Advances in Immunology Volume 35

ADVANCES IN

Immunology VOLUME 3 5

CONTRIBUTORS TO THIS VOLUME

DENNIS J . BEER STEPHENT. CREWS RICHARDDOUGLAS PATRICIA J. GEARHART LEROY HOOD DAVID R. JACOBY NELSON JOHNSON STEVENM . MATLOFF NADINENIVERA ROBERTJ. NORTH LARSB. OLDING MICHAELB. A. OLDSTONE ROGERM. PERLMUTTER Ross E. ROCKLIN JOHN ROGERS GREGSORENSEN HANSL. SPIEGELBERG RANDOLPHWALL

ADVANCES IN

Immunology BY

EDITED HENRY G. KUNKEL

FRANK J. DIXON

The Rockefeller hiversify New York, N e w York

Scripps Clinic and Research Foundation La Jollo, California

VOLUME 35

1984

ACADEMIC PRESS, INC. (Harcourt Brace Jovonovich, Publishers]

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London

Slo Poulo

COPYRIGHT @ 1984,

BY

ACADEMIC PRESS,INC.

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LIBRARY OF

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NUMBER:6 1 - 1 705 7

ISBN 0-12-022435-6 PRINTED IN THE UNITED STATES OF AMERICA 84 85 86 87

9 8 7 6 5 4 3 2 1

CONTENTS

CONTRIBUTORS ....................................................... PREFACE ............................................................ HENRYG K U N K E L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix xi

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The Generation of Diversity in Phosphorylcholine-Binding Antibodies

ROGERM. PEHLMUTIXR,STEPHENT. CREWS,RICHAHI)DOCJGLAS, SORENSEN, NELSONJOHNSON, NADINENIVERA, IIOOD PATRICIA J. GEARHAR'I', AND LEROY

GREG

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. Restricted Nature of the Anti-PC Response. . 111. Clonotypes of Anti-PC Antibodies . . . . . . . . .

IV. Hidden Diversity of the Anti-PC Response. . . . . . . . . . . . . . . . . . . . . . . . V. T h e Structure of the Variable Regions of Antibodies Which Bind Phosphorylcholine and the Molecular Basis of Their Diversity . . . . . . . ...................... VI. Heavy Chain Variable Regions VII. Somatic Diversification of Heavy Chain Variable Regions. . . . . . . . . . . . VIII. Somatic Diversification Can Be Extensive an Boundaries of the VII G e n e . . . . . . . . . . . . . . . IX. Somatic Diversification Probably Occurs by a ..................... Mechanism. . . . . . . . . . X. Soniatic Mutation Prob That Is Localized in and around t h e V,, G e n e . . . . . . . . . . . . . . . . . . . . . XI. Light Chain Variable Regions . . . . . . . . . . . . . XII. Somatic Diversification of Light Chain G e n e s . . . . . . . . . . . . . . . . . . . . . XIII. T h e Pattern of Variation by Somatic Hypermutation ......... XIV. Soniatic Mutation Is Correlated with Innnunoglobuli XV. V H , D, and J I I Segments a n d Junctional Diversity XVI. Diversity in the VII S e g m e n t . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . ...................... XVII. Diversity in the JtI Segment XVIII. Diversity in the D Sekment.. . . . . . . . . . . . . . , ......................................... XIX. T h e N Region XX. J. G e n e Segments and Junctional Diversity . . . . . . . . . . . . . . . . . . . . . . XXI. Summary of Diversity of Antibodie XXII. Selection of Variant Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XXIII. Molecular Basis of the T I 5 Idiotype. . XXIV. T h e T15 VIIG e n e Family . . . . . . . . . . . . , . . . , . . . . . . . . . . . . . . . . . . . . . ..................... XXV. Evolution of the T15 G e n e Fariiily XXVI. Structural Diversity in Antihodies to P C . . . . XXVII. Future Research ......................................... ...., .... References . . . . . . . . . . . . . . . . . . . . . . . .

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V

6 7 7 15 16

17 18 19 20 22 22 23 23 25 25 26 26 27 29 29 30 31 33 35

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CONTENTS

Immunoglobulin RNA Rearrangements in 6 Lymphocyte Differentiation

JOHNROGERSAND RANDOLPH WALL I. 11. 111. IV. V. VI. VII. VIII. IX.

.

................................... Introduction. . . . . . . . . . Immunoglobulin Structrir ................................... Innnunoglobdins as Antigen Receptors on B Cells . . . . . . . . . . . . . . . . . Two mRNAs with Different 3' Ends Encode Membrane and Secreted p Heavy Chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . All Immunoglobulin Heavy Chain Genes Have Membrane Gene Segments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Model for the Transmembrane Insertion ............. Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Membrane and Secreted Heavy Chain mR Complex Transcription Units. . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . Coexpressed p and 6 mRNAs Are Coded by a Very Complex Transcription Unit. . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary: Developmental Regulation of Heavy Chain Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. .

.

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39 40 42 43 46

47 50 52 55 56

Structure and Function of Fc Receptors for IgE an Lymphocytes, Monocytes, and Macrophages

HANSL. SPIECELBERC Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Affinity for IgE and Structure of FceR on Lymphocytes and M+ . . . . . . Rosette Assays for Detection of FcsR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . FcsR+ Cultured Lymphocytes and MI$ and FUR+ Leukemic Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. FcsR+ B and T Cells in Nonatopic Healthy Humans. . . . . . . . . . . . . . . . VI. FcsR+ B and T Cells in Atopic Patients. . . . . . . . . . . . . . . . . . . . . . . . . . . VII. FcsR+ Lymphocytes in Normal and Parasitically Infected Rats and

I. 11. 111. IV.

...................................

VIII. FcsR+ Monocytes in Nonatopic and Atopic Humans . . . IX. FceR+ Rat and M O L I SM+. ~ . . .. . . . . . . . , . . . .. . . . . . . . . . . . . . . . . . . . . X. FcsR+ Eosinophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI. Conclusions: Induction and Function of FcsR on Lymphocytes and . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

61 62 65

68 69 71 74

77 79 80 81 85

The Murine Antitumor Immune Response and Its Therapeutic Manipulation

ROBERTJ. NORTH I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Analysis of Antitumor Immunity by Adoptive Immunization against Established Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. T h e Meaning of the Adoptive Immunization Assay . . . . . . . . . . . . . . . . .

..

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CONTENTS

IV . Analysis of Concomitant Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Tumor Innuunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii 122 133 142 153

Immunologic Regulation of Fetal-Maternal Bolance

DAVIDR . JACOBY. LARSB. OLDING. AND MICHAELB. A . OLDSTONE 1. Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I1 . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Proposed Mechauisms for Maintenance of the Fetus . . . . . . . . . . . . . . . . IV . Fetal Expression of Histocompatibility Antigens . . . . . . . . . . . . . . . . . . .

V . Immunologic Basis of Lymphocyte Interactions between Mother and Fetus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Human Maternal and Neonatal Lymphocyte Interactions . . . . . . . . . . . . VII . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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157 158 159 170 172 180 199 202

The Influence of Histamine on Immune and lnflammotory Responses

DENNIS J . BEER. STEVEN M . MATLOW. A N D Ross E . ROCKLIN

I . Histamine as an Autacoid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Histamine Modulation of Polymorphonuclear Inflammatory Cells . . . . . I11. Histamine Modulation of Iniinune Effector Cells . . . . . . . . . . . . . . . . . . . IV. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . INDEX ............................................................... CONTENTS OF PREVIOUS VOLUMES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

209 215 223 262 263 269 273

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CONTRIBUTORS

Nrimbers in parentheses indicate the pages on which the authors' contributions begin.

DENNIS J. BEER(209), Pulmonary Medicine Section, Evans Memorial Department of Clinical Research, Boston University School of Medicine, Boston, Masscichusetts 02118 STEPHENT. CREWS~ (l),Division of Biology, California Institute of Technology, Pasadena, Californici 91 125

RICHARDDOUCLAS~ (l),Division of Biology, California Institute of Technology, Pasadena, California 91 125 PATRICIA J. GEAHHART' (I),Divi-sion of Biology, California Institute of Technology, Pnsadenci, California 91 125 LEnoY HOOD(l),Division of Biology, California lnstitute of Technology, Pasadena, California 921 15 DAVID R. JACOBY (157),Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037

NELSONJOHN SO^ (I), Division of Biology, California Institute of Technology, Pasadena, Cnlifornia 91125 STEVENM , MArmtw (209), Allergy Division, Department of Medicine, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111 NADINE NIVERA~ (l),Division of Biology, California Institute of Technology, Pasadena, Californici 91 125 ROBERTJ. NoRm (89), Trudeau Institute, Inc., Saranac Lake, New York 12983

' '

Present address: Department of Pathology, Stanford University, Stanford, California 94305. Present address: Integrated Genetics, Framingham, Massachusetts 01707. -iPresent address: Department of Biochemistry, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205. Present address: Department of Biochemistry, State University of New York at Stony Brook, Stony Brook, New York 11790. Present address: Deparhnent of Biochemistry, Johns Hopkins University School of Hygiene and Public Health, Baltiniore, Maryland 21205.

'

ix

CONTRIBUTORS

X

LARSB. OLDING(157), Department of Pathology, University of Goteborg, Goteborg, Sweden MICHAELB. A. OLDSTONE (157),Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037 ROGER M. PERLMUTTER (l),Division of Biology, California Institute of Technology, Pasadena, Calqornia 91 125

Ross E. ROCKLIN(209), Allergy Division, Department of Medicine, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 021 11

ROGERS(39), MRC Laboratory of Molecular Biology, University Medical School, Cambridge CB2 2QH, England

JOHN

GREGSORENSEN (l),Division of Biology, Cal$ornia Institute of Technology, Pasadena, California 91 125 HANSL. SPIEGELBERC (61),Depnrtment of Immunology, Research l n stitute of Scripps Clinic, La Jolla, California 92037

RANDOLPHWALL(39), Molecular Biology Institute, and Department of Microbiology and Immunology, UCLA School of Medicine, LOS Angeles, California 90024

PREFACE

Progress in the field of inmunology continues at an ever-increasing rate and at every level of investigation. The once mystifying maneuvers of DNA as a prelude to antibody formation and the manipulation of RNA in the course of carrying out orders from the immunologic genome are now reasonably well understood. No longer do we need to be primarily concerned with the basis of antibody diversity nor with the mechanisms of translating genetic information into antibody molecules. The complicated events underlying the manifestations of immunologic diseases are becoming better understood in terms of the cell types involved, their regulation provided largely by products of immunocytes and their effector mechanisms. The complex interrelationship between a host and a partially incompatible graft in the form of either a conceptus or a neoplasm is also being elucidated. The most effective defense of a fetus against an immunologically sensitized mother appears to be conducted by fetal suppressor T cells, which fight their battle in the trenches of the placenta. As we learn more about the nature of antitumor immune responses and the reasons for their relative ineffectiveness, possibly strategies may be devised that can influence the outcome of the host-tumor struggle. These are the subjects addressed in this volume, and they represent exciting excerpts from the broad spectrum of immunologic research. Drs. Roger Perlmutter, Leroy Hood, and associates have chosen the murine antibodies directed against phosphorylcholine as a model system for studying the generation of antibody diversity, a subject to which they have been major contributors. In the first article they review the various elements contributing to diversity including the combinatorial association of heavy and light chains and joining of germline gene segments, the variable joining within gene segments, the appending of additional nucleotides to the D segment, and finally the somatic hypermutation operating coordinately on all V, J, and D gene segments. Together these mechanisms can generate a diverse repertoire of similar but distinct antibody specificities from a single germline V gene. The operation of these different diversity producing events is considered in the general context of B cell maturation, plating both molecular and cellular events in perspective. The role of posttranscriptional RNA processing in the regulation and differentiation of B lymphocytes is reviewed in the second article by Drs. John Rogers and Randolph Wall. Discovery of the membrane gene segment established the p heavy chain gene as the first example of a complex transcriptional unit in chromosomal DNA. This unit proxi

xii

PREFACE

duced two heavy chain p mRNAs with different 3' spliced structures coding for secreted and membrane bound forms. The authors, who were key movers in this development, predicted and later found a similar complex transcriptional unit responsible for secreted and membrane forms of all immunoglobulin heavy chains. The coexpression of IgM and IgD on the surfaces of early B cells was also found to involve a complex transcription unit encoding both p and 6 mRNAs. This transcription unit was developmentally regulated by the choice of multiple polyadenylation sites and by selective recognition and use of RNA splicing sites. Thus, posttranscriptional processing appears to be intimately involved in the changes in p and 6 expression by maturing B cells. Recent demonstration of similar mechanisms operating in species as different as yeast and rat would seem to establish the generality of posttranscriptional RNA processing in eukaryotic gene regulation. Although the binding of IgE to basophils and mast cells has been recognized for some time, the association of IgE with lymphocytes, monocytes, and macrophages is a more recent discovery. In the third article, Dr. Hans Spiegelberg summarizes the available data on the structure and function of Fc receptors for IgE on various immunocytes. Not only is the chemical nature of receptors for IgE on immunocytes quite different from that on mast cells but the strength of binding to the former is several magnitudes lower. The function of IgE receptors on immunocytes is not Entirely certain. However, the number of receptor positive cells, and probably receptors per cell, parallels the levels of extracellular IgE, suggesting that they are a part of the IgE response. On macrophages and monocytes, IgE receptors promote phagocytosis and killing of IgE coated targets and, in the presence of IgE complexes, induce release of phlogogens. On lymphocytes the role of IgE receptors is less clear, but there is some evidence for the hypothesis that receptor positive T cells may be involved in down regulating IgE synthesis by B cells. One of the great challenges in the field of immunology is the development of means to enhance host antitumor immunity. I n spite of a few promising but inconsistent leads, there is no generally successful antitumor immunostimulatory measure. One of the major difficulties in this field is our lack of precise knowledge of the immunologic hosttumor interaction during oncogenesis and tumor growth. In the fourth article Dr. Robert North proposes concomitant antitumor immunity, i.e., the development of transient, early T cell antitumor immunity that is soon negated by the generation of suppressor T cells, as a rational model for the analysis of natural tumor immunity and for the development of appropriate therapeutic manipulations. H e presents convincing evidence for the existence of such an antitumor response

PREFACE

xiii

in mice and then proposes means of potentiating or facilitating it to achieve the elimination of established syngeneic tumors. Several factors probably contribute to the persistence of a histoincompatible fetus during a long gestation period in an immunocompetent maternal host. However, since the mother clearly becomes sensitized to a variety of fetal histocompatibility antigens during pregnancy, and since maternal immunocompetence is not systemically suppressed, it seems likely that the mechanisms primarily responsible for fetal maintenance act locally at the placenta inhibiting the action of sensitized maternal lymphocytes. In the fifth article Drs. David Jacoby, Lars Olding, and Michael Oldstone review this field, focusing on the potent suppressor effects of fetal lymphocytes, a subject to which they have been leading contributors. Apparently fetal lymphocytes, via their suppression of maternal immune functions at the site of placentation, are the major protectors of the conceptus during gestation. In addition to its long recognized role as a vasoactive amine producing symptoms of allergic disease, histamine is now considered, together with prostaglandins and beta-mimetic catecholamines (the autacoids), as a regulator of both immune and inflammatory events. In the final article Drs. Dennis Beer, Steven Matloff, and Ross Rocklin review this field that has largely developed within the past decade. Histamine can be derived not only via the interaction of antigen with specifically sensitized mast cells, as in IgE reactions, but also by stimulation of sensitized effector T cells to make histamine releasing factor, which may provide a source of histamine in the absence of IgE mediated responses. Once available, histamine may act to modulate the immune response by activation of either or both suppressor and contrasuppressor cells with the result depending on the ratio of these two cell types activated. The effects of histamine on inflammation can also b e pro or anti. Its phlogogenic effects are achieved at least in part via T cells; these are stimulated to produce chemoattractant and migration inhibitory lymphokines that attract and hold lymphocytes and eosinophils at sites of inflammation. The antiinflammatory effects of histamine are achieved both by directly suppressing the action of cytotoxic T cells, natural killer cells, neutrophils, and eosinophils and indirectly via suppressor T cells. The latter may augment the production of prostaglandins by macrophages and monocytes, resulting in inhibition of effector T cells and thereby dampening cell-mediated immune reactions. As always, the editor wishes to thank the authors, who have given generously of their time and effort, and the publisher, whose staff does much to ensure a volume of high quality. FKANK J. DIXON

HENRYG. KUNKEL (1916- 1983)

HENRY G. KUNKEL (1916-1983)

Henry Kunkel’s untimely death has left a void in the field of immunology which will be felt in many ways. Among the people who will miss his advice and guidance most will be those of us involved with the Advances in Immunology, a series he coedited since 1967. As an editor, his ability to recognize the most significant movements in immunologic research, to identify those most expert in the area, and then to prevail upon them to write scholarly reviews was unexcelled. Much of the success enjoyed by this series is owed to his efforts. It is appropriate that in this volume of Advances we present a close and rather personal view of this remarkable man’s scientific career, and Dr. Hans Miiller-Eberhard, a long time associate and friend of Dr. Kunkel, has joined in its preparation. To a large extent Henry Kunkel was a self-made immunologist and clinical investigator. H e had no formal training in immunology or biochemistry, nor did he have any clinical specialty training. H e started his career at the Rockefeller Institute for Medical Research in 1945 in the field of liver disease. After the untimely death of his laboratory chief, he continued these investigations for several years making fundamental contributions to the diagnosis, prognosis, and treatment of liver cirrhosis. His interest in y-globulin, which was to continue to the end of his life, originated with the study of this disease. To acquire expertise in protein separation by electrophoresis and to prove himself worthy of appointment to senior rank at the Institute, he took a leave of absence and worked at the Biochemical Institute in Uppsala under Arne Tiselius. After a most successful year with Tiselius, Henry Kunkel was promoted to full Member of the Rockefeller Institute and was given a laboratory of his own at age 36. Henry Kunkel’s art of conducting science was to establish a fact by simple technology. His laboratory was austere, containing only the necessary basic equipment. The intellectual input was all that counted. He was exceedingly well read in the biomedical science literature and had a penetrating and critical mind. By association of seemingly unrelated facts and by informed intuition he was able to identify potential breakthroughs in immunology. In the first decade of his career as an immunologist, he showed that myeloma proteins are immunoglobulins, that 7 S and 19 S y-globulins are related but immunologically and chemically distinct proteins, and that rheumatoid facxv

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HENRY G. KUNKEL

tor is an autoantibody to IgG. H e discovered idiotypy of human antibodies and, in an interlude between phases of strict immunologic research, he described henioglobin A2 and its relationship to thalassemia. All these early advances were accomplished merely employing precipitin techniques and starch block electrophoresis and ultracentrifugation as the only high-technology tool. Henry Kunkel was an ingenious mentor of his research trainees and associates. Particularly in the earlier years h e set an example skillfully experimenting at the bench. H e was gifted in inspiring his people through long and frequent discussions conducted individually. He was a proponent of training in the philosophy of research which, he felt, involved questions concerning discipline of thought, intellectual integrity, respect for the written word, and the ethics of research work itself. He was masterful in creating an atmosphere in the laboratory in which fellows were compelled to go forward to eventual success or hopelessly fall behind. Tension in the laboratory was high at times and the admonishing reprimand “they will beat you to it” was a hard experience for a beginner in research and meant longer hours at the bench and greater mental effort. For some it meant humiliation and anguish. But good work, the exciting results of a “key experiment” that would “advance the field significantly,” were always met with a beaming face and eventually would lead to true recognition, respect, and often lasting friendship. Henry Kunkel trained many young physicians and scientists and he did so with a phenomenal success rate. It seems a fair estimate that at least twenty senior professors of leading medical schools and research institutions, among them one Nobel Laureate and four members of the National Academy of Sciences, trace back the beginnings of their careers to Henry Kunkel’s laboratory. Henry Kuiikel wrote lucidly, often pondering for a considerable time over the precise formulation of a sentence. His numerous publications attest to his talent as an author of scientific prose. Yet his spoken word could be sketchy, even vague, as though he was expecting the other person to know what he was talking about or to read his mind. He was impatient with ignorance, especially in relation to publications from his own laboratory. He was indignant with “shoddy” work published in the literature since his own high standards of performance did not allow publication of work unless it was thoroughly substantiated and documented: “one of our finest traditions in sci,, ence, h e wrote, “concerns the sanctity of the written word and the special pride involved in the avoidance of error. We should preserve it at all costs.”

HENRY G . KUNKEL

xvii

Henry Kunkel became a formidable leader and pioneer in the investigation of immune complex and autoimmune diseases in man. His abiding interest in antibody structure, function, and genetics which led to the elucidation of much of what is known today in this field, was later extended to studies of B cell-associated immunoglobulins and recently to the T cell antigen receptor. In recognition of his many fundamental contributions to immunology and medicine he received numerous awards and honors. He held an endowed chair at the Rockefeller University, the Abby Rockefeller Mauze Professorship, and he had been president of two learned societies, the American Society for Clinical Investigation and the American Association of Immunologists. Yet neither the honors bestowed on him nor his natural dignity and high self-esteem prevented him, when the occasion arose, from joining his associates and friends in merry socializing. He delighted in playfully poking fun at them and being the target of their humorous attacks. Such situations revealed the engaging warmth and the humanness of his personality. Henry Kunkel will be remembered as the gifted teacher and scientist he was, endowed with the drive and ability to be creative and to be productive throughout his life. He was dedicated to and excited by science. As he put it, “scientific inquiry is a sort of opiate that once experienced is not readily shaken off.” Those who knew him well in the scientific community, his students, colleagues, and friends, will behold his memory with the admiration and deep affection they had for him. HANSJ. MULLER-EBERHARD FRANK J. DIXON

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ADVANCES IN

Immunology VOLUME 3 5

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ADVANCES IN IMMUNOLOGY, VOL. 35

The Generation of Diversity in Phosphorylcholine-Binding Antibodies ROGER M. PERLMUTTER,' STEPHEN T. CREWS,2 RICHARD DOUGLAS,3 GREG SORENSEN, NELSON JOHNSON: NADINE NIVERA? PATRICIA J. GEARHARTt5AND LEROY HOOD Division of Biology, California Institute of Technology, Pasadena, California

I. 11. 111. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII. XVIII. XIX. XX. XXI. XXII. XXIII. XXIV.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . Restricted Nature of the Anti-PC Response. . Clonotypes of Anti-PC Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . Hidden Diversity of the Anti-PC Response. . . . The Structure of the Variable Regions of Anti Phosphorylcholine and the Molecular Basis o Heavy Chain Variable Regions. . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . Somatic Diversification of Heavy Chain Variable Regions Somatic Diversification Can Be Extensive and Extends Be Boundaries of the VII Gene. Somatic Diversification Prob Somatic Mutation Probably Occurs by a Hypermutational Mechanism That Is Localized in and around the VIIGene . . . . . . . . . . . . . . . . . . . . . . Light Chain Variable Regions. . . ........................ Somatic Diversification of Light es . . . . . . . . . . . . . . . . . . . . . . The Pattern of Variation by Somatic Hypermutation . . Somatic Mutation Is Correlated with Immunoglobulin Vll, D, and Jll Segments and Junctional Diversity.. . . . . . . . . . . . . . . . . . Diversity in the VII Segment.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diversity in the J11 Segment ........................ . ......... Diversity in the D Segment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The N Region. ............................................. J. Gene Segments and Junctional Diversity . . . . . . . . . . . Summary of Diversity of Antibodies That Bind PC . . . . . Selection of Variant Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................. Molecular Basis of the T15 Idiotype . ............ The T15 VII Gene Family . . . . . . . . . . . . . . . . . . . . .

6 7 7 15 16

17 18

19 20 22 22 23 23 25 25 26 26 27 29 29

Recipient of New Investigator Award AI-18088 from the National Institute of Allergy and Infectious Diseases. Present address: Department of Pathology, Stanford University, Stanford, California 94305. Drs. Crews and Perlmutter contributed equally to this review. Present address: Integrated Genetics, Framingham, Massachusetts 01707. Present address: Department of Biochemistry, State University of New York at Stony Brook, Stony Brook, New York 11790. Present address: Department of Biochemistry, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205. 1 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-0224356

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ROGER M. PERLMUTTER E T AL.

XXV. Evolution of the T15 Gene Family.. .............................. XXVI. Structural Diversity in Antibodies to P C . . ......................... XXVII. Future Research., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ....................................................

30 31 33 35

1. Introduction

The ability of higher vertebrates to mount an immune response to a seemingly infinite variety of distinct antigens has attracted the attention of biologists for decades. I n particular, immunologists have struggled to explain the extraordinary diversity of antibody molecules. During the early part of this century, Landsteiner’s classic analysis documented the exquisite specificity of immunoglobulins, which, for example, could clearly distinguish between identical chemical structures substituted at different positions on a phenol ring (Landsteiner, 1945). These early results prompted “instructionist” theories which viewed antigen as a template around which antibodies would fold. Beginning around 1960, structural analysis of antibody polypeptides defined the kappa, lambda, and heavy chain families, and the sequence studies of Hilschmann and Craig (1965) and Putnam (reviewed in Putnam et al., 1971) identified light chain variable (V) and constant (C) regions. Viewing these data, Dreyer and Bennett suggested with admirable foresight that antibody heavy and light chains are encoded by more than one gene, thus anticipating the noncontiguoils nature of eukaryotic genes and the DNA rearrangements which are central to the formation of antibody coding regions (Dreyer and Bennett, 1965). More detailed structural analysis revealed that the amino terminal variable regions of both heavy and light chains contain three short segments of hypervariability (Wu and Kabat, 1970; Capra and Kehoe, 1974). These hypervariable regions were shown by X-ray crystallography to comprise the antibody combining site (Padlan et al., 1973; Amzel et al., 1974), whereas the remaining portions of the variable region are relatively invariant in structure and hence are called “framework” regions. In the early 1970s the problem of antibody diversity was approached at the nucleic acid level. Hozumi and Tonegawa (1976), using cDNA probes for murine kappa light chains, were the first to show that the constant and variable region-encoding segments of antibody genes are separated by intervening DNA in the germline but are more closely juxtaposed during B cell differentiation. Four separate coding regions, leader (L), V, J, and C, for lambda genes were identified (Brack et al., 1978) and a similar analysis was carried out on

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3

murine kappa chains (Seidman et al., 1979; Sakano et al., 1979).Hood and co-workers demonstrated that heavy chain genes include an additional gene segment, the D or diversity segment (Schilling et al., 1980) which encodes a portion of the third hypervariable region and thus two separate rearrangement events are required to generate a functional heavy chain gene (Early et al., 1980; Sakano et al., 1981). In addition, the expression of multiple isotypes with the same variable region structure was found to involve another type of DNA rearrangement, the class switch, which juxtaposes a fully assembled VH-D-JH unit with a 6, y, a,or E heavy chain constant region gene located 3’ to the p heavy chain gene on chromosome 12 (Davis et al., 1980). Thus germline DNA, typically isolated from sperm cells, contains multiple dispersed gene segments which undergo rearrangement in B lymphocytes to yield a functional immunoglobulin coding region. Cells of the lymphocyte lineage are the only known cells which demonstrate immunoglobulin gene rearrangements. Examination of antibody genes clearly demonstrated the elegant strategies which permit a limited number of coding sequences to direct the synthesis of millions of different antibodies. Protein sequence and DNA or “Southern” blotting experiments [which permit the identification of restriction endonuclease-digested DNA fragments containing immunoglobulin genes via electrophoretic separation and subsequent hybridization with appropriate 32P-labeledprobes (Southern, 1975)] indicate that only a few hundred light and heavy chain variable region gene segments probably exist in germline DNA. Each variable region gene is constructed through the combinatorial joining of the V and J, in the case of light chains, or the V, D, and J gene segments, in the case of heavy chains. Thus combinatorial joining of gene segments further expands the germline repertoire. Flexibility in the site of joining of the V, D, and J gene segments provides a second mechanism by which the germline information encoding antibodies is amplified (Max et al., 1979; Sakano et al., 1979; Weigert et al., 1980). Finally, the combinatorial association of thousands of different heavy and light chains provides an additional level of information amplification in the formation of antibody combining sites. The significance of these strategies for the generation of antibody diversity is outlined in Table I. Differentiation of pleuripotent lymphocyte stem cells into pre-B cells is marked by rearrangement of heavy chain genes, the earliest event in the formation of B cells (Maki et al., 1980). Light chain gene rearrangement occurs subsequently with K chain rearrangement apparently preceding A chain rearrangement (Hieter et al., 1981). The

4

ROGER M. PERLMUTTER ET AL.

TABLE I GERMLINE AND COMBINATORIAL STRATEGIES FOR THE GENERATION OF ANTIBODY DIVERSITY Germline gene segments -250 V, 4 J. 2 VA 3 JA -250 VII -10-20 D 4 Jii Combinatorial joining 250 V, X 4 J x = 1000 V, genes 250 VII x 10 D x 4 J I I = 10,000 VIIgenes Combinatorial Association

1000 K x 10,000 H = lo7 antibody molecules

resultant B cells, once stimulated with antigen, may terminally differentiate into plasma cells secreting large quantities of antibody protein. The structural analysis of antibody genes was achieved for the most part using murine myeloma tumors, generally induced by intraperitoneal administration of mineral oil (Potter, 1972), as a source of cells “frozen” at the level of plasma cell differentiation. More recently, hybridomas generated through the fusion of a nonsecreting plasmacytoma with hyperimmune spleen cells have served as a ready source of monoclonal antibodies (Kohler and Milstein, 1976). In order to examine the generation of antibody diversity in greater detail, we studied a model immune response to a simple, well-characterized immunogen. The murine antibodies directed against phosphorylcholine (PC) which we discuss in this article proved an ideal system for delineating the fundamental mechanisms which generate the immunoglobulin repertoire of higher vertebrates. II. Restricted Nature of the Anti-PC Response

Phosphorylcholine is the immunodominant determinant in vaccines derived from certain rough pneumococcal strains (e.g., R36A, Leon and Young, 1971). Early serologic analyses of murine antibodies raised against pneumococcal vaccine showed that the heterogeneity of anti-PC antibodies is restricted in several respects: (1) greater than 90%of the induced antibody is IgM (Lee et al., 1974), (2)the affinities

DIVERSITY IN

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5

of these antisera for PC are nearly homogeneous as measured by hapten inhibition of plaque formation (Claflin and Davie, 1974a), and (3) the majority of induced anti-PC antibodies bear idiotypic determinants related to those present on a prototype PC-binding BALB/c plasmacytoma protein, T15 (Lieberman et al., 1974). Furthermore, neonatal administration of anti-T15 idiotypic antibodies completely abrogates the anti-R36A pneumococcal response in mice (Cosenza and Kohler, 1972). Taken together, these data suggested that the murine response to PC is essentially monoclonal. 111. Clonotypes of Anti-PC Antibodies

Using more refined analyses based on idiotypy and isoelectric focusing of antibody light chains, Claflin, Davie, and others were able to define several related clonotypes of anti-PC antibodies in mice immunized with pneumococcal vaccine or with PC coupled to protein carriers via a hydrophobic spacer (e.g., PC-KLH, Gearhart et al., 1975a; Claflin and Rudikoff, 1976). Lieberman et al. (1974) generated an allogeneic anti-T15 serum which identified the majority of anti-PC antibodies derived from Ig-la allotype mice (e.g., BALB/c) but which failed to recognize anti-PC antibodies raised in mice of other allotype groups (the allotypes are serologically defined polymorphisms in heavy chain constant region genes). Close linkage of this immunoglobulin heavy chain variable region marker with a well-defined heavy chain constant region marker reinforced the speculation that the T15like antibodies were encoded in germline DNA. A complementary antiserum was later raised against anti-PC antibodies from Ig-1'' mice (Lieberman et al., 1981). Other idiotypic markers include a hapteninhibitable xenoserum which identifies most anti-PC antibodies in all mouse strains (Claflin et al., 1974b) further emphasizing the structural homogeneity of these molecules. Four serologically distinct PC-binding plasmacytomas have been isolated, and immunization of mice with pneumococcal vaccine yields low levels of anti-PC antibodies resembling three of these, M511, M167, and M603, as well as the majority of induced anti-PC antibodies which resemble T15 (Ruppert et al., 1980). Light chain isoelectric focusing provided further evidence of a limited clonal repertoire of anti-PC antibodies encoded in the germline (Claflin and Rudikoff, 1976). IV. Hidden Diversity of the Anti-PC Response

Although hapten inhibition profiles of murine anti-PC antisera were consistent with the thesis that the PC antibodies constitute a highly

6

ROGER M. PERLMUTTER ET AL.

restricted family of antibodies, studies of monoclonal anti-PC antibodies generated using a splenic focus technique revealed a large number of different antibodies distinguishable by affinity for PC and PC analogs and by idiotype (Gearhart et al., 1975a). Under these circumstances, anti-PC antibodies of IgGl and IgGz, subclasses could be detected as well (Gearhart et al., 197513) although anti-PC antisera contain mainly IgM and some IgG3 antibodies (Perlmutter et al., 1978). Thus while serologic analyses indicated that a small family of murine antibodies encoded by germline genes formed the entire antiPC response, limited clonal analysis suggested that the total antibody repertoire was rather heterogeneous and perhaps in part generated by somatic mutation (Gearhart et al., 1975a). In this context, we chose to apply protein and DNA sequencing strategies to anti-PC hybridomas in hopes of elucidating the structural basis of antibody diversity. Determination of the complete variable region structure of M603 (Rudikoff and Potter, 1976), a PC-binding myeloma protein, and the construction of a high resolution electron density map of the Fab fragment of this antibody (Padlan et al., 1973) added further impetus to our study since the sites of antigen-antibody interaction could be determined. Specific contact residues in the hypervariable regions of the heavy and light chains (situated at the loops of the immunoglobulin fold) interact with the charged phosphoryl and choline moieties of the PC hapten. Of particular importance are tyrosine, glutamic acid, and arginine residues at positions 33, 35, and 52 of the heavy chain. Changes in antibody primary structure can be correlated to some degree with alterations in the affinity of PC binding of variant antibody molecules. V. The Structure of the Variable Regions of Antibodies Which Bind Phosphorylcholine and the Molecular Basis of Their Diversity

Further understanding of the structural diversity of PC-binding antibodies required the isolation and sequencing of monoclonal antibody variable regions and the isolation and sequencing of the genes which encode these antibodies. Fortunately, monoclonal antibodies that bind PC are available in abundance from myeloma and hybridoma sources. In addition, recent advances in recombinant DNA technology coupled with efficient methods for sequencing DNA have expedited the structural characterization of both the germline gene segments as well as the rearranged expressed genes encoding anti-PC antibodies.

DIVERSITY IN

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7

VI. Heavy Chain Variable Regions

Initially, the sequences of nine heavy chain variable regions from myeloma immunoglobulins that bind PC were determined by our group and by others (Rudikoff and Potter, 1976; Hood et al., 1977a,b). As shown in Fig. 1, four of these sequences are identical to a prototype sequence found in the heavy chain of the T15 myeloma protein. The remaining five sequences have from 1 (H8) to 14 (M167) amino acid substitutions compared with T15, half of which (15/29) are located in a region encoded by the D and JH gene segments; however, there is also considerable diversity scattered throughout the V H segment which encodes position 1-101 in the protein sequence. While five of the VH segments are identical, four have substitutions from the T15 prototype sequence. The variants differ from one another as well and include from one to eight substitutions compared with T15. Much of the diversity localized between residues 100 and 115 probably results from the flexible mechanism of V-D-J joining which we discuss in more detail below. In the following section, we focus on the mechanism that generates substitutions within VH segments.

VII. Somatic Diversification of Heavy Chain Variable Regions

Two general explanations might account for the observed sequence differences in VH segments: the variants might each be derived from distinct germline gene segments, or they might result from somatic diversification operating on one or a small number of germline gene segments. In order to distinguish between these alternative explanations, it was necessary to isolate and sequence all of the germline gene segments which might encode these variant protein sequences (Crews et al., 1981). Our laboratory constructed a cloned copy of the heavy chain variable region mRNA from the S107 myeloma tumor, the immunoglobulin of which shares an identical primary sequence with T15 (Early et al., 1979). Since all of the PC-binding VH segment protein sequences are quite similar, utilizing this clone as a hybridization probe we could detect all of the germline genes that could encode these VkI segments. A Southern blot of BALB/c sperm DNA digested with EcoRI and probed with labeled S 107 cDNA revealed four dominant hybridizing bands (Fig. 2). We utilized this S107 probe to screen a recombinant library of BALB/c sperm DNA cloned into a bacteriophage vector and

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DIVERSITY IN

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FIG.2. Southern blot analysis at the T15 VII gene family. At right is shown the pattern obtained when 10 pg of BALB/c sperm DNA is completely digested with EcoRI and the resulting fragments analyzed by Southern blotting (Southern, 1975) with a 32Plabeled probe complementary to the T15 VIIgene segment. A similar analysis using EcoRI digests of recombinant phage clones corresponding to the four major genomic bands is shown in the left lane. Band sizes are indicated in kilobases and the k clones containing each band are identified at the far left of the figure (modified from Crews et az., 1981).

were thus able to obtain at least one clone for each of the four major bands observed on the genomic Southern blot. Figure 2 also shows the comigration of the EcoRI fragments homologous to the S 107 probe in our phage clones when compared with the bands in the genomic blot. Comparison of the intensity of hybridization of known amounts

10

ROGER M. PERLMUTTER E T AL,

of the cloned DNA to the intensity of the bands on the genomic blot indicated that each genomic band contained at most one or two homologous VII gene segments. The complete nucleotide sequences of the cloned VH gene segments identified with the S107 probe were determined and the translated protein sequences of these were compared with our previously obtained myeloma heavy chain sequences (Fig. 3). One gene segment, labeled V1, encodes a protein that is identical to T15, thus confirming that this prototype heavy chain variable region is indeed a germline sequence (Early et al., 1980). Three additional, independently isolated clones with identical restriction maps also were found to contain identical V1 DNA sequences. This result adds support to the conviction that each hybridizing band on the genomic Southern blot contains only a single VH sequence complementary to the S107 VH probe. The remaining three germline gene segments isolated from the BALB/c library (designated V3, V11, and V13 in order of their characterization) are each greater than 13% different in amino acid sequence from T15. None of the variant myeloma heavy chain sequences is identical to any of these three VH gene segments and all are much closer to V 1 than to the other three gene segments (Fig. 3). Since we are confident that we have cloned all of the germline gene segments that could encode these VH regions, we conclude that the variant antibody segments are the result of somatic diversification processes operating on the germline V1 gene segment. Studies to be discussed subsequently have verified this conclusion. A more complete picture of the murine immune response to PC was developed through the analysis of a large number of anti-PC hybridomas (Gearhart et al., 1981).Unlike the mineral oil-induced plasmacytomas which produce mainly IgA antibodies, the hybridoma anti-PC antibodies include both IgM and IgG classes. Figure 4 shows the complete amino acid sequences of 11 hybridoma heavy chain variable regions in addition to the previously examined myeloma sequences. N-terminal sequences of a total of 42 heavy chains from anti-PC antibodies are shown in Fig. 5. All but one of these heavy chains are clearly encoded by the V 1 gene segment and those that differ from the germline sequence have undergone minor somatic diversification. As shown in Fig. 6, the single exception is HPCG15 which is similar to the translation of the V11 gene sequence and probably resulted from somatic diversification of that gene segment, differing from V11 at five positions of 90 residues positively identified. Thus although V1 is the principal VH gene segment utilized in the anti-PC response, with sufficient somatic alteration the V11 gene segment can encode a PC-

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DIVERSITY IN

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A-go%) of adult peripheral T lymphocytes; OKT4 reacts with a subset that provides T cell help in functional assays and comprises 50 to 60% of adult peripheral T cells; OKT8 marks a nonoverlapping population of adult peripheral T lymphocytes that are suppressor/cytotoxic in functional tests (Reinherz and Schlossman, 1980). Unfractionated peripheral blood lymphocytes from mothers at term, their infants, and unrelated adults have been analyzed for their distribution of these T cell phenotypes by indirect immunofluorescence and cytofluorographic analysis (Jacoby and Oldstone, 1983). Levels of OKT3+ T cells were similar in 26 pairs of maternal-cord blood lymphocyte samples and those from 33 unrelated adults. Of note, maternal-newborn samples differed significantly in the circulating levels of OKT8+ T lymphocytes. Of the 26 pairs investigated, 21 newborns had lower OKT8+ levels than their mothers. The lymphocytes from newborns were, on average, 20.5% OKT8+ compared to a 29.0% OKT8+ value obtained from their mothers. In contrast, OKT4+ levels did not differ significantly between newborns and their mothers; however, these values exceeded the proportion of OKT4+ lymphocytes in the peripheral blood of unrelated, nonpregnant adults. These studies corroborate those of Yachie et al. (1981)and Hayward and Kurnick (1981), who described significantly higher levels of OKT4+ lymphocytes and lower levels of OKT8+ cells in cord blood compared to peripheral T cells in unrelated adult samples. The OKT4+ : OKT8+ ratio is elevated in newborns relative to their mothers and unrelated adults, and gradually declines to adult levels as the infant reaches approximately 1.5 years of age. The decrease in this ratio parallels the progressive loss of suppressor activity exerted by newborns’ lymphocytes in uitro. The majority of resting peripheral T lymphocytes from adults do not express class I1 histocompatibility antigens (HLA-DRIMT loci), but may express these antigens after stimulation with mitogens, alloanti-

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DAVID R. JACOBY ET AL.

gens, or soluble antigens (Indiveri et al., 1980; Winchester and Kunkel, 1980; DeWolf et al., 1979). Monoclonal antibodies specific to this antigen framework (OKIal) demonstrate that only 3% of unstimulated adult T lymphocytes express DR antigens by immunofluorescence. This level increases to nearly 50% when T lymphocytes are cultured in the presence of PWM (Miyawaki et al., 1982). The expression of DR is largely restricted to the OKT4+ subset, because the reciprocal OKT8+ subset neither proliferates nor expresses these antigens after PWM stimulation. Cord T lymphocytes do not express class I1 histocompatibility antigens either in an unstimulated state, or after culture with PWM for 3 to 21 days (Miyawaki et al., 1982; Jacoby and Oldstone, unpublished results). Stimulated T lymphocytes from newborns at varying ages from birth onward begin to express DR antigens, reaching adult levels at approximately 2 to 3 years of age. Yachie et al. (1982) have shown in adults that OKT4+DR+lymphocytes are critical in activating OKT8+ suppressor cells in PWM-stimulated cultures. Although cord blood has a greater proportion of OKT4+ T cells, it appears to lack the OKT4+DR+ subset. C. ASSAYS OF SUPPRESSION BY CORDT LYMPHOCYTE SUBSETS 1 . Separation by Fc Receptors Human T lymphocytes in the peripheral blood have been shown to have receptors for the Fc fragment of IgG (Ty) or IgM (Tp) (Moretta et al., 1977). Adult Tp cells when added to B lymphocytes in PWMtreated cultures are capable of inducing differentiation and immunoglobulin synthesis. Ty cells added to these cultures suppressed immunoglobulin synthesis, even if PHA, Con A, or PWM was the mitogen (Moretta et al., 1977; Pichler and Broder, 1981). Oldstone et al. (1977), using depletion and reconstitution procedures, demonstrated that cord blood lymphocytes bearing the Ty phenotype were responsible for the suppression of maternal immunoglobulin synthesis. Using a 5- to 7-day PWM culture system, they showed in all eight of the mother-baby pairs studied that purified Ty cells caused the same increment of suppression as did the total cord blood lymphocyte population. Further, addition of Tp cells and macrophages did not enhance the degree of suppression. With a different assay, Hayward and Lydyard (1978)noted that Tp cells suppressed adult lymphocyte immunoglobulin synthesis. This apparent discrepancy of Ty and T p suppressors was resolved by experiments of Durandy et al. (1979), who demonstrated that both Ty and Tp cells suppressed adult B cell differentiation in PWM-stimulated cultures. Tp lymphocytes exerted

REGULATION OF FETAL-MATERNAL

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189

transient suppression on proliferation and PFC formation that ceased after 18 hours of in nitro cultivation. In contrast, Ty cells from newborns inhibited adult PFC formation by 85% in similar cultures, and the activity persisted during incubation for 5 to 7 days. Furthermore, cord blood Ty but not Tp cells inhibited the division of maternal lymphocytes in PHA-stimulated MLR. Hence, in the newborn, both Ty and Tp subsets define suppressor populations. The Tp suppressor cell is activated early and its action is transient. Suppressor activity of Ty cells occurs later and is longer lasting. In the adult, Ty cells can be induced to become suppressor cells only after the in vitro fixation of immune complexes (Moretta et al., 1977). Ty lymphocytes in cord blood are already in an active state, suggesting that immune complexes in maternal circulation (Masson et al., 1977) may activate fetal suppressor cells in vivo. However, treatment of neonatal Ty lymphocytes with trypsin or pronase to remove bound immune complexes did not abolish suppression of adult lymphocyte proliferation. Also, incubation of adult T lymphocytes in maternal or newborn sera did not induce suppressor activity, suggesting that cord blood suppressor lymphocytes may be primed by other mechanisms. 2. Fractionation b y Monoclonal Antibodies Cord blood T lymphocytes specifically depleted by monoclonal antibody and complement of cells expressing the OKT4 antigen (OKT4-, i.e., enriched for OKT8+ cells) are unable to exert a suppressor effect on IgG synthesis by maternal lymphocytes in PWM-treated cultures (Jacoby and Oldstone, 1983) (Fig. 3B). This remains true after incubation for 3, 5, 8, or 12 days and when the ratio of added cord blood T lymphocytes :maternal lymphocytes varies from 0.1 : 1 to 4 : 1. To the contrary, when cord blood lymphocytes are treated with OKT8 antibody and complement (OKT8-, enriched for OKT4+ lymphocytes) and cultured under similar conditions, potent suppression of IgG synthesis follows. This phenomenon is significant at low ratios of added neonatal T lymphocytes : maternal lymphocytes and increases as greater numbers of newborns’ T cells are added. The maximal degree of suppression observed is an average of 86% at the highest dose of added cord OKT8- cells (Table IV). These results and experiments by Yachie et al. (1981) indicate that cord OKT8- lymphocytes suppress B cell differentiation in mitogen-stimulated cultures. This outcome contrasts with those in functional assays of negatively selected adult lymphocytes in nitro. Adult OKT4- lymphocytes suppress proliferation, B cell differentiation, and immunoglobulin synthesis. Conversely, adult OKT8- lymphocytes provide soluble factors required for immunoglo-

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a Graded amounts of cord T cells added to 5 x lo5 maternal PBL in the presence of 15 pl/ml PWM. Cultured in U-bottom 96-well plates for 8 days in IgG secreted into the supernatant measured by human IgG ELISA. See Jacoby and Oldstone (1983)for experimental details. Suppression index (%) is calculated as 1 - (immunoglobulin synthesis of maternal PBL + cord T lymphocytes/immunoglobulin synthesis of maternal PBL) x 100. Negative indices indicate an elevated level of IgG synthesis. Mean 2 SD of four experiments.

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191

BALANCE

bulin synthesis when added to purified B lymphocytes, and boost the immunoglobulin synthesis of peripheral blood lymphocytes when added to PWM-treated cultures. Similar results with cord blood lymphocyte subsets were obtained from positive selection experiments. The functional profiles of newborn and maternal T cell subsets selected in this fashion differ substantially. OKT4+ lymphocytes and to a lesser degree OKT3" lymphocytes (total T) from mothers or unrelated adults, when added to autologous PBL, mediate a boost in IgG synthesis, indicating a predominate helper activity (Table V). In contrast, several ratios of OKT3+ and/or OKT4+ lymphocytes from newborns suppress the IgG synthesis by lymphocytes from their mothers. This suppression is comparable to that in the negatively selected OKT8- subset. Positively selected maternal OKT8+ cells suppressed autologous IgG suppression when added to cultures, confirming that maternal T lymphocyte subsets are functionally similar to those in nonpregnant adults tested in vitro. However, cord OKT8+ lymphocytes do not suppress IgG synthesis of maternal lymphocytes in uitro. The IgG secretion levels do not significantly differ when maternal lymphocytes are cul-

TABLE V T CELLSUBSETSFROM MOTHERS AND THEIRNEWBORNS HAVEDIFFERENT

FUNCTIONAL PROPERTIES Source of positively selected T cells

Ratio of'T cells : maternal PBL" in culture

0.5 Cord (baby)

Autologous (mother)

OKT3' OKT4' OKT8+

OKT3+

-

OKT4' OKT8+ -

Negative control (media alone)

ng/ml

1975 t 275" 1070 f 310 2165 5 370 1380 2 265 1245 f 180 1480 2 175 1650 f 275

2 p value