTIBODYRESPONSES ~

(CBA X C57BL/6)F1

Cell donors

Treatr mento

Cells transferred

Cell recipients

ALS

2. CBA sensitized to C57BL/6 skin and S. typhosa H antigen

NRS

3. CBA sensitized to C57BL/6 skin

-

4.

a

125 Million spleen and lymph node cells/recipient 125 Million spleen and lymph node cells/recipient 125 Million spleen and lymph node cells/recipient

CBA (600 R) (H titer logz)

~_________

~

1. CBA sensitized to C57BLj6 skin and Salmonella typhosa H antigen

No.

Average Splenic spleen index weight (mg./gm.)

(CBA X C57BL/6)Fi

178

6.4

-

-

14.7

10.1

-

-

15.2 -

CBA (600 R)

10

(CBA X C57BL/6)Fi

10

CBA (600 R)

10

(CBA X C57BLj6)Fi

10

272

10.2

(CBA X C57BL/6)Fi CBA (600 R)

20 10

121 -

4.3

272

ALS, antilymphocytic serum. See text for details of timing and dosage. NRS, normal rabbit serum.

-

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Advances in Immunology Volume 45

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