Molecular Genetics of Immunoglobulin (New Comprehensive Biochemistry)

MOLECULAR GENETICS OF IMMUNOGLOBULIN

New Comprehensive Biochemistry

Volume 17

Generml Editors

A . NEUBERGER London

L.L.M. van DEENEN Utreclzt

ELSEVIER Amsterdam New York

*

Oxford

Molecular Genetics of Immunoglobulin

Edifors

F. CALABI and M.S. NEUBERGER Medical Research Council Ltihoratory of Molecular Biology, Hills Road, Combridge C B 2 2 Q H , U K

1987 ELSEVIER Amsterdam New York

. Oxford

0 1987, Elsevier Science Publishers

B.V. (Biomcdical Division)

All rights reserved. No part of this publication may hc rcprocluced. stored i n a retrieval system, or transmitted i n any form or by any means, electronic. mechanical. photocopying. recording or otherwise, without the prior written permission of the Publisher. Elsevicr Science Publishers B.V. (Biomedical Division). P . O . Box 1527, 1000 BM Amsterdam. The Nethcrlands. No responsihility is assumed hy the Publisher for any injury and/or damage to persons or property as a matter of products liahility. negligence o r otherwise. or frcm any use or operation of any methods. products, instructions or ideas contained in the material herein. Because of the rapid advances in the medical sciences, the Publisher recommends that independent verification o f diagnoses and drug dosages should be made. S p e d rc7guluriotz.s f i i r reciders i i i rlic U S A . This publication has been registered with the Copyright Clearance Center. Inc. (CCC). Salem, Massachusetts. Information can he obtained from the CCC about conditions under which the photocopying o f parts of this publication may he madc in the USA. All other copyright questions. including photocopving outside of the USA. should be referred to the Publisher.

ISBN 0-444-XO915-5 (volume) ISBN 0-434-80.303-3 (series) Published by: Elsevier Science Publishers B.V. (Biomedical Division) P.O. Box 211 1000 A E Amsterdam The Netherlands Sole distributors lor thc USA and C;ln;da: Elsevier Science Publishing Company. Inc 51- Vandcrbilt Avenuc New York, NY 10017 USA Library of Congress Cataloging in publication Data Molecular genetics of inimunoglohulin (New comprehensive hiochcmistry ; v . 17) Includes bibliographies and index. 1, Immunoglobulins--Genetics. 2. Gene exprcssion. I . Calahi, F. (Franco) 11. Neuberger, M.S. (Michael S.) 111. Series. [DNLM: I . Gene Expression Regulation. 2. Immunoglobulins--genetics, WI NE372F v.17 / QW 601 M7181 QD4IS.N-IX ~ 0 1 . 1 7 571.19'2 s [616.07'9] 87-24302 (QR186.71 ISBN 0-444-80915-5 (U.S.) Printed in The Netherlands

V

Preface Immunoglobulin genes are not just of interest to immunologists. An understanding of the way in which DNA rearrangement and somatic mutation contribute to antibody diversity is of importance to a wide range of biologists. The cell-type specificity of immunoglobulin gene expression is of concern to many who are interested in gene expression in mammals. Furthermore, the immunoglobulin superfamily itself presents important questions to those interested in evolution. The analysis of immunoglobulins and of their genetics has advanced rapidly since the mid-l970s, mainly as a result of the application of recombinant DNA and monoclonal antibody technologies. The essential features of the molecular anatomy of both antibodies and their genes have been largely identified; this has resulted in significant insights into the way antibody diversity is generated. Clearly, much still remains to be elucidated in these areas, whilst studies both of regulation and of phylogeny are still in their infancy. We felt nevertheless that it was a good time to draw together what we do know about the molecular genetics of immunoglobulin. We wish to thank the authors for contributing to this volume and the publisher for prompt publication. Cambridge

Franco Calabi Michael S. Neuberger

This Page Intentionally Left Blank

Contents Preface .................................................................................................... . . List of abbreviations ................................................................................ Chapter I Structure and function of antibodies D.R. Burton (Sheffield, U K ) . . . . . . . . . . . . . . . . . I. 2.

Introduction .... .... ....................... Structure of IgG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. General considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Domain structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Structure of Fab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Antigen recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Structure of Fc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. The hinge: IgG subclasses . . . . . . . . . . . . . . . . . 2.7. Isotypes, allotypes and idiotypes. . . . . . . . 3. Functions of IgG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . ...... 3.2. Interaction with protein A . . . . . . . . . . . . ......................... 3.3. Complement activation ................................. 3.4. Interaction with cellular .................................. 3.5. Other functions of IgG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Rheumatoid factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Membrane or surface IgG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. Structure-function relationships in IgG: domain hypothesis . . , . , . . , . . , , . , , . , . , 4. Structure o f other immunoglobulins in relation t o IgG . . . . . . . . 5 . Structure and function of I g M . . . . . . . . . . . . . . . . . . . . . . . . . ......... 5.1. Structure of IgM. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Functions of IgM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Membrane IgM . . . . . . . . . . . . . . . . . . . . . . . . . ........ 6. Structure and function of IgA . . , . . 6.1. Structure of serum 6.2. Structure of secret 6.3. Functions of IgA ...................

v XII

1 1 3 3 6 6 8 11

21 21 22 24 26 27 27 28 36 37 39 39 39 41 41

VIII 7 . Structure and function of IgD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . Structure and function of IgE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . l . Structure o f IgE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 . 2 . Functions of IgE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

41 43 43

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

46 47

Chapter 2 Genes encoding the immunoglobulin constant regions M . Briiggemann (Cambridge. UK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

51

1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Chromosomal localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Organization of constant region genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Mouse heavy chain genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Mouse light chain genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . 3 . Human heavy chain gencs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Human light chain genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Other species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Switch regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Membrane exons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......................................

1 constant region genes . . . . . . . . ............. 4.1. Heavy chain genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2. 4.3. 4.4. 4.5. 4.6.

Light chain genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polymorphism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pseudogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aberrations and malignancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

52 52 53 54 55 55 58 61 62 62 64 64 67 68

69 70 72

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

75 75

Chapter 3 Genes encoding the immunoglobulin variable regions P.H. Brodeur (Boston. MA. USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

81

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . V gene structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Gene families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Mouse VI, families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Human V,, families. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Mouse V, families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Human V, families ............................................ 3.5. Mouse V, families . . . . . . ................................. 3.6. Human V, families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

86 87 88 89 89

IX 4.

Genenumber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Number of mouse V genes 4.2. Number of human V genes

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

. ......

.. ...,.

, ,

..,.,

, , ,

.. ... .. .. ..,

. .. ..

5. Chromosome assignment .................. ... . .. .. ... . .. ... 6 . Gene organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Igh locus organization . . . . . . . . . . . . . . , . , . . , . . , . , , . , . . . . . . . . . , . , . . . . . . .. .. .... 6.2. I g K locus organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Igh locus organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Polymorphism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements , , , . . . . . . . . . . . . . . . . . . . . . . , . . . . . , , . , . . . . . . . . . . __ .. . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

90 90 02 94 95 95 99 101 101 105

105 106

Chapter 4 Assembly of immunoglobulin vuriuble region gene segmeitls M. Reth and L. Leclercq (Cologne, FRG and Paris, France) . . . . . . . . . . 111 1.

Introduction mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.1. Joining signals , . . . . . . . . . . . . . . . . . . . . . . . . , , . , , , , , , . . . . . . . . . . 2.2. Joining models . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . 2.3. Control of joining. . . . . . . . . . . . . . . . . . . . . . . , . , , , , , . , . . . . . . . . . . 3. Order of rearrangement events during B cell tlcvelopment . . . . . . . 3.1, Rearrangements at the IgH locus . . . . . . . . . . . . . . . . . . . . . 3.2. Rearrangements at the light chain loci . . . , . . . . . . . . . . . . . . 4. Allelic exclusion of immunoglobulin gcne expression . . . . . . . . . . .

.. .. ..

111 112 112

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

I14

.. ....

129

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . , , . . . . . . . . . . . . . . . . Kefcrences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13 1

Chapter 5 Immunoglobulin heavy chain cluss switching U. Krawinkel and A. Radbruch (Cologne, FRG)

I17

131

135

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

135

. ... . .. .. . .. . , .. ..

.

139

. . . . .. . .. ... . . . .. . ... . . . . . ... .. . . .. . . .. . .. . . . ... ... .. . .. ... . . ..... .. . .. . . .. . . . . .. ... .. . ... . ... .. .. ... .. .. ... , , , , . . . . . . . . . . . . . , . , . . . . . . . . . . . . . . . .

142 145

....

4.

3.2. Isotype commitment . . Molecular analysis . . , . . .

4.2. 4.3. 4.4. 4.5.

Long transcripts , . . . . . . . Class switch recombination Switch sequences . . . . . . . Switch recombination sites.

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

.. ... .. ... .. ... .. .,,

... ... . .

140

146

X 5 . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

147

Reviews . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

149 149

Chapter 6 Immunoglobulin gene expression G.P. Cook. J.O. Mason and M.S. Neuberger (Cambridge . UK) . . . . . . . 153 I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ 2 . Tumours as models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Patterns of immunoglobulin gene expression during B cell ontogeny . . . . . . . . . . . . . . . . 3.1. Changes in chromatin structure . . . . . . .......................... 3.2. Expression of productively rearranged lo .......................... 3.3. Expression of aherrantly rearranged loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Expression of unrearranged loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Processes regulating immunoglobulin gene expression . . . . . . . . . . . . . . . . . . . . . . 4.1. Promoter upstream elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Enhancer elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Other promoter elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Transcription termination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. RNA cleavageipolyadenylation . . . . . . . . . . . . . . . ............... 4.6. RNA splicing . . . . . . . . . . . ............................... 4.7. Messenger R N A turnover . ................................ 4.8. Translational and posttransl ation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . Major aspects of cell-type specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Restricted cell-type specificity of immunoglobulin gene transcription . . . . . . . . 5.2. Control of the difference in mRNA abundance between B and plasma cells . . . . . . . 5.3. The relative abundance of membrane and secreted immunoglobulin . . . . . . . . . . . . . 5.4. Co-expression of two immunoglobulin classes . . . . . . . . . . . . . . . . . . . . . . . . . . . .

153 153 154 154 155 156

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

173

Chapter 7 The generation and utilization of antibody variable region diversity T . Manser (Princeton. NJ. USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

156

157 157 159 164 164 165 166 167 167 168 168 169 170 173

177

177 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 2 . Antigen independent diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 2.1. Combinatorial diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 2.2. Junctional diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 2.3. The multiplicative potential of combinatori 3 . Antigen dependent diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 3.1. The evidence for somatic mutation: a histor . . . . . . . . . . . . . . . . . . 186 188 3.2. Somatic mutation and the immune respons 190 3.3. Mechanistic considerations regarding somatic mutation . . . . . . . . . . . . . . . . . . . . . . 194 3.4. Somatic mutation and clonal selection theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 3.5. T cells and somatic mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 3.6. Antibody diversity and B cell subsets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

XI 4.

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Referenccs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 8 The immunoglobulin superfamily F . Calabi (Cambridge. UK) . . . . . . . . . . . . . . . . . . . . . . .

19X 198 198

203

1 . Introduction . . . . . ........ 2 . The immunoglobulin 3. The T cell receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. The a/p T cell receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. The $8 T cell receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Relationship between the expression of the aip and of the $8 T cell receptors . . . . . 4 . The major histocompatibility complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Overall structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Structure of the variable region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Structure of the constant region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Genetic basis of diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . Accessory molecules of the a/p T cell receptor (CD4 and CDX) .............. 6 . Other members . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Thy-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Poly-immunoglobulin receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Neuronal cell adhesion molecule (NCAM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Myelin associated glycoprotein/ncuronal cytoplasmic protein 3 (MAG/Ncp 3) . . . . . . 6.5. Viral members . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 . Evolutionary considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

203

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

233 233

Subject index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

241

206 207 215 218 219 221 221 224 224 226 228 229 230 230 231 231 231

XI1

List of abbreviations Ars BiP C region CDR D segment Fab

Fc FcR FR

H Hm Hs HAT HIV Ig IL-4 J segment J chain L LPS LTR MAG MHC N region NCAM N K cell

p-azophen ylarsonate immunoglobulin heavy chain binding protein constant region complementarity determining region diversity segment antigen binding fragment from papain digestion of immunoglobulin antigen binding fragment from pepsin digestion of immunoglobulin crystallizable fragment from papain digestion of immunoglobulin immunoglobulin Fc receptor framework region immunoglobulin heavy chain membrane form of immunoglobulin heavy chain membrane form of immunoglobulin heavy chain hypoxanthine, aminopterin, thymidine medium human immunodeficiency virus immunoglobulin interleukin 4 joining segment joining chain immunoglobulin light chain bacterial lipopolysaccharide long terminal repeat myelin associated glycoprotein major histocompatibility complex nucleotide region neuronal cell adhesion molecule natural killer cell

4-hydrox y-3-nitrophenylacetyl 2-phenyl-5-oxazolone PC phosphorylcholine Poly-IgR poly-immunoglobulin receptor RFLP restriction fragment length polymorphism RS rearranging sequence ( K locus) S region switch region T cell receptor TcR tk thymidine kinase V regionisegment variable regionisegment

NP

ox

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CHAPTER 1

Structure and function of antibodies DENNIS R. BURTON Department of Bioc-lzcwristry, Universi~yof Sheffirld, ShrJjjield S I O 2TN. U K

I . Introduction Antibody molecules are essentially required to carry out two principal roles in immune defence: (i) to recognise and bind to foreign material (antigen). In molecular terms this generally means binding to structures on the surface of the foreign material (antigenic determinants) which differ from those o f the host. Such antigenic determinants are usually expressed in multiplc copies on the foreign material, e.g. proteins on a bacterial cell surface. The host needs to be able to recognise a wide variety of different structures - it has been estimated that a human being is capable of producing antibodies against more than 10" different molecular structures. This is described as antibody diversity. (ii) to trigger the elimination of foreign material. In molecular terms this involves the binding of certain molecules (effector molecules) t o antibody-coated foreign material to trigger complex elimination mechanisms, e.g. the complement system of proteins, phagocytosis by cells such as neutrophils and macrophages. The effector systems are generally triggered only by antibody molecules clustered together as on a foreign cell surface and not by free unliganded antibody. This is crucial considering the high serum concentration of some antibodies. The requirements imposed on the antibody molecule by the functions (i) and (ii) are in a sense quite opposite. Function (i) requires great antibody diversity. Function (ii) requires commonality, i.e. it is not practical for Nature to devise a different molecular solution for the problem of elimination for each different antibody molecule. In fact the conflicting requirements are elegantly met by the antibody structure represented in Fig. 1. The structure consists of three units. Two of the units are identical and involved in binding to antigen - the Fab (fragment antigen binding) arms of the molecule. These units contain regions of sequence which vary greatly from one antibody t o another and confer on a given antibody its unique binding specificity. The existence of two Fab arms greatly enhances the affinity of antibody for antigen in the normal situation where multiple copies of antigenic determinants are presented to the host. The third unit - Fc (fragment crystalline) is involved in binding to effector molecules. As shown in Fig. 1. the antibody molecule has a four-chain structure consisting o f two identical heavy chains spanning Fab and Fc and two identical light chains associated only with Fab.

7

Antigen binding

Antigen binding

Complement triggering Cell receptor binding (Rheumatoid factor binding)

Fig. I . A schcinatic rcprcsentation o f antibody structure emphasising the relationship between structure ;ind function. The antibody molecule can be thought of in terms ol three structural units. Two Fab arms bind antigen and arc therefore crucial for antigen recognition. The third unit (Fc) binds effector inolecules triggering antigen elimination. The antihody niolccule thus links antigen recognition and antigen elimination. The structure is composed of four chains. Two identical heavy (H) chains span Fah and Fc regions and two identical light ( L ) chains arc a w x i a t c d with Fah alonc.

The five classes o f antibodies o r immunoglobulins termed immunoglobulin G (IgG), IgM, IgA. IgD and IgE differ in their heavy chains termed y, p, a , 6 and E , respectively. T h e differences are most pronounced in the Fc regions of the antibody classes and this leads to the triggcring of different effector functions on binding to antigen, e . g . IgM recognition o f antigen might lead to complement activation whereas IgE recognition (possibly of the same antigen) might lead to mast cell degranulation and anaphylaxis (increased vascular permeability and smooth muscle contraction). Structural differences also lead to differences in the polymerisation state of the monomer unit shown in Fig. 1. Thus, IgG and IgE are generally monomeric whereas IgM occurs as a pentarner. IgA occurs predominantly as a monomer in serum and as a dimer in seromucous secretions. T h e major antibody in the serum is IgG and as this is the best-understood antibody in terms of structure and function we shall consider it shortly. T h e other antibody classes will then be considered in relation to IgG. First, however. a very brief overview of the structure and function o f the different immunoglobulins will be presented [ 11. IgG is the major antibody class in normal human serum forming about 70% of the total immunoglobulin. I t is evenly distributed between intra- and extravascular pools. IgG is a monomeric protein and can be divided i n t o four subclasses in humans. It is the major antibody of secondary immune responses. IgM represents about 105k of total serum immunoglobulin and is largely confined t o the intravascular pool. I t forms a pentameric structure and is the predominant antibody produced early in an immune response, serving as the first line of defence against bacteraemia. As a membrane-bound molecule on the surface of B lymphocytes it is important as an antigen receptor in mediating the response of these cells to antigenic stimulation.

3 1gA forms about 15-20% of total serum immunoglobulin where it occurs largely as a monomer. In a dimeric complex known as secretory IgA (sIgA) it is the major antibody in seromucous secretions such as saliva, tracheobronchial secretions, colostrum, milk and genitourinary secretions. IgD represents less than 1% o f serum immunoglobulin but is widely found on the cell surfaces of B lymphocytes where it probably acts as an antigen receptor analogously to IgM. IgE though a trace immunoglobulin in serum, is found bound through specific receptors on the cell surface of mast cells and basophils in all individuals. It is involved in protection against helminthic parasites but is most commonly associated with atopic allergies.

2. Structure of IgG 2.1. Gerierul considerutions

In IgG the Fab arms are linked t o thc Fc via a region of polypeptide chain known as the hinge. This region tends t o be sensitive to proteolytic attack generating the basic units of the molecule as distinct fragments. The discovery of this action by Porter in 1959 [2] provided the first great insight into antibody structure. In 1962 Porter proposed a four-chain structure for the IgG molecule [ 3 ] .Since then, chemical and sequence analyses, notably by Edelman [4]. have confirmed a four-chain structure consisting of two identical heavy (H) chains of inolecular weight approximately 50000 and two identical light (L) chains of molecular weight approximately 25 000. The molecular weight of IgG is thus typically approximately 150000. The light chains are solely associated with the Fab arms of the molecule whereas the heavy chains span Fab and Fc parts as shown in Fig. 2. A single disulphide bond connects light and heavy chains and a variable number, depending on IgG subclass (see below), connects the two heavy chains. The latter connection is made in the hinge region of the molecule. Papain cleaves heavy chains to the amino-terminal side of these hinge disulphides producing two Fab and one Fc fragment. Pepsin cleaves to the carboxy-terminal side producing a single F(ab’), fragment and smaller fragments of Fc including a carboxy-terminal pFc’ fragment. The light chains exist in two forms known as kappa ( K ) and lambda (A); the forms arc distinguished by their reaction with specific antisera. In humans, K chains are somewhat more prevalent than A. in mice. A chains are rare [ S ] . The heavy chains can also be grouped into different subclasses. the number depending upon the species under consideration. In humans there are four subclasses having heavy chains labelled y l , y2, y3 and 74 which give rise to the I g G l , IgG2, IgG3 and IgG4 subclasses. In mouse there are again four subclasses denoted IgG1. IgG2a, IgG2b and lgG3. The subclasses - particularly in humans - have very similar primary sequences, the greatest differences being observed in the hinge region. The cxistence of subclasses is an important feature as they show marked differences in their ability to trigger effector functions. In a single molecule, the two heavy chains are identical as are the two light chains; hybrid molecules are not found.

4

5 Sequence comparison [6] of monoclonal IgG proteins, either myeloma proteins or more recently antibodies generated by hybridoma technology, indicates that t h e carboxy-terminal half of the light chain and roughly three quarters of the heavy chain, again carboxy-terminal, show little sequence variation between different IgG molecules. In contrast, the amino-terminal regions of about 100 amino acid residues show considerable sequence variability in both chains. Within these variable regions there are relatively short sequences which show extreme variation and are designated hypervariable regions. There are three of these regions or ‘hot spots’ on the light chain and three on the heavy chain. Since the different IgGs in the comparison recognise different antigens, these hypervariable regions are expected to be associated with antigen recognition and indeed are often referred to as complementarity determining regions (CDRs). The structural setting for the involvement of the hypervariable regions in antigen recognition is discussed below. Sequence comparison also reveals the organisation of IgG into 12 homology regions or domains [4] each possessing an internal disulphide bond. The basic domain structure is central to an understanding of the relation between structure and function in the antibody molecule and will shortly be taken up in some detail. However, the structure in outline form is shown in Fig. 2(b,c). It is seen that the light chain consists of two domains, one corresponding to the variable sequence region discussed above and designated the V , (variable-light) domain and the other corresponding to a constant region and designated the C, (constant-light) domain. The IgG heavy chain consists of four domains, the VH and CHI domains of the Fab arms being joined to the CH2 and CH3 domains of Fc via the hinge. Antigen binding is a combined property o f the V Land ~ V, domains at the extremities of the Fab arms and effector molecule binding a property of the C,2 and/or cH3 domains of Fc. From Fig. 2(b,c) it is also clear that all of the domains except for cH2 are in close lateral association with another domain: a phenomenon described as domain pairing or truns-interaction. The CI,2 domains have two N-linked branched car~~~~~

.

~~~

~~~

~

~~

- .

~

~~

Fig. 2(a-c). The 4-chain structure of IgG. ( a ) Linear rcpresentation. Disulphide bridges link the two heavy chains and the light and heavy chains. A regular arrangement of intrachain disulphide bonds is also found. Fragments generated by proteolytic cleavage at the indicated sites are represented. This representation should be interpreted in terms o f Fig. 2(b,c) for a fuller understanding. (b) Domain representation. Each heavy chain (shaded) is folded into two domains in the Fab arms, forms a region of extended polypeptide chain in the hinge and is thcn folded into two domains in the Fc region. The light chain forms two domains associated only with an Fab arm. Domain pairing leads to close interaction of heavy and light chains in the Fab arms supplemented by a disulphide bridge. The two heavy chains are disulphide bridgcd in the hinge (the number of bridges depending on IgG subclass) and are in close domain-paired interaction at their carboxy-tcrmini. (c) Domain nomenclature. The heavy chain is composed of V , , , C,,1, C,,2 and Cl13domains. The light chain is composed of V, and C , domains. All the domains are paired except for the CF,2 domains which have two branched N-linked carbohydrate chains interposed between them. Each domain has a molecular weight of approximately 13000 Icading to a molecular weight of -50000 for Fc and Fab and 1.50000 for the whole IgG molecule. Antigen recognition involves residues from the V I , and V,. domains, complement triggering the C,2 and Fc receptor binding the C,,2 and possibly the C,,3 domain (see text).

6

bohydrate chains interposed between them. The domains also exhibit weaker cisinteractions with neighbouring domains on the same polypeptide chain. Fig. 2 shows human IgGl in a Y-shaped conformation with the Fab arms roughly coplanar with the Fc. This choice is for illustration only - the relative orientations of Fc and Fab and the involvement of the hinge in such orientations is a complex problem with possible significance for the function of IgG subclasses as discussed below. As described in detail in Chapter 2. the three constant domains and hinge of the heavy chain and the constant domain of the light chain are encoded by separate exons. The variable domains arise from genetic recombination events.

2.2. Domain structure Crystallographic studies of whole IgG and fragments of IgG [7-91 have revealed that each domain has a common pattern of polypeptide chain folding depicted in Fig. 3 . This pattern, the 'immunoglobulin fold', consists of two twisted stacked psheets enclosing an internal volume of tightly packed hydrophobic residues. The arrangement is stabilised by an internal disulphide bond linking the two sheets in a central position. One sheet has four and the other three antiparallel p-strands. These strands are joined by bends o r loops which generally show little secondary structure. Residues involved in the P-sheets tend to be conserved while there is a greater diversity of residues in the joining segments. Fig. 3 shows the chain folding for a constant domain. The @-sheets of the variable domain are more distorted than those of the C domain and the V domain possesses an extra loop. 2.3. Structure of Fab The four individual domains are paired in two types o f close truns-interaction (Fig. 4) [103,17]. The V, and VL,domains are paired by extensive contact between the /

N el b6

Face Y (fy)

Fig. 3. Peptide chain folding o f a constant domain. The segments fxl-4 (unshaded) and fyl-3 (shaded) form two roughly parallel faces of antiparallel /3-pleated sheet linked by an intra-chain disulphide bridge (filled rectangle. Cys-L31-Cys-2(K) (C,I, human IgG I ) . Cys-261-Cys-321 (CJ), Cys-367-Cys-425 (CJ)). Between the p-pleated segments arc other scgments (bl-6) forming helices, bends and other structtires. Segments fx3. 1x4, f y l and b4 arc foreshortencd in this three-dimensional representation after Beak and Feinstein [76].

7

-I1

Fig. 4. Structure of a complex of the Fab fragment of an antibody molecule with antigen. The complex was formed between hen egg-white lysozyme and the Fab fragment of a mouse monoclonal anti-lysozyme antibody. The diagram shows the crystal structure at 2.8 resolution. Alpha carbon atoms only are shown. thick lines being used for lysozymc and the heavy chain o f Fab and a thin line for the light chain. The tight pairing of V,, and V, and of C,,1 and C, domains of Fdb is clearly seen in this diagram. The area of interaction between antihody and antigen is large. approximately 20 X 30 A. The region of Fab in contact with the antigen includes hypervariable loops from both heavy and light chains with more interactions involving the former. However. the combining site is not a simple cleft enclosed by the hypervariable loops but extends beyond them. The antigenic site recognised o n lysozyme is not a linear amino acid sequence - rather it is an arrangement of amino acids in three dimensions provided by different parts of the linear sequence. There is no cvidence for significant conformational changes occurring in either antigen or Fab on complex formation. (This diagram was very kindly provided by Dr. S . E . V . Phillips.)

two respective three-strand P-sheet layers (face Y , Fig. 3 ) and the C,l and C, domains by contact between the two four-strand layers (face X, Fig. 3). In both cases the geometry of pairing is such that the two domains are related by an approximate two-fold axis of symmetry. The interacting faces of the domains are predominantly hydrophobic and the driving force for domain pairing is thus the removal of these residues from the aqueous environment. The Fab arrangement is further

8 stabilised by a disulphide bond between CHI and C, domains. This bond covalently links the carboxy-terminal region of the C, domain with the e2 segment (human IgG1) or the b l segment (human IgG2,3,4) of the CH1 domain. These latter positions, although widely separated in terms of CHI sequence, are close in space conserving the essential structure of the molecule. V,-CH1 and V,-C, cis-interactions are very limited allowing flexibility about the V-C switch region or 'elbow bending' [11,12]. In the crystallographic analyses of Fab structures this is reflected in an elbow angle, i.e. angle between the V,-V, and CH1-C, pseudo two-fold axes, varying between about 137" and 180" [8].

2.4. Antigen recognition Further contact between V,, and V, domains in Fab is made by loops from each domain - the hypervariable loops or complementarity determining regions (CDRs) - which come together in space to constitute the antigen binding site [14,15,17]. It is essentially the extreme variability of these loops on the common framework of the immunoglobulin fold which provides for the enormous diversity of antigen recognition by antibodies while retaining the same basic structure. Fig. 5 illustrates variability in human light and heavy chain V regions to highlight the relationship between framework regions and CDRs. This traditional view of antigen surrounded by CDRs may be somewhat simplified. Thus, it is predicted for example that one of the light chain CDRs is nat generally in the antigen binding site [18], that framework residues can be involved in binding (see below) and that buried residues distant from the binding site may contribute to specificity [ 181. Nevertheless a 'CDR replacement' experiment using genetic engineering techniques indicates, for hapten binding at least, thk overwhelming importance of CDRs [112]. Thus, the CDRs from the heavy chain variable region of a mouse monoclonal antibody binding NP-cap (4-hydroxy-3-nitrophenacetyl caproic acid) were substituted for the corresponding CDRs of a human myeloma protein and the hapten affinity of the 'humanised' mouse antibody found to be very similar to that of the original mouse antibody. This elegant experiment also opens up the possibility of constructing human monoclonal antibodies from the corresponding mouse antibodies. Antigen binding sites have also traditionally been viewed as clefts in the antibody structure. There is n o a priori reason why this should be so - the antigen binding site could, for example, protrude and the cleft be found in the antigen molecule itself. The traditional view has probably arisen from studies on myeloma proteins where the natural antigen was not known but, by extensive screening, small molecules were found which bound to the antigen binding site. This view is challenged by the recently solved crystal structure of the complex of the Fab fragment of a mouse monoclonal anti-lysozyme antibody and lysozyme [16,17] shown in Fig. 4. The area of interaction between antibody and antigen is large, approximately 20 x 30 A, and is formed by relatively flat complementary surfaces on the two proteins. The region of Fab in contact with antigen includes CDRs from both heavy and light chains, with more interactions involving the former, but also extends to

9 All

human light chains

..

70

x

c

.-0 L

p

40 30 20 10 0 0

I0

20

30

40

50. 60

70

90

80

100. 110

Residue #

All

human heavy chains

1 I0

100 90 80 70 60

x

r -

n

50 40

’ 30 0

20

0

10

20

30

40

50

60

Residue

70

80

90

100

110

120

#

Fig. 5. Amino acid variability in the variablc domains of human immunoglobulin heavy and light chains. For a given position variability is defned as the ratio of the number of different amino acids found at that position to the frequency of the most common amino acid. The three CDRs are apparent as peaks in both plots and the four framework regions (FRs) as separating regions of relatively low variability. (This diagram from [6] is reproduced with the kind permission of Dr. E . A . Kabat.)

some framework residues. The third CDR of the heavy chain makes a particularly large contribution to antigen contact. The antigenic site recognised on lysozyme is not a linear amino acid sequence - rather it is an arrangement of amino acids in three dimensions provided by different parts of the linear sequence (‘topographi-

10 cal’ determinant). Whilst in this case there is no evidence for significant conformational changes occurring in either antigen or Fab upon complex formation, limited changes have been reported in a different Fab-protein antigen complex [130]. At the present time, the structure of only two Fab-antigen complexes have been solved and one has to be wary in drawing general conclusions. However, a number

Fig. 6 . Structure of the Fc fragment of human IgG. (*), Alpha carbon positions: (0). approximate centres of carbohydrate hexose units. Coordinates were obtained from the Brookhaven Data Bank (after Deisenhofcr [23]).The pairing of C,,3 domains and the position o f carbohydrate between C,,2 domains is clcarly seen in this view. The contact between carbohydratc chains is much more extensive in rabbit Fc. Note that the heavy chains are described only from residue 238: residues 725-238 do not show welldefined electron density.

11

of other complexes are currently being studied, and it is likely that general principles for the details of antibody-antigen recognition will emerge in the next 2-3 years. One general principle that has been suggested is that antibodies recognise mobile regions of protein antigens [ 19,201. Others have suggested that it is surface accessibility or protrusion which is the primary requirement for antigenicity [21,22]. The correlation of accessibility or protrusion and mobility then leads to the reported correlation of mobility and antigenicity.

2.5. Structure of Fc

In the Fc of IgG [9,23] the two cH3 domains are paired in a pattern similar to that found for the CHl-C,- interaction (Fig. 6). The two CH2 domains show no close interaction but have interposed between them two branched N-linked carbohydrate chains which make little contact between one another in human Fc [23] but more extensive contact in rabbit Fc [30]. In the pairing of the C,3 domains, approximately 1000 A' of surface per domain is involved in the interaction. In the Ck,2 case the carbohydrate provides a substitute for the domain-domain contact and helps to stabilise the C,2 domain. However, the CH2-carbohydrate contact area is only about half that of, for example, the cH3-cH3 contact so that one might expect a lower inherent stability for the cH2 domain. Indeed the cH2 domain is more sensitive to proteolytic degradation than the other domains of IgG [24]. Domain stability has also been related to the apparent 'softness' of parts of the cH2 domain most remote from the CH2-cH3 interface as indicated by large temperature factors or missing electron density in the crystal analysis of human Fc [ 2 3 ] .A corresponding 'softness' has not, however, been found for rabbit Fc (Sutton B.J.. personal communication). Tables 1 and 2 show a comparison of known cH3 and c H 2 sequences. The carbohydrate chains of the IgG C,2 domains are not a single oligosaccharide moiety but consist of a set of about 20 structures based on a mannosyl-chitobiose core which can be represented [13] as:

+ ~

i(h')

(-5')

(4')

Siaa2-+6Galpl~4GlcNAc~l+2Mana 1

2 Fu c a l

1 h

1 (3)

(2)

(7

(1)

? GlcNAc~1+4Man~l+4GlcNAc~l-+4GlcNAc-Asn-297 3

12 TABLE 1 Comparison of IgG C,,3 domain sequences Human IgGl [113], IgG2 [114], IgG3 11201, IgG4 (1151, mouse l g G l (1161, lgG2a ‘a’ allotype (1171, IgG2b ‘a’ allotype [1181, IgG3 (1261 and rabbit IgG [119] are translated from nucleotide sequences. Guinea pig (G.pig) IgGl and IgG2 [59] were protein sequenced. lgGl Eu numbering [6] is used throughout this chapter. A dot indicates no residue at the position corresponding to the numbered human IgGl residue. The right-hand column ( e l , fxl, etc.) indicates the approximate domain location of residues in human Fc and should be compared with Fig. 3. Alternate (mostly hydrophobic) residues iii the P-strands tend to be buried and show greater degree of conservation than residues o n bends. Cys-367 forms an intrachain bridge with Cys-425 close t o Trp-381. The asterisked arginine (R’) at position 435 in IgG3 highlights the involvement o f this position as an allotypic marker important in protein A (Section 3.2.) and rheumatoid factor (Section 3.6.) binding. Other positions at which allotypic substitutions occur in human IgGs are 356, 358, 379, 384, 392, 431 and 436 [123].

Human

Mouse

G.pig

G1

G2

G3

G4

G1

G2a

34 1 342 343 344 345 346 347 348 349 350

G Q P R E P Q

G Q P R E P Q

G Q P R E P Q

G Q P R E P Q

G R P K A P Q

G S V R A P Q

G L V R A P Q

Y T

Y T

Y T

Y T

Y T

Y V

35 I 352 353 3.54 355 356 3.57 358 359 360

L P P S R D E L T K

L P P S R E E M T K

L P P S R E E M T K

L P P S Q E E M T K

I P P P K E Q M A K

361 362 363 364 365 366 367 368 369 370

N Q

N Q

N Q

N Q

S L T

S IT

S L T

L v K

L v K

L v K

v

v c

v

v c

v

v

c

G2b

G3

G1

Rabbit G2

G

R A Q T P Q

G P P R I P Z

G A P R M P D

G Q P L E P K

Y I

Y T

Y L

Y T

Y T

L P P P E E E M T K

L P P P A E Q L S R

I P P P R E Q M S K

L P P P R B Z L S K

L P P S R D E L S K

M G P P R E E L S S

D K

K Q

K D

K K

K K

S K

S

S L T

S L T

T L T

S L T

S L T

S L T

S

T

S L T

L v K

M I T

M v T

L v V

L v T

M I T

L I I

M I N

v

v

c

v

v

c

v

v

c

v

v

c

G

v

v

c

v

v

c

v

v v

c

.el

>fxl

v

R

v c

< .bl

i >fx2

13 TABLE I (continued) Human

37 I 372 373 374 375 376 377 378 379 380

G2

G3

GJ

G F Y P S D

G F Y P S D

G F Y P S D

G F Y P S D

Rabbit

G.pig

Mousc

GI

GI

G2a

G2b

G3

GI

G2

G

r)

D F M P E D

cr

N F F

G F Y P A D

N F F P A D

G F Y P S D


fyl

F F 1’

E D

F N P G D

s

E A

I

I

I

l

l

I

I

I

I

I

I

A v E

A v E

A v E

A

7‘

v E

S v E

S V E

N v E

H v E

S

V E

Y v E

w

w

w

w

w

w

w

w

w

w

E S S G

E

S

N G

E S N G

0’1- ‘ I ’ E

w

E

N G

W N

D S S E

E K N G

386 387 388 389 390

Q P E N N

Q P E N N

Q P E N N

Q P E N N

Q P A E N

P

A S N R V P V

D . .

E K E

K A E D N

39 1 392 393 394 395 306 397 398 399 400

Y K T T P P V L D S

Y K T T P P M L D S

Y N T T I’ P M L D S

Y K T T I-’ P V L D S

Y K N T 0 P I M N T

Y K N

401 402 403 404 40s 406 407 408 409 410

D G S F F L Y S K L

D G S F F L Y S K L

D G S F F L Y S K L

D G S F F L Y S R L

N S Y F V Y S K L

D G S Y F M Y S K L

D G S Y F I Y S K L

411 412 413 414

T V D K

T V D K

T V D K

T V D K

N V Q K

R V E K

N M K T

38 1 382 3x3 384 385

S

C;

Ci

v E

N N G

S N G

R N

K

H T E E N

E L E Q D

Y K D T A P V

Y K N ‘I. P P I L D S

Y K N T P P V F D S

Y K N T P P I E D A

Y K T T P A V L D S

D T Y F L Y S K L

D E T F F L Y S R L

D G S Y F L Y S K L

D G S Y F L Y N K L

T V D T

K V D T

T V D K

S V P T

‘r E L N

.r E P V L D S

1-

D S

G

G

s

s

.b2


fx3


fx4

< .12s

14 T A B L E I (continued)

Mouse

Human

41s

416 417 418 419 420

42 I 422 423 424 425 426 427 428 429 430 43 1 432 433 434 435 436 437 438 439 440 44 1 442 443 444 435 446 447

G.pig

Rabbit

GI

G2

G3

G4

GI

G2a

G2b

G3

GI

G2

G

S R

S R

S R

S R

S N

K N

S K

D S

N A

S A

S E

Q

Q

Q

Q

E

V

E

L

N

D

Q

Q G

Q

Q G

E G

A Ci

E R

K

Q G

D G

Q G

R G

E

'r

w

w G

w

w

N

N

N

N

V

V

I

v

F

F

F

F

s c

s c

N

' F

s Y

w

7'

D

s

F

c

S

S

S

S

S

S

N

M H

M H

M H

M

L

V

R

E A

v

s c

N

7

w

s c

v

s c

w

v

v

-r

v

v

s

c

w

E

w

w

D V V > f y 2

1

s

F 1'

T

c.

c

'r c

S

S

S

M

M

F

v

v

v

V

w

Y

v

F T

c S

v

M

E

E

H E

H E

H E

H E

H E

H E

H E

H E

c

A L

A

A

A

.b6

H

L

H N H H T E K S

I4 N I1 H T O K N

L P N H

L

N H Y T Q K S

G L K N Y Y L K K T

L

N H Y T Q K S

G L H N H H T T K S

A

H

A A L L H H N N R ' H F Y T T Q O K K S S

G

L

H N €I

I Q K S

O K A

H N H Y T Q K S

L

L

L

L

F

I

L

I

I

I

L

H

R

R

R


fxl

i .hl

>fx2

v v v

v v v

v v v

v v v

v v v

v v v

c'

c v v v

c v v v

v v v

C

c v v v

c v v v


fx3

Q

B

K

R P P

29 I 292 293 294 295 296 297 298 299 300

P R E E Q Y N@ S T Y

P R E E O F N@ S T F

P R E E Q Y N@ S T F

P R E E Q F N@ S

P R E E O F N@ S T F

T H R E D Y N@ S T L

T H R E D Y N@ S T I

P R E A Q Y N@ S T F

V L Z Z Z F N@ G T F

P K V E Q Y N@ T T F

L R E Q Q F N@ S T I


fx4

R

s

w


fyl

w

< .b5

17 TABLE 2 (coininued) Human

G.pig

Mousc

Rabbit

G1

G2

G3

G4

GI

G2a

G2b

G3

G1

G2

G

316 317 318 3 19 320

G K E Y K

G K E Y K

G K E Y K

G K E Y K

G K E F K

G K E F K

G K E F K

G K E F K

u u u u

G K E F K

G K E F K

32 1 322 323 324 325 326 327 328 329 330

c

c

c

c

c

c

c

c

u

c

c

K

K

K

K

K

K

K

K

u

K

33 I 332 333 334 335 336 337 338 339 340

K

v

v

v

v

v

v

v

v

u

v

v

S N K A L P A

S N K G L P A

S N K A L P A

S N K G L P S

N S A A F P A

N N K D L P A

N N K D L P S

N N K A L P A

u u u u u U u

Y N K A L P A

H N K A L P A

P

P

P

s

P

P

P

P

u

P

P

l

I

I

l

l

l

l

I

I

I

I

E K T

E K T

E K T

E K T

E K

E R T

E R T

T R T

E K T

E K T

I

I

I

T

.r

E R T

l

I

l

I

I

l

>ry2

I

< .b6

>fy3

s

s

s

s

s

s

s

s

s

s

s


IgG2a > IgGl [40]. Similarly guinea pig lgG2 shows greater segmental flexibility than IgGl [42]. The flexibility has been associated particularly with the upper hinge [25,41]. There is an association between hinge flexibility and effector function seen in its extreme form by the lack of flexibility and loss of effector function associated with hinge-deleted IgG. However. flexibility is closely related to the proximity of Fab and Fc and it is difficult to establish the causal factor in modulating effector function. This will be discussed in greater detail below. The equilibrium conformation of the monomeric human IgG subclasses have

TABLE 3 Comparison of hinge sequences of IgG Sequence sources as in Table 1. Residues are aligned so that the positions of the first and last interheavy disulphide bridge cysteines coincide. In human IgGl, mouse IgGl and rabbit IgG, Cys-220 (Eu numbering) forms a disulphide bridge to the light chain and thus the residues 216220 belong structurally to Fab rather than the hinge. Crystallography indicates that Pro-238 is the first residue forming part of the folded C,2 domain so that the residues from Ala-231 to Gly-237 are assigned structurally to the hinge. Papain cleaves human IgGl at position 224, pepsin at position 234. The sequence of human IgG3 corresponding to the above is given in Table 6. I

I

;Human IgGl Human IgG2 Human IgG4 Mouse IgGl Mouse IgG2a Mouse IgG2b Mouse IgG3 G.pig IgG I G.pig IgG2 Rabbit IgG

216 E P E R E S V P E P E P E P Q S E P A P

K K K R R S R W I S

I 224 S C D K T H T . .

.

.

.

.

.

.

.

.

.

.

.

.

.

Y D G G I G R T

G C P P P H T C

. G T I K T P S

.

.

. .

. .

. .

. .

.

. I T . P M

. N P . B .

.

. . p . . p G S S . . . . . P

. . I K S T P S . . Z B K P

. P . . .

.

1

Middle

I-

.

.

.

C C C C C C C C C C

P C P K C P . P T .

P V P P P P . P C .

1st

inter-heavy

s-s

. . . . . E C P P . . . . . . C I . . . P K . . . C K E C H . . . . . C I P , . P K . . . .

.

.

.

.

.

.

C C C C C C . C C

.

.

. . . .

K .

.

P P P T P P P G P P

A A A V A A P A P P

P E P P P E P . P N P N G N P Z P E P E

234 L L V A F L . E L L L E I L L L N L L L

I I

G G G . G G . V G G G G G G G G G G G G

P P P S P P P P P P

last inter-heavy

s-s

I

I

Genetic hinge

II

I

I I

I -

After Burton. 1985 [2S] and Feinstein et al

I

Lower

I

I p p

I

I

'I -

Upper

,

1986 [41]

W

20

Fig. 7. Models proposed for the solution conlormations o f human IgG subclasses. The models are proposed on the basis of sedimentation data with supporting small-angle X-ray scattering data [48]. The hinge-delcted IgGl Doh protcin is used as a reference. IgG2 and IgG4 arc shown to resemble Dob in the close approach of Fat7 and Fc: for IgG2 thc Fab arms are suggested to fold back. In IgGl the hinge is apparent and the Fab arms are non-colinear. as illustrated. I n IgG3. the central or middle hinge (Table 6) is about 90 A long. i n agreement with electron microscope studies 1491.

been investigated by physical techniques including hydrodynamic and small-angle X-ray and neutron scattering studies [44,48]. There is more or less general agreement that IgG3 is a very extended molecule as predicted from modelling studies [9,49] with a long middle hinge (Table 6), although an alternative model has been suggested [46]. We have recently proposed the structures presented in Fig. 7 [48]. These structures, although average conformations for monomeric IgGs, do show interesting correlates with the functional activity of IgG subclasses in an associated state. For example, complement activation is most efficient for IgG3 especially and also for IgGl where the Fab arms are less likely to interfere with binding sites on Fc than in IgG2 or IgG4.

21

2.7. Isotypes, allotypes und idiotypes T h e variability of antibodies is often conveniently divided into three types. Isotypes a r e variants present in all healthy members of a species: immunoglobulin classes a n d subclasses are examples o f isotypic variation involving the constant region of t h e heavy chain. Allotypes are variants that are inherited as alternatives (alleles) with not all healthy members of a species therefore inheriting a particular allotype. Allotypes occur mostly as variants of heavy-chain constant-region genes, in man in all four IgG subclasses, IgA2 a n d IgM. T h e nomenclature of human immunoglobulin allotypes is based o n the isotype o n which it is found (e.g. G l m defines allotypes on a n IgGl heavy chain, Km defines allotypes o n K light chains) followed by a n accepted WHO numhering system. The positions of some allotypes a r e noted in t h e legends t o Tables 1-7. T h e variable region of an antibody can act as a n antigen, and the unique determinants of this region that distinguish it from most other antibodies of that species a r e termed its idiotypic determinants. T h e idiotype o f an antibody, therefore, consists of a set of idiotypic determinants which individually are called idiotopes. Polyclonal anti-idiotypic antibodies generally recognise a set of idiotopes whilst a monoclonal anti-idiotype recognises a single idiotope. ldiotypes are usually specific for a n individual antibody clonc (private idiotypes) but are sometimes shared between different antibody clones (public, recurrent o r cross-reacting idiotypes). A n anti-idiotype may react with determinants distant from the antigen binding site. it may fit t h e binding site a n d express the image of the antigen o r it may react with determinants close to the binding site and interfere with antigen binding. Sequencing of an anti-idiotypic antibody generated against an antibody specific for the polypeptide GAT antigen in mice revealed a CDR3 identical to that of the antigen, i.e. the anti-idiotype contains a true image of the antigen [ l l l ] .

3. Functions of IgG 3.1. Introductiori

T h e function of I g G in simplified terms is to recognise antigen and trigger its elimination. The molecular basis of antigen recognition has been discussed above. Here we shall discuss t h e interaction with molecules related to the elimination of IgG, principally those of the complement system and cellular receptors. These molecules interact primarily with the Fc part o f IgG, a n d strong variations in subclass reactivity is a notable feature. First we consider the interaction of IgG with staphylococcal protein A which, although not a functional interaction for the host, is a n Fc interaction which is understood in detailed molecular terms.

3.2. Interaction with protein A Protein A is a major cell wall component of most strains of Staphylococcus aiivem which binds to the Fc regions of immunoglobulins of a variety of subclass and spe-

22 cies with varying affinity (251. This reaction has been widely exploited in immunoand histochemistry. The protein consists of five homology regions binding to Fc and another region that does not bind to Fc but binds to cell walls [ 1241. Trypsin digestion can be used to isolate active fragments each corresponding to an homology region and of molecular weight about 7000 (SO]. The fragments bind to Fc with a stoichiometry of 2: 1. The multivalency of both IgG and protein A with respect to one another means that if the two are added to one another in the correct proportions they will form extended complexes and precipitate. The structure of a complex of one of the fragments (fragment B) with human IgG Fc has been solved crystallographically to 2.8 A [23] and indicates that protein A binds at a site between C,2 and C,3 domains. This binding is observed for the human IgG subclasses IgG1, IgG2 and IgG4 and also for IgG3 proteins bearing allotypic markers characteristic of Mongoloid populations ( 5 I ] . In IgG3 proteins from Caucasian populations the protein A contact residue His-435 of the above IgGs is replaced by Arg. This lengthy side chain prevents the formation of favourable IgG-protein A contact so that such IgG3 proteins do not bind protein A . Protein A binding at the C1.,2-C,,3 interface is not perturbed by deletion or reduction and alkylation of the hinge or by aglycosylation of IgG [25,26,24].

3.3. Coniplement activation The classical pathway of complement is a cascade system generating a variety of potent biological molecules including anaphylatoxins and chemoattractants and leading ultimately to lysis of antibody-coated cells [ S 2 , S 3 ] . In health. foreign cells will be the primary target. I n disease, host tissue may be attacked on a large scale. e.g. in autoimmune disorders. The pathway is triggered by the interaction of the first complement component, C1, with IgG in an associated state, i.e. coating a target cell or aggregated by antigen in an immune complex. The pathway is clearly not triggered by monomeric IgG which is at high concentration in the serum. C1 is a complex of the complement components C l q , C l r and Cls. It is the subcomponent C l q which interacts with the C,2 domain of IgG to initiate the enzymatic process of the pathway. Clq is a molecule having the appearance of a ‘bunch of tulips’ [52] and it is multivalent in its binding to IgG (Fig. 8). This multivalency is probably the key to why complement is only triggered by IgG in an associated form. Binding of C l q to monomeric IgG is only weak ( K , - lo4 M-’), whereas binding to associated IgG and the consequent use of two or more of the tulip heads makes binding much tighter (K;, 10’ M-’). and allows t h e activation process to proceed [25]. Theories of activation which involve binding of antigen to the Fab arms of IgG and the induction of conformational changes which are passed down the molecule to Fc, thus affecting the interaction with C l q , are now rejected by most workers [54,25]. The reasons for this rejection are many. In the first instance, C l q multivalency in itself appears an adequate explanation of activation by associated IgG and this is highlighted by the observation that isolated C l q heads bind to IgG aggregates with a similar affinity as intact Clq to monomeric IgG (5.51. Further, C l q binding and CI activation occur whether the association of IgG is

-

23

Fig. s(a.1~).(a) A schematic view of the binding of complement Clq to two IgG molecules on a cell surface. C l q is a hexavalent molecule o l molecular weight approximately 460000. It adopts a structure likened to a hunch o f tulips in which six collagenous stalk regions are connected to six globular head regions which contain thc IgG binding site. The dimensions for C l q used here correspond to longer 'arm' regions than those originally proposcd from electron microscope studies. There is evidence from physical measurements that the arms of CIq posscss some tlexibility. although this appears less in C1 than isolated C l q . Any flexibility of Clq may complement that of Fc in reducing steric requirements in the Clq-IgG interaction. (h) A model of C l q binding to dislocated IgG molecules. In this speculative model [lOS] it is suggested that hinge flexibility allows dislocation ol the Fah arms of the IgG molecule out of the plane of the Fc allowing Fc-Fc interaction. The model has similarities to that envisaged for Clq binding to IgM based on electron microscope studies.

achieved by antigen, heat or chemical cross-linking and, in the case of chemically cross-linked IgG, they are unaffected by combining site occupancy [25].Attempts to identify a conformational change in IgG upon antigen binding which correlates with Clq binding, have not been successful [54]. Finally, in the case of human IgG3, it is particularly hard to visualise II common conformational change being passed through the extended hinge region as the result of the binding of a wide variety of different antigens at the extremities of the Fab arms. The flexibility of Fc is complemented by some flexibility in the arms of the C l q molecule [56,58] which may be important in complement triggering. Thus, flexibility reduces the stringency of steric requirements when Clq binds to an array of IgG molecules. There is general agreement about the importance of charged groups in the interaction of IgG and Clq but controversy over the precise location of the Clq binding site on IgG [25].The domain responsible for C l q binding is concluded to be c H 2 [25] as the Facb fragment of rabbit IgG (lacking the C,3 domains) binds C1 with an affinity comparable to IgG and isolated CH2 domains bind C1 with an affinity comparable to Fc. Early interest in the cH2 domain centred on Trp-277 and nearby residues, but this was quenched by the demonstration that Trp-277 is

24 buried in the crystal structure of Fc. Three Clq binding sites have more recently been proposed. The first [S9] involves residues in an extended chain region, between Lys-290 and (3111-295 in human IgG1. the second [60] involves residues in the overlapping extended chain region between His-285 and Arg-292, and the third [82] involves residues on the last two anti-parallel /?-strands of the C,2 domain (Gln-318, Lys-320, Lys-322, Pro-331, (3111-333, Thr-335, Ser-337). IgGs of different subclass and species show differing affinity for Clq. and the above proposals attempt to account t o some extent for this in terms of sequence differences between isotypes. However, there is an added complication in that it appears that the proximity of Fab arms can modulate the expression of the Clq binding site on Fc [25]. Thus hinge-deleted IgGl does not bind C l q [34]. Further and more strikingly, IgG4 does not bind C1 whereas its Fc fragment (Fc4) does [61]. This is consistent with the close approach of Fc and Fab in IgG4 visualised in Fig. 7. In comparing isotype behaviour with respect to complement it is important to distinguish C l q binding. C1 binding, C1 activation and whole complement activation. The most widely used assay is the measurement of the end-product of the whole complement cascade, i.e. cell lysis. The inability of an IgG to promote efficient lysis does not necessarily indicate an inability to bind Clq. A later stage may be implicated. For example, it appears that Clq binding is not always directly related to C1 activation [62,63] and. furthermore. later components of complement, e.g. C4b, C3b also interact with IgG. A further complication is that a small change in C l q binding affinity may, through the amplification nature of the complement cascade process. produce a large change i n whole complement activation measured as cell lysis. Comparison of the human IgG subclasses provides the following view [64]. All the subclasses in a monomeric state bind Clq with measurable affinity with the order of binding constants IgG3 > lgG1 > lgG2 > lgG4. IgG3 and IgGl activate C1 and whole complement efficiently. IgG2 is less efficient in complement activation. IgG4 does not appear to bind C1 and does not activate complement. In other species, mouse IgG3, IgG2a and IgG2b, guinea pig IgG2, rabbit IgG, rat IgG2b, lgGl and IgG2a. bind Clq and activate complement. Mouse IgG1, guinea pig IgGl and rat IgG2c bind C l q weakly. if at all, and do not significantly activate complement [25,65,66]. Whilst it is clear that C1 interacts with associated IgG primarily through C l q , there have been suggestions that C l r and/or Cls may also interact weakly with sites on IgG (e.g. [67]) but this has not been clearly demonstrated [25]. Activated forms of C3 and C4 also bind to IgG [53] via covalent interaction with residues in the heavy chain of Fab. The former interaction is important not only in the classical pathway but also for IgGs such as rabbit IgG which activate the alternate pathway.

3.4. lnteractiori with cellular Fc receptors Receptors for the Fc region of IgG are found on a number of cell types and are associated with a variety of functions including phagocytosis (monocytes, macrophages, neutrophils), antibody-dependent cellular cytotoxicity (monocytes, mac-

25 rophages, lymphocytes), maternofoetal transport (trophoblast) and possibly immunomodulation (lymphocytes). ‘Fc receptor’ is an operational term and does not imply that the same molecular species is found on the different cell types [68]. Indeed, there is good evidence that a number of molecular species are involved. Human leucocyte IgG Fc receptors (FcR) fall into three categories [69] as defined by a number of criteria but most especially by reactivity with specific monoclonal antibodies. FcRI is a 72000 molecular weight receptor found on monocytes which binds monomer IgG with high affinity ( 5 x 10’ M - ’ ) . FcRII is a 40000 molecular weight receptor found o n monocytes, granulocytes, platelets and B cells which binds well to associated IgG but only very weakly to monomer IgG ( IgG4. The subclasses are shown to bind to the same receptor by competition experiments. IgG2 does not bind. The subclass specificity of FcRII and FcR,, has not been definitively demonstrated at this stage. Murine leucocyte Fc receptors show both similarities to and differences from human Fc receptors [69]. FcRl. found on mononuclear phagocytes, shows relatively high affinity (10’ M-I) for monomeric mouse IgGZa. FcRII, found o n mononuclear phagocytes, granulocytes and B cells, preferentially binds aggregated IgG2 and IgGl. This receptor has recently been cloned and found to belong to the immunoglobulin supergene family [129]. A third receptor binds mouse IgG3 specifically. Until recently, the conventional wisdom was that complement interacts with the cH2 domain and cellular Fc receptors with the cH3 domain of IgG. This has been challenged by a number of workers [25]. The binding of IgG to the human monocyte Fc receptor (FcRI) will be briefly discussed. The cH3 domain was originally favoured, owing to the (weak) ability of the pFc’ fragment of IgG (cH3 domain dimer) to inhibit the interaction of IgG and monocyte Fc receptor. Using domain-specific anti-human IgG monoclonal antibodies this has been shown to arise from contamination of pFc’ preparations by small amounts of parent IgG [3S]. In contrast, cH2 domain involvement in receptor binding is favoured by the loss of binding associated with hinge deletion [35] and aglycosylation of IgG [27]. Further, anti-human IgG monoclonal antibodies specific for the cH3 domain and the CH2icH3 domain interface do not inhibit the Fc receptor-IgG interaction and are still able to bind to receptor-bound IgG [70]. Antibodies specific for a cH2 epitope are inhibitory and do not bind to receptor-bound IgG. It has, therefore, been suggested that cH2 is the critical domain for monocyte receptor binding [70,71]. A similar conclusion is reached for mouse IgG2b binding to mouse macrophage Fc receptor. based on the use of mutant deleted proteins [72]. There is, however, still controversy in this area, which is complicated by the existence of the different types of Fc receptor [2S,69]. Based on a sequence comparison of IgGs showing differing affinity for the monocyte Fc receptor, the model shown in Fig. 9 has been proposed [71]. The IgG binding site is suggested to coniprise residues of the lower hinge. i.e. those between the hinge disulphides and the

26

Fig. 9. A model proposed for the interaction of IgG and human monocyte Fc receptor I. The model is proposed primarily on the basis o f a comparison of sequences of IgG of different species and subclass, showing differing affinity for monocyte Fc receptor [71]. The region o f interaction on Fc is suggested to involve that between the interheavy disulphides and the folded Ci12 domain. The monocyte Fc receptor has a molecular weight o f approximately 70000 and is represented as a globular protein of appropriate size. An antigenic surface is included in the diagram t o show the close approach of foreign cell and monocyte required by the interaction. although most studies have been carried out using unliganded monomeric IgG.

folded cH2 domain between Leu-234 and Pro-238 (Table 3), and possibly those on a nearby bend (Leu-328-Pro-33 1). Finally molecules secreted or shed by T cells which have specificity for IgG (IgG binding factors, IgG-BF) have been described [ 1081. 3.5. Other functions of 1gC

3.5.1. Clearance The molecular mechanisms by which IgG is removed from the circulation are referred to as clearance. The number, nature and relative importance of the molecular species interacting with IgG in this process are unclear as is the nature of ‘damaged’ or altered IgG subject to clearance. However, it is apparent that the cH2 domain is critical in control of clearance [25]. 3.5.2. Fc-fragment active peptide A number of immune responses including polyclonal antibody production, T-cell responses such as antigen and alloantigen-induced proliferation and cell-mediated

27 cytotoxicity can be mediated by subfragments of Fc (251. A peptide corresponding to residues 335-358 of human IgG1, a sequence which spans CH2 and C,,3 domains, has been shown to induce polyclonal antibody production [73]. 3.5.3. Fc-Fc interactions in immune precipitation Classical immunology explains the precipitin reaction in terms of the formation of lattices through the bridging of antibody combining sites by multivalent antigens. Recent evidence, however, also implicates Fc-Fc interactions as being important in immune precipitation. Thus it is found that IgG and its F(ab’), fragment behave very differently in immune complex formation under certain conditions. although avidity for antigen is identical [25]. 3.6. Rheumatoid factors Rheumatoid factors are autoantibodies directed against epitopes expressed in the Fc region of IgG. Although most patients with rheumatoid arthritis show a heterogenous population of rheumatoid factors, a common specificity has been detected in most rheumatoid sera. This antibody is of the IgM class and reacts with the CH2-C,3 interface region, showing many features in common with protein A binding [74]. 3.7. Membrane or surface I g C A small percentage (about 1%) of peripheral blood B lymphocytes express detectable levels of membrane-bound IgG on their cell surfaces, which functions a s an antigen receptor. In the case of mouse IgG, an additional 71-residue carboxyterminal segment is found on membrane IgG compared to secreted IgG [7S]. I t is suggested that a 17-residue highly charged region links the C,3 domain to a 26residue transmembrane region followed by a terminal 28-residue intracellular domain. 3.8. Structure-function relationships in I g C : domain hypothesis The domain hypothesis of Edelman et al. [4] states that ‘The internal homologies and symmetry of the molecule (IgG) suggest that homology regions may have similar three-dimensional structures each consisting of a compact domain which contributes to at least one active site’. The structural statement of the hypothesis is well validated. The functional statement is also broadly validated depending on interpretation of ‘contributes’. It does not, however, appear that the IgG molecule is functionally simply the sum of independent constitutive units. Thus. t h e C,,2 domains are critically dependent on the CH3 domains for stability. and Fc function can be modulated by hinge/Fab arm conformation as discussed above. The view that each domain of IgG has evolved for a separate and independent function is not validated. It seems more likely that, as IgG has evolved through gene duplication, new functions and new ways of controlling these functions have appeared with the increasing complexity of the molecule.

28

4. Structure of other immunoglobulins in relation to IgG All immunoglobulin classes are based on a four-chain structure consisting of two identical heavy chains and two identical light chains. In all cases the chains are organised into domains formed around the immunoglobulin fold. The immunoglobulin class is determined by the nature of the heavy chain or, more precisely, by the constant part of the heavy chain. This is so because of the origin of the heavy chain gene in recombination events, as discussed in Chapter 5 . As shown in Fig. 10, IgG, IgA and IgD have a similar arrangement of three constant domains and a hinge region. For IgM and IgE the hinge is replaced by an extra domain. With the involvement of tail pieces, IgM and IgA are able to form polymeric molecules. IgM is found almost exclusively as a pentamer whereas IgA is found principally as a monomer or dimer. Comparison of constant domains [76] reveals general conserved features, such as cysteines associated with the intradomain disulphide bridge and hydrophobic residues in alternating positions characteristic of /3-pleated sheets. Comparison also allows grouping of constant domains. Thus, the IgG cH2 ( C J ) domain, for example, is best related to the IgM cH3 (C,3) domain, and the IgE C,3 (C,3) domain in terms of sequence and in terms of a common carbohydrate attachment site. It seems reasonable to argue that the latter feature will lead to non-paired C,3 and C,3 domains as for C+? in IgG. The CJ domain is best related to the C,4 and C,4 domains and all three are expected to be paired in the conventional manner for constant domains. In the absence of crystallographic structures, arguments such as the above are used below to construct schematic models for the structures of the other immunoglobulins based on the data available for IgG. Tables 4-6 group together sequences of human immunoglobulin domains and hinges for ready comparison. In general, discussion below refers to human immunoglobulin unless specifically stated otherwise.

Light (L)chain A or

K

chain

L = V,

+ C,

domains

Heavy (H)chain y = V,

*= V, V, 6 = V,

(Y=

E =

V,

+ C,1 + C1,

+

+hinge+ C2 ,

+C,2+ C,3 CHI +hinge+ C,2

+ C,3

(IgG)

+ C4, + tail piece (IgM)

+ C,3 + tail piece (IgA) + CHI +hinge+ C,2 + C,3 + tail piece (IgD) + C,1 +C,2+ C,3 + C4, (IgE)

Fig. 10. Domain composition of immunoglobulin chains. Membrane-bound immunoglobulins have extra C-terminal segments. IgD composition is that of human IgD: mouse IgD lacks the C,,2 domain.

29 TABLE 4 Comparison o f human immunoglobulin domains \iinilar t o IgG C,,3 and C,,I Alignment is after Beale and Feinstein (761 and sccks to align /3-strands (compare Table 1 ) with insertions and deletions tending to be associated with bends. The C,,1 sequenccs are aligned as far as possible with those of IgG Cl13.Eu numbering refers strictly to IgG Ci,3 and C,,I but for convenience is extended t o the other immunoglobulins. A dot indicates no residue at the position corresponding to the numbered human IgGl residue. N@ indicates N-linked carbohydrate. Intradomain disulphides link Cys-367 ( o r Cys-144) to Cys-42.5 (or Cys-200) with Trp-381 (or Trp-158) nearby. C,4 and CJ have a n extra tailpiece cysteiiie involved in polymer formation.

c,I

C,1

A S T K G P

Y A

IIX 119 120 121 122 I23 12J 125 126 127

V F P

G S A S A P T L F P

A S P T S P K V F P

A P T K A P D V F P

L L A S S D P

F A T P E W P

128 I20 130 131 132 133 134

L A P S S K S

L V S C E N S

L S L C S T Z

I I S G C R H

P E A

G S R

13s

T S G

B P S

P .

P K

C$

C,4

CJ

C,3

c',4

34 1 342 343 344 345 346 347 348 349 350

G Q P R E P Q

V A L H R P D

G N T F R P Q

A Q A P V K L

G P R A A P E

Y T

Y L

H L

L N

35 I 352 353 354 35.5 356 357

L P P S R D E

358 359 360

L T K

L P P A R E Q L N L R

L P P P S Q Q L A L N

v

v

v

36 1 362 363 364 365 366 367 368 36Y 370

N Q V S L T

E S A T 1 T

Q L V T L T

L V K

L V T

L A R

37 1 372 373 374

G F Y P

G F S P

37s 376

S D

A D

c

c

s

136 137

S

D

S P S

V F P L T

N

R C C K N I P S @ A T S V T L G C L A T

G T A A L G C L V K

S T V A V G C L A Q

G B V V I A C L V Q

148

G F F P

G Y H P

G Y F P

T S

E P

L L C E V

o

138 139 140 141 142 143 144 135 146 147

G F S P

G F S P

N F M P

149 150 IS1

D Y F P

D F L P

K D

P N

E D

152 I53

E P

D S

s

B

Q

N S P V V L A C L I T

D K R 1' L A C L I

c

A S W

v

A S T

Q Q P

T A B L E 4 (confinued)

CJ

c,4

CJ

C,3

c,4

377 378 379 3x0

I A V E

V F V Q

V L V R

I L L M

I S V Q

381 382 383 384 385 386 387 388 389 390

w

w

w

w

w

E S N G Q P E N N

M Q Q R G Q P L S P E K Y V T S A P M P E P

L Q G S Q E P R E K

Y L T W A S R Q E P

L L E H D N O E R V E Q V L N @ P T D S A G R F A H P S A T R T P Q P P P R Q K P ?

Q

S

A P G

Q G

G S

391 392 393 394 395 396 397 399

Y K T T P P V L D

400 40I 402

S D G

398

T

I

'

I57

s

s

L S V T

V T V T

V M V T

I 58

w

w

159

w

w

w

N S G A L T S G

K Y K N@ N S D I

S E S G Z G V T

Y M G T Q

D T G S L N@ G T

I60 161

I62 I63 I64

I65 I66

S Q P

S

S 167

I Q R

K G S

176 177 I7X

S S G

G G .

R D S

L S G

G F F V F

I79

L Y S L S

K Y A A T

A S G B L Y T T S

Y Y M T S

H Y A T I

1x4 185 186

s

s

s

s

s

V V

Q V

Q L

Q L

L L

187

'.r

1x8

V P

L L P

T L P A T Q C

S T P L Q Q

T V S G A W

s

s

1

L

V L

R L

T V D K S R

T V S E E E

R V A A E D

R V P A P P

E V T R A E

s

I T F

Z R

s

w

V T V

S

s

w

154 155 156

170 171 172 173 I74 175

I L

w

C,I

Q R T F P E

K L

41 I 412 413 414 415 416 417

C81

A R B F P P

s

T F W A W

C,,I

T K G F P S V L R

R Y F A H

S F F L Y

c,1

V H T F P A V L O

T T F A V T

403 404 405 406 407 408 409 410

c,1

w

168 169

1 xo

181 182 I x3

I 89 I90 191

192

s

S S

s

K D V M Q

T M T L P A T T L T

T A B L E 4 (conlitwed)

c,1

c1 ,

C,,I

c,1

c,1

L G T

G T N

L A G

W R Q

A K .

c

I06 107 lox I99 200

Q T Y I

E H V V

K S V l

G E Y K

Q M F T

R A V H E

20 I 202 20.3 204 20.5

N V N H K

K V Z H P

A L P L A F T Q K T

D A S A R S T P L S L Q N @ T A V S Q R K

206

P S N

B G B

212 213 214

T K V D K K

K E K N V P

V D K S T G K P T L Y N@ V

I D R L A G K P

s

v

215

V

L E V S Y V T D H G P M

S V N P G K

L P V

s

s

L V M S D T A G T

V V M A O V D G T

Y

Y

c,3

c,4

c,,3

c,3

c,4

418 419 420

Q Q G

N T G

K K G

P Q P

E Q K

19.3

42 1 422 423 424 425 426 427 128 429 430

N V F S

E T Y T

D T F S

A T Y T

c

D E F I

S V M H E

V V A H D

M V G H E

V V S H E

43 I 132 433 434 435 436 437 438 439 440

A L H N H Y T Q K S

A L P N R V T E R T

44 1 442 1‘43 444 445 446 441

L S L S P G K

c

c

c

I94 19.5

207 208 3JO

210 21 I

c

c

c

€

c

I V K H .

V Q H T

. Y T

A S K

N P

P S S

I K E I F

R V A H 7’

S K

S O H V

c

V

T ) W V D N@

A

c

‘r

H V N@ V

c

The data as from reference\ [ 113. 6 . 107, 9.3 atid 791

T V

K T F S

32 TABLE 5 Comparison of human immunoglobulin domains of the IgG C,,? type and IgM and IgE C,,2 domains ‘IgG C,,2 type’ refers to non-paircd domains with interposed carbohydrate chains attached to Asn-297. C,2 lacks this carbohydrate but has a chain at Asn-258 which could function similarly, as discussed in the text. C,2 and C,2 are included separately tor the sake o l the completeness. They probably more closely resemble IgG CHI or C,,3 than C,,2. A dot indicates no residue at the position corresponding to the numbered human IgGl residue. N@ indicates N-linked carbohydrate and S@ 0-linked carbohydrate. The intradomain bridge is formed between Cys-261 and Cys-321. In IgA, Cys-236 forms an intradomain bridge with Cys-296 effectively linking the bottom of the structural hinge (to which Cys-236 probably belongs) to the tip of the C,,2 domain. Cys-235 and Cys-298 form interheavy bridges which have been suggested to be Straightforward or crossed. Cys-309 in C,,2 is unaccounted for and may be involved in polymerisation and interaction with secretory component. Cys-309 in C,3 has been suggested in the IgM pentanier to form a disulphidc bridgc to the corresponding cysteine in another monomeric unit, although this is disputed. An alternative involvement in polymerisation has been proposed. I n C,2, Cys-340 forms an interchain bridge to the corresponding cysteinc. In C,2, Cys-248 and Cys-339 form two interheavy chain bridges. These two residues are suggested to be close together in space and may form a ‘crossed’ linkage system.

v 23 1 232 233 234 235 236 237 238 239 240

A

I

P E L L G G P S V

A

24 1 242 243 244 245 246 247 248 249 250

F L F P P K P K D T

F

25 1 252 253 254 255 256 257 258 259

L M I S

I F L T K S T K L

R T P E V

Q D T A I R V

A I

P P S F A S

P S@ C C H P R L

S H T

S L H R P

Y L L T P

S A Y L S

F

A L

A V

R

Q

Q D

D G F F G

D

L L L G S E A N@ L

Q P L G V

L W L R D K A T

F

A

D S N P

R G V

P S P F

E L P P

K V S

V

C S

R D

F

T

P P T V

V

K I

P P

L

R

Q S S C D G

D L F I

N

R

P

K S P T I

R

H F P

K S K L

T I Q

G G

P

33 TABLE 5 (continued)

c,3 T

T

T

I

C

C

C

C

C

L V T D L T T Y

T L T G L R D

Q

V V G S D L

L V V D L A P

260

T

T

26 1 262 263 264 265 266 267 268 269 270

C V V V D V S H E D

27 1 272 213 274 275 216 217 27 8 219 280

P E

D S

V K

V

F

N W

Y V D

F

S

K

A

S G V T F T W P

K D A H L T W E V

Q

S

T I S W T R

A T G F S P

L L C L V S G Y T P

R Q I Q

G T I

V S W

A

G T V N@ L T W S R A

I T W L E D

L R E

N@

281

G

D

T

G

S

G K

G

282 283 284 285 286 287 288 289 290

V E V H N A K T K

G E

S G K

K V P T G G V E E

G K

Q V G S G V T T D

Q V M

29 1 292 293 294 295

P R E E Q

T

T A S T T

296 291 208

Y S

T R K E E K Q R N@ G

Q V

I S E S H P N@ A

G L L E

299 300

T Y

T F

N@

A V K T H

N@

S A V

Q G P

P E R D L

C G C

R H S

N@ G S

Y

Q

P V N H S

T L

Q A E A

K E S G P T T Y

D V

D L S

Q E G E L A

30 1 302 303 304 305 306 307 308 309 310

C? R V V S V L T V L H

31 1 312 313 314 315 3 16 3 17 318 319 320

Q D W L N G K

32 1 322 323 324 325 326 327 328 329 330

C K V S

33 1 332 333 334 335 336 337 338 339 340

E Y K

S

T S T L P V G

T

T

E

S L W N A G T S V T

R D

S D W L S

C

C R V T H P H L P R

C R V D H

A L M R S T T K T S

T F

T

Q Q

E D S T K K C

D D W N

E P W

S

H G K T F T

G

E A

S

I

C E

G

E R F T

N

C

C T

T A A Y P E

K A L P A

S

P I E K T

P L K Q T I S R P K G

I S K A K

K V

S

V T H T D L P

N

T V

S V S S V L P G C A

A V

Q H

S R L T L P R

T L

N

H P

S

S L P P

K

Q

T

P

L

T A T L S K S

Nomenclature and sequence sources as in Table 4.

R L M A L R E P A

W

I E G E T Y

Q

S

T L T I K

Q S M F T

S T

Q S E

L T L S Q K H W L S D R T Y T C

Q V

T Y

R G

Q L

N@

A S S

M C V P D

G H

F

35 TABLE 6 Comparison of hinge sequences of human immunoglobulins

216 217 218 219 220 22 1 222 223 224 225

Yl

P

Y3

Y4

E P K

E R K

E L K

E S K

S

.

T

Y

C

.

D

.

P L G

G P P

D T T H

. . .

K

.

T H T

. . .

T

a1

.

s R

. P

P E

a2

.

P

c

P V P S@

T P P

c

R V P P< P P P

7'

P

w

s

Q

A

S@

S

V

P T A Q P Q A E

S@

P

S@

T

P P T

P
V,III and V,IV probes and a set of approximately 12 genes detected by a V,II probe. Pech et al. [78] have recently reported evidence that most, and perhaps all, of the human K locus is duplicated. Therefore, there may be twicc the number of germline V, genes as originally thought; that is. there are at least 40 and perhaps 80 human V, genes. depending o n the extent of the large duplication within this locus. Until very recently. there has been relatively little data available regarding the number of human VH genes. Rabbitts et al. (371 argued that the VHIII protein subgroup is encoded by about 20 V,, genes a n d , b y assuming that all four V, subgroups then recognized [79] were of comparable complexity, they suggested that the human VH locus contains approximately 80 V,, genes. Subsequently, the five proteins which made up subgroup IV have been reclassified as VHIII sequences [ 3 5 ] . If t h e assumption of comparably sized V, gene families is applied, the estimate for t h e human V, locus is reduced to approximately 60 V,, genes. Two features of V region genes could make such estimates unreliable. First, the protein sequence subgroups d o not always correspond to sets of homologous genes [26,43]. and second, the number of genes within ii family can vary by more than ten-fold P61. Honjo’s laboratory has recently reported a direct cloning approach to study the structure of the human V, locus. Kodaira et al. [40] isolated 23 cosmid clones containing 1000 k b of the V H locus. Hybridization of these cosmid clones with three V H gene probes, each representing o n e of three V, families, identified 61 V, gene segments. It should be noted that work from both the Rabbitts and Honjo laboratories is based o n the idea that the three classic V, subgroups defined using protein sequence data [3S] a r c equivalent to three VH gene families and represent the entire germline human V,, repertoire. Although there is some evidence to suggest a reasonable correlation between V, subgroup and V, gene families, there has been no systematic survey designed to identify new V, gene families. Nevertheless, the extensive analysis of t h e human IgH locus by Honjo’s laboratory will n o doubt lead to a detailed knowledge of this locus in the near future. O n e of the cosmid clusters cloned by Kodaira et al. [40] was examined in great detail. This particular cluster (called cluster 71) contained seven V, genes, five of

94

which were shown t o be non-functional by sequence analysis. These results extend previous work in both mouse and man, which showed that approximately 40% of V, genes a r e pseudogenes [ 11,80-821. T h e human V, locus is quite diverse. A s discussed above (Section 3.6.), hybridization studies with probes for the V,I, I11 and VI subgroups, as well as V,O, indicate that each subgroup is composed of about ten genes. Based on the limited sequence homologies between these four probes. it is likely that they identify distinct, non-overlapping gene families. T h e human A locus probably contains a minimum of 40 V, sequences based on available Southern blot data. Probes for the genes encoding the V A I I ,V,IV and V,V subgroups are not yet available but will be necessary t o determine the size a n d diversity of the human V, germline repertoire.

5. Chromosome assignment Genetic analyses showed that the genes encoding immunoglobulin chains belong to three unlinked loci in m a n , mouse, and rabbit [83,84]. Such studies o n the inheritance of immunoglobulin polymorphisms led to the assignment of the mouse K locus t o chromosome 6 in tight linkage with the T cell alloantigen Lyf-2,3 [8S]. Further mapping of the murine and human immunoglobulin gene loci was accomplished using somatic cell hybrids. Using mouse-mouse hybrids and Robertsonian translocation markers, Hengartner e t al. [86] confirmed that the expression of K light chains requires a locus on chromosome 6, whereas the expressiori of heavy chains required chromosome 12. T h e formal demonstration that the structural K and f g h loci reside on chromosomes 6 and 12, respectively, was obtained by the analysis o f mouse-hamster hybrids using Southern blot hybridization [87,88]. A similar approach was used by D’Eustachio e t al. [X9] to assign the A locus to mouse chromosome 16. D’Hoostelaere et al. I901 have recently mapped the K locus 8 centimorgans from the mouse T-cell receptor beta locus (TcKP). By setting up a three-point cross, K and TcRP were mapped with respect to the morphological marker hypodactyly (Hd) allowing the chromosome order centromere-TcRP-K to be determined [90]. T h e chromosomal orientation of V, and C , genes has not been determined. T h e mouse f g h locus is located on the telomeric end of chromosome 12 [91]. However, the orientation of the heavy chain locus is controversial. T h e order centromere-V,-C, was originally proposed based on recombination frequencies [92] and an analysis of the Harwell translocation T(S;12)31H [91]. More recently, a mouse plasmacytoma (5558) carrying a translocation between the f g h locus on chromosome 12 and the c-myc locus on chromosome 15 has been fused with Chinese hamster cells by Erikson et al. [93]. These investigators concluded that the orientation of the mouse f g h locus is centromere-C,,-V,. Additional studies will be necessary to resolve this point. T h e expression of human immunoglobulin heavy chains in mouse-human hybrids was shown to be correlated with the presence of chromosome 14 [94]. Hob-

95 art et al. [95] formally demonstrated that the human lgh locus maps to chromosome 14 by nucleic acid hybridization using heavy chain constant region probes. The use of in situ hybridization allowed Kirsch et al. [96] to localize the lgh locus to the tip of the long arm of chromosome 14 at band 14q32. Based on an analysis of Burkitt lymphoma cells having X;14 translocations (between c-myc and l g h ) , Erikson et al. [97] have concluded that the chromosomal orientation of the lgh locus in man is centromere-C,-V,,. The human A locus is located o n chromosome 22. As in the case of the human Igh locus, the A genes were first tentatively mapped by studies measuring immunoglobulin gene expression in somatic cell hybrids [91] and the chromosome assignment later confirmed by direct hybridization studies [99]. The human K locus was mapped to chromosome 2 based on an analysis of human-rodent hybrids with nucleic acid probe hybridization [W] and localized close to the centromere on the short arm of chromosome 2 by in situ hybridization of normal fibroblasts and lymphocytes [ 1001.

6. Gene organization The organization of immunoglobulin gene loci has been examined using several approaches: classical Mendelian genetics, analysis of deleted DNA sequences within rearranged immunoglobulin gene loci and direct analysis of cloned DNA segments. Although the organization of no immunoglobulin gene locus is completely known, enough detail is available to outline the organization of the heavy, K and A loci in both mouse and man. Fig. 3 summarizes current views of immunoglobulin gene organization. While some features have been examined directly, much of Fig. 3 is quite speculative. 6.1. lgh locus organization

DNA cloning studies of the mouse lgh locus have provided a detailed map of the D, J H and CH regions. As discussed in Chapter 2, eight C, genes occupy a 200 kb region of chromosome 12 in the order 5’-C,-C8-Cy3-Cy,-Cy,,-C,;,-C,-C,-3’ [ 1011. Approximately eight kb 5‘ of the C, cluster are four functional JH genes (5’-JHlJH2-JH3-JH4-3’)[102]. A single D segment (D-Q52) is located 700 base pairs 5’ of JH1 [103]. The other 11 identified germline D segments (9 D-SP2 family members and 2 D-FL16 family members) have been physically linked within a 60 kb region located 20 kb 5‘ of JHl by Tonegawa and co-workers [104,105]. The DNA cloning studies from Tonegawa’s and Honjo’s laboratories [ 101,1051 together have shown that the D, JH and C, coding regions all have the same transcriptional orientation. It has been generally assumed that the V, genes are 5’ of the D-JH-C, region. Evidence for the order ~ ’ - V H - D - J H - C Hhas - ~ ’recently been obtained by the identification of a mouse strain carrying a recombinant Igh locus. This strain, C.BIR5, was constructed by Riblet by crossing strains having the lgh haplotypes a and b [106]. The cross-over event has been localized to the region between the D-FL16ID-

96 IgH LOCI VH

M.

D

(FL16 SP21

CH

JH

...... (-200) D

VH

VK

JK

JH

CH

cK

M. iN3-..-.---* (- 300)

VK

*JK

CK

....... Ha 7 - 1 0 0 ,

Fig. 3. Organization o f murine a n d human inimunoglohulin loci. Each pancl shows the murine (M.) and humnn ( H . ) immunoglobulin locus organizatioii. Much ol this figure i b speculative. See text for dctails and rclcrences. Details o f c', gcne organization are reviewed by Honjo IS] and elsewhere in this volume.

,

SP2 region and C,, giving rise to a recombinant f g h locus having the V, and D segments of the u haplotypc and the C , genes of the h parent (Brodeur and Riblet. in preparation). Therefore, the Igh haplotype of C.B/RS, V~"-D"-/-CH"(where / indicates the cross-over), provides direct evidence for the general organization of the mouse f g h locus. There are three lines of evidence which suggest that, in general, mouse V, genes belonging to the same V, gene families are clustered. This was first suggested by the cloning of DNA fragments large enough to contain more than one V, gene. Kemp et al. [22]examined three distinct clones each bearing a pair of V, genes. One clone had two members related to V,,S107, while the other two bore two V, genes which hybridized to t h e same V, probe, V,76 (V,J606 family). Bothwell et al. [ l l ] and Givol et al. [69] reported similar findings for members of the very large VHJ558family. Although no unrelated V, genes were detected among these ' 3 '

97 clones, it should be emphasized that detection of ‘non-family’ V, sequences in these experiments always relied on low stringency hybridization using probes representing only two or three unrelated Vk, families. Several mouse plasmacytomas have been useful in studying the organization of V, genes using ‘deletion mapping’ techniques. The rationale of such experiments is that the completion of a VH-D-JII rearrangement deletes all DNA sequences between the rearranged V,, and the J,, segments [24]. Therefore, B cell lines which have rearranged V, genes on both alleles are potentially useful in determining V, gene order. Deletion mapping, carried out by Kemp et al. [2S] and Rechavi et al. [ 1071, demonstrated that the V,, gene families examined were clustered; that is, homologous V,, sequences were deleted as a group. Using classical genetic recombination, coupled with restriction fragment length polymorphism (RFLP) analysis, Brodeur et al. [27] have obtained mapping data for much of the V, region. Several panels of fgh-recombinant mouse strains were constructed by Riblet [ 1061 to preserve chromosomes with recombinant fgh loci. These strains each have an independent genetic cross-over between the V, gene locus encoding the DEX idiotype. Igh-DEX, and the constant region (allotype). Since the mice were bred from parental strains having polymorphic fgh loci, the parental origin of each V, gene family could be ascertained by Southern blot analysis [%]. The v, gene families vH36-60,v,,3609, V,,J606, vHx24 and V,,J558 have not been separated from each other using these strains. Since multiple independent cross-overs between v,J558, which contains fgh-DEX and vHs107 have not separated the VH36-6O/3609/J6(j6/x24group. it was originally presumed (but not proven) that the V,,J558 family is immediately 5’ to V,,S107 [27]. Using the deletion mapping approach described above, further analysis of V, gene family organization is currently under way [Brodeur, unpublished]. A panel of over 30 Abelson-virus transformed pre-B cell lines has been established from mice heterozygous at the f g h locus. Each cell line has at least one VH-D-J, rearranged allele, allowing the presencc or absence of V,, genes on the rearranged allele to be determined by Southern blot analysis. Preliminary results based on deletion mapping have confirmed the map obtained using fgh-recombinant strains: 5’-VHS107-V,QS2N-V,7183-D-C,-3’.with vHJ558, VH36-60, vHJ606 and vH360c) mapping 5’ of v1,s107. The current map, based on deletion studies, is: ~ ’ - ~ ~ ~ 6 ~ ~ 9 - ~ ~ 107-V~Q52N~ 6 ~ ~ 6 - ~ ~ 3 6 V,7183-D-C,-3’. The position of the complex VHJSS8 family is not yet certain. However, at least some V,.lSSX sequences appear to be more distal to the C H region than vH3609. Further studies are in progress to determine whether the entire v H J % 8 gene family maps in this region. The organization of the mouse V, gene families, based on recombination and deletion studies, is shown in Fig. 4. Although the members o f mouse V, gene families appear to be generally clustered, there is evidence that some degree of interspersion may occur. Deletion mapping has indicated interspersion of V,, sequences belonging to the two adjacent families VHQ52N and V,7183 in NIHiSwiss mice [IOX]. Similar results have been obtained in the CBA mouse strain (Brodeur, unpublished) suggesting that interspersion of V,,QS2N and V,7183 members may exist in all mouse strains.

98 Centromere 12

--. -.

r

*x, , ,x\

J558

3609

36-60 Q52N 7183 J606 S107

Fig. 1. Organization of the mouse Zgl7 locus. The order or V,, gene families is based on /g/i recombinant mouse strains [27] and dcletion mapping studies [Brodeur. unpublished]. Approximate location of genetic cross-overs are designated hy ‘X‘. Distance (molecular and rccombinational) between or within V,, gene families are unknown. The map position of the V,,JSS8 family is tentative (see text).

The overall genetic size of the mouse fgh locus has been estimated by analyzing seventy recombinant inbred strains for cross-over events within the V, region (Brodeur and Riblet, in preparation). The frequency of recombinants among such strains can be used to calculate map distances [109]. Only three recombinant loci were identified among seventy strains, indicating that the fgh locus is one to two centimorgans in length, a ‘genetic distance’ which is estimated to represent 1-2000 kb of DNA [110].The estimate of one to two centimorgans for the total Igh locus is consistent with extensive back-cross analysis (over 5000 mice tested) by R . Riblet (personal communication) which maps the lgh-DEX idiotype marker 0.5 centimorgans from the C , region. Igh-DEX is encoded by a germline V, [ l l l ] gene belonging to the V,JSS8 family. Furthermore. the estimate of 100G2000 kb for the mouse Igh locus is consistent with 100-200 V, genes spaced 10-20 kb apart [151. The current picture of the mouse V, region is that at least 200 V, genes are grouped in nine sets (families) of highly homologous VH sequences - the families ranging in size from 2-100 (or more! [73]) VH sequences. The f g h locus occupies one to two centimorgans (1 000-2 000 kb) of the 70 centimorgan long mouse chromosome 12 [27]. The V, genes are 5’ of the D-JH-C, region and appear to be generally clustered, although some interspersion between two adjacent families has been noted [108]. The J, region in man consists of six functional and three pseudo-gene segments. As in the mouse, there is a single D segment immediately 5’ to the most 5‘ functional JH segment [l-91. The organization of the three kb JH cluster is 5’-+JH1-D-

It is presumed that the remaining D segments are 5’ of t h e J H cluster, although this has not been shown. Siebenlist et al. [113] cloned a 33 kb segment of the human D region and identified four D segments spaced approximately nine kb apart. Since most human heavy chain CDR3 sequences do not use any of the four germ-

99

line D segments reported by Siebenlist et al. [113], it is assumed that additional germline D segments exist. V H genes in man may be organized differently from mouse. Cosmid cloning of human V, sequences in Honjo’s laboratory [40]has revealed extensive interspersion of non-homologous V H genes. Clones were isolated using probes from each of the three defined V, families (V,,l,VHII, VHIII) which correspond to the classic V, subgroups defined by Kabat et al. [35]. Hybridization studies allowed the identification of 61 V, gene sequences spanning approximately 1000 kb of DNA. A detailed analysis of one cluster of about 40 kb (cluster 71) revealed seven V, genes arranged in the order ~‘-v,II~-v,II-vHIII-vHII-vHI-v~III-vH~-3’, suggesting extensive interspersion of V,, gene families in man. Interestingly, only two of the seven V, genes of cluster 71 appear functional by sequence analysis, and these two are both Vl,lImembers having 99% sequence homology. Further analysis of this V, cosmid collection by Honjo’s group will no doubt show whether the high frequency of V, pseudogenes and widespread family interspersion is a general feature of the human V, region. There are limited data available concerning the overall size of the human Igh locus. Johnson et al. [114]analyzed families for a restriction fragment length polymorphism detected with a vHI1 probe. This analysis was used to estimate, by logarithm of odds score, that the VklIlgenes were approximately four centimorgans from the C , gene cluster. This computation is valid only if crossing over has a uniform frequency over the entire chromosome. If the four centimorgan estimate is accurate, it suggests that the human Igh locus spans at least 4000 kb of DNA and that the 1000 kb cloned by Kodaira et al. [40]may represent only a fraction of the entire human heavy chain locus. 6.2.

IgK

locus organization

Several initial reports utilized ‘deletion mapping’ to examine mouse V, gene organization [47,115].However, this approach was no longer pursued following reports that the v, rearrangement mechanism may operate by sister chromatid exchange [116,117]or inversion [118],either of which would make deletion mapping of the K locus difficult or impossible to interpret. Gibson and colleagues [ 1191 have sought to examine mouse K gene organization using recombinants and recently reported the first instance of recombination between K locus markers. The markers used, Igrc-Efl and IgK-EfZ [120]were detected by isoelectric focusing of K chains. The genes encoding these polypeptides were shown to be 0.45 centimorgans apart. One of the EfllEj2 recombinants, ‘NAK’, was preserved as a strain homozygous for the recombinant K locus. D’Hoostelaere and Gibson [48]recently analyzed NAK and two additional K recombinants using seven distinct v, probes for Southern blot analysis. By considering the most intensely hybridizing polymorphic restriction fragments, no examples of family member interspersion was observed, indicating that V, gene families are not extensively interspersed. The map order inferred from these results is (V,19,V,21)-Efl-(V,4,V,8,VK10)-(Ef2,V,9,VKll ,V,24). A tentative map of the mouse K locus is diagrammed in Fig. 5 .

(VK

1.9.11.24)

(V, 4.8.10)

Ef1

(V, 19,21) C,

Fig. 5. Organization of the mouse K locus. Approximate locations of genetic recombinations are designated by ‘X’. This map is based on data and discussion of D’Hoostelaere and Gibson [48] and D’Hoostelaere et al. [W]. The orientation of V, and C , relative t o the centromere is unknown.

A detailed cloning and sequence study of the V,21 gene family of BALBic mice also showed that highly homologous V, genes are physically clustered [46]. It should be stressed, however, that family member interspersion of some degree has not been ruled out by any experiments to date. The mouse K locus consists of four functional J, segments (3,1,2,4,5), a single C, gene and about 300 V, genes. The J,3 segment appears to be inactive, most probably because of a mutation in its RNA splice signal [18]. There are likely to be 10-20 V, gene families and highly homologous sets of V, genes appear to be clustered. It is not known whether V, genes are 5’ or 3‘ of C, (or both). Current models of K locus rearrangement via inversion [121] would allow V, genes to be on either side of the K constant region gene. Indeed, at least one functional T cell receptor V, gene is located 3’ of C, and appears to rearrange via chromosomal inversion [ 1221. Bentley and Rabbitts [49] initially proposed the number of human V, genes to be relatively small. Their estimate was based on the finding that approximately 25 V, genes, as counted on Southern blots, constituted three of four human V, subgroups. Given the rather small apparent size of the V, locus, Zachau and colleagues [53,123] have initiated studies of V, gene organization using overlapping cloned DNA fragments. Pech et al. [S3] initially reported a cloned region (now termed cluster A) containing five V,I sequences and one pseudogene homologous to VJI subgroup members. Subsequently, Pech and Zachau [123] reported that this 80 kb region also encodes two V,III genes. both potentially functional on the basis of sequence analysis. The presence of V,IV sequences was ruled out by hybridization experiments using a V,IV probe. The order of V, genes within this region was determined to be S’-V,I-V,I-V,III-V,II-V,I-V,I-V,III-V,I-3’. Therefore, extensive interspersion of human V, gene family members exists, as in the case of human V, genes. In both cases, all V genes examined are in the same transcriptional orientation. Pech et al. [78] have recently identified a second cluster of V, genes, termed cluster B, which contains six V, genes belonging to three different subgroups (families). Interestingly, cluster B appears to be a duplicated copy of the previously reported 80 kb region (cluster A ) . The two clusters have not been linked and the extent of the duplication is not yet known. Although the orientation of V, genes

within each cluster is the same, the orientation of the two clusters relative to each other or to C , has not been determined. I t is possible, for instance. that the entire V, region has been duplicated and may be present in two different transcriptional orientations. Lotscher et al. 1551 have recently shown by somatic cell genetics that human V, genes are dispersed among several chromosomes. These investigators estimate that about 10% of human V, gene sequences may be located o n chromosomes other than chromosome 2, to which t h c functional K locus maps.

6.3. I g A locus organization

In addition to the two mouse V, genes, molecular analysis of the BALBic locus has identified four J, gene segments and four C, genes organized in two similar clusters: 5’-JAz-CAZ-JAj-C,,-3’and 5’-J,,-C,,-J,,-CAI-3’ [124,12S]. J,, is most likely a pseudogene due to several anomalies [126,127] and probably accounts for the lack of any observed productive or non-productive JA4CA4 rearrangements [ 1281. The two A clusters have not been psysically linked so that the distance between the J,,-C,z-J,,-C,j cluster and the JAA-CA3-JAL-CAI cluster is unknown, as is the relative orientation of the clusters. Furthermore, the location of the V, genes is unknown except that the V, and C, genes are tightly linked on chromosome 16 [89]. Despite the lack of direct evidence, a model of the mouse A locus organiztion has been proposed based on the highly restricted pattern of V, to J, gene rearrangements [ 1281. The structure ~ ’ - ~ , ~ / / ~ , Z - ~ , ~ - ~ , , - ~ A j / / ~ A ~ / / ~ A ~ - is ~ , 3 - ~ , l - ~ , , consistent with the observation that rearranged V,2 genes are nearly always joined to JAz,while rearranged V,1 genes are always joined to JA3or J A I The . model is also consistent with the finding that a given plasmacytoma can retain rearranged and germline forms of both V,1 and V,2 [ 1241. Since overlapping clones failed to provide evidence of the proposed organization of the h locus (Jim Miller, personal communication), definitive mapping of this locus may have to await linkage analysis using pulsed-field electrophoresis [ 1291. The first report regarding the organization of the human A locus was from Hieter et al. [13O] who identified six C, genes tandeinly arranged on SO kb of DNA. Udey and Blomberg [131] have recently shown that C,,, C,, and Ch3each have a single J, segment located approximately 1.3-1.7 kb upstream. In addition, hybridization studies have indicated the presence of at least one J, segment 5’ to each of the other three C , genes (C,,, CA5,C,(J [131]. There is, as yet, no information regarding the organization of the V, genes and V, gene families in man. It does not appear, however, that V, genes are interdigitated among the JAC, cluster [ 13I , 1321.

7. Polymorphism Immunoglobulin variable region genes are extraordinarily polymorphic, displaying a degree of genetic diversity paralleling that of the major histocompatibility com-

102

.. U

EcoRl

. u

Hindm

Fig, 6. Restriction fragment length polymorphism within the mouse Igh locus. Southern blots of BALBic (1gh") and AiHe (Igli') liver DNA hybridized with a V,,JS58 gene family probe. Dxl I [26]. DNA samples (IS& were digested to completion with either EcoRl or HiiidIII. separated by agarose gel electrophoresis, blotted and hybridized as previously described [XI.

plex (MHC) [ 1331. This extensive polymorphism presumably contributes to the role immunoglobulins and MHC products play in protecting the individual and the species against a continuously varying antigenic environment. V, gene polymorphism among inbred mouse strains can be readily seen by comparing restriction fragment patterns on Southern blots hybridized with V, probes

103 TABLE 2 Iglz haplotypes of some inbred mouse strain\

lgh-V,i"

Igh-Clf

Igh

allotype"

haplotype'

Strain

BALB, C58, C57L C57BLI6, SJL SM DBAIZ, R F AKR NZB

V,,X24" VI,J606, V,,J558, V,,SlU7. V, ,7 183, V,,36OY

V,136-60 V,,052N D'

a

a

a

1

a

d

a

b

b

b

b

b

b

b

-

a

a

a

12

C

C

c

C

j j

J I e e f a g g

j j e e f

d d e e f

d

d n e

0

0

f

a f f

f gr

g

R

e e f a g g

b c d n e

br

C

I

i

I

!k

c C

f I

e g I

l

I

e e

C

g

P

P

I

I

AiHe

AL CE RIII BSVS SWR C3H. PL

V , allele designations are based on Southern blot analysis of EcoRl digested liver DNA using V,, gene probes except for VIlX24,which is based on blots of RglII digested gcnomic DNA. VI,X24 data are from Hartman et al. [134]. Vl13609 data are from Brodeur and Riblet (unpublished). All other V,, gene family data are from Brodeur and Riblet [26]. Strains SM and PL not examined with V,,X24 prohc. ' Alleles for D region were assigned using a D-FLl6.l 5' flanking region probe (Brodeur, unpublished results) which hybridizes to members of the D-FLlh and D-SP2 families [ 1041. " Serologically determined heavy chain constant region allele (allotype) designations are as described by Lieberman [135] and Huang et al. [ 1361. The C,, allotype designation is used for thc complete ( V l , and Cl,) I@ haplotype except for recombinant haplotypes such as SM and RIII. Thew arc distinguished by adding an 'r' for recombinant.

(Fig. 6). These patterns display considerable variation among strains; for example, a survey of 18 strains revealed six to cight different patterns when Southern blots were hybridized with any one of seven V, gene probes. Mouse strains with the same restriction fragment patterns for a particular V,, gene family were considered to have the same 'allele' at that region of the locus. Using this approach, Igh-V region haplotypes were assigned to each of the 18 strains examined [26]. Table 2 lists these and includes more recent information regarding the vHx24 [134] and VH3609 [Brodeur and Riblet, unpublished] families. Table 2 also lists the Igh-C region haplotypes of these strains. I n general, strains of the same C,, haplotype also have the same restriction fragment patterns with V, probes. Thus, the V , haplotype, based on restriction fragment polymorphisms, and C,, haplotype, based on serological analyses [ 1351, together define a 'complete Igh haplotype' [ 137,261. Since V, genes are generally separated by 10 kb or more [ 5 ] , most restriction fragment length polymorphisms are due to differences within the DNA flanking V, genes. The underlying assumption is that such polymorphisms among different

104 haplotypes are associated with differences in V-gene coding regions, and therefore in primary structure of antigen-combining sites of immunoglobulins. Data to support this assumption are not easily obtained due to the inherent difficulty of determining allelic relationships among genes within cvolutionarily complex multigene sets. For instance, Loh et al. [82] attempted to define the BALBic alleles of the NP" V, gene family of the C57BLi6 strain, which had been previously analyzed by Bothwell et al. [ l l ] . The NPh gene family is a subset of t h e V,J55X family. which includes the V, gene utilized by C57BLi6 mice in the antibody response to the hapten (4-hydroxy-3-nitropheny1)acelyl(NP) [ 111. Loh et al. [82] isolated and sequenced five 'NP" equivalent' V, genes from BALBic mice. Based on strong hybridization to the NP" VH gene probe and restriction map similarities, these five BALBic genes were considered to be the BALBic V,, genes most closely related to t h e C57BLi6 NPh V, gene set. Sequence comparison of these 'NPh-equivalent' BALBic genes with the seven C57BLi6 NP"-family genes did not allow allelic relationships to be established. Loh et al. [82] suggested that the origins of these BALBic and C57BLi6 gene sets. which share 8 5 9 6 % nucleotide sequence identity, may be obscured by frequent recombination and gene conversion events. Furthermore, the dominant response to the NP hapten in BALBic mice (the NP" idiotype [138]) was shown to use a V, gene quite distinct from the BALBic NP"equivalent set. On the other hand, studies of the antibody response to phosphorylcholine (PC) and p-azophenylarsonate (Ars) have provided unambiguous examples of V, alleles among Igh haplotypes. Rudikoff and Potter [ 1391 proposed that the nearly identical sequences of PC binding myeloma proteins from BALBic (Igh") and C57BLi6 (Igh") mice are encoded by allelic forms of the same germline gene. These PC associated V, genes are members of the small V,,S107 gene family [17]. Perlmutter et al. [140] have compared the four V,S107 genes of C57BLi10 with the four BALBic VHs107 genes and established allelic pairs. The genes encoding the anti-PC responses in each strain differ at only five positions, four of which are replacement substitutions. Near et al. [31] examined allelic forms of a vH36-60 family member utilized in responses to Ars in BALBic (Igh") and AiJ (Igh') mice. These allelic germline V, genes differ at only two nucleotide positions, both of which are replacement substitutions. Data from the anti-PC and anti-Ars systems suggest that, in certain responses, structurally and functionally allelic forms of a V, gene are expressed and encode differences at the protein level. Strain-specific antibody responses to other determinants, such as the anti-NP response of BALB/c and CS7BLi6 mice, may instead utilize V, genes which are clearly non-allelic, and for which allelic relationships among homologous germline V, sequences are unclear. Therefore, the restriction fragment polymorphisms which have been identified among various Igh haplotypes most likely reflect differences in the functional antibody repertoire. It is possible that V gene polymorphisms represent an additional source of antibody diversity for the species as a whole. The functional significance of V gene polymorphism, however, is unclear. It should be appreciated that, as discussed by Kelsoe and Farina [141], the influence

105 of natural selection on a given V gene is probably minimized by the great capacity of somatic recombinatorial diversification. With respect to the evolution of immunoglobulin loci, therefore, it may only be important for an individual, or species, to have a suitably large and diverse starting library of V genes (and D and J segments) with which to somatically construct a large antibody repertoire. Polymorphism among V genes may simply reflect the immune system’s remarkable ability to construct an effective repertoire using starting libraries which, although similar in complexity, are distinct collections of germline sequences.

8. Conclusions During the last decade, much detailed information has been obtained describing the content and organization of immunoglobulin loci. In addition, a more general picture has emerged from studies of chromosome location, gene family structure and the overall size and structure of heavy and light chain loci of mouse and man. In general, it appears that a typical mammalian immunoglobulin locus consists of up to several hundred V genes, a small number of constant region genes (from one to about a dozen), a small number of J segments (about half a dozen) and, in the case of IgH loci, a set of D segments (one dozen in mouse, an unknown number in man). Obviously, evolution has provided exceptions, such as the mouse A locus with only two V genes. A striking feature of immunoglobulin locus organization is the variety of arrangements which have evolved (Fig. 3). There are no obvious mechanistic constraints requiring, for example, all J segments to be clustered (IgH and K loci versus A loci). Recent studies on the organization of T cell receptor genes (1421 and on the chicken A locus [143] further underscore the genetic diversity of antigenspecific receptors. As noted above. studies investigating the germline V gene repertoire are still in progress. Information concerning the content and organization of V gene loci will allow the analyses of the immune repertoire during ontogeny [144], within B lymphocyte subsets [145], and in the context o f autoimmune disease [146] to be made within the framework of genetic organization and germline diversity.

Acknowledgements I am indebted to Dr. Geronimo Terres for his many useful comments on this manuscript. I thank Drs. Franco Calabi and Michael Neuberger for their excellent editing (and extreme patience!). It is a pleasure to thank Ms. Julie Dzengeleski for her professional and cheerful preparation of this manuscript. The author is supported, in part, by USDHHS Grant R01-GM36064.

106

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F. Calahi and M.S. Ncuherger (Eds.) Mo/ec,rr/orG c v r r ~ r i c su/ /,rir,iitnoR/ohir/i,i

0 1987 Elscvier Science Publishers B.V. (Biomedical Division)

111 CHAPTER 4

Assembly of immunoglobulin variable region gene segments MICHAEL RETH" and LISE LECLERCCPb .'Institute f o r Genetics, University of Cologne, Weyertal 121, 0-5000 Cologne 41, FRG and 'lnstitut Pastrur, 25 rue du Docteur Roux, 75015-Paris, France

I . Introduction Cells of the immune system of vertebrates carry on their surface variable receptor molecules which are able to bind an unlimited variety of determinants thus protecting the organism against many different pathogens. To reach the high variability necessary for this task, the genes coding for the variable part of the receptor molecules have evolved a unique recombination mechanism which allows their somatic assembly from gene segments. On B cells, the variable receptor molecules are immunoglobulins consisting of a heavy and a light chain. The genes encoding the variable part of these chains are assembled during B cell development from different gene segments which are VH, D, JH for the heavy chain variable region and V, and JL for that of the light chain. The somatic reorganization of the V-gene segments was first described in 1976 by Tonegawa and his colleagues [ 1-41, who found that the location of V, gene segments in mature B-cell lines (myelomas) differs from that in the germline. Cloning and sequencing analysis have since revealed the organization of the V gene segments at the IgH and IgL loci (see Chapter 3). However, a dynamic picture of the order and control of rearrangement events during B cell development has only recently been obtained by the study of Abelson pre-B cell lines. Abelson cells are obtained after an infection of mouse foetal liver or bone marrow cells with Abelson-murine leukaemia virus (A-MuLV) which specifically transforms early pre-B cells with incompletely rearranged V genes [5-71. During their growth in culture, many Abelson cells continue to assemble their V genes, thus allowing a detailed study of this process [7,8]. The first part of this chapter summarizes the current knowledge of the rearrangement process with a focus on studies of Abelson lines. We then describe the different rearrangement events, according to their order of occurrence during the B cell developmental pathway. In this second section, we hope to demonstrate how proliferating pre-B cell clones are using the rearrangement process and a multiple joining strategy to fulfill two tasks: the generation of a highly diverse set of variable region genes and the expression of a functional antibody molecule on each B cell. The final section of this chapter summarizes the

112 recent data concerning the feedback mechanism responsible for the allelic exclusion of immunoglobulin gene expression.

2. The rearrangement mechanism 2. I . Joining signals Most of the enzymes mediating the rearranging process have not yet been identified and characterized in detail. Thus, our knowledge of the DNA cutting and religation events that take place during joining is only indirect. However, the sequence analysis of gene segments before and after their rearrangement has given us some insight into the order and mechanism of the joining process. Each V gene segment is flanked o n one or both sides by conserved sequences consisting of a heptamer abutting the end of the segment and a nonamer which is separated from the heptamer by a spacer region of either 12 or 23 base pairs. These sequences are called joining signals or recognition sequences [9]. Joining signals flanking the 5' end of a segment have the consensus sequence GGTTTTTGT-spacer-CACTGTG, while those flanking the 3' end of a segment have the consensus CACAGTGspacer-ACAAAAACC (Table 1). Notice that the two sequences are inverted complements of each other: thus an inverted 5' joining signal is identical to a 3' joining signal. The conservation of the joining signal is not absolute. Nonamers always contain an A or T rich core but their sequence can vary in five out of nine positions. The heptamer sequence is more strongly conserved; indeed the four nucleotides (TGTG or CACA) flanking the segment are nearly invariant, which suggests that these sequences play an important role in the recognition or cutting reactions of recombinase enzymes (see below). The sequence of the spacer region between the heptamer and nonamer is not conserved; however, its length has to be either 1222 or 2 3 k 1 base pairs. The highly conserved spacer length corresponds to one or two turns of a-helical DNA and maintains precise spatial orientation between blocks of conserved sequences within the heptamer and the nonamer. Thus similar recombinase enzymes should be able to bind to either a 12 or a 23 base pair joining signal on the DNA helix. Analysis of many V gene rearrangements revealed the general rule that two segments are joined together only if they are flanked by joining signals of different spacer length (12/23 joining rule). T o account for this rule, the following molecular mechanism was suggested [9]: the 12 or 23 base pair joining signals would be detected by different recombinases which, after binding their respective recognition sequence, would come together to form the active recombination complex (see below). An exception to the 12/23 joining rule was recently found in rearrangement events which involved a segment flanked by a 22 base pair spacer joining signal and a segment flanked solely by a heptamer [lo-151. In these rearrangements, an isolated heptamer was obviously recognized in the absence of a nonamer by t h e recombinase proteins. These findings suggest that the heptamer is the most important part of the joining signal, a notion supported by the strong conservation of its se-

113 TABLE 1 Conserved joining signals 5' joining signal

Segment

D-FL16 D-SP2 D-Q52 D-I1XIA Dpl v,,7 183 V, , 0 5 2 V1,441 v,,s107 V,,S58 V,,XIA V,2 1B VA 1 VA2

V,?B4 VJH gv,, V,10.8 Consensus

Nonamer

Spacer

Heptamer

Ref.

I? 12 12 21 23 23 2.1 23 23 23 23 12 22 23 23 21 24 23

The exainplcs above come from the immunoglobulin and T cell receptor loci of different species

114 quence. Furthermore, the heptamer consensus sequence is palindromic around the central nucleotide, a characteristic of many sequences detected or cut by DNA binding proteins. 2.2. Joiriing models The rearranging mechanism is a special form of non-homologous recombination which so far has only been found at the J clusters of B or T lymphocytes. In fact, the same recombinase system may be responsible for rearrangement events at all variable gene loci in T and B cells as suggested by the ability of an Abelson preB cell line to rearrange introduced T cell receptor gene segments [16]. Furthermore, in mice with severe combined immunodeficiency a genetic defect involving the recombinase system results in abnormal joining events in both developing B and T cells [17]. The phylogeny of the rearranging mechanism is presently unknown; however, it seems to have been present from the beginning of vertebrate evolution since sharks, one of the most primitive members of this group, have V, D and J segments flanked by joining signals (Table 1) identical to those found in mammals [18,19]. The observation that two joints are formed during each V gene rearrangement has provided a more detailed picture of the rearrangement mechanism [20]. One of the joints is the ‘coding joint’ between the two segments and the other is the ‘reciprocal joint’ in which the heptamers of the two segments are fused together [21,22]. Reciprocal joints were first discovered at the K locus [23] where, after a V,-J, assembly, both joints are often retained on the same chromosome (see Section 3.2.1.). The two joints differ in one intriguing aspect from each other. While the reciprocal joint is a precise ligation of the two heptamer sequences, the coding joint is imprecise. Nucleotides are generally lost from the ends of the segments before these are ligated together. These deletions can be as large as 20 nucleotides [24]. Furthermore, many D to JH or V, to DJH coding joints contain sequences which are neither present in the germline segments nor do they derive from sequences elsewhere in the genome. These sequences are called N sequences [20] and seem to be added to t h e segment ends in a template-free fashion (see below). The existence of a reciprocal and a coding joint o n the same chromosome cannot be easily explained by early joining models such as the copy and insertion mechanism [25-271. The model which accounts best for the different observations is that first proposed by Alt and Baltimore [20] and is depicted in Fig. 1. Once proteins have detected and bound two segments with different joining signals, a protein-DNA complex is formed in which both gene segments are held stably together (stage 1). In this complex, double-stranded DNA cuts occur precisely at the heptamer segment border, after which the heptamers are fused directly together (stage 2). The ends of the two segments are not immediately ligated but are first exposed to modifying enzymes such as double-strand exonuclease or terminal transferase which will delete or introduce nucleotides at the segment ends prior to the final ligation (stages 3-5). The modifications at the coding joint play an important role in the generation of antibody diversity. They explain why most joints

115

D

9 GCAAAAACC CGTTTTTCG

CACTGTG CACACTC GTGACAC CTGTCAC

GCAAAAACC CCTTTTTCC

J double-strand exonuclease 7 loss of bases

terminal transferase base addition

DNA polymerase and ligase ? base replication and strand joining

G C C C 1.C C C T C C CCA GCGA

0

J

Fig. 1. Joining model of a D to J segment joint. For explanation, see the text. (Taken from [ I ] and 1201)

differ from one another even when they use the same germline segments. However, the modifications also have one disadvantage: due to the flexible nature of the joint, the two segments are often (statistically in two out of three cases) not connected in the right reading frame and thus generate non-functional V genes. We will learn in a later section that a multiple joining strategy has developed which may overcome this disadvantage and favour the generation of a functional V gene (see Section 3.1.3.). The observation of chromosomal translocations in some leukaemias, in which a J H segment is brought in the vicinity of a cellular oncogene, suggests that immunoglobulin gene rearrangement may play a role in the oncogenic transformation of lymphocytes [28,29]. In rare cases, an active recombinase

116 complex containing a double-stranded DNA cut at the heptamer-segment border may be disrupted prior to the ligation step; the free DNA ends can then recombine elsewhere, resulting in a chromosomal translocation and possibly in the transcriptional activation and deregulation of oncogenes. The formation of two joints during each V gene rearrangement provides a satisfactory explanation for the occurrence of deletions and/or inversions of intervening sequences after a joint [20-22,30,31]. If the joining signals used are pointing towards each other, their fusion generates a circle of intervening sequences which is subsequently lost from the genome (Fig. 2, a). If, however, the two joining signals used have the same orientation, the coding joint and the reciprocal joint are retained on the same chromosome and generate a total inversion of the intervening sequence (Fig. 2, b). The D elements are flanked on each side by 11 base pair joining signals, either of which can be used in the joining to a JH segment. Depending on which l l base pair joining signal is used, the D to JH joint will be accompanied by either a deletion or an inversion of intervening sequences. Even though one case of inversion has been reported [20], most D to JH joints are accompanied by deletions [24] demonstrating that the 3' signal is predominantly used in this joint. In V, to J, joints, on the other hand, inversions are generally found [30,31] suggesting that most V, segments are inverted on the chromosome with respect to the J, cluster (see Section 3.2.1.). An inversion joint has also been described at the T cell receptor p chain locus and, in this case, the inverted orientation of the V, and DJ, segments prior to joining was directly proved by mapping and sequence analysis [32]. An extreme case of inversion of nearly half the chro-

Fig. 2. Deletion and inversion joining. The deletion (a) o r retention (b) of intervening sequences after a joint is dependent on the chromosomal orientation of the two segments involved. The segments are indicated by boxes and the joining signals by triangles. Two fused triangles represent the reciprocal joint. Arrows indicate the transcriptional orientations of the gene segments. (The Fig. was modified from [20].)

117 mosome occurred during the abnormal rearrangement of an immunoglobulin VH segment to the T cell receptor J, locus [33]. Of all the enzymes thought to be involved in the rearranging process (doublestrand DNase and exonuclease, terminal transferase, DNA polymerase and ligase) only the terminal transferase has been identified and characterized. The enzyme adds deoxynucleotides to the 3' end of DNA and shows a preference for dGTP [34]. The frequent presence of this nucleotide in N sequences (see above) suggests that such sequences are generated by the terminal transferase [20]. The enzyme activity is high in developing T and B cells, and was first detected [35] in the organs where lymphocyte development takes place (the thymus or the bursa of Fabricius in chickens). During B cell development, the expression of terminal transferase declines. This may be the reason why N sequences are predominantly found in early joints, such as D to JH or some of the VH to DJH rearrangements [24,36], but only rarely in VL genes which are assembled at a later stage of B cell development [31,37]. Studies of V gene rearrangements in Abelson lines have provided more direct evidence for the generation of N sequences by terminal transferase [16,36]. In one experiment, a plasmid containing D and J segments from the T cell receptor p chain locus was introduced into a foetal liver-derived Abelson line, and daughter cells were analysed for rearrangements of the introduced segments. Several of the DJ complexes formed were analysed, and N sequences were only found in complexes isolated from daughter lines that expressed terminal transferase [ 161. In addition, the above experiment demonstrated that the recombinase system of the B cell is perfectly able to recognize and rearrange T cell receptor gene segments. Thus, the same recombinase system seems to be responsible for rearranging variable region gene segments in both T and B cells. The characterization of other proteins involved in the joining process is still in its infancy. There have been reports of a double-strand DNase activity in cells or organs of B cell development [38-401. However, none of these enzymes is highly specific for the heptamer-segment border and they also cleave several unrelated TG-rich sequences [40]. The failure, so far, in identifying more of the enzymes involved in the rearranging process suggests that such enzymes either have a low abundancy or are only active in a structured protein-DNA complex which is difficult to study by classical biochemical approaches. Furthermore, our limited knowledge of the topology of the DNA at the stage where joining occurs is also hampering our understanding of the molecular mechanism of joining. Thus, at present, it is impossible to prove or disprove the details of the joining model described above by enzymological studies. 2.3. Control of joining The manner of the activation of V gene segments in the rearrangement process shows similarities to that of the expression of eukaryotic genes. Variable region gene rearrangements are tissue- and stage-specific events. So far, they have been found only in B and T lymphocytes and are restricted to a certain developmental stage of these cells. The similarities between gene expression and gene segment

118 activation suggests that the two systems possess common mechanisms. One of these mechanisms seems to be the controlled ‘opening’ of the chromosome as put forward in the accessibility model of joining [16,41]. Most regions of eukaryotic DNA are covered by proteins (chromatin) which protect the genome from uncontrolled expression. The expression of a gene requires the opening of the chromatin structure and the same has been proposed for the rearrangement of V gene segments [42]. A plasmid containing unrearranged D and JH elements linked to the thymidine kinase (rk) gene as a selectable marker was introduced into a t k - Abelson cell line. The frequencies of rearrangement of the introduced D and JH segments could be drastically increased if the cells were grown in selective (HAT) medium. Obviously, only cells expressing rk survived the selection and the transcription of the transfected tk gene ensured an open chromatin structure for the adjacent situated D and JH segments; these thus became accessible to the recombinase enzymes. The correlation between accessibility and rearrangement was even more directly shown in another experiment in which a plasmid containing V, and J, segments was introduced into an Abelson line [43]. Eleven copies of this plasmid were stably integrated at different locations in the genome but rearrangement occurred on only one of these copies during culture. The chromatin structure of the integrated DNA was tested by a DNase I sensitivity assay [16,43]. Of all the integrated plasmid copies, only the one which could rearrange in culture was DNase I sensitive, thus indicating that an open chromatin structure is one of the requirements for joining to occur. During B cell development, the specific opening of the chromatin structure at the three immunoglobulin loci is indicated by their transcriptional activity; this is observed shortly before or at the same time as rearrangement. Such transcripts derive either from unrearranged V gene segments or from around the J clusters; they are called germline or sterile transcripts because they never encode a mature immunoglobulin polypeptide 144-481. Whether these transcripts play a direct role in the rearranging process itself, is presently unknown. Their role could be more indirect, for example by either opening an immunoglobulin locus and/or by ensuring that it stays open. Accessibility is probably one of the most important control mechanisms of the rearrangement process. However, additional control elements have to be postulated to explain the behaviour of the gene segments during V gene assembly. At the IgH locus, for instance, all rearrangement events are focussed on the JH region. VH to D joints, although possible according to the 12/23 joining rule [9], are only observed in the form of a V, to DJ, joint once a D element has been appended to a J H segment [8]. Likewise. VH to VH joints are theoretically possible (see Section 3.1.3.) but are only seen as VH to VHDJH replacement joints (14,151. This order of joining cannot be solely explained by the accessibility model. In an Abelson line, which simultaneously performed D to J H and V, to DJH rearrangements, VH and D elements were clearly both accessible and nevertheless no V, to D joints were found [24]. Thus, the rearranging process may be controlled in such a way that an active recombinase complex can only be formed at the JH locus. Another phenomenon that still lacks an explanation is the ordered usage of J,

119 elements during V, gene assembly. At the K locus, four functional J, segments (J,1 to J,4 in a 5' to 3' order) are located on a 2 kb region of DNA which is transcribed in J, rearranging cells [46,47]. This suggests that all four segments are situated in an open chromatin domain and should thus be equally accessible to recombinases. However, J,l and J,2 are used much more frequently than J,3 and J,4 in the V, to J, joining process [49,50]. Furthermore, an analysis of B cells carrying V,J,2 complexes revealed that these cells had previously formed a VJJ complex [51,31]. Thus, during V, assembly, the J, segments seem to be used sequentially in a 5' to 3' direction. This J, usage pattern can be explained by a tracking model of joining [52] which postulates the existence of proteins that glide along the DNA helix until they find an appropriate joining signal to which they then bind. The ordered J, usage pattern may be the result of such a tracking mechanism if one assumes starting sites for tracking 5' of the J, locus and a 5' to 3' direction for the search. At the IgH locus, a similar 5' to 3' usage pattern is seen for D and J H elements [24]; this could be due to the same molecular mechanism (see Section 3.1.1.).

3. Order of rearrangement events during B cell development 3.1. Rearrangements at the IgH Iociis During B cell development, J H rearrangements occur in an ordered fashion. The assembly of D to JH segments precedes V, to DJH joining which can itself be followed by a V, to VHDJH joint (V, replacement; Fig. 3). These different rearrangements will be described in detail in the following three sections. 3.1.1. D to .IHjoints A D segment encodes most of the third complementary determining region (CDR3) of the V, domain. It is only 1G23 nucleotides long and is flanked on both sides by 11 base pair joining signals [53-561. Twelve D segments are located within 80 kb upstream of the four J H elements of the mouse [52].These D segments are clustered in homology groups and have the following 3' to 5' order: the single DQS2 segment lies SO0 base pairs 5' of J H 1 followed by nine D-SP2 segments and, finally, by two D-FL16 segments, which are the most upstream of the known D segments. A D to JH joint is the first rearrangement observed at the IgH locus. It seems to happen very early during B cell development. Indeed, all Abelson cells analysed so far that represent early pre-B cells, already carry a DJH complex on both IgH alleles [S]. This may indicate that the first D to JH joint occurs in a precursor B cell which cannot yet be transformed by the Abelson virus. It is even possible that the first D to JH joint takes place in a pluripotent precursor that is able to give rise to both T and B cells [57]. Between 10 and 20% of T cells carry DJH rearrangements although they never assemble a VHDJH complex [54]. Such T cells may be derived from a common TIB precursor [S7]. Alternatively, it is also possible that D to JH rearrangements take place occasionally during T cell development.

120

/

0-0 stem-cell

B- precursor

\

r

w

\

/

'\,-

;

- ,*/ \

time Fig. 3. J , , rearrangements during B cell development. Thc different rearrangement events during the clonal expansion of a B precursor are indicated on the top. Representative cells are drawn as circles indicating the rearrangement status of each cell. Notice that the p-positivc pre-B cell stage can he reached following different routes. The possible branching points to the T cell differentiation pathway are drawn as dashed lines.

A D to J H joint can occur several times on a single allele. This point became clear from the analysis of Abelson lines which originally carried a D-SP21J.3 complex on both JH alleles [24,58]. One of these lines (300-19) frequently used an upstream D element and the free JH4 segment to replace the previously formed DSP2/JH3complex (Fig. 4). The CDR3's encoded by the newly formed DJH4 complexes differ greatly from those encoded by the original DJH3 complexes (Fig. 4, c). Thus, secondary D to J H joints, which occur in 10-20% of the subclones analysed [24], may play an important role in the generation of antibody diversity. Theoretically up to four D to JH complexes can be sequentially produced on a single allele if the four JH segments are used in a 5' to 3' order, in a manner analogous to J, segments. However, once a V, segment is appended to the DJH complex, no further D to JH joint can occur because all other D segments are deleted ~~~~

~~~~~

~~~~~~~~~

Fig. 4. Secondary D to J , , rearrangement. The two DJtI3 complexes of the Abelson line 300-19 are replaced in one of its subcloncs (300-19 P4-12) by DJ,,4 complexes. (a) Location of D and J,, elements on the two IgH alleles (A and B) of 300-19. The secondary D to J,,4 rearrangements of P4-12 are indicated by arrows. (b) Sequences o f the DJ,, complexes o f 300-19 and P4-12 are compared to those of gerrnline D and J,, elements [9,54.55]. Crossover points arc marked and N-sequences are boxed. Heptamers of the S' o r 3' joining signal are underlined. (c) CDR3 sequences potentially encoded by the DJ,, complexes of 300-19 and P4-12. Amino acids determined by N-sequences are boxed and J,-sequences are underlined. (Taken from [24].)

121 during the VH to DJH joining. A DJ, complex is also fixed once the most upstream D (D-FL16) and the most 3' J,, elements (JH4) are used. To understand the biological function of multiple D to JH joining, it is important to realize that all rearrangement events occur in rapidly dividing pre-B cell clones [59,60]. How many different stem cells are giving rise to the pre-B cells generated

DFLl6-1

DSP-1 DSP-2

DSP-3

DSP-4

DSP-5

DSP-GJH~

I DSP-1J H ~

300- 19 Paaen_t

@

A

DSP-4

TACTGTGCCTAC

B

DSP2.6 DSP-6

TACTGECCTACTATAGTTACT

A

DFL16.1 DFLlG-1

TACTGTGTTTATTACTACGtiT TACTGTGTITATTACTACGAT

GGCCAAGGGACTCTGGTCATC (JH3) GGGACTCTGGTCATC (JH3)

CTATGGACTA CTATGGACTA

(JH4) (JH4)

LeuLe&GluGlySerThkGlyGln

(JH3)

B DSP-1

300-19 Pacent

@

A

DSP-4

B DSP-6

P r o T h r I l e V a l T h r ~ T r p P h e A l a T y r T r p G l y G l n (JH3)

5mL1LmePP$-12_ A

DFL16-1 T y r T y r T y r A s p G l y S e r T y a T y r A l a M e t A s p T y r T r p G l y G l n

(JH4)

B

DSP-1

(JH4)

SerMetMetValThrSerTyrTyrAlaMetAspTyrTrpGlyGln

122 TABLE 2 Usage of D and JI, segments in unselected VHDJHcomplexes (155,681 and Dildrop et al.. unpublished)

D-QS2 D-SP2 D-FL16 X Z (total)

Jlll

5112

5143

3114

Z

0

2 3 5 3 13

0 4 4 3

0

2 12 21 14

11

18

0 6 1 7

S

6 7

Data were obtained from myelomas or hybridomas from B cells activated with bacterial lipopolysaccharide. X. D element used could not be identified.

daily in the mouse bone marrow is presently unknown. Their number may be small and many of them may have already performed a first D to JH joint (see above). Without further joining abilities, the progeny of these stem cells would be restricted in their DJH variability. Taking into account the flexibility of the joint (see Section 2.2.), one may predict that secondary D to J H joints occurring throughout the clonal expansion of a pre-B cell will yield a population with diversified DJH alleles. However, secondary D to JH joining will only be profitable provided it occurs at the same developmental stage and with a similar frequency to that of VH to DJHjoining; otherwise, a newly generated DJH complex will be replaced before it has a chance to be stably integrated into a VHDJH complex. In one Abelson line analysed, secondary D to J H and VH to DJH rearrangements indeed occurred with roughly equal frequency [24]. Evidence for frequent DJH replacement during B cell development has also been obtained from an analysis of the D and JH usage in VHDJH complexes of myeloma or unselected hybridoma proteins (Table 2). The J,1 segment is found less frequently and, even then, only in association with a member of the most 5' D family (D-FL16). Thus, most DJHl joints (with the obvious exception of D-FL16/JH1see above) may have been replaced by another DJ, complex before VH to DJH joining occurs. Multiple joining events also occur at the K locus and seem to be a general mechanism during V gene assembly. This notion is supported by the fact that most V, D and J segments are present in multiple copies, thus allowing this type of joining. An extreme case is the T cell receptor a locus which contains at least 50 J elements, although multiple V to J joints at this locus have not yet been demonstrated [62]. The analysis of VHDJH complexes in myelomas and hybridomas mentimed above showed another peculiarity. The D-FL16.1 element was used more frequently than any of the nine D-SP2 elements. One reason for D-FL16 dominance may be the fact that the D-FL16/JH complex cannot be replaced by another D to JH joint. However, in Abelson lines, dominance of D-FL16 is also due to the preferential usage of upstream D segments. D-FL16 was used in eight out of 14 cases while the usage of the other (D-SP2) element declined in a 5' to 3' gradient [24]. The DJH complexes are transcribed in pre-B cells. The 5' region of most D ele-

123 ments contains a functional promoter; after a D to JH joint, this promoter is activated and the DJH complex gives rise to a D p mRNA containing the DJH complex and the C, exons. Such transcripts are translated into D p protein if a D to J, joint places the AUG start codon of the D segment in the same reading frame as the JH coding sequence [63]. The amino terminal sequence of the D p protein shows strong homology to leader sequences. The presence of a leader sequence along with the glycosylation of the D p chain suggests this protein enters the endoplasmic reticulum [45]. The role played by the D p transcript and/or by the DF protein during B cell development is still unclear. However, similar transcripts occur at the T cell receptor /3 locus where a DJ, complex is transcribed and, presumably, translated [64].

3.1.2. VH to DJH rearrangement There are 100-1 000 V, segments located at an unknown distance 5' of the D segments. These V, segments can be grouped in nine V, families whose members share more than 75% sequence homology (see Chapter 3 and [65-68]). The V, segments of different families are, in general, organized in separate clusters [67,69]. An exception is found in the elements of the most 3' families VHQ52 and V,7183 which are interspersed [24]. Studies of recombinant inbred mice [66] and a deletion analysis of Abelson lines bearing different VHDJH rearrangements [24] have allowed the order of these V, families on the chromosome to be determined (see Table 3 ) . How the different V, segments are activated for a V, to DJH joining is still unknown. This activation seems to require control elements active in B but not in T cells because the latter cells may undergo D to JH but never V, to DJH rearrangement [54]. Germline V, transcripts have been found in pre-B cells and may be part of the activation process [70]. A sequence analysis of rearranged V, segments found in Abelson lines that have been derived from foetal liver of BALBic mice revealed that the V, segments are not used randomly but in a highly restricted manner [71]. Most VHDJH complexes contained a V, segment of the V,7183 family. In particular vH81x, the most 3' ( J H proximal) functional V, segment of this family was predominantly rearranged in several independent Abelson lines [36,71]. The same preferential usage pattern was subsequently found in un-transformed foetal liver cells and hybridomas made from foetal liver pre-B cells [71,73]. Preferential V, usage was also analysed in an Abelson line (300-19) derived from the bone marrow of a NIH/Swiss mouse [24]. In this mouse strain, V,Q52 segments are the most 3' elements of the V,, cluster and, indeed, they are predominantly used for rearrangement in daughter cells of the 300-19 line. However, V, segments of other V, gene families were also rearranged in this line, although with a much lower frequency. Whereas V, elements of the nine Vkl families are not randomly used in foetal liver- or bone marrow-derived pre-B cells, resting splenic B cells or splenic B cells activated with bacterial lipopolysaccharide carry VHDJH complexes that contain V, segments of all the different families [74]. The V, family usage in these cells is roughly proportional to the size of each family and does not show any prefer-

124 TABLE 3

V,, usage of assembled V,,DJ,, complexes in pre-B and B cells [24.67,70.74] V,,family

V,,606

Vf136-60

VHSS8

V,,441

V,,S107 VFlQS2

Family size

10

2

4

12

12

3

9 4

100

Pre-B cells B cells

1 33

1

2 1

13 1

25 1

3

V117183

V,l families are given according to their presumed S‘-3‘ order

ence for those V elements situated at the 3’ end of the locus (Table 3). How this transition from a highly restricted pattern of V, usage in pre-B cells to a broader V, usage in B cells happens is an intriguing problem to which we do not have the answer. A VH usage program may exist which opens the V, locus in a 3’ to 5’ direction during B cell development; all Abelson cells as well as normal foetal liver cells may be restricted to the early stages of this program. Alternatively, a less abundant pre-B cell population with rearrangements of the more 5‘ located V, segments may develop into B cells and thereafter be selected by the immune system, thus dominating the B cell compartment in the spleen. However, it is also possible that the V, to VHDJH rearrangement mechanism (see below; [14,15]) is influencing the shift in usage pattern (see Section 3.1.3.). A V, to DJH joint is, in general, accompanied by deletion of all intervening sequences on the same allele including all free D segments [S]. Thus, in contrast to the D to JH joint, a V, to DJH joint can occur only once on a given allele. Like all the coding joints, the V, to DJH joint is flexible and is not restricted so as to ensure that V, and J H coding sequences remain in the same reading frame. Statistically, one could expect only 33% of the VH to DJH joints in pre-B cells to be in frame and to result in the expression of a p-polypeptide. Indeed, an extensive study of VH to DJ, rearrangements occurring during culture of an Abelson line showed that productive VHDJH complexes were assembled with roughly the expected frequency of 33% [24]. However, one should keep in mind that the frequency of productive V, to DJ, rearrangements in the pre-B cell population may even be lower if the D sequence used contains a stop codon in the appropriate reading frame [45,75] or if the V, segment used is a V, pseudogene with crippling mutations [24,36]. 3.1.3. VH to VHDJH joint As mentioned above, a VH to DJH joint is productive only in at most 33% of the cases and can occur only once per chromosome, generating up to 50% of pre-B cells with non-functional VHDJH complex on both alleles. These ‘null’ pre-B cells were formerly regarded as dead-end products of the B cell developmental pathway. However, recent studies have revealed that these cells have another joining option (14,151. Most VH segments contain within their coding sequence a heptamer joining signal lying just seven nucleotides upstream of their 3‘ end. This VH heptamer has the same sequence as the heptamer located at the 5’ side of a germline D segment and is still present after a V, to DJH joint. Thus, similarly to a

125 DJ, complex, a VHDJH complex carries a potential 5’ joining signal and can be joined to an unrearranged VH segment that is located upstream and that carries a 3‘ signal (Fig. 5 ) . Products of such a V, to VHDJ, joint have indeed been found [ 14,151. In two different null cell lines carrying non-functional VHDJ, complex, such a V, replacement resulted in the construction of an ‘in phase’ VHDJH complex and subsequent production of p chain. Because most VH segments carry an internal heptamer, a V, to VHDJ,, joint can occur several times on a chromosome, thus increasing the chance of the formation of a productive VHDJH complex. However, a VHDJH joint can also occur on a productive VHDJ, complex with or without destroying its ability to produce p polypeptide. The internal V, heptamer is highly conserved in most mouse and human VH segments, suggesting that V, to VHDJH joining may play an important role during B cell development. This role may be to rescue production of p chain in null preB cells or to diversify the V, segment repertoire. The contribution of V, to VHDJH joining to the 3‘ to 5’ shift of V, segment usage observed in a comparison of preB cells and B cells (see Section 3.1.2.) has not been demonstrated from data that are presently available. Indeed, in the cases analysed so far, the replacing and replaced v, segments were either neighbours or were found to lie in close proximity on the same chromosomes, suggesting that the V, to VHDJH joint is a locally restricted event. Thus, upstream v, segments may be activated for the joining to VHDJH complexes by the nearby situated IgH enhancer element, which provides an open chromatin structure in its surrounding [7&78]. ,CACAGTGI

,TACTGTG,

I

1

VH

her

gmer

9mer

7mer

D

JH

Fig. 5 . V,, to VI,DJ,, joining. A V, to DJ,, joint is shown for comparison on the top. Heptamer and nonamer of a joining signal are indicated by black triangles. The internal V, heptamer is shown as a white triangle. The location of the IgH enhancer (E) is indicated. Notice that V,, to VHDJ,, joints could occur repeatedly on the same IgH allele.

126

3.2. Rearrangemerits at the light chain loci Immunoglobulin light chains are encoded by two different IgL loci ( K and A) situated on separate chromosomes of the mouse. Assembly of light chain variable regions occurs later during B cell development than that of heavy chains, as evidenced by studies of immunoglobulin expression in liver cells of mouse embryos at different stages; light chains are always found later than heavy chains [79-811. The later assembly of V, genes is also established by analyses of foetal liver hybridomas and Abelson pre-B cell lines, all of which carry JH but only rarely J, rearrangements [7,81,82]. A recent study of an Abelson line, which assembled its VH gene as well as its V, genes during culture, indicated that the delayed V, assembly is due to regulation [83]. Rearrangements of V, to J, were only found in those daughter cells which carried a productive VHDJH complex and expressed a p chain. Thus, p chains seem to have a positive regulatory function in inducing the rearrangement process at the J, locus. The activation of the two different IgL loci is ordered in that V, to J, rearrangements always precede V, to J, rearrangements [84]. The activation of the A locus seems to be dependent on rearrangements (RS rearrangements - see below) which render the two J, alleles non-functional [ 11-13]. Thus, analogously to rearrangements at the IgH locus, the rearrangements at the light chain loci occur in an ordered and controlled fashion.

I I

.

2" VKto JK

1" VKto JK I

RS

Vx to J i

& &

@ KYKO

pre-Bcell

Bcell

time

Fig. 6. J, rearrangements during B cell development. The different rearrangement events during the clonal expansion of a pre-B cell are indicated on top. Circles indicate representative cells which have different rearrangement status. The ordcr o f RS (see Fig. 7) and V, to J, rearrangements is hypothetical: thus, only one of the many possible pathways of rearrangement at the A locus is illustrated. The loci are symbolized: K O , germline I g K locus; K + , productively rearranged and K . , non-productively rearranged I ~ alleles. K

127 3.2.1. V , to J , rearrangement Four functional J, segments are found several thousand base pairs 5’ of the C, exon [85] and 100-200 different V, segments are available for the V, to J, joint. As already mentioned (see Section 2.3.), the frequency of usage of each J, segment during V, gene assembly seems to follow a 5’ to 3’ gradient with J,1 being the most frequently and J,4 the least frequently used J, segment [49,50]. The ordered J, usage allows a p-positive pre-B cell to undergo several V, to J, joints on each J,-allele. Multiple V, to J, rearrangements may not only ensure the assembly of a productive VJ, complex; they may also allow each pre-B cell to assemble and test several productive V,J, complexes until one of them produces a functional K light chain capable of binding the pre-existing heavy chain with high enough an affinity to form an antibody molecule. Such a molecule is thought to induce feedback control (see Section 4.), stopping any further V, to J, rearrangements [86,87]. Multiple V, to J, rearrangements could thus be regarded as a program to find the best fitting light chain for the pre-existing p heavy chain. Such a program would explain why, in some B cells, a productive V,J, complex is replaced by another [31] and why the heavy and light chains isolated from the same cell tend to have a higher affinity for each other than randomly recombined heavy and light chains from different B cells [88]. The assembly of K variable regions seems to involve an inversion joining mechanism (see Section 2.2.). It is, therefore, likely that many or all mouse V, segments have an inverted orientation relative to the J, cluster and it is even possible that V, segments are not situated 5’ but 3’ to the J, locus on mouse chromosome 6. A detailed study of rearranged J, alleles in B cells actually demonstrated that both inversions and deletions frequently occur during assembly of K variable re-

Fig. 7. Possible rearrangement events at the J, locus. V, segments are assumed t o have an inverse orientation to that of the J, segments. The 22 or 11 base pair joining signals are indicated by black o r white triangles, respectively.

128 gions [22,31]. For example, the V,J,1 reciprocal joint is, in most cases, retained whereas the V,J,1 coding joint is often deleted from the K locus [51]. These findings are best explained by multiple joining events, as depicted in Fig. 7. The first V, to J,1 joint inverts all sequences between the two segments used; this inversion can include V, segments which then lie in the same orientation as the J, segment. Any further rearrangement involving these V, segments is accompanied by a deletion of the intervening sequences. A retroviral vector containing the J, cluster and an inverted V, segment was introduced into an Abelson line that was actively rearranging its own K gene segments. In this experiment, the introduced segments rearranged frequently, thus convincingly demonstrating the occurrence of multiple inversion and deletion joinings at the K locus [21,22].

3.2.2. RS rearrangements Multiple V, to J, joinings occur on each K allele ensuring the expression of a functional K light chain in most B cells. However, the K locus can be rendered nonfunctional by a further rearrangement event involving a sequence (RS, for rearranging sequence) which lies 10-20 kb 3‘ of the C, exon (Fig. 7) and which is flanked by a 22 base pair joining signal [ll-13,891. The RS element can be appended to a V, segment, a joint allowed by the 12/23 joining rule because V, segments are flanked by joining signals with a 12 base pair spacer. More frequently, RS elements are rearranged into the J,-C, intron through a joint involving an isolated heptamer. Both types of rearrangement result in the deletion of the C, exon, thus preventing any K chain production (Fig. 7). RS rearrangements are regularly found in mouse or human A-producing B cells [10,11]. In these cells, both K alleles are in most cases destroyed by an RS joint while K-producing B cells only occasionally carry even a single RS rearrangement. These findings suggest that RS rearrangements play a role in the isotype switch from K to A production in developing B cells. The order of events is not yet clear. The K locus may be destroyed before or after the A locus is activated. An intriguing possibility is that the RS rearrangements are causally related to activation of the A locus. For example, a regulator gene preventing A activation may be situated between the J, and RS segments and would, therefore, be deleted after RS rearrangements. Alternatively, after disruption of the K locus, the free /-t chain may activate the A locus in the same way as it had previously activated the K locus [83]. 3.2.3. V , to J , reurrungements The A locus of the mouse carries only two V, segments but four C, exons which are organized in two clusters (CA3-CA1 and CA2-CA4) ([90,91], for a review see [92]). A single J, segment is found 1.3 kb 5’ of each C, exon. The location and orientation of the V, segments with respect to the two C, clusters is presently unknown. However, because V,1 is in most cases appended to J,1 or J,3 while V,2 is connected to J,2, it is assumed that V,1 lies 5’ of the C,, cluster and VA2 lies 5’ of the C,, cluster [92]. In rare cases, a V,2 to J,3 or J,1 joint was found [93]. The existence of these joints suggested that the V,2-CA2 cluster lies 5‘ of the V,l-C,, cluster. In the mouse, only 5% of all immunoglobulin-positive B cells ex-

129 press A light chains, most of which are VA1CAL. Less frequently expressed isotypes are A2 and A3 while A4 is a pseudogene and never expressed [94]. The sequence of VAto J, rearrangement during B cell development has not been studied so far. Many Al-expressing myeloma carry the A2 gene segments in the germline configuration, while those expressing A2 frequently have JA1 rearrangements. Taking these data together with the fact that A 1 is more frequently expressed than A2 or A3, it is likely that rearrangement of J,1 occurs prior to that of J,2. Because a single vA1 segment is present on each allele, mouse B cells have only two chances to generate a productive A 1 light chain; this happens with a one in three chance on each allele. However, a sequence analysis of productive V,1 genes revealed that several of them may have been derived from non-functional (out of phase) V,lJ,l complexes by an, as yet unknown, mechanism deleting one or two nucleotides close to the VA-J, joint [95].

4. Allelic exclusion of immunoglobulin chain expression Even though each mouse B cell can theoretically produce two different heavy chains (one from each of its IgH alleles) and up to six different light chains (one from each of its K and two from each of its A alleles), immunoglobulin chain expression in B cells is restricted to one functional heavy and light chain. This was shown in an early analysis of F1 mice whose B cells carried a different marker ( a and b ) on each IgH allele [96]. Staining of these cells revealed that they produced either an a or b type heavy chain but never u and b together in the same cell. Thus, the expression of one IgH allele excluded the expression of the other allele, hence the name ‘allelic exclusion’. Allelic exclusion is important for the function of the immune system because it ensures that each B cell expresses only one type of heavy and one type of light chain and, thus, makes an antibody molecule with a unique binding specificity. This enables the specific activation of B cells by the relevant antigens, as demanded by the clonal selection theory [97]. Studies of immunoglobulin chain expression in pre-B and B cells showed that allelic exclusion is established by a control of the rearranging process [41,42]. According to a model proposed by Alt and Baltimore [20], this control consists of a feed-back mechanism whereby the product of a successful rearrangement on one allele stops further rearrangements on the second allele. This model was tested in a p-positive Abelson line which carried a productive VHDJH complex on one allele and a DJH complex on the second allele. After several weeks of culture, the Abelson line was subcloned and daughter lines analysed for further rearrangements. N o VH to DJH rearrangements were found in this line, although secondary D to JH joints occurred on the DJ, allele and a VH to VHDJH joint was found on the VHDJH allele (Wiese and Reth, unpublished). Thus, production of p chain seems to prevent any further VH to DJ, joint, but it still allows D to JH and VH to VHDJH rearrangements to occur. The target of the feedback control, therefore, does not seem to be the recombinase enzymes themselves, but rather the mechanism responsible for activating VH segments for a VH to DJH joint. This regulation may, for example,

130 influence the accessibility of the V, locus. Indeed, no V, germline transcripts were found in p-producing Abelson lines, suggesting that once a productive V,DJH complex is formed, the V, locus becomes inaccessible [70]. The regulatory influence of the p chain was also demonstrated in experiments where a functional p chain gene was introduced into the germline of the mouse. The introduced gene was expressed as a mature p chain in many B cells of the recipient mouse, and this expression stopped or delayed rearrangements at the JH cluster [98-101]. In this case, even the D to JH rearrangement was inhibited in 40% of the Abelson pre-B cell lines obtained from the transgenic mouse. Thus, if present before the JH locus is activated, a p chain may prevent all JH rearrangements, but once a D to J H joint has occurred, the p chain regulates the V, to DJH joint. By demonstrating that expression of the p chain controls the allelic exclusion, the above experiments rule out earlier models regarding allelic exclusion as a consequence of an extremely low rate of productive V, to DJH rearrangements [lo21 or of the destruction of IgH double-producers [103]. However, in rare cases where the rearrangement control may fail, other mechanisms could possibly contribute to the allelic exclusion of immunoglobulin chain expression. Such mechanisms could be the deletion of one IgH allele which occurred in 10% of the clonal progeny of an Abelson line [24] or the inactivation of a productive VHDJ, complex by the hypermethylation of its DNA [104]. As mentioned in Section 3.2., the p chain seems to have a dual regulatory function: the cessation of V, gene assembly and the activation of the V, to J, joint [83]. Whether both types of regulation are based on the same molecular mechanism is presently unknown. The allelic exclusion of K light chains, however, seems to follow the same principle as that of the IgH chains. The only difference appears to be that further VL to JL rearrangements are stopped by the expression of a complete antibody molecule rather than by the expression of the light chain on its own. The regulatory role of the complete antibody molecule was proposed [86] after several myeloma cells had been found that apparently violated allelic exclusion, and that contained two light chains, although only one of them bound efficiently to the heavy chain [105,106]. Further support for the regulatory role of the complete antibody molecule was provided by an experiment in which the expression of a transgenic K chain inhibited J, rearrangements only in those B cells where it was bound to a heavy chain and thus assembled into a complete antibody molecule [87,107,108]. Thus, during B cell development, productive V, to J, or V, to J, rearrangement may occur on both alleles until a light chain is expressed which binds the pre-existing heavy chain with a high affinity. Heavy chain binding protein (BiP) may be involved in this control [ 103,109,110]. BiP is expressed in pre-B cells and seems to bind to the same part of the p chain as that involved in the interaction with the light chain. The binding to BiP seems to prevent the surface expression of the p chain. Only light chains with a greater affinity for the heavy chain than BiP can efficiently bind to the heavy chain, thus allowing the cell surface expression of an antibody molecule; this cell-surface expression may itself be the stop-signal for further J, rearrangements.

131 chromosome 12

chromosome 6

chromosome 16

allele A

chromosome 12

chromosome 6

chromosome I6

allele B

Fig. 8. Regulation of immunoglobulin chain expression. The expression of an immunoglobulin chain after productive V,, to DJ,, or V, t o J , rearrangement is indicated by a solid line. The regulatory influence of the IgH chain or the complete immunoglohulin molecule is indicated by a dashed line.

In summary, the expression of a functional antibody molecule on each B cell requires several regulatory circuits where the product of a given locus can either stop rearrangements of the second allele or activate rearrangements at other loci (Fig. 8). The elucidation of the molecular basis of this regulation is presently under way and may provide a fascinating illustration of gene regulation in eukaryotic cells.

Acknowledgements The authors are grateful to Eva Siegmund who helped typing this manuscript and to Udo Ringeisen for excellent graphic work.

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F. Calabi and M.S. Ncubcrgcr (Eds.) Muleculiir (;cncrics of Ir,lrntrrlog/obrt/irr (Biomedical Division)

01987 Elsevier Scicncc Publishers B.V.

135 CHAPTER 5

Immunoglobulin heavy chain class switching U. KRAWINKEL and A. RADBRUCH Institute for Genetics, University of Cologne, Weyertal 121, 0-5000 Cologne 41, FRG

I. Introduction Whilst the variable region of an immunoglobulin molecule carries the antigen binding site and regulatory idiotypic determinants, the constant region is responsible for the effector functions of the molecule. These functions include transmembrane signalling and secretion signals, agglutination of antigens, complement activation and binding to Fc receptors (reviewed in [l]).Immunoglobulin molecules may carry a number of different constant regions (classes which are serologically defined as isotypes) with different functions. This functional diversity enables the immune system to shape the response to different antigens. In 1964 Nossal and coworkers observed that individual activated B cells could switch the heavy chain constant region of the antibody that they produce, while retaining the antibody’s specificity [ 2 ] .Since then, considerable effort has been directed at understanding the molecular basis of heavy chain class switching. The regulatory steps and the initial phases of switching are still unclear. More is known about the later events. The comparative analysis of switched and unswitched cells has clearly shown structural rearrangements in the heavy chain gene loci of switched cells. Thus, recombination plays an important role not only in the formation of active immunoglobulin gene transcription units (V-D-J-joining) but also in the course of class switching. Switch recombinations, at least in plasmablasts (B cells activated by antigen), generate the functional diversity of secreted antibodies. They occur at high frequencies and have typical sequence requirements. The existence of switch (S) regions in front of the constant region genes suggests that specific recombinatorial mechanisms are involved. The differences in the structures of the various switch regions raise the possibility of class-specific switch recombination, which would make no sense without a corresponding regulatory mechanism. It is known that the frequency of cells expressing various immunoglobulin classes can be regulated by T cell derived lymphokines [3-91, although no lymphokine has been clearly demonstrated to induce the switch directly rather than to expand switched cells or switch precursors. In this chapter we shall review the data available from the cellular and molecular analysis of class switching.

136

2. Frequency of class switching 2.1. Avian B cells The bursa of Fabricius plays a crucial role in avian B cell ontogeny because surface immunoglobulin bearing B cells are generated there. Bursectomy studies have shown that this organ is indispensable for class switching. Chickens bursectomized at day 19 d o not have any IgG- or IgA- but only IgM-expressing B cells. Bursectomy at day 21 (hatching) results only in depletion of IgA [lo]. Thus, no detectable switching occurs in prebursal or postbursal B cells but the IgM-, IgG- or IgAexpressing B cells are generated in bursa1 follicles. Although activation by antigen and induction of immunoglobulin secretion may also occur in the bursa, it is still unclear whether the class switch is occurring only in the context of activation or independently of it. Detailed studies on the molecular basis of switching in chicken have not yet been published.

2.2. Mammalian B cells 2.2.1. Pre-B cells As in birds, the first immunoglobulin heavy chain class to be expressed in mammals is IgM; this is characterized by p heavy chains, the constant region of which is encoded by the C, gene. This is the CH gene located immediately 3' of the JH cluster. During B cell ontogeny, C, is the first CH gene to be expressed in pre-B cells upon successful VH-DH-JH joining. The frequency of normal pre-B cells that express other CH genes is below the level of detection in foetal liver and in adult bone marrow [ 111. 2.2.2. Naive B cells In small B cells, prior to activation by antigen, surface IgM and IgD become concomitantly expressed. This particular switch is of special interest because no switch recombination is involved and the p and 6 heavy chains are produced by differential splicing of long transcripts [12,13]. B lymphocytes that express classes other than IgM or IgD can be found at very low frequency (below 1% [14]). It is likely, however, that the switched small B cells are not naive cells but are memory B cells. Okumura et al. have been able to demonstrate that a particular IgG2 memory was conferred by surface IgG2-bearing B cells [15]. In any case, apart from the p to pi6 switch, class switching rarely, if ever, occurs in B cells prior to activation. 2.2.3. Activated B cells Switching to classes other than IgD is most obvious in activated B cells. Extensive studies have been performed on mouse B cells that have been polyclonally activated by bacterial lipopolysaccharide (LPS) [2,1&18] or by both LPS and 'switch factors', i.e. certain lymphokines produced by T lymphocytes [&9]. Antigen stimulation in vitro (e.g. [19]) and mitogenic stimulation of human B cells (e.g. [20])

137 have also been studied. It has been shown in these systems that single, activated B lymphocytes switch from IgM directly to IgG, IgA or IgE production (saltatory switch) and that this switch requires proliferation (see above and [21-241). The frequencies of switched cells have been determined over the time course of in vitro stimulations and found to increase from below 1% to well over 30%. The frequency of class switching within, for example, LPS cultures, can be calculated to be in the order of 1-10% per cell per generation - assuming continuous switching over the period of culture. generation times of 12 to 24 h and equal proliferation of IgM+ and IgG+ cells [18,25]. Among the switched cells, secondary switches can be observed at equally high frequencies. Between 1 and 5% of the switched cells express two IgG isotypes in the cytoplasm [181, suggesting sequential switching from IgM to an IgG, then from this IgG subclass to another IgG. Nevertheless, the majority of switched cells switches only once, from IgM directly to IgG, IgA or IgE. This is also evident from suppression of LPS activation by anti-immunoglobulin antibodies. Whereas anti-IgM suppresses the generation of any isotype, anti-IgG3 suppression effects only the production of IgG3 but not of IgG1, IgG2b or IgG2a expressing cells [26]. A second type of activated B cell is the ‘memory’ cell, an obscure cell type defined by its function. The experiment of Okumura et al. [15], in which the IgG2 memory was transferred with sorted surface IgG2+ B cells, is the strongest evidence so far for the notion that memory B cells already switch upon their generation. 2.2.4. Plasma cells Whether the high frequency of class switching in activated B cells, i.e. plasmablasts, also occurs at the terminal stage, the plasma cell, is not clear. Plasma cells are only distinguished from late plasmablasts by the fact that they no longer proliferate. Since switching in plasmablasts requires proliferation, switching in plasma cells is unlikely. It is unclear whether long-lived plasma cells from the bone marrow cannot be reactivated to proliferate and then switch again. In summary, IgH class switching in mammalian B cells seems to be closely associated with B cell activation and occurs only rarely, if at all, prior to activation. 2.2.5. Transformed B lineage cells Much of the confusion as to when and at what frequency B cells switch stems from the analysis of lymphomas and plasmacytomas. Since such transformed cells can be cloned and adapted to grow in vitro they have been studied extensively. Most of the information on DNA rearrangements in switched cells is derived from analysis of B cells that became transformed after switching. The transformation had immortalized the switched phenotype. With few exceptions, switching within transformed cell lines of the B lineage is a rare event. It has been observed in murine pre-B cell lines [27-291, human pre-B and B lymphomas [30]. the murine B cell lymphoma 1.29 [31] and in several murine myeloma and hybridoma cell lines ( e . g [32]). In general, the frequencies of switching in these lines do not reflect the switch frequencies in the corresponding B cells from which they are presumed to be derived.

138 In most lines the frequencies of switching per cell per generation are below lo-'. Exceptions are the pre-B cell line 18-81 and the B cell line 1.29, where switching can be induced by LPS to occur at frequencies similar to those of LPS blasts [27,31]. The observation that class switching in pre-B lymphomas occurs at an even higher frequency than in plasmacytomas contrasts with what is found in untransformed cells where switching is apparently induced upon B-cell activation (see above). However, it is unclear whether pre-B lymphoma cells are representative in all aspects of the pre-activation stage of B lymphocytes. It is entirely possible that with regard to class switching they are already 'activated'. The B lymphoma 1.29 is the perfect example of an inducible B cell line that, although transformed, upon activation by LPS or anti-immunoglobulin antibodies switches class at high frequency and goes on to immunoglobulin secretion as evaluated by cytoplasmic fluorescence [31]. The low frequency of class switching in cloned myeloma and hybridoma cells (below lO-'/cell/generation, see review [C]), points to a remarkable stability of isotype expression in plasma cells. Although most detailed analysis has been performed on plasmacytoma P3X63 and hybridomas derived from it, a few other myelomas, such as M P C l l [33]and 5606 [34] also show only low frequency switching. This is a first hint that switching requires specific signals and is not an automatic consequence of the activated state of a B cell as reflected by proliferation and immunoglobulin secretion of plasmacytoma cells. Detailed analysis of the low frequency switches in plasmacytomas has revealed that they are different in kind to those found in normal B cells. The plasmacytoma cells usually switch from expression of one C, gene to expression of the next CH gene downstream. The most striking examples are three IgM producing hybridomas, where no IgG-, IgA- or IgE-producing variants could be detected in lo6 cells screened. Instead IgD-producing variants were found [35,36], a switch that is extremely rare in normal murine B cells [37] presumably because the murine Cs gene lacks a switch region (see Section 4.4.). A saltatory switch from IgM to IgGl has been described only for the hybridoma line PC140 [38].

3. Switch competence and regulation Although upon activation B cells switch at very high frequency, it is clear that not all cells switch - even after long culture periods (e.g. [39]). Moreover, upon a particular mode of activation not all IgG, IgA and IgE isotypes are expressed at equal frequency. Thus, stimulation of murine splenic B cells with LPS over up to 2 weeks results in a population containing as much as 30% IgG3 but few IgG1-, IgA- and IgE-expressing cells. On the other hand, many in vivo immune responses are dominated by IgGl or IgA or IgE plasma cells, IgG3 being a rare isotype (reviewed in [A]). The questions are (a) whether all B cells or only a subpopulation (committed B cells) can switch, and under optimal conditions will do so, and (b) whether the observed isotype frequency imbalances are the result of (1) the selective activation of cells committed to certain switches, (2) the selective induction of cer-

139 tain switches or (3) the selection of cells expressing certain isotypes from a pool of randomly generated isotype producers. These questions can only in part be answered by cellular immunology, important clues coming from the analysis of the molecular events accompanying class switching (see below).

3.1. Switch commitment In no cellular system described up to now could all B cells be driven to switch. This could merely reflect the lack of optimal activation signals or the fact that the activated B cells die before they get a chance to switch. On the other hand, the analysis of the clonal progeny of one activated B cell by use of limiting dilution LPS cultures has shown that switching takes place in about 80% of these clones [18]. Nevertheless, nearly all clones contained IgM+ cells, i.e. not all siblings had switched. The picture emerging from this and related studies [9] as well as from work on the 1.29 lymphoma [31] is that most, if not all, LPS-reactive B cells are switch competent because they produce offspring, some of which may switch. The observation that not all B cells do switch can be taken as an argument for incomplete but high frequency switch induction during a short time window. 3.2. Isotype commitment The generation of several isotypes in the clonal progeny of a single murine splenic B cell following activation with LPS [18] shows that the precursor cell was not committed to switch to a single specific isotype, although some restriction probably would have gone unnoticed. There is, however, a gross imbalance - in cellular terms - in the overall frequency of the various isotypes (see above) and this imbalance is dependent on the quality of B cell activation. Thus, polyclonal activation in vitro with ‘T cell independent’ mitogens such as LPS is usually dominated by IgM and IgG3 (see review [A]) while mitogen activation in the presence of T cell derived lymphokines and ‘T cell dependent’ antigenic activation give high IgGl and low IgG3 titers ([40], review [A]). Other regulatory T cells have been described that lead to dominance of IgA and IgE [3-51. In all of these cases it is not yet clear whether the T cells induce the specific switch or whether they merely selectively expand clone size and stimulate secretion of already switched cells of certain isotypes. It is difficult to demonstrate the induction of an isotype switch at the cellular level. The first phenotypic consequence should be the expression of switched surface immunoglobulin, although it is difficult to exclude the possibility that precommitted cells were already expressing low levels of the novel isotype. In the case of LPS activation, one group has seen the expression of surface IgGl+ plasmablasts in the absence of T cell lymphokines [6]. According to these workers, the T cell help can then induce IgGl secretion by surface IgG1+ cells. Other groups claim the induction of both surface and secreted IgGl by the T cell lymphokine IL-4 [7,9]. Probably the best evidence for switch induction is the decrease in the frequency of IgG3 cells that accompanies the increase in IgGl cells upon addition of IL-4

140 [9,41]. This suggests that cells that are committed to switch can be induced to go to C,, rather than C,. But even the detailed analysis of Layton et al. [9] could not distinguish between induction of the specific switch to IgGl and the selection of precursor cells committed to the IgGl isotype, because an identifying marker for the latter cells was missing. In any case, the isotype commitment would have had occurred after activation of the yet uncommitted splenic B cell. Isotype commitment seems to be the rule particularly for transformed cells. In the murine pre-B cell line 18-81 and some other pre-B cell lines switching occurs from p to y2b at high frequency [27] whereas 1.29 cells mainly switch from IgM to IgA or IgE [31].

4. Molecular analysis The molecular events that lead to the expression of a switched immunoglobulin heavy chain have been the target of extensive studies. Nevertheless, the initial stages of class switching remain obscure. Most of the available data concern the comparative structural analysis of the IgH loci of switched and un-switched cells. 4.1. Early steps

Due to experimental difficulties, little is known about the initial phase of class switching. Specific inductive stimuli for switching have not even been hypothesized for mammalian cells except for the isotype determination by IL-4. In the cell line 1.29 partial demethylation of the C,, C, and C, genes has been found in a mixed population of switched and un-switched cells [42]. In addition, short transcripts of C,, C,, C , in 1.29 cells [42] and C,, in 18-81 cells [28] have been identified. This has been interpreted as evidence for an opening of the chromatin at those CH genes in the initial phase of switching. It could, however, equally well reflect the open-ness of switched CH genes after switch recombination (see below). Demethylation of switched CH genes has been shown in plasmacytomas [43]; furthermore, the short transcripts found in 1.29 and 18-81 could resemble the sterile C, transcripts found in a variety of B cell lines [44]. Another early event could be recombinations within switch regions (see below) and these might precede the recombinations between switch regions which are found in transformed switched cells [85]. This indicates recombinations within S , prior to the actual switch recombination. In addition, the fact that several rounds of proliferation are required for the switching of polyclonally activated and immunoglobulin secreting plasmablasts [21-231 suggests that DNA rearrangement precedes the phenotypic switch.

4.2. Long transcripts The argument has been made that the first steps of class switching in cells not yet secreting immunoglobulin involve the differential splicing of long transcripts span-

141 ning several unrearranged CHgenes [4S]. Such splicing has been shown for the simultaneous expression of c, and C6 from the same IgH locus by naive murine splenic B cells [13]. Likewise the simultaneous expression of IgM and IgG, IgA or IgE on the surface of B cells would argue for a switch without switch recombination. However, such double-expressor cells are not easy to demonstrate because they are so rare and they are hard to distinguish from B cells that have passively adsorbed serum IgG, IgA or IgE to their Fc receptors. At present one cell line is available expressing C, and C, from one IgH locus [46,47]. Long transcripts containing several C, genes have not been demonstrated directly in these cells. In another cell line the secretion of IgG by switch variants could lead to presentation of passively absorbed IgG on IgM' cells which have all CH genes in germ-line configuration [48]. Two groups have isolated normal murine B cells with surface IgG, IgA or IgE and directly examined the IgH locus organisation of the isolated cells by restriction analysis [4S,49]; in one case the heavy chain mRNA was also analysed (491. Yaoita and Honjo [45] looked at splenic B cells co-expressing surface IgM and IgE from SJA mice that had been infected with the nematode Nippostrongylus brasilierzsis. Such IgM+/IgE' cells make up about 10%. of the splenic B cells and they can be isolated by fluorescence-activated cell sorting. When examined by restriction analysis, the C, and C, genes of the IgM '/IgE+ cells were found in germ-line configuration on both chromosomes. This led the authors to suggest a two-step class switch model: in the first phase, there would be transcription of several CH genes but with differential splicing to yield co-expression of two isotypes on the cell surface; in the second step, deletion of C,, genes would result in the exclusive production and secretion of the new isotype. In the absence of apparent rearrangement of the C, and C, genes in the IgM+/IgE+ cells, the argument relies, however, on the exclusion of IgM' B cells harbouring passively adsorbed surface IgE, since most splenic B cells have Fc receptors. Yaoita and Honjo argue that in SJA mice no IgE-secreting plasma cells can be found and thus there would be no IgE available in the serum for passive adsorption. Recently, however, Katona et al. [ 5 0 ] found IgE-secreting plasma cells in some SJA mice and it was only in such mice that they found large numbers of surface IgM'IIgE' B cells. The surface IgM'IIgE' B cells could be stripped of IgE with acid, a clear indication that it was passively adsorbed. The controvcrsy about the nature of the cells analysed by Yaoita and Honjo could probably best be resolved by the direct demonstration of transcripts containing both C, and C,. The direct demonstration of long transcripts containing C, and C,/C, in murine splenic B cells has been reported by Perlmutter and Gilbert [49]. Splenic B cells were separated according to the class of surface immunoglobulin by cell sorting. The RNA in these cells was analysed by a hybridization technique in which the RNA was hybridized to CYIor C, probes immobilized on nitrocellulose filters; the filters were then probed for RNA containing C, sequences. RNA from sorted IgG1+ cells was C,- and CYI-but not C,,-positive while RNA from sorted IgA' cells was C,-, CY1-and C,-positive. Although confirmation of these results by other groups is lacking, this experiment represents a direct approach to demonstrate that the first step of class switching is controlled at the RNA level.

142

4.3. Class switch recombination The second step of class switching according to Yaoita and Honjo [45] would be the recombinational joining of two switch regions. This step is obviously easy to demonstrate if enough cells can be obtained for restriction endonuclease analysis of their IgH loci.

4.3.1. Activated B cells This analysis has been largely performed on human B cell lines transformed with Epstein-Barr virus and on murine plasmacytoma cells where class switch recombination was first detected [51]. These cells presumably had class switched before they became transformed and were isolated as IgG-, IgA- or IgE-expressing cells. In all of them, the expressed CH gene was no longer in germ line position but was juxtaposed to the VHDJH complex by deletion of intermediate CH genes. This deletional recombination - the class switch recombination - was apparently closely associated with DNA regions of remarkable structure, i.e. the switch regions (see Sections 4.4. and 4.5.). Frequently, class switch recombination could also be detected on the inactive, allelically excluded IgH locus resulting in the deletion of the same or other CHgenes [52-541. Structural reorganisation of switched IgH loci is found in all transformed cells analysed so far. Possibly, it may be the only way to ensure efficient transcription of reasonably short units in immunoglobulin-secreting terminally differentiated cells. For the analysis of switched normal (non-transformed) immunoglobulin-secreting B cells, it has proved difficult to obtain enough cells for molecular analysis. At present it has only been possible to isolate switched progeny of many independent switching events, e.g. all cells that had switched to IgGl in a polyclonal B cell stimulation carried out using LPS. This poses an additional problem. Independent blot

scanner

A ! B

-

Fig. 1. Quantitative restriction analysis for the evaluation of class switch recombination in polyclonally activated B cells. The rearrangement of the S,, region is reflected by the disappearance of the S?,,germline restriction fragment ( ). This is measured by determination of the ratio of hybridisation intensities of s,, and a reference probe. The loss of s,, is calculated from the comparison of intensity ratios in lane A (liver DNA) and B (activated B cells). Further details are given in Section 4.3.1. of this chapter.

143 TABLE 1 Class-switch rearrangement of the IgH loci of polyclonally activated B cells Time"

Phenotypeh

S, c, s73 SYI SyZh ~~-~~ i' a' i' a' i' ac i' a' i'

Early stimulation

IgM' IgG3' IgG I

RIG'' R/G R RIG R RIG

IgM+/IgGIgG3' IgG I

Ci

+

Late stimulation

+

:,'

R R

G R R

G -

-

G -/G -/G

G R -

G RIG -/G

G G R

G G RIG

G G G

G G G

G R -

G G R

G G R

G G G

G G GIR

G

G

-

-

G R

-

-

-

.' Stimulation of murine splenic B cells with LPS with or without IL-4 for about 3 days (early) to 9 days (late). " Production of immunoglobulin of the respective isotype. ' Active (a) and inactive ( i ) refer to the productive and allelically excluded IgH loci of a cell. 'I Rearrangement (R). deletion (-) or germ-line configuration (G) of EcoRI restriction fragments.

switch recombinations could be (and indeed are) heterogeneous with respect to recombination sites. There will be multiple restriction fragments that contain the novel DNA joint created by such switch recombination and these will be scattered over the whole length of a Southern blot track so that the individual bands will disappear in the background (Fig. I). Therefore, the analysis can only show the disappearance of germ-line restriction fragments carrying a given C, gene or switch region. The principle of this quantitative restriction analysis is illustrated in Fig. 1. The D N A from unstimulated cells gives the control value for the proportion of IgH loci in germ-line configuration in all cells. Using this method Hurwitz and Cebra [55] have shown that there is more rearrangement of C, genes in LPS blasts expressing little cytoplasmic IgM compared to those expressing a lot. We have sorted IgM-/IgG3+ LPS blasts and have shown that the intensity of C,-gene hybridization decreases to about 50% on day 6 and C, is practically completely absent on day 9 (Table 1, [39]). Cells that are surface IgM+/IgG- o n day 9 show no C,-gene deletion and little if any rearrangement of C enes [41]. Thus, class switch recombination is restricted to switching cells, where it happens on both alleles. It does not happen on the inactive IgH locus of cells that remain IgM producers. Furthermore, in switching cells class switch recombination preferentially appears to join the same switch regions on both alleles. To demonstrate this, we stimulated splenic heterozygous B cells with LPS and the lymphokine IL-4 (BCDFy), and isolated cells expressing the CYIgene of one of the parental allotypes. Restriction analysis showed that the majority of them had performed switch recombination to C,, on both the active and the inactive IgH loci (Table 1, [41,85]). The molecular analysis of recombinant switch regions also sheds light on switch induction and isotype commitment. Such studies reveal that, upon activation, class switching is induced in some cells but not others. Otherwise, recombinations on the excluded IgH allele would have been found in late IgM expressing cells; this

144 has not so far been found (Table 1). Furthermore, switching cells must be programmed to switch to certain isotypes because both IgH alleles perform the same type of recombination. In polyclonally activated B cells class switch recombination precedes or rapidly follows the switch in CH-gene expression. Switching by a differential RNA splicing mechanism would apply for a very short period, if at all. The frequency of cells expressing surface IgG3 begins to increase after day 3 of LPS stimulation and already by day 6 about half of the IgH loci of these cells have performed switch recombination. 4.3.2. Plusmacytomas Class switch recombination is also found to take place in plasmacytoma and hybridoma cells. These rare variant cells can be detected in many cell lines at frequencies of lo-' to lo-' (see Section 2.2.5.). In all cases analysed so far intermediate CH genes have been deleted from the active IgH loci. Frequently, however, the recombination sites were outside of the switch region - most drastically in the IgD variants (see review [C]), there being no switch sequence in front of C, [56]. This could be the reason why in normal B cells switching from IgM to IgD is rare upon activation. Furthermore, the recombinations in switch variants, unlike those in normal B cells, are always restricted to the active IgH allele [32]. Thus, it is likely that switching in plasmacytoma cells is different from physiological switching, not only by the criteria of frequency and pattern (see above) but also by that of type of recombination. Probably, it reflects the rate of spontaneous deletions within the IgH locus rather than that of switch recombinations.

4.3.3. Chromosomal translocutioris Murine plasmocytomas and human Burkitt's lymphomas contain chromosomal translocations which involve the c-myc protooncogene and, in most cases, the IgH locus. Typically, the transposed c-myc joins one of the IgH switch regions in a headto-head fashion [57-591. The removal of the oncogene from its normal context may play an essential role in tumorigenesis. Why are the IgH loci favoured recipients of the c-myc translocation? There are no structural homologies between the 5' region of c-myc and switch regions. Klein and Klein [60] suggest that c-myc translocates into chromosome loci that provide an open chromatin region, a high level of gene expression and ongoing DNA rearrangements. Only the genes of the immune system and in particular the IgH locus, are presently known to fulfill these conditions. The predominance of S, in plasmacytoma translocations and of S, in those of Burkitt's lymphomas presumably does not indicate a particular structural quality of the murine S,, and human S, regions but rather reflects the type of the cells analysed. Mouse plasmacytomas usually originate from the B cells of the omentum and the mesentery which are believed to switch to a-chain production at an early stage of maturation [3,4]. The translocation of c-myc to the S, region of the allelically excluded IgH locus may thus occur as an accident of the S,-S, recombination on both IgH loci and just resemble programmed switch recombi-

145 nation (see above). The precursor of Burkitt’s lymphomas is a pre-B or early B cell that has not yet started to switch. The S, region of these cells appears to represent a more active chromatin region than that of other heavy chain genes, as indicated by hypomethylation and DNase I sensitivity [43,61,62]. This open region seems to be a preferential target for c-myc translocation. The fact that all c-myc translocations observed so far involve the allelically excluded IgH loci probably just reflects the experimenters’ selection of IgA and IgM producing plasmacytomas and lymphomas as a subject of investigation. Why is c-myc, as opposed to other oncogenes, regularly translocated in plasmacytomas and Burkitt’s lymphomas? There is no particular sequence homology which could favour the interaction between c-myc and switch regions. It is possible that it is the activation of c-myc but not of other oncogenes that induces tumorigenic behaviour in B cells at the maturation stage characteristic for the plasmacytoma and Burkitt’s lymphoma precursor cell.

4.4. Switch sequences The class switch is mediated by a genomic rearrangement which moves a V,DJ,segment from its original location 5‘ of C, to a position 5’ of another C, gene (Cy3, C,,, C,,, C,,, C, or (2,). As discussed above, in B cells such rearrangements occur at switch regions, resulting in the deletion of the segment between the site of recombination in S, and the new S region [51,63-661. Switch regions are 2-10 kb long and composed of simple sequences such as AGCT, TGAGC, GAGCT, TGGGG, GGGGT, GAGCTGGGG, ACCAG, GCAGC [54,64-SO], repeated in tandem array (Fig. 2). Comparison of longer stretches has yielded consensus rather than identical sequences. The S,, S, and S, regions are quite homologous to each other but share less homology with the S, regions. However, weak sequence homology and weak cross-hybridization [73] indicate a distant structural relationship between s, and s,. Multiple tandem repeats of the sequence motifs mentioned above are found in S,, S, and S,, but only

tandem repetition of 49 mers ct long identical repeats

.1*,.2 3 4

....

11000bp’

1234

Sy2b

Fig. 2. Structure and superstructure of S,, S,>, S,, and S,,,,. Thc S, region carries highly repetitive sequences which frequently undcrgo deletions ( A ) prior to switch recombination. The enhancer region is designated E. S Y 3 , S,, and S,,, regions contain tandem repeats of 49mers (hatched regions). S,, and S,,, show long identical repeats (arrows) within the 49mers. The 4Ymers in S,, are flanked by direct rcpcat sequences (DR). The core of the S,, region has not yet bccn analyscd (cross-hatched region).

146

S, exhibits a regular pattern of repeated 30 base pair long sequence blocks characterized by a consensus sequence [80]. The four s, regions are homologous to each other and carry multiple repeats of a 49 nucleotide consensus [73,77]. The 49mer is repeated in tandem from 44 to more than 100 times [73,74,77]. The y2b and yl switch regions both contain long perfect repeats which do not themselves comprise reiterated short repeats [76,81]. The S,, region carries two sets of four long identical repeats each 782 base pairs apart. The higher order structure of ST1 consists of two directly repeated units of a non-49mer sequence (DRI and DRII; D R , direct repeat) interspersed by many repeats of the 49mer consensus [76]. In addition, two long identical repeat units (indicated as 1 and 2 in Fig. 2) are found in the core of S,, within the 49mer repeats. The overall organization of S,, is maintained among IgH haplotypes despite extensive length polymorphism which can be largely attributed to variation in the number of 49mers [76]. Two copies of both D R elements are maintained in all IgH haplotypes. The organisational scheme of the murine S,, region may thus be abbreviated as DRL(49) DRII-DRIr-(49),,-DRII'where n varies between 40 and 160 amongst different mouse strains. The evolution of SYImay have started from a primordial S region which can be abbreviated DRI-(49),-DRII. Duplication of this unit then generated the tandem array characteristic of the present S,, region. Amplification of the 49mers by unequal cross-over between tandemly repeated elements [82] would result in homogenization of the repeat units. A comparative analysis of all murine S regions indicates that S,, has diverged more from S, and S y Z h than these two S segments have from each other. The S,, region shows the largest length variation among the IgH haplotypes and is the most homogeneous of the four S, regions, whereas S, shows the smallest length variation and is the least homogeneous. The y2b and y2a switch regions fall somewhere between S,, and S, in both size and sequence homogeneity. The S,, and SyZh regions share a similar organisation of 49mers into repeat structures whereas S, lacks higher order structures. Too little sequence information is available for S,;,, S, and S, to identify possible higher order structures. No ordered pattern of repeated sequence units could so far be detected in the S, region. 4.5. Switch recombination sites The conservation of repetitive elements in S regions has led to the working hypothesis that such elements play a central role in the switch rearrangement. About 20 DNA sequences of the recombination junctions of various class-switches have been determined in order to get information on the sequence requirements of switch recombination [63-80,83,85]. Comparison of these sequences makes it clear that the enzymes involved in class-switching do not recognize an obviously well conserved sequence. Switch recombination sites share only little and patchy sequence homology at variable distances from the actual breakpoint of recombination. Sequence motifs such as GAGCT and G G G G T [79] or YAGGTTG [84] have been postulated to play a specific role in switch recombinase recognition. However, these hypotheses

147 are essentially unverifiable because thesc sequences are ubiquitous in all S regions. Wu and co-workers [81] have suggested that the higher order structures of S regions direct isotype switching. In all switches to S,, studied the first set of long identical repeats in S,, (1, 2, 3, 4 in Fig. 2) is always deleted and part of the second set ( l ' , 2', 3', 4') is always retained, i.e. the breakpoint is always located between the two sets of long identical repeats (Fig. 2). In the S,, region, however. the switch recombination sites studied so far [69,76,85] do not correlate with the superstructure of the region. It should be noted that the S, regions exhibiting higher order structures, namely S,, and S,,, seem to be preferentially used in certain subsets of switching B lymphocytes. Abelson virus transformed pre-B lymphocytes preferentially switch from p to y2b [27-291 and most splenic B lymphocytes which are driven into class-switching by stimulation with LPS and IL-4 (see Section 3.2.) switch from p to y l . In the S, region, switch recombination may occur everywhere between the 3' end of a V,DJ, segment and the 5' end of the C, gene [36,86]. Switch recombinations also occur, but less frequently, within that portion of S, in which the many short repeats are found. This region is the one which frequently undergoes in vitro recombination catalyzed by Escherichin coli extracts [87]. Deletions which seem to precede the recombination of S, to another S region (see Section 4.1.) are frequently observed within S, (Fig. 2). These deletions are not artefacts of molecular cloning ([74], and own results) and seem to be characteristic of IgH loci which are poised for switch recombination. It would seem from the present findings that the specificity of class-switch recombination is not mediated by consensus sequences in the S regions. It is more likely that the control of class-switching operates at the level of chromatin structure which may be influenced by repetitive sequences.

5. Conclusion The construction of switch models suffers from the lack of information on the early steps of switching. Nevertheless, one can try to sketch a simple picture of the classswitching process in mammalian B cells (Fig. 3). Two types of switching can be defined: first the switch from expression of IgM in early B cells to expression of IgM and IgD in naive B cells. This switch does not involve switch recombination but differential splicing of a long transcript; t h e regulation of this process is unclear. This switch happens at a defined stage of B cell ontogeny and presumably provides the naive B cell with a receptor (IgD) required for the activation by antigen. Upon activation, the naive B cell stops to express IgD, starts secretion of IgM and, at a high frequency, is induced to a second type of switching (Fig. 3). This switch from IgM to any other immunoglobulin class is performed by repeated recombinations within and between switch regions through several rounds of replication. It occurs only in induced cells and on both IgH alleles. Lymphokines switch factors - may be required to determine which switch regions are open for

148

Fig. 3. Programmed class switch recombination, Upon activation the B cell starts to secrete IgM, proliferate and perform class switch recombination. In early stages recornbinations within S, and probably other S regions are found (not shown). In later rounds of replications recombinations betwzeen switch regions accompany the phenotypic switch. These recombinations occur o n both, active and inactive, IgH loci of a cell. The active locus is not necessarily rearranged first. Frequently both IgH loci show recombination between the same switch regions, indicating a ‘programming‘ inductive event.

recombination. Only after switch recombination is the new C , gene expressed. Switched plasmablasts could be selectively stimulated by lymphokines to proliferate, leave the place of activation and home to the bone marrow as long-lived plasma cells, and serve as memory cells [88]. They probably would not switch again.

149

Reviews A Cebra, J . J . , Komisar, J.L. anti Schweitzer, P.A. (1984) Ann. Rev. Immunol. 2. 493-548. B Burrows, P.D. and Cooper, M.D. (1984) Mol. Cell. Biochem. 63, 97-111. C Radbruch, A . , Burger, C . , Klein, S. and Muller, W. (1986) Immunol. Rev. 89, 69-83.

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151 79 Nikaido. T.. Yamawaki-Kataok;i. Y. a n d Honjo. T. (19x2) I . Biol. Chem. 257. 7322-7329. Yamagishi. H.. Tnkahashi. N.. Yamawaki-Kataoka. Y . . NiXO Obata. M.. Kataoka, T., Nakai kaido. T.. Shimizu. A . and Honjo, ‘I. (19x1) Proc. Natl. A c x l . Sci. USA 78. 2437-2441. X I Wu. T.T.. Reid-Miller. M.. Perry. f1.M.. Kabat. E . A . (1984) E M B O J . 3. 2033-2040. X2 Smith. G.P. (1973) Cold Spring Harbor Symp. Ouiint. Biol. 3X. 507-513. 83 Kim, S.M., Davis, M.. Sinn. E . , Patten. P . a n d Hood. L. (1081) Cell 27. 573-578. 84 Marcu. K.B., Lang, R.B.. Stanton. L.W. and Harris. L.J. (19x2) Nature 298. 87-89. 85 Winter. E.. Krawinkel. U. and Radhruch, A . (19x7) E M B O J . 6 , 1663-1671. 86 Apuilera. R . . Hope. T.J. and Sakano. H . (19x5) EMBO J . 4. 3689-3693. 87 Kataoka. T., Takedn. S.I. and H o n j o . T. (19x3) Proc. Natl. Acad. Sci. USA 80. 26662670. 88 MacLennan, I.C. and Gray. D . (19x6) Iiiimunol. Rev. 91. 61-85,

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153 CHAPTER 6

Immunoglobulin gene expression GRAHAM P. COOK, JOHN 0. MASON and MICHAEL S. NEUBERGER Medical Research Council Laborator! of Molec,ukir Biology, Hills Roud, Carnbridge CBZ ZQH, U K

1. Introduction Immunoglobulin genes a r e exprcssed in cells of the B cell lineage and t h e detailed patterns of expression a r e characteristic o f the particular stages of ontogeny. Studies of the regulation of immunoglobulin gene expression are, therefore, predominantly concerned with identifying the mechanisms that ensure that the immunoglobulin gene loci are active only in lymphoid cells as well as those that govern thc different patterns of immunoglobulin gene expression characteristic of the various stages of B cell differentiation. This review is based largely on results obtained from the study of mouse lymphoid cell lines and it is therefore worth considering the validity of applying conclusions obtained from such data to thc situation in vivo.

2. Tumours as models Many lymphoid cell lines exist and these are taken to reflect some of the various stages of B cell ontogeny. Thus, prc-B cell lines (which, in the mouse system, are frequently obtained by transformation with Abelson murine leukaemia virus) are taken to correspond to B cells at an early stage of ontogeny which are undergoing o r have recently undergone assembly of the variable region genes, and which at most express only intracellular heavy ( p ) chain. B cell lymphomas are analogues of a diverse group of cells that bear membrane immunoglobulin. Plasmacytomas a n d hybridomas are taken to be analogues of immunoglobulin-secreting plasma cells. Obviously, tumours cannot be a perfect reflection of the normal in vivo situation. Plasma cells are terminally differentiated cells that d o not divide and which contain a n enormously extensive array of endoplasmic reticulum; the same preponderance of intracellular membranes is not apparent in plasmacytoma and hybridoma cells maintained in culture, presumably because this would not be consistent with rapid proliferation. Similarly, primary cells can often be induced t o differentiate in response to mitogens o r interleukins, whilst this is true t o a much lesser extent of cell lines. Nevertheless, despite such limitations, the technical ad-

154 vantages of working with cell lines and, therefore, with essentially homogeneous cell populations, is so great that much of the work associated with understanding the molecular mechanisms that regulate gene expression has been carried out with such systems. In many cases, the conclusions have subsequently been tested on primary cells, the results usually being in agreement. The ability to establish lines of transgenic mice may allow the conclusions reached by use of cell lines to be tested in vivo.

3. Patterns of immunoglobulin gene expression during B cell ontogenY The DNA rearrangement required for the production of a functional immunoglobulin gene can also yield aberrant integrations of the variable region gene segments that are incapable of directing the synthesis of antibody polypeptide chains. Nevertheless, productively rearranged, aberrantly rearranged and germ-line loci are usually all transcribed in lymphoid cells. Thus. quite apart from the DNA rearrangement, the structure of the chromatin of the immunoglobulin gene loci in lymphoid cells differs from that in other cell lineages. Before considering transcription itself, we will discuss the structural changes that are observed in the chromatin of the immunoglobulin gene loci. 3.1. Changes in chromatin structure

The structure of the chromatin at the immunoglobulin gene loci in lymphoid cells differs from that found in other cell types. Evidence comes from investigations into the degree of nuclease sensitivity and of DNA methylation. In other systems it has been observed that active genes are generally in a nuclease sensitive structure and are undermethylated at CG dinucleotides. These correlations also apply to immunoglobulin genes. Thus, in liver, the loci are found to be DNase I resistant [14] and hypermethylated [1,5-71 whereas in B lymphomas and plasmacytomas the C, and C, genes on both alleles are DNase 1 sensitive and hypomethylated. However, nuclease sensitivity does not simply correlate with active transcription. The cell line 7 0 2 / 3 harbours both a productively rearranged and a germ-line K allele; both are normally transcriptionally silent. Nevertheless, both are DNase I sensitive. Induction of K transcription in these cells by treatment with a mitogen (bacterial lipopolysaccharide) does not appear to affect the general DNase I sensitivity of the gene but does result in the appearance of a DNase I hypersensitive site in the region of the K enhancer on both chromosomes [8,9]. An actively transcribed K gene also contains DNase I hypersensitive sites in the region of the V, promoter and around the C, polyadenylation site [2,4,6,10]. Similarly, hypersensitive sites in the heavy chain locus in lymphoid cells have been identified in the region of the enhancer and of the switch region [ 11,121. There is a correlation between the general demethylation of the immunoglobulin gene loci and their transcriptional activity [1,6,7,13], although clearly the de-

155 methylation of all HpaIIIMspI recognition sites is not a prerequisite for transcription [7,13-151. Whilst the rearranged V, segments in plasmacytomas are hypomethylated and DNase I-sensitive, most of the unrearranged V, segments in plasmacytomas are hypermethylated and DNase-resistant [ 1.31. Similarly, whilst the productively rearranged C, genes in a plasmacytoma are nuclease sensitive and demethylated [5,6],the exons of the unrearranged yl gene in an IgM-expressing plasmacytoma are hypermethylated [ 5 ] . Class switching appears to correlate with the demethylation of the incoming CH gene. In fact, work on the 1.29 B cell lymphoma suggests that demethylation may occur prior to class switching. Sub-clones of 1.29 that express IgM and which yet are committed to switch either to IgA or to IgE, have already demethylated the relevant incoming C, gene [ 161. The situation with regard to the state o f the chromatin of the C, exons is somewhat different from that of the other C,, genes located 3' of C,. Firstly, it is much closer to C, than are the other C,, genes and secondly there are many IgM-expressing B cell lines that transcribe through or into the 6 locus. Studies of different IgM-expressing B cell lymphomas and plasmacytomas indicate that the C, locus is methylated in some lines and demethylated in others; the degree of C, methylation in different lymphomas does not, however, correlate with the extent of 6 transcription [ 5,171.

3.2. Expression of productively rearrariged loci Productively rearranged IgH genes first appear at the pre-B cell stage. Such cells contain a relatively low abundance of p mRNA - some two orders of magnitude less than in plasmacytomas. Immunoglobulin p polypeptide is detected inside the cell, presumably located in the endoplasmic reticulum, and is degraded intracellularly. Such intracellular turnover can also occur in plasmacytoma cells, although the extent of turnover may depend on the heavy chain class expressed [27]. Productive light chain rearrangement allows the synthesis of membrane immunoglobulin, although the DNA rearrangement itself is not sufficient to ensure light chain expression. Several cell lines have been described that harbour productive K gene rearrangements but which require induction by treatment with mitogens or other stimuli in order to achieve K expression [ 18,191. The most immature B cells express membrane IgM. However. such cells often contain mRNAs for both the membrane and secreted form of the p polypeptide chain (termed pm and p,,, respectively). The ratio of p,,, to p, mRNA varies considerably amongst B cell lines. Sometimes the two are in equimolar amounts, though often pm mRNA predominates. Nevertheless. whilst many B cell lines contain ps mRNA, secreted IgM is often not readily detectable; this indicates the importance of translational or posttranslational regulation in determining the cell phenotype. More mature B cells often co-express surface IgM and IgD and there are rarer cells that show other Combinations of heavy chain class expression. The later stages of B cell maturation and particularly the development into an IgM-secreting plasma

156 cell, are accompanied by the appearance of mRNA for the immunoglobulin J chain (see [20] for a review). The J chain polypeptide is detected in all plasmacytomas regardless of the heavy chain class expressed [21,2?]; it is presumably degraded intracellularly except in IgM and IgA expressing cell lines where it forms part of the secreted antibody [22]. Plasma cells contain a much larger amount of mRNA for both heavy and light chain than is found in B cells; this mRNA is found largely on membrane-bound polysomes [23]. Pulse-chase experiments indicate that the heavy chain mRNA is very stable with a cytoplasmic half-life of at least 6 h and probably more like 20 h [24,25]. Pulse-chase experiments at the protein level indicate that the heavy and light chains are assembled very shortly after synthesis [26]. Oligosaccharides are added in the rough endoplasmic reticulum soon after polypeptide synthesis; the immunoglobulin then proceeds to the Golgi apparatus and the sugars are only trimmed immediately prior to secretion [26].

3.3. Expression of aherrantly rearranged loci In many lymphocytes it is found that, whereas one allele encodes the expressed immunoglobulin polypeptide chain, the other has undergone some aberrant or incomplete DNA rearrangement leading for example to an out-of-frame V-(D)-J joining or to a D-JH integration that lacks an appended VH segment. Similarly. Aexpressing cells usually harbour some aberrant rearrangement at their K loci, although the A genes are normally found in the germ-line configuration in K-expressing cells (discussed in Chapter 4). Transcripts are often detected from the excluded allele, although both the rate of transcription and the level of mRNA accumulation varies from cell line to cell line, depending upon the nature of the aberrant rearrangement (see [28] and references therein). In several cell lines, aberrantly rearranged alleles have been found to be translated into an immunoglobulin polypeptide fragment that is degraded within the cell and does not contribute to the expressed antibody [29,30]. In the case of several D-JH integrations, transcripts have been detected that initiate a few nucleotides upstream of the rearranged D segment [31,32]. Some of these DJH-C, transcripts have been shown to be translated into polypeptides that are presumed to be synthesized on membrane-bound polysomes as the nucleotide sequences suggest the presence of a hydrophobic leader at the amino-terminal end of the polypeptide [31] and, at least in one cell line, the protein has been shown to be glycosylated [32]. 3.4. Expression of unrearranged loci Whilst transcription of the endogenous immunoglobulin gene loci in cells outside the haematopoietic lineage has not been described, transcription of unrearranged gene segments has been observed in B, T and myeloid cell lines [32-351. Thus, in the case of IgH loci that retain the germ-line configuration, sterile p transcripts (i.e. transcripts that d o not initiate at a V gene promoter) have been observed in B and T cell lines and these transcripts initiate in the region of the IgH enhancer

157 [32,36].Interestingly, the abundance of these transcripts is not significantly higher in plasma cells than in B cells [37].Similarly, nuclear transcripts of the mouse C, locus have also been detected in plasmacytomas that retain a germ-line K configuration; these transcripts initiate some 3.5 kb upstream of the J, segments. These polyadenylated sterile C, transcripts are not processed into mRNA, although they contain apparently functional J,-C, splice junctions. Rather, they are degraded within the nucleus [38]. Transcription of unrearranged V, segments of the mouse VHII sub-group (the JS58 family) has been described in pre-B cell lines that have not produced a functional heavy chain rearrangement [39]. Transcripts derived from unrearranged V, genes belonging to other sub-groups have not been detected, but this may simply reflect the low abundance of these transcripts as the V,II sub-group is larger than the other mouse V, sub-groups (see Chapter 3). With occasional exceptions (e.g. see [40]) the germ-line V, transcripts are not normally detected in more differentiated cells of the B lineage [41] and it has been proposed that these transcripts reflect an ‘opening-up’ of the unrearranged V, segments that is a prerequisite for joining [39]. Transcripts have also been observed from the unrearranged V, segments in K-expressing cell lines [42].

4. Processes regulating immunoglobulin gene expression Like most genes transcribed by RNA polymerase 11, immunoglobulin mRNA molecules are capped at their 5‘ end and have a 3‘ poly(A) tail. The 5’-untranslated regions vary in length upwards from three nucleotides depending on the V gene employed [43,44]. The 3’-untranslated regions vary in length between 100 and 200 nucleotides for light chain and secreted heavy chain mRNA. The mRNA of membrane forms of the heavy chain are often longer and employ multiple cleavage sites in the generation of their 3‘ ends (see Chapter 2). Our understanding of how immunoglobulin genes are expressed in a cell type specific manner has largely resulted from the study of cis-acting DNA sequences that control gene expression. More recently, studies have focussed on the factors which bind to these sequences. Classically, the cis-acting DNA sequences have been grouped into those located near the transcription start site (promoter upstream and TATA elements) and those located far from the start (enhancer elements). Whilst all of these elements can be considered to be components of the promoter, we shall discuss them separately for the sake of convenience.

4. I . Promoter upstrewn elemerirs At the 5‘ end of practically all V genes so far sequenced a homology is found to the TATA consensus (see [44]). The role of the TATA is thought to be in determining the position of transcriptional initiation by RNA polymerase I1 [45]. Little study has been made of immunoglobulin gene TATA elements and there are no published data concerning factors that interact specifically with them.

158 Sequences upstream of the TATA are implicated in the regulation of transcription. Transfection experiments, in which upstream sequences have been fused to heterologous genes, have shown that the variable regions of both the heavy chain 146,471 and of the K and A light chains [48,4Y] possess promoter elements that are preferentially active in cells of the lymphoid lineage. The exact degree of cell type specificity observed varies according to the details of the assay used. Both V, and V,, promoters have been observed to be only weakly active in fibroblasts [50,51] but strongly active in plasmacytomas as well as in pre-B and B cell lines [4&52]. There is also evidence that V, promoters show activity in T cells 1.531. A detailed functional analysis of the immunoglobulin promoter elements is not available. However, comparison of sequences from a number of immunoglobulin promoters revealed the presence of a well-conserved octanucleotide consensus (ATGCAAAT) in all V, promoters and its complement (ATTTGCAT) in all V, promoters as well as in the heavy chain enhancer [54,55]. This octanucleotide has been shown to be essential for transcription in both VH and V, promoters, as deletion of it abolishes promoter activity [46,55,56]. Furthermore, in the presence of the IgH enhancer, the octanucleotide is a sufficient V, upstream promoter element as measured in transfection assays [57]. A seven out of eight match to the octanucleotide is found in the promoter region of another gene encoding a lymphoid specific protein, the immunoglobulin J chain (581. However, the octanucleotide element is not restricted to lymphocytespecific genes. It is also found in the promoters of the herpes simplex virus thymidine kinase gene 1591, histone H2B genes [60.61] and both U1 and U2 small nuclear RNA genes [62-651 - all of which are active outside the lymphoid lineage. Moreover, it has been demonstrated that the octanucleotide is a functional part of these promoters [5Y,62-65]. A seven out of eight match to the octanucleotide is found in the SV40 enhancer. Indeed, the fact that the SV40 and the IgH enhancers use common factors is suggested by competition assays performed both in vivo [66] and in vitro [67]. Furthermore, the SV40, polyoma, IgH and K enhancers are all repressed by the adenovirus E 1A gene product 168-701. A nuclear factor designated NF-A which binds to the octanucleotide in both V, and V, promoters as well as in the IgH enhancer has been identified by gel retardation assays 171.721. This factor is found in nuclear extracts of fibroblasts as well as in cells of lymphoid origin. Nuclear extracts from these various cell types give indistinguishable DNase I footprints on the octanucleotide [7 11. Transcription of a K gene in vitro using different cell extracts has been shown to occur in a cell type dependent manner [73], this discrimination requiring upstream promoter sequences that include the octanucleotide. Similar experiments have shown that the octanucleotide upstream of the human histone H2B gene binds a nuclear factor present in HeLa cells [74] and these sequences in the H2B promoter region, which include the octamer, are required for optimal in vitro transcription of H2B [75]. Thus, there is an apparent paradox in that V, and V, promoters show a lymphoid cell type specificity, and yet the main component of the promoter that has been identified is an octanucleotide which is an essential component of many nonlymphoid-specific genes that in vitro can bind a nuclear factor present in many cell

159 types. Furthermore, oligomerisation of a short region of the SV40 enhancer which includes the octanucleotide motif, creates a lymphoid-specific enhancer when assayed on an enhancerless p-globin gene [76]. Possible solutions to this paradox are given by the suggestions that specificity is provided by the combination of promoter and enhancer [59,77], as well as by the indications that there are multiple NF-A components (NF-A1; NF-A2) binding to the octanucleotide, one of which (NF-A2) is lymphoid specific [78,79]. Apart from the octanucleotide no other strongly conserved consensus sequences have been identified in VH gene promoters, though this does not exclude the presence of other cis-acting elements. A second consensus is found in the promoter of V, genes [%I; this sequence (TGCAGCGTG) is also found in the heavy chain enhancer. However, this element is less well conserved than the octamer and mutations within it do not appear to alter the activity of a V, promoter in transfection assays [52,56].N o nuclear factor has yet been found which binds this element, and its significance remains unclear. 4.2. Enhancer elements Enhancers are classically defined as DNA sequences which stimulate transcription of a linked gene from its authentic start site. This stimulation is observed with the enhancer placed upstream or downstream of the gene, at a distance of several thousand base pairs and in an orientation-independent manner (for a review see ")I). Enhancers were originally described in viruses but have since been identified in a number of cellular genes. The first cellular enhancer to be found was in the major intron of the mouse heavy chain locus [81-831 and subsequently in the mouse K [84,85] and human IgH loci [86]. The IgH enhancer is spread over some 400 base pairs and is located between the J H cluster and the p switch region such that canonical VH-D-JH joining or heavy V ?

I

-

D

-)i*WWHll "1

"2

JH

CP

IgH mRNA

?

v3

\/ \/

GERMLINE

-

to

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t

Fig. 1. Immunoglobulin gene rearrangement and expression. A highly simplified view of the mouse IgH locus is presented which is neither to scale nor arc the full complements of gene segments depicted. The IgH enhancer is indicated by the symbol E.

160 chain class switching leave the enhancer intact (Fig. 1). It is envisaged that somatic recombination brings a V, gene promoter within the activation distance of the enhancer. thus potentiating its transcription; activation of germ line V, genes located immediately 5' to the productively rearranged V, segment has also been observed (401. Immunoglobulin enhancers differ from previously identified viral enhancers in their spectrum of activity, enhancers from SV40 or polyoma virus being active in a variety of cell types [87,115,161,162]. Transfection experiments have established that both the IgH [81,82], and the K enhancers (841 are preferentially active in plasmacytoma cells. Although weak activity has been described in fibroblasts, this weak activity manifests a strong dependence on distance [88,89]. The fact that some deletions of the IgH enhancer increase its activity in non-lymphoid transfectants has been used to infer the existence of repressor molecules in non-lymphoid cells (a) 301 ATTAAGTTTAAAATATTTTTAAATGAATTGAGCAATGTTGAGTT

N F-pE 1

NF-pE 3 421

GGAAGGGA~AATAAA_A_C_CACTAGGTAAACTTGTAGCT~~G-G-T~~~AAGAA~~~~~T 0

481 0 0 AACACTCTGTCCAGCCCCACCAAACCGAAAGTCCAGGCTGAGCAAAA 0

-

p

b

ATTTGCAT TCTAAAATAAGTTGAGGATTCAGCCGAAACTGGAGAGGTCCTCTTTTAACT

NF-A1 N F-A 2 601 TATTGAGTTCAACCTTTTAATTTTAGCTTGAGTAGTTCTAGTTTCCCCAAACTTAAGTTT

661

Fig. 2(a,b). The mouse IgH and K enhancers. ( a ) 'The IgH enhancer. Open circles above and below the sequence indicate G residues on the top and bottom strands t h a t were protected against dimethyl sulphate modification in the genomic footprinting experiments described in [%I: tilled circles indicate G residues that exhibited enhanced reactivity. The main protected clusters have heen grouped into four regions (El-E4) that show homology to the consensus CAGGTGGC [Y3]: these are boxed. The conserved octanucleotide is boxed and doubly underlined. Factors that have been described to interact specifically with the consensus sequcnces are indicated helow the relevant boxes. Sequences that fit to the proposed enhancer core sequence (TGG;?PPG [163]) are indicated with a broken line. The se-

161

[88,89]. Such repressor molecules have been proposed in other work [go] where transcription of a human yl gene transfected into mouse L cells appeared to be strongly stimulated by treatment of the cells with the protein synthesis inhibitor cycloheximide. Experiments in which the IgH enhancer is linked to a heterologous gene and incorporated into the mouse germline [91,92] have shown that the enhancer is capable of conferring rigid lymphoid cell type specificity, even when placed 3' to the test gene. In these transgenic mice, activity within the lymphoid lineage is largely confined to the B cell pathway, supporting earlier conclusions from transfection experiments [46]. This B cell specificity agrees with genomic footprinting data [93,94] in which, following treatment of cells with the methylating agent dimethyl (b)

3687 AGCTTTTGTGTTTGACCCTTCCCTGCCAAAGGCAACTATTTAAGGACCCTTTAAAACTCT

3147 TGAAACTACTTTAGAGTCATTAAGTTATTTAACCACTTTTAATTACTTTAAAATGATGTC

3807 AATTCCCTTTTAACTATTAATTTATTTTAAGGGGGGAAAGGCTGCTCATAATTCTA

3867 TTTTCTT"G_T_A_A_A~AACTCTCAGTTTCTGTTTTACTACCTCTGTCACCCAAGAGTTGGCA

3927

CAGTTGCTTAAGATCAGAAGTGAAGT

NF-KB 3987 CTGCCAGTTCCTCCTAG

CAGATTACAGTTGACCTGTTCTGGTGTGGCTAA

4047

4107 TCTGGACACCCAAATACAGACCCTGGCTTAAGGCCTGTCCTGTCCATACAGTAGGTTTAGCT quence within the arrows contains full enhancer activity as judged from transfection assays. The scquence is numbered with nucleotide 1 being at the XhaI site immediately 3' of J,,4. ( b ) The K enhancer. Sequences (El-E3) that are homologous to the E consensus proposed for the IgH enhancer [93] are boxed ah is the NF-KB binding site. Squares indicate G residues on the two strands which, when methylated, interfere with factor binding in vitro [ 1051. NF-pE3 has been observed to hind to the K E3 consensus [ 1051. Sequences within the iirrows have f u l l enhancer activity as judged from transfection assays [85,84]. Broken lines indicate hotnology to the enhancer core sequence [ 1631 and the sequence is numbered following Max et al. [ 1641.

162 sulphate, several regions of the enhancer were shown to be resistant to methylation only in cells of the B lineage. Such resistance to methylation suggests that, in B lymphocytes, there are factors that are specifically bound to these regions. The protections from dimethyl sulphate modification were not observed in fibroblasts or in two T cell lymphomas (Fig. 2a). Although transfection and transgenic mouse experiments carried out with the IgH enhancer linked to heterologous genes indicate that enhancer activity is limited to B cells, rearranged IgH genes introduced into the mouse germ line are often expressed in both B and T cells of the transgenic animals [53,95]. This apparent paradox is discussed more fully in Section 5.1. of this chapter. However, one possible solution is that the IgH enhancer is active early in lymphoid ontogeny (before separation of the B and T lineages) and this is sufficient to allow subsequent expression of the transgenic heavy chain genes (but not of heterologous genes) in descendent T cells without the enhancer itself exhibiting activity. This explanation presumes that IgH transcription only manifests a transient requirement for the enhancer. Such a model is supported by the existence of several cell lines in which aberrant heavy chain class switching has resulted in deletion of the IgH enhancer [9&99]; these lines retain active heavy chain gene expression. Indeed, when DNA from the enhancer-deleted gene was cloned out of these cell lines and reintroduced into plasmacytoma cells, expression of the transfected gene was dependent on provision of an enhancer; these experiments have been interpreted as suggesting a role for the enhancer in establishing but not in maintaining stable transcription complexes [100,101]. The cell type specificity of the IgH enhancer observed in vivo can, to a certain extent, be mimicked in vitro [ 1021. Stimulation of heterologous gene transcription occurs to a greater extent with nuclear extracts from lymphoid cell lines than from fibroblasts if the enhancer is placed immediately upstream of the test gene [102,103]; not surprisingly, however, this stimulation is much lower than that observed in vivo. At present, there are no reports of complementation of fibroblast extracts with fractionated lymphoid extracts. although such experiments would, presumably, identify putative cell type specific factors. The existence of such factors is implied by experiments in which fibroblasts containing a transfected. productively rearranged heavy chain gene were shown to express this gene following fusion with red cell ghosts previously loaded with nuclear extracts from a variety of B lymphoid lines. This activation required the IgH enhancer to be linked to the transfected heavy chain gene [104]. Genomic footprinting [93,94]. as already discussed, has shown the presence of cell type specific interactions with the enhancer (Fig. 2a). These contacts are mainly clustered into four regions ( E l to E4). all showing homology to the consensus CAGGTGGC. The ATTTGCAT octanucleotide lies immediately adjacent to the fourth homology region (E4) and is included in these protections. Binding of nuclear factors to the octanucleotide in the enhancer has been demonstrated by gel retardation assays and by dimethyl sulphate and DNase I footprinting [71,105]. Gel retardation experiments have been used to identify other factors binding to the heavy chain enhancer [105,106]. Two factors have been identified, NF-pE1 and

163

NF-pE3, which bind to the E l and E3 elements respectively. No factors have yet been described that bind in vitro to E2 or E4. Thus, despite the fact that elements E l through E4 show homology to one another, NF-pE1 and NF-pE3 show distinct binding specificity as shown by competition experiments [105,106]. Analysis of the dimethyl sulphate interference patterns obtained for NF-pE1 shows that the binding site extends beyond the E consensus. Both NF-pE1 and NF-pE3 are found to have a widespread distribution amongst mammalian cell types. Analysis in vitro of proteins binding to the mouse K enhancer [lo51 shows that a sequence ( K E ~ which ), is related to the E3 element of the heavy chain enhancer, binds the same factor (NF-pE3) as deduced from competition experiments. A second factor has been identified which binds to the K enhancer, NF-KB;this factor binds to the sequence GGGGACTTTCC [ 1051. Genomic footprinting around the region assumed to encompass the enhancer in the human K locus reveals a factor contacting a homologue of the NF-KB recognition sequence [107]. Induction of K transcription in the pre-B cell line 70Z/3 by treatment with bacterial lipopolysaccharide has been shown to be accompanied by an induction of NF-KBas detected by gel retardation assays [ 1081. This induction occurs in the absence of new protein synthesis, suggesting that NF-KB is formed by modification of an inactive precursor [108]. The combined use of protein synthesis inhibitors and of mitogens super-induces the factor. Treatment of 702/3 cells with phorbol esters has also been shown to activate K transcription [ 1101, and this also correlates with induction of NF-KB.The induction by phorbol esters implies a role for protein kinase C in t h e activation pathway. Although NF-KBwas originally found exclusively in cells of the B lineage, treatment of other cells such as HeLa cells (human fibroblast) with phorbol ester or Jurkat cells (a human T cell lymphoma) with phytohaemagglutinin, results in NFKB induction [108]. The NF-KB binding site is not restricted to the K enhancer. Although not found in other immunoglobulin gene regulatory elements, it constitutes part of the SV40, cytomegalovirus and human immunodeficiency virus enhancers. Interestingly, the SV40 enhancer has been shown to be activated by phorbol esters in a manner independent of protein synthesis [ 1111. These results imply that, during the differentiation of B cells, cell type specificity of immunoglobulin gene expression is in part achieved by specific modification of a precursor factor that is of widespread tissue distribution. A role for NF-KB in the activation rather than in t h e maintenance of K gene expression has been proposed following studies carried out on the S107 plasmacytoma [109]. S107 expresses its endogenous K gene but does not contain NF-KB as judged from gel retardation assays carried out using extracts from untreated cells. The plasmacytoma also fails to express transfected K genes despite transcription of the endogenous loci. No enhancer has yet been found in the mouse A locus, although transfection experiments indicate that expression of a transfected, rearranged A , light chain gene in certain plasmacytoma lines manifests an enhancer requirement (unpublished observations). However, the mouse A locus possesses only two V, genes and, therefore, there may be no requirement to place an enhancer downstream of the

164 start sites; thus, it is possible that if an enhancer does exist, it is not located in a major intron. An enhancer has been identified in the human A locus but is not, as yet, well characterised [112].

4.3. Other promoter elements There are indications that elements other than the promoter and the enhancer may be involved in the regulation of immunoglobulin expression. Grosschedl and Baltimore [47] found that sequences other than the enhancer but which are also located within the gene, have a small effect on the amount of IgH mRNA that is produced. However, it is not clear where such sequences are located nor whether they are active at the transcriptional or posttranscriptional level. Evidence that the activity of the promoter depends upon intragenic sequences apart from the enhancer comes from experiments in which some or all of the introns of a rearranged IgH gene are removed. When such mutant genes are tested in transfection assays, the amount of mRNA they produce is often considerably diminished; this effect may reflect a general intron requirement for the production of IgH mRNA [113]. A mutant mouse myeloma line has been described which produces reduced levels of a chain mRNA [114]. Examination of the a locus in this line reveals that at least 4 kb of sequence 3’ to the body of the gene is deleted and replaced with sequences of unknown origin. Nuclear run-off assays show that the polymerase loading on the a gene is less than in the parental line. This reduced level of a transcription might be due to the loss of a positive regulator from the a locus, or to the introduction of a negative one from elsewhere in the genome.

4.4. Transcription termination Little is known about the process of termination in RNA polymerase I1 transcription units. Most of the evidence concerning termination of immunoglobulin gene transcription applies to the p-6 region of the heavy chain locus and comes from nuclear run-off assays [17,37,116,117]. In IgM-secreting plasmacytomas, which contain p but not 6 mRNA, nuclear run-off assays indicate that polymerase loading falls off in the region between C, and C,, such that transcription of the C, exons is essentially undetectable [ 17,371. No specific sites of transcription termination have been found, but rather RNA polymerase loading seems to fall off over a region of several thousand base pairs between the C,M exons and CJ. In some cell lines, however, there is also some unloading between the C,4 and C,M exons

WI.

In contrast to IgM-secreting plasmacytomas, IgH transcription in B cells that express membrane IgM often continues into the C, exons whether the B cells express both membrane IgM and IgD or only membrane IgM [17]. Thus, the main difference in transcription termination in the p-6 locus occurs on differentiation of a B cell into an IgM-secreting plasma cell, as in the plasma cell there is little transcription read through into the C, exons. In the case of IgG expression, it has been shown that both in B cell lines that express membrane IgG2a and in myelomas

165 that secrete IgG2a transcription of the CyZagene is found to terminate well downstream of the y2a membrane exons [118]. The findings that, in B cell lines, RNA polymerase density within the p-6 locus falls off gradually, rather than at a discrete point and that the density on the Cis gene does not correlate simply with the abundance of 6 mRNA, have also been demonstrated in primary cells [116,119]. Whereas neonatal B lymphocytes contain significantly less 6 mRNA than adult B cells, in both cell types a similar proportion of polymerase molecules continue through C, into C , [119]. Also consistent with results obtained using cell lines is the observation that B cells treated with lipopolysaccharide show an altered pattern of transcription termination in that an increased proportion of polymerase molecules now unload between C, and C , [116]. In summary, in antibody-secreting cells that contain little mRNA for the membrane form of the heavy chain, transcription of the heavy chain locus nevertheless continues into and beyond the membrane exons. Similarly, in cells expressing membrane IgM, RNA polymerase molecules often continue transcribing into the 6 locus; there does not appear to be a simple correlation between the amount of C, transcription and the steady-state level of 6 mRNA. Caution must be exercised in assigning any regulatory role to transcription termination, as it is possible that termination is largely a consequence of cleavageipolyadenylation. It has been found [120] that an 800 base pair fragment from t h e 3' flanking region of the mouse P-globin gene which includes the region within which transcription termination normally occurs is not sufficient to cause termination when inserted into the adenovirus E1A gene. However, a larger fragment containing the P-globin polyadenylation site in addition to the termination region does cause transcription termination in the same assay. In further support of such an hypothesis, an a-globin gene with a mutant polyadenylation site has been described [ 1211; it is found that not only do transcripts from this gene not polyadenylate correctly, but transcription continues for several hundred nucleotides further than usual, terminating after a cryptic polyadenylation site which is located downstream of the mutant site [121]. Thus, although some differential transcription termination is seen during B cell development, it is possible that the altered pattern of termination is a consequence (rather than a cause) of differential polyadenylation site usage. A role for termination as a modulated, regulatory step in immunoglobulin gene expression, therefore, remains to be established. 4.5. R N A cleavageipolyadenylatiori Cleavage of the primary transcript and its polyadenylation are thought to be tightly coupled events in vivo, since all accurately processed 3' termini are polyadenylated. However, in vitro studies [ 1221 show that the processes can be separated, at least for adenovirus-2 L3 mRNA. All immunoglobulin gene mRNAs contain the usual highly conserved sequence AAUAAA some 20 nucleotides upstream of the poly(A) tract, but no other highly conserved sequences that specifically identify all immunoglobulin gene polyadenylation sequences have been described. Multiple mRNAs are produced from a specific immunoglobulin heavy chain lo-

166 cus both in the co-expression of two heavy chain classes from the same chromosome and in the production of the mRNAs for the membrane and secreted forms of the same heavy chain class. Presumably, these events require the processing of a common primary transcript into different mRNAs, each utilising a different polyadenylation site. However, regulation of these events by controlling the usage of polyadenylation sites has not been demonstrated. 4.6. R N A splicing Whilst significant advances are now being made in our understanding of the molecular mechanisms of RNA splicing [123,124], there is little information at this level that specifically applies to immunoglobulin genes. Attempts have been made to deduce the pathway of removal of the introns from immunoglobulin gene transcripts by looking at the steady-state levels of mRNA precursors in the nucleus [ 125-1271, While the techniques used had insufficient resolution to determine the order of removal of the small introns, it was found in an IgM-secreting line that the J,,-C, intron was normally removed before the C,4-CPMl intron (consistent with processing in a 5'-3' direction) [ 1261. However, in contrast to this finding, a transcript from the mouse a locus has been described which contains the large V,C, intron, but lacks the C,,-C,M intron [ 1271, indicating that splicing of the primary a transcript does not always proceed in a 5'-3' direction. The main problem raised by the splicing of immunoglobulin gene transcripts arises from the co-expression of two heavy chain classes from the same chromosome. A large proportion of B cells co-express IgM and IgD on their surface; in any one cell, the two heavy chain classes are expressed from the same chromosome and use the same rearranged variable region. Cell lines that exhibit a similar phenotype have been described (for example, see [125,128]). It has been generally assumed that the mRNAs for the p and 6 heavy chains are derived from a common precursor by differential splicing; the critical choice would be whether to splice from the rearranged J H segment to the C,1 exon or to the C,1 exon. Splicing from J, to C,1 would require the six splice acceptor sites of the C, region being ignored. As yet, there are no experimental data concerning how this splicing is achieved. Indeed, the existence of a common p-6 primary transcript (which would be at least 25 kb long) has not been formally demonstrated. It is worth noting that in the case of the C, gene - unlike the genes for the other heavy chains (see Section 5.3. of this chapter) - the production of the mRNAs for t1;z membrane and secreted forms of the heavy chain involves differential RNA splicing. This is because the carboxy-terminal ends of the secreted and membrane forms of the 6 polypeptide are encoded in distinct exons located 3' of the C,3 exon. The question of stable co-expression of C, and other CH genes has also been raised. In particular, primary B cells have been described in which p and E are coexpressed from the same chromosome [ 1291. As lymphocytes have Fc receptors for IgE, it is difficult to exclude t h e possibility that the IgM' IgE' cells seen in this work are not in fact IgM ' cells that have passively adsorbed secreted IgE onto their surface. B cells have also been identified that co-express IgM and IgA on their

167 surface [130]. A primary transcript extending from a rearranged V, gene through C, and on to C,, would be about 200 kb long (slightly longer than the transcript for the blood-clotting factor, factor VIII). Sandwich hybridization studies have been used to support the existence of an RNA containing both p and a sequences [130]. Considerable weight has been given to the idea that p and other heavy chain classes apart from 6 can be expressed from the same primary transcript by the isolation of a variant of the IgM-expressing mouse B lymphoma line BCL, that stably coexpresses IgM and IgGl on its surface; these immunoglobulins contain the same V, region and derive from transcripts from the same chromosome [131,132]. Nuclear run-off assays and sandwich hybridization studies on this line are consistent with both classes being produced from a common precursor transcript. 4.7. Messenger R N A turnover

The half-life of immunoglobulin gene messenger RNA in plasmacytomas has been estimated from pulse-chase experiments to be certainly greater than 6 h and probably between 20 and 40 h [24,25]. There is no corresponding estimate for the halflife in B cells. However, by following the decay of p mRNA after its induction by heat-shock in cells transfected with a p gene under heat-shock control, it has been estimated that the half-life of p mRNA in a plasmacytoma (more than 15 h) is considerably greater than in a B cell lymphoma ( 3 to 4 h) [133]. The molecular basis of this differential mRNA turnover has not been identified. 4.8. Translational and posttranslational regulation

In resting B cells, the level of p,, mRNA is about ten-fold higher than that for the corresponding a,,, mRNA [ 1341, yet IgD is expressed on the membrane of these cells at a higher density than IgM [ 1351. This suggests that the relative expression of these two immunoglobulins must be controlled at the translational or posttranslational levels. It has been found that although the rate of a,,, polypeptide synthesis in B lymphocytes is about 10% of that of pm,surface IgM turns over faster than surface IgD, resulting in higher net expression of IgD [135]. There is evidence for translational or posttranslational control in the expression of the membrane forms of p [136]. y2a [137], and E [138] heavy chains. In each case, immunoglobulin-secreting cells are also found to contain mRNA for the membrane form. However, there is no corresponding expression of surface immunoglobulin. Even when more RNA for the membrane form is present than is found in analagous membrane immunoglobulin bearing cells, these secreting cells do not express immunoglobulin polypeptide on the membrane. Cells lacking membrane IgE have been shown nonetheless to be actively synthesising intracellular E , chains [ 1381, implicating posttranslational regulation. It is attractive to speculate that it is the proliferation of intracellular membranes that accompanies B cell to plasma cell differentiation which may impede membrane immunoglobulin from reaching the cell surface; much of the membrane immunoglobulin would then get degraded within the cell [139]. This type of phenomenon might also explain the

168 decrease in the expression of other surface proteins that accompanies B cell differentiation. A number of B cell lines contain p5 mRNA, often equimolar with the amount of p m , and yet do not secrete immunoglobulin [140]. Consistent with this is the fact that resting B cells that contain both p,,, and ps mRNAs translate both forms, but the ps polypeptide is not correctly processed or secreted [141]. Thus, resting B cells can exert posttranslational control over ps production. Some B cells [142] and B lymphomas [ 1431 which contain p5 mRNA do secrete p5 chains, although this may simply reflect their being at a later developmental stage. It is possible that the posttranslational inhibition of IgM secretion in B cells is correlated with the absence of immunoglobulin J chain in these cells. However, it is notable that nonlymphoid cells (which do not express J chain) that have been transfected with constructs that ensure production of mRNA for immunoglobulin p and light chains, are nevertheless capable of assembling and secreting IgM, despite the absence of J chain polypeptide [144]; furthermore, some human myeloma IgMs have been described that lack J chain (see [145]).

5. Major aspects of cell-type specijkity In Section 3 of this chapter, the patterns of immunoglobulin gene expression that are characteristic of different stages of differentiation were described, and Section 4 summarises our present knowledge about the various levels at which expression is regulated. In this final section, the ways in which the various regulatory elements and mechanisms contribute to the overall pattern of immunoglobulin expression in different cell types are considered.

5.1. Restricted cell-type specijicity of immunoglobulin gene transcription Normally, productively rearranged immunoglobulin genes are only present in cells of the lymphoid lineage. However, from transfection experiments it is clear that rearranged immunoglobulin genes are only correctly expressed in lymphoid lines [46,50,14&148]. Similarly, transgenic mice carrying rearranged immunoglobulin genes integrated into the germ line only express these genes in lymphocytes [53,95,149-1511 - although, in several of these reports, expression was found in both B and T cells. The expression of transgenic K genes does, however, appear to be restricted to cells of the B lineage [149]. How is this cell-type specificity achieved? As described in Section 4 in this chapter, transfection and transgenic mouse experiments have shown that both the enhancers and the promoters of immunoglobulin genes confer cell-type specificity. The molecular mechanism by which these elements confer cell-type specificity remains to be identified. Nevertheless, it seems likely that it is the cell-type specificity of these transcription elements that accounts for the inability of non-lymphoid cells to express transfected immunoglobulin genes. However, it is notable that, whereas using both cellular transfection and transgenic mouse assays, the IgH en-

169

hancer (when linked to hetevologous test genes) manifests activity which is largely restricted to cells of the B lineage [46,91,92,152], productively rearranged transgenic IgH genes are often active in both B and T cells. It may be that there is synergy between promoters and enhancers [77] such that the IgH enhancer could activate expression of immunoglobulin genes in T cells, but is unable to potentiate transcription from the promoters of heterologous genes when transfected into the same cells. Alternatively, as discussed in Section 4.1. l . , active IgH transcription may only require transient activity of the IgH enhancer. Thus, if the enhancer is active early in lymphocyte ontogeny, but is no longer fully active in mature T cells, the transient early activity of the enhancer may nevertheless be sufficient to ensure transcription of the transgenic IgH genes in mature T cells. Activity of the IgH enhancer early in lymphocyte ontogeny might explain why many T cells are found to harbour D-JH integrations and contain sterile IgH transcripts initiating in the region of the enhancer, as activation of the IgH locus might render it accessible to factors involved in both IgH transcription and variable gene segment rearrangement (see Chapter 4 and [34] as well :is references therein). Furthermore, the accessibility of the IgH locus in normal T cells is indicated by the fact that there is a DNase I hypersensitive site in the region of the p switch region in many T cell lines [12]. It is interesting in this context to note that the data discussed here suggest that it is the regulation of V, to DJH joining (and therefore, presumably, the control of accessibility of the germ line V,, cluster) that accounts for the lack of immunoglobulin heavy chain gene expression in T cells of normal mice. Although transgenic IgH genes are often expressed in both B and T cells, expression of transgenic K genes is B cell restricted [95,149]. This observation could be explained by a model in which rearrangement of the germ line K locus is correlated with its transcriptional activation. As the K locus does not normally rearrange until after productive heavy chain rearrangement and as T cells do not usually express complete heavy chain polypeptides, transgenic K genes would not be expected to be expressed in T cells. It should, however, be noted that assigning a role to the immunoglobulin enhancers in ‘activation’ of regions of the chromosome is premature. The immunoglobulin gene enhancers have always been assayed as transcriptional activators; they have not, as yet, been demonstrated to act as foci for regulatory events associated with altering local chromatin structure - as may be assayed, for instance, by regional DNase sensitivity. 5.2. Control of the difference in rnRNA abundance between B and plasma cells Plasma cells and their tumour analogues contain large amounts of mRNA for immunoglobulin heavy and light chains, the abundance being up to 100-fold higher than in B cells (see, for example, [153]). Several lines of evidence suggest that posttranscriptional events play a major role in the regulation of this change. The steady-state levels of heavy chain mRNA in the nuclei of B and plasma cells are much more similar than in the cytoplasm. Thus, 3-6-fold differences have been

170 found in the nuclear IgH RNA levels of cell lines which have 100-500-fold differences in cytoplasmic IgH mRNA levels [133,153]. A similar conclusion comes from nuclear run-off assays [37,133,152], where the polymerase loading across the IgH locus either appears to be comparable amongst representatives of both cell types or is certainly not sufficient on its own to account for the difference in mRNA levels. Therefore, the difference in the amount of mRNA accumulation is not solely due to a difference in the rate of production of the primary transcript. It is not known at which stage the differential regulation occurs, but the similarity of immunoglobulin nuclear RNA levels in B cell lymphomas and plasmacytomas suggests that it must be a cytoplasmic or late nuclear event [133,153]. It is probable that differential half-life of cytoplasmic mRNA for the immunoglobulin polypeptides is a major contributor [133]. It is conceivable that this differential half-life of immunoglobulin mRNA may be coupled in some way to translation. For instance, the increase in immunoglobulin mRNA in plasma cells may be correlated with the proliferation of endoplasmic reticulum or other changes associated with the differentiation into a secretory cell. In support of this idea, it has been found [37] that sterile IgH transcripts are not subject to differential posttranscriptional control in the same way as authentic IgH transcripts; in other words, the level of sterile transcripts (i.e. those lacking the variable region and its associated leader sequence) is similar in B cells and plasma cells. These sterile transcripts originate either from cryptic promoters upstream of DJH joints or in the region of the enhancer. The latter category cannot be translated [36]. Measurement of the ratio of membrane-bound to free cytoplasmic C, mRNA in B and T cell lines has shown that only about 50% of the sterile transcripts present in T cell lines are located on membrane-bound polyribosomes [34]. It is to be presumed that these are the sterile transcripts that originate upstream of the DJH joints [31,32]. Nuclear run-off assays have been carried out on splenic B cells [116,119,154]. In contrast to the findings in cell lines, the RNA polymerase density on the p gene was found to increase when resting B cells were stimulated with bacterial lipopolysaccharide, in one case by as much as 8-10-fold [116]. However, it should be remembered that lipopolysaccharide treatment not only causes B cells to differentiate but also causes them to leave the resting state and start proliferating, thus making it difficult to draw direct comparisons with cell lines.

5.3. The relative aburzdurice of membrane and secreted immunoglobulin Whereas membrane IgM bearing B cells contain either more pn,than ps mRNA or roughly equimolar amounts of the two forms, plasma cells contain a vast excess of pS mRNA. Examination of the organisation of the C, gene suggests various possible mechanisms for regulating the ratio of the production of the two mRNA forms (Fig. 3 ) . Four types of mechanism can easily be envisaged: (a) regulation of transcription termination such that much of the transcription in plasma cells terminates before the p,,, polyadenylation site; (b) regulation of cleavage/ polyadenylation such that, for example, the primary transcript is efficiently cleaved at the p\ polyadenylation site in plasma cells but not in B cells; (c) regulation of

171 L VDJ

$1

cP

*

CF4

s

M1 M2

1 as,

AS

Am

(not to scale)

Fig. 3 . Production of k,and p,,)rnRNAs.

splicing such that there is competition between the splicing out of the CP4-C,M1 intron and the use of the p5 polyadenylation site; (d) a regulated cleavage of the primary transcript at some point between the two polyadenylation sites which would therefore preclude the production of p,,,mRNA. Analysis of nuclear RNA has shown that the precursors of the membrane and secreted forms in the nucleus are present in the same ratio as the mRNAs in the cytoplasm [126]. This demonstrates that the relative production of p,,,and ps mRNAs is determined in the nucleus, although, as described in Section 4.4., the overall ratio of membrane IgM to secreted IgM produced is also controlled at the translational and/or posttranslational levels. Several groups have used nuclear run-off assays to ascertain whether transcription termination could play a role in determining the production of membrane versus secreted immunoglobulin [37,116-1181. Whilst no evidence of a specific termination site has been found, the data suggest that, whereas in B cell lymphomas most polymerase molecules continue considerably beyond the C,M polyadenylation site, in IgM-secreting plasmacytomas polymerase loading does fall off in the region of the membrane exons. This off-loading varies considerably between cell lines; in most lines tested the membrane exons are transcribed almost as actively as the C,4 exon, but in a few lines up to 80% of polymerase molecules off-load before reaching the membrane exons [37]. Comparison of lines that express the membrane or the secreted form of IgG2a indicates that, in both cell types, RNA polymerase continues substantially beyond the C,, membrane polyadenylation site [118]. Thus, termination of transcription may play a role in the regulation of p,,, versus p, production in some cell lines, but it is not clear whether this is generally true. Certainly, there is enough transcription through the p m polyadenylation site in plasmacytomas to ensure that mutant genes that have a defective p s polyadenylation site produce high levels of p mRNA, all of which is polyadenylated at the membrane site [155]. Furthermore, as discussed in Section 4.2. of this chapter, the

172 difference in polymerase loading in the region 3‘ of C,4 that is found on comparing B cell lymphomas and plasmacytomas, could be a consequence (rather than a cause) of differential cleavage/polyadenylation. Thus, it seems unlikely that the differential production of the mRNAs for membrane and secreted forms of immunoglobulin heavy chain is solely regulated at the level of transcription termination. There is evidence which supports the proposal that it may be possible to polyadenylate the primary IgH transcript at a membrane polyadenylation site and then subsequently cleave and polyadenylate at a secreted site. A small RNA has been described that is polyadenylated at the a,,, polyadenylation site and that appears to have a 5’ terminus close to the a, site [127]. Similarly, an RNA has also been described which is polyadenylated at p m and contains the C,M exons but not the other exons of the p gene [156]. However, these RNAs have either been described in B lymphomas or in a T cell lymphoma/plasmacytoma hybrid but they have not been found - as one might more reasonably expect - in several immunoglobulinsecreting plasmacytomas analysed [ 127,155,1561, Therefore, the significance of sequential cleavageipolyadenylation in the generation of mRNA for the secreted form of IgH polypeptides remains unclear. Whilst it has been popular to ascribe the differential production of pm and p< mRNAs to regulation at t h e level of polyadenylation (e.g. see [157]), it is notable that there is no direct evidence in support of this. Indeed, experiments in which the p m and p5 polyadenylation sites are used to substitute for the normal polyadenylation site of the human a-globin gene do indicate that the p,,, site is stronger than the ps site [ M I ; however, transfection of the chimaeric genes into B and plasma cell lines does not reveal any difference in the relative strengths of the two polyadenylation sites when comparing the two cell types. Thus, t h e relative production of p,,, and ps mRNAs is unlikely to be simply determined by the polyadenylation sites. Indeed, Peterson and Perry [159] found that normal p , , , / ~reg~ ulation was abolished when the p,,, site was brought 900 base pairs closer to the p\ site. In this case, both B and plasma cells produced roughly equimolar quantities of the two transcripts. Normal regulation was restored by the insertion of 900 base pairs of ‘miscellaneous’ DNA. These results suggest a model in which the relative production of the two forms of p mRNA is determined by competition between polyadenylation at psand splicing of C,4 to C,M1. This could be regulated, for example, by a trans-acting factor specific to plasma cells which inhibits splicing of C,4 to C,Ml. Alternatively, changes in the ratios of polyadenylation and processing effector molecules could be involved, such that polyadenylation is more efficient in plasma cells than B cells or splicing is more efficient in B cells than in plasma cells [160]. In conclusion, the relative production of p,,, and p5 is controlled at the level of RNA processing in the nucleus. An attractive model for the regulation of pm/ps production involves competition between cleavageipolyadenylation at the p, site and splicing of the C,4 and C,Ml exons.

173

5.4. Co-expression of two immunoglobulin classes A number of B cells have been described that co-express p and 6, p and y , p and a or p and E (see Section 4.6. of this chapter). Such co-expression requires alternative splicing of a common primary transcript. We do not know how such splicing occurs, although there is as yet no demonstration that such splicing is regulated in a cell type specific manner. It could be that the splicing pattern is simply a consequence of the three-dimensional structure of the primary transcript and that the production of mRNAs for multiple IgH classes is ultimately regulated at the level of production of the primary transcript by control of transcription termination or cleavageipolyadenylation. Finally, it is worth noting that, at least in the case of B cells co-expressing IgM and IgD on the membrane, the ratio of the amounts of the two heavy chain classes expressed o n the cell surface is considerably affected by translational or, more likely, posttranslational mechanisms ([ 134,1351 and see Section 4.8.).

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177 CHAPTER 7

The generation and utilization of antibody variable region diversity TIM MANSER Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA

1. Introduction It is now well established that the vertebrate immune system is capable of synthesizing an enormous number of distinct antibody variable region structures. In recent years the genetic basis for this ability in the mouse has been elucidated. Diversity of the V region arises from four sources: (a) diversity encoded directly in the germ-line genome in the form of V region heterogeneous multigene families; (b) diversity created by the somatic rearrangement of different combinations of gene segments to form functional V region genes, and the association of different V, and V, polypeptides to form the heterodimeric V domain (combinatorial diversity); (c) diversity created at the junctions of V gene segments due to apparent addition and deletion of nucleotides during segment joining (junctional diversity) ; and, finally, (d) diversity created by somatic replacement of nucleotides in expressed V, and V, genes (somatic mutation). What now remains to be determined is the manner in which this potential for generating diversity is utilized toward the formation of antibody specificity and immunity. In this chapter, I review what is known about the genetic mechanisms that create diversity and discuss recent experiments that provide insights into the question of how this diversity is utilized during an immune response. The majority of research in this area has used the mouse as an experimental system, and 1 will draw upon this data base nearly exclusively. Data obtained from other systems will be discussed only when they appear to conflict with what has been learned from the mouse. The ability to create a large number of different V region structures should be of little value to the organism if most or all of these structures are not functionally distinct - that is, have different binding specificities. Nevertheless, it is possible that the function of antibody diversity in some instances is not related to specificity for antigen, or that some diversity has little or no function. I therefore endeavor to point out the evidence for the functionality of diversity, paying particular attention to observed differences in antigen specificity. In recent years evidence has accumulated strongly suggesting that the expression of both combinatorial-junctional diversity and of somatic mutational diversity varies as a function of the developmental stage of the B cell. Gene segment rear-

178 rangements that create combinatorial and junctional diversity seem to occur nearly exclusively at the pre-B cell stage of ontogeny - that is, during the antigen independent phases of B cell differentiation. During these stages of differentiation each pre-B cell combines and expresses a single set of segments and thus the resulting B cell expresses a single V region structure. It appears that this form of diversity remains largely invariant throughout the subsequent antigen dependent stages of differentiation. Conversely, there is a strong correlation between the time elapsed after antigenic stimulation of the B cell population and the degree of somatic mutational diversity expressed by this activated population. This suggests that the majority of mutational diversity may be created during the antigen dependent stages of differentiation. Unfortunately, the degree to which cellular selective processes, acting on either the ‘naive’ or immune B cell population, influence the degree of expression of these two forms of diversity, is not well understood. Antigen dependent and independent phases of B cell ontogeny nevertheless provide two convenient conceptual frameworks in which to review the formation and utilization of diversity. The developmental regulation of expression of the different forms of V region diversity could have a profound impact on the character of the preimmune and immune antibody repertoires and on the manner in which the former is transformed into the latter.

2. Antigen independent diversity 2.1. Combinatorial diversity An active light chain variable region gene is created by the somatic juxtaposition of two gene segments, the V, and J, segments. A functional heavy chain variable gene is constructed by the fusion of at least three gene segments, the V,, D and J, segments (see [l] and [2] for reviews). Here I briefly review the origins of the resulting combinatorial diversity and refer the interested reader to Chapters 2 and 3 for a more detailed discussion of the structure, number and genomic organization of immunoglobulin V and C region genes. The V, and VL segments encode the majority of their respective V region polypeptides - the amino-terminal 95-97 amino acids. The JH and JL gene segments encode the carboxy terminal 12 to 17 amino acids and the D segment encodes a region between V, and J, consisting of between 1 and 15 amino acids. There are two classes of light chain variable region genes in mammals, K and A. Gene segment ‘counting’ experiments that rely on Southern blotting, Cot analysis and cloning and sequencing suggest that there are approximately 100 V, and 300 V, segments in the mouse genome [3-61. Only two V, gene segments are present in the genome of most inbred strains of mice [7] and the concentration of A light chains in normal mouse sera is only 5% of the K concentration. A single study of the D region segments encoded in the genome of the BALB/c mouse revealed 12 different segments which could be grouped into two families (D-FL16 and D-SP2), and a single segment (D-Q52) [8]. Sequencing analyses of expressed V, genes have provided evidence for several more germ-line D

179 segments [9.10, T. Manser, unpublished results]. Sequencing of the DNA upstream of mouse C, and C, revealed four functional JH and four functional J, segments [ 11-14]. Sequence analysis of expressed variable region genes has confirmed that these segments are probably the only functional J segments in the genome since every expressed sequence can be accounted for by one of the already characterized germline J’s. The molecular map of the BALB/c V, locus reveals that only two V, and 4 J, segments are present. Of the eight VA-J, combinations that are theoretically possible, only three are predominantly found to be expressed among myelomas and hybridomas [7]. Studies on the rearrangement status of V gene segments in fetal liver (the fetal site of B cell development) and in Abelson virus transformed pre-B cell lines that spontaneously undergo D to JH, VH to DJH and V,- to JL recombination in vitro suggest that a temporal pattern of V gene segment rearrangement occurs during the development from pre-B to B cell (see Chapter 4 of this volume for a more detailed discussion). The deduced sequence of joining events is: D to J, at both chromosomes; VH to DJH at one allele, followed by a second such event at the other allele if the first event creates a non-functional gene (e.g. results in an out of frame joint); V, to J, (at both alleles if necessary) and, finally, V, to J, if both V,-JK’s are non-productive or the productive V,J, has been deleted [15]. It has been proposed [ 161 that this temporal pattern results from positive and negative regulation of rearrangement events mediated by the protein products of productively rearranged gene segments (e.g. an intact p heavy chain inhibits subsequent VH to DJ, joining and promotes V,-J, joining). It is suggested that such regulation is necessary to ensure that the expression of heavy and light chain genes is allelically excluded (see Chapter 4). Due to the order and orientation of the VH and V, gene segments in genomic DNA (see Chapter 3 ) it is possible that in some instances a given product of gene segment fusion could be replaced due to secondary joining events. In the case of heavy chain variable region gene segments, where the order of segments along chromosome 12 of the mouse is: centromere-VH family-D family-J, family [6], and the D’s and JH’s and possibly all the V,’S are in the same orientation [17], a primary DJH joint could be replaced by subsequent rearrangement of an upstream D to a downstream J H . Analogously, a joined V,J, could be replaced by an upstream V, in the same orientation, or a downstream V, in opposite orientation, to a downstream J,. Such secondary rearrangements may increase the frequency of productive joints on a per cell basis during B cell ontogeny and may serve a purpose during antigen dependent stages of differentiation as well (see below). The mechanism by which V region gene segments become juxtaposed has only recently received experimental attention [18-221. I refer the interested reader to Chapter 4 of this volume for a more detailed discussion of these matters. Evidence does exist, however, suggesting that certain gene segments and combinations of segments are expressed more predominantly than others in the B cell population. Since such biases could clearly influence the amount of functional V region diversity expressed by the immune system, the evidence for such biases will now be considered. Alt and his colleagues have observed that of the more than 100 V,,

gene segment exons present in the V, locus of the BALB/c mouse [6], only a few are predominantly expressed in fetal liver [16,23]. These gene segments are members of the V,7183 family and are those most proximal to the JH-CH locus [16]. This observation, combined with the observation that D to J, recombination precedes V, to D recombination [24,25], has led these workers to propose a 3’ to 5‘ ‘tracking’ model for V, gene segment rearrangement [23]. Other workers have confirmed that members of the VH7183 family are involved in the majority of V, rearrangements in fetal liver [26]. Such a bias in segment usage at the stage of immature B cells would be expected to have an influence on V, segment usage in the mature B cell population and thus on the degree of functional diversity expressed by this population. Several lines of evidence suggest, however, that this fetal bias in V, use is not reflected by the V region repertoire of the adult mouse. Klinman and others have evaluated the V regions expressed by the B cell population at various stages of mouse development [27-311. The results suggest that while an extreme fetal bias in V, segment usage appears to exist, this bias is not carried over into the B cell population of the mature animal. These studies have also shown that members of the VH7183 family are not the only V, sequences prevalently expressed in fetal liver, members of the V,36-60 family are also disproportionately expressed [29,31]. Since the V,36-60 family has been mapped as one of the most distal to the J H - C H locus [32], this observation makes the 3’ to 5’ ‘tracking’ model far less appealing. Manser and Gefter have also shown that both the V,36-60 gene segment and a particular member of the V~J.558family (VHIdCR) are each productively rearranged and expressed at a frequency of approximately 1/350 adult B cells [33]. Further, the V,IdCR segment is productively rearranged to a diverse group of D segments and to all the J, segments at roughly equal frequencies [34]. In toto, these data suggest that during the developmental transition from fetus to adult, the V, repertoire is transformed from being highly biased in VH segment expression to being, to the first approximation, random. How is this transition accomplished? A possible molecular explanation has been recently provided by the work of Weigert and his colleagues [35] and Reth and his coworkers [36]. Both of these groups have been examining V region expression in transformed B cells. Both groups have obtained evidence that a phenomenon termed ‘V, replacement’ spontaneously occurs in members of these transformed lines. That is, the V, exon segment which was originally part of a VHDJH complex has been ‘replaced’ by another, upstream V, exon segment, leaving the DJ, region of the original VHDJH gene undisturbed. While the mechanism whereby this replacement takes place is not understood, the site of VH to DJ, recombination seems to occur at a position in the ‘acceptor’ V, where a cryptic heptamer sequence is found. This heptamer in combination with a nonomer sequence is found in highly conserved form flanking all functional germline V, and VL segments examined to date, and is thought to constitute part of the recognition sequence for the putative ‘recombinase’ enzymes that mediate gene segment joining (reviewed in [1,2] and in Chapter 4 of this volume). If V, replacement occurs in pre-B cells which initially express a member of the V,7183 family, and the donor V, is chosen from any of the up-

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stream VH’s with similar frequency, it may explain why the fetal repertoire is highly skewed toward expression of the V,7183 family while the adult repertoire is combinatorially more diverse. This observation also has important implications for the mechanism of gene segment joining (see Chapter 4 in this volume). While the adult VtjDJH repertoire appears to be rich in combinatorial diversity. the work of Coleclough and Honjo and their colleagues suggests that this is not true of the adult V,J, repertoire [37,38]. Both groups of investigators have found that the J,1 and J,2 gene segments (first and second most distal segments to the C, exon, respectively) are predominantly expressed among splenic B cells. N o evidence suggesting skewed use of certain V, gene segments has been obtained, and since the four functional J, segments encode the highly conserved fourth framework region of the V, domain it is unclear at present whether the biased use of J,1 and J,2 has any major effect on the diversity of specificity expressed by the mature B cell population. While there may be biases in the use of certain V exon segments by the mature B cell population, the number of distinct antigen-binding specificities expressed by this population exceeds lo7 [39,40]. It is generally assumed that the contribution of combinatorial diversity to this figure is substantial, but there is little direct evidence supporting the idea that a large fraction of combinatorial diversity is functional. Affinity labelling experiments have been used to show that both V, and V, contribute to the formation of the antigen combining site [41]. However, the specific contribution of individual gene segments to specificity has not been systematically studied. This problem is further complicated by the observation that, while hybridomas isolated after immunization with a particular antigen often display similar antigenic specificities, their only structural commonality may be that they share a single V, or V, segment [10.42-531. It is obvious that different combinations of gene segments most often encode V domains with different antigen specificities (e.g. see [42,54,55]). Nevertheless, knowledge of the physical basis for this difference in specificity is restricted to the few V domains for which the X-ray crystallographic structure of an antibody-antigen complex has been solved [56,57]. In most of these cases the eliciting antigen is not known. Therefore, while it is clear that some measure of the expressed combinatorial diversity must be related to antigenic specificity, the possibility must be considered that some fraction of this diversity is non-functional [58] or provides a function unrelated to antigen specificity (e.g. see reference [59]). In this regard it is possible that V genes are in pieces largely to ensure that junctional diversity (see below) can be created. Having a large number of different copies of each type of segment may not only allow diversity to be stored in the germ line but may also increase the chance that junctional diversity is created productively. For example, since four functional J, segments are present in the genome, a maximum of four V,J, rearrangements can presumably occur per cell per chromosome. A single pre-B cell has a maximum of eight chances to make a productive V,J, rearrangement. Since an out of frame join will on average occur twothirds of the time, a cell has eight chances to beat one in three odds. Three or more joining events are required to produce a functional V, gene representing one

182 in nine odds of productive joining. In the absence of multiple V segments the efficiency of pre-B to B cell transition would be very low. A further question concerning the functionality of combinatorial diversity is raised by the fact that the bulk of a heavy or light chain variable region gene is encoded in either the V, or V, segments - only 10-20% of the carboxy-terminal information is donated by the JL or D and JH segments. Four of the complementarity determining regions (CDR’s) are encoded in the germline (within the V, and V, segments) and only two are partially created by somatic rearrangement. If it is assumed that, on average, all six CDR’s participate in the formation of an antigen combining site, the specificity of an antibody is largely determined by germ-line information. Perhaps the structure of germ-line encoded CDR‘s has been selected in evolution for ‘general antigen binding’ characteristics while the somatically constructed CDR’s provide fine specificity of binding.

2.2. Junctional diversity It was initially observed by Max et al. [60] that a joining event between two gene segments could occur at different nucleotide positions within these germ-line elements. Now that hundreds of rearranged VH and V, genes have been sequenced, it has become clear that the mechanism(s) responsible for both VL-JL and VH-DJH joining are highly ‘imprecise’. It appears that a large fraction of joining events are out of frame with respect to D , J, or JL downstream coding sequences. It has been suggested that the allelic exclusion of expression of heavy and light chain genes can be largely explained by non-functional joining events [61]. A recent observation by Karjalainen and Coleclough, however, suggests that an out-of-frame joining event may sometimes be compensated for by a deletion of one or two base pairs upstream of the joint [62]. The ‘imprecision’ of gene segment joining not only generates lack of function but also generates VH and VL polypeptides with novel amino acids and length differences in junctional regions. That the resulting antibody diversity is functional in the case of light chains has been directly demonstrated by Eisen [63] and Capra [64] and their colleagues. These investigators used heavy and light chain reassociation experiments in which a heterologous light chain differed in amino acid sequence from the homologous chain at the VLJ, junction. In both cases the resulting reassociated antibodies lacked affinity for a haptenic determinant characteristic of the homologous antibody. In the case of V, genes it appears that not only does the imprecision of V, to DJH and D to J, joining create diversity, but de novo addition of nucleotides at both junctions and possible D to D joining events amplify this diversity. Nucleotides are often present at VH-D and D-JH junctions that cannot be accounted for by germ-line sequences at the 3’ cnd of the VH segment. the 5’ end of the J, segment, or the 5’ and 3’ cnds of the putative D gene segment. Data presented in Fig. 1 serve to illustrate this point. Alt and Baltimore have proposed that the lymphocyte specific terminal transferase (a template independent DNA polymerase) adds these nucleotides de novo during D to JH and VH to DJ, joining, and they

have termed such sequences ’N’(nucleotide) regions [65]. The observation that N regions tend to be rich in G residues has been cited by these investigators a s support for the role of terminal transferase in N region addition since this enzyme adds dGMP preferentially to free DNA 3‘ hydroxyl ends in vitro. Recent experiments [66] using Abelson virus transformed pre-B cell lines in which VH to DJH and D to J, joining spontaneously occurs have lent support to this hypothesis and are discussed in Chapter 4. Comparison of the ‘core’ D region sequences found in rearranged V, genes with the sequences of the characterized germ-line D elements suggests that D to D joining events may also increase the diversity in this region o f the V, gene. It is D

VH

-

JH

....... .

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

. . _AGA __

ACC TAT GGT/GGT A

.....AGA

TAT AGT AAC AC

C TAT GCT ATG

_ _ _ _ _ AGA

GAN NAT GGT TAN CC

G TTT GCT TAC

.....AGA

GNN GAT GAT GGT TAC TCG CT

._... AGA

AAG ATC TAT GAT/GGT TAC G

.....AGA

M G GGA/GGN

.....AGA

TCG GGG GGT TAC GAC GGG

.....AGA

GAC TAT AGT GAC TAC CTG T A

- - -AGA

GC TAC TGG TAC

GAT/TAT AGT AAC TAC GGC C

T GCT TAC GG TTT GCT TAC CC TGG T T T GCT TAC TGG C TAC TTT GAC

..

ACC TCC TCC TAT GAT/GGN GAC CTC T

CC TGG NTT GCT

.....AGA

TCT GGG/TAT

GT GNC TGG NTT

.....AGA

GNC AGN TCG G G N TAC CCC CTG

.._.. AGA

TCC CCT TAN/AGT AAC TAC CT

..._. AGA

TCG ATG G/TT ACT AC/T

GAT G

CTA T

GCT ATG GAC TAC T TAC TAT GCT AT TAC TAT GCT

Fig. 1. Nucleotidc sequences in the VllD.II,region of several functional V,, genes that arc partially cncoded by the same V,, segment. The sequences arc aligned at the 3’ terminal AGA codon donated by the V,, segment and are prescntcd in triplet form. Nucleotides that can be accounted f or by either germline V,,, D o r J , I gene segments arc shown in plain type. those that cannot arc shown in italics (i.c. putative ’N’ regions). The boundarics between V,,. D and J,, coding regions are indicated by gtps in the sequence. Nucleotidcs that have not been unambiguously identified are represented by an .N’. Slashes represent locations in which homology with one germ-line D element ends and homology with another such element begins (i.c. putative sites of D-D joining). Homologies o f less than 4 nucleotides were considered insignificant for the purposes of this analysis. All o f these penes are expressed by hybridomas derived from the splenic B cells of AiJ mice. The spleen cells were mitogcnically stimulated in vitro with either bacterial lipopolysaccharide or goat anti-mouse IgM antibodies prior to hyhridoma formation. The top 8 sequcnccs are from [ 3 S ] which provides a detailed description of the isolation and characterization of thcse hybridomas. The hottom 5 sequences have not been previously published. (T. Manser. unpublished ohscrvntions.)

184 often observed that the sequence in the D region of expressed V, genes can be best accounted for by assuming that two germ-line D elements have contributed information (for examples see Fig. 1). Kurosawa and Tonegawa originally proposed that D to D joining events might account for such 'hybrid' D regions [8].It must be said, however, that it is also possible that a number of germ-line D segments remain to be identified or that the D segment repertoire varies in different strains of mice since several investigators have characterized V, genes that contain D regions whose structures cannot be accounted for by any known BALBic germline D element [9,10, T. Manser, unpublished results]. Recent experiments by Gefter and his colleagues [67] and Capra and Tucker and their colleagues [68,69] have provided more detailed evidence that D to D joining events may occur. Both groups of investigators noticed that a serine residue recurred at the VHD junction in antibodies synthesized by hybridomas derived from NJ mice immunized with I-'-"zophenvlarsonate (An)-protein conjugates. All these antibodies are Ars specific and are partially encoded by a V, segment whose germline sequence is known. Gefter and his colleagues used site directed mutagenesis to show that if this serine residue is changed to alanine, Ars binding activity is lost [70]. Thus, it is possible that antigen selection of randomly generated junctional amino acids is responsible for the recurrence of serine at the V,-D junction. However, several lines of evidence argue that this is not the case. First, if the serine is changed to threonine by site-directed mutagenesis no alteration in the affinity for Ars can be detected in the resulting antibodies [70]. Threonine has never been observed at this position in the over 30 independently isolated Ars binding antibodies which are encoded by this single VH segment. Second, in all such antibodies the junctional serine residue is encoded by the TCX group of serine codons, AGT or AGC serine codons are never observed. Examination of the 3' flanking sequences of the encoding germline V, segment [71] shows that the terminal codon of this segment is immediately flanked by CAC, thus no part of the TCX serine codon could be donated by 3' flanking sequences of this segment. The possibility that another V, segment, identical in all respects to the characterized germ-line V, except in containing a 3' flanking TCX codon, is present in the A/J genome, has been effectively ruled out using Southern blotting analyses [67]. Finally, the amino acid codon immediately 3' to the TCX serine codon is highly variable, while the next 3' codon can be usually accounted for either by a characterized AiJ germline D element [69] or, more interestingly. by a J,, segment (direct V, to J, joining should not be possible if the currently accepted rules of gene segment joining are correct). This evidence strongly suggests that a fourth segment contributed to the formation of the heavy chain variable region gene that encodes this family of antiArs antibodies. An alternative hypothesis is that the coding V, segment 'replaced' (see above) another serine encoding gene segment during the formation of these V, genes. Two likely candidates for these 'acceptor' segments that are members of the V,7183 family do not contain a T C dinucleotide sequence at their 3' ends [23]. Four heavy chain variable region gene segments may be utilized during other rearrangement events as well since VH-D junctional amino acids have been observed to be highly conserved among antibodies elicited with several other antigens [43].

2.3. T h e multiplicative potential of comhinatorial and junctional diversity If we assume that, to the first approximation, V gene segments and their ultimate polypeptide products independently assort during the formation of V domains in the B cell population, then 10’ different combinations (100 V,’s x 20 D’s x 4 J,’s x 300 V,’s x 4 J,’s) can be expressed by this population (ignoring the contribution of V,,). A conservative estimate concerning the number of different junctional amino acids increases the number of possible V region structures to greater than loy. There are approximately 1OX B cells in a mouse. Any individual combination of segments and junctions. therefore, has a probability of less than 1 in 10 of being expressed at any one time. These calculations, although probably oversimplifications, serve to make two points: ( 1 ) the number of different antibody structures that can be created by combinatorial and junctional processes alone is enormous, and (2) whether or not an antibody encoded by any single combination of segments and junctions will be expressed during an immune response depends not only on whether the resulting antibody has specificity for the immunogen, but also on the probability that this combination will be assembled as well. Due to the large number of possible variable region domains as compared to total B cells, genetically identical individuals will express. at any one time. very different antibody repertoires. Is this amount of diversity required for immune function? Many different combinations of segments and junctions are clearly capable of encoding antibodies with similar specificities, that is. the V region repertoire is redundant [43-531. Why, then, are so many gene segments carried in the germ-line? A possible explanation for this apparent paradox is provided by the ‘priming problem’ argument [72] - extensive diversity is required to ensure that the immune response begins within a reasonable time after antigen is encountered. A potential for diversity that is both redundant in terms of antigen specificity and greater than can be expressed at one time, may serve to buffer the animal against the possibly deleterious effects of both the stochastic assortment of gene segments and the ‘imprecision’ of joining. That is, the probability that a given antigenic specificity will reside in the ‘null set’ is minimized. It must also be considered that the excess diversity is functioning in an immuno-regulatory capacity [59] and is not directly related to antigen specificity.

3. Antigen ‘dependent’ diversity In the previous sections I have discussed issues concerning the forms of antibody diversity that are expressed by both the ‘antigen naive’ and the immune B cell populations. I now turn to a discussion of a form of diversity that is predominantly expressed by only the immune B cell population, namely somatic mutation.

186

3.1. The evidence for sornutic nzutution: u historical perspective Prior to there being any data concerning the primary structure of immunoglobulins, many theorists noted that the mammalian genome was simply too small to encode, in a colinear fashion, the diverse repertoire of antibodies any individual animal was capable of producing [73].I t seemed inescapable that a large fraction of diversity was created by somatic events. A number of these theorists proposed that somatic mutation of immunoglobulin genes was the most plausible way to generate antibody diversity. With the advent of biochemical procedures for determining the primary structure of proteins it became possible to directly address the question of the structural basis of the diversity of antibody binding specificity. Initially, the structures of human antibodies were studied d u e to their availability in essentially homogeneous form from patients with multiple myeloma. These studies confirmed that the antibody molecule was composed of variable and constant regions and demonstrated that there were areas of hypervariability within the variable regions [74]. These studies demonstrated little, however, about the genetic origins of antibody diversity. T h e seminal studies of Weigert and Cohn provided the first insights into this question (75-771. They determined the primary structures of the V regions of a large number of mouse light chains of the A l type that were synthesized by myeloma tumors that had been induced in BALBic mice by a procedure developed by Potter’s group [7X]. It was known that only a minor fraction of norm‘kiiiil. M.. Kcarney. J . F . . Baltimore. D . and Alt, F.W. (1984) Naturc 31 1 , 727-733. 24 Alt. F. W.. Yancopoulos. G . D . . Blackwell. 'T.K.. Wood. C . , 'l'homas. E.. Boss. M . , Coffman, R.. Rosenberg. N . . Tonegawa. S. and Baltimore. 11. (1984) E M B O J . 3. 1209-1219. 25 Yaoita. Y.. Matsunami. N.. Choi. C . Y . . Supiy;iin;i, H.. Kishimoto. T. and Honjo. T. (1983) Nucl. Acids Res. 11. 7303-7316. 26 Perlmuttcr, R.M.. Kearncy. J.F.. Chang. S.1'. and Hood. L. (1985) Science 227. 1597-1601. 27 Cancro. M.P.. Wylie. D . E . . Gerhard. M'. aiid Klinman, N.R. (1979) Proc. Natl. Acad. Sci. USA 76. 6577-6581. 28 Sigal. N.H.. Pickard. A . R . . Metcalf. E.S.. Cicai-hai-t.P.J. and Klinman, N . R . (1977) J . Exp. Med. 146. 933-048. 29 Riley. S.C., Connors. S.J.. Klinman. N . K . and Opata. R . T . (19x6) Proc. Natl. Acad. Sci. USA 83. 2589-2593. 30 Teale, J . (1985) J. Immunol. 135. 953-958. 31 Teale, J. and Kcarney, J.F. (1986) J . Mol. Cell Immunol. 2, 2x3-292. 32 Brodeur. P. and Riblet. R. (1984) i n : Kepulation o f the Immune System, pp. 444-453. Liss. New York. 33 Near. R . . Manser. T. and Gefter. M 1.. (I0X.S) J . Immunol. 134. 20042009. 34 Manser. T.. Huang. S.-Y. and Gefter. M.I.. (1984) Science 226. 1283-1288. 35 Kleinfield, R.. Hardy. R . . Tarlinton. D . . Dangl. J . . Herzenberg. L. and Weigert, M. (1986) Nature 322. 843-846. 36 Reth. M . . Gehrmann, P . . Petrac. E . mid Wiese. P. (1986) Nature 322. 84C842. 37 Wood. D . L . and Coleclough. C. (1984) Proc. Natl. Acad. Sci. USA 81. 4756-4760. 322 Nishi. M . . Kataoka. T. and Honjo. T. (1988.5) Proc. Natl. Acad. Sci. USA 82, 6399-6403. 39 Sherman. L . A . , Vitiello. A . and Klinmai. N . R . (1983) Annu. Rev. Immunol. I . 63-88. 40 Owen. J . A . , Sigal, N . H . and Klinman. N.R. (1082) Nature 295, 347-348. 41 Kindt. T. and Capra. J . D . (1984) in: The Antihody Enigma, Plenum Press. New York. 42 Capra. J . D . and Fougereau. M. (1983) lmmunol. Today 4. 177-179. 43 Boersch-Supan. M.E.. Agarwol. S.. White-Scharff. M.E. a n d Imanishi-Kari, T. (1985) J. Exp. Med. 161. 1272-1292. 44 Dzierzak. E.A.. Brodeur. P.. Marion. '1. .. Janeway. C . A . and Bothwell. A . (1985) 162. 14941511. 45 Rocca-Serra. J . . Matthcs. H . W . . Kaartinen. M . . Milstein. C., Theze. J . and Fougercau, M. (1983) EMBO J . 2. 867-872. 46 Perlmuttcr. R . . Klotz, J.L.. Bond. M . W . . Nahm. M.. Davie. J . M . and Hood, L. (1984) J . Exp. Med. 159. 179-192.

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203 CHAPTER X

The immunoglobulin superfamily FRANC0 CALABI M R C Laboratory of Moleciilrir Biology. Hills Road, CB? 2 Q H Carnbririge, U K

I . Introduction lrnmunoglobulin polypeptide chains are composed of homology units [ 1,2]. Related units are found in a large set o f non-immunoglobulin proteins. Thus all these proteins, belonging to several functionally diversified families. can be grouped into ;I single superfamily, the immunoglobulin superfamily, based exclusively o n structural homology [3]. Such homology between members of different families is h i ited as a rule to the protein structure. homology being virtually non existent at the DNA level, except for a very general principle of organization (each homology unit is usually encoded by a single exon). However. within individual families, DNA sequence homology is found to pseudogenes and to gene sequences which are not known to b e expressed. These gene sequences must be regarded as members of the immunoglobulin superfamily. since they may play o r may have played iniportant roles in its evolution. Any unifying hypothesis is bound t o introduce some degree of distortion to the experimental data, and this may be very significant when. as in the case of the immunoglobulin superfamily, the unifying criterion can often be only partially verified. However, such an hypothesis is justified in so far as it makes specific predictions which are testable [4]. The analysis o f how variations of a basic structural module a r e achieved during evolution through the operation of a variety of genetic mechanisms and of how such variations can serve different functional purposes is clearly of both great theoretical and practical interest. Furthermore, structural relatedness can be assumed t o reflect relatedness to an ancestral function. Thus, the investigation of the evolutionary relationships amongst members of the superfamily may lead to predictions for members o f unknown function. In this respect, in choosing immunoglobulins as a structural standard (which is clearly dictated by the quality a n d the quantity of the information available). it must be appreciated that the function they are serving has arisen late in phylogeny and may thus be significantly different from the ancestral function.

2. The immunoglobulin homology unit T h e qualifying feature of the immunoglobulin homology unit is its molecular architecture. Delineation of this structure has resulted from a considerable amount

204

Fig. 1. Polypeptide chain folding in immunoglobulin V and C units. Thick arrows: p-strands; thin lines: loops. Filled arrows (p-strands A.B,D.E) lay on the plane of the sheet. whilst hatched arrows @-strands C,(C').F,G) lay on a parallel plane behind. A lilled circle on p-strands B and F indicates the cysteines which establish the intra-unit disulphidc. The filled square on @-strand C indicates the tryptophan residue which is an invariant component o f the unit core [ h ] .

of sequence and crystallographic data [ 5 ] .Basically, the unit has been likened to a sandwich, consisting of two stacked antiparallel @-sheetswhich enclose an internal space filled mainly by hydrophobic amino acid side chains; this structure can be viewed as consisting of three elements [6]: (1) a pin, including an invariant tryptophan and the two invariant cysteines which link facing @-sheets; (2) a @-sheet core, including conserved (mainly inward pointing) residues in @-sheet strands; and ( 3 ) a periphery, including some non-conserved @-strand structures as well as the loops which connect individual @-strands in a characteristic sequence. Whilst in different immunoglobulin units extensive insertions and deletions as well as replacements are found in the periphery, especially in the loops. the core shows mainly replacements, which are at times extensive but are mostly conservative in nature. Two types of immunoglobulin units can be distinguished: V and C ([7], Fig. 1 and Table 1). Seven @-strands(A to G) are found in both types. However, V units contain two additional @-strands(C' and C") as part of an extra hairpin loop between the C and D strands. V, and V,, units are also distinguishable on the basis of a few critical residues [8.9]. In immunoglobulins, units of the same type are found paired in domains. Such pairing occurs through the 4-stranded @-sheetsin C domains and the 5-stranded psheets in V domains and involves a variety of specific inter-unit interactions [lo]. Whilst pairing of VH units has never been found. both V,-V, and V,-V, pairings occur, the latter one possibly resulting in a different geometry of the binding site. To what extent can any linear polypeptide sequence be expected to fold in a three-dimensional structure characteristic of the basic immunoglobulin unit? Given our very limited knowledge of the rules governing protein folding, four minimal criteria have been suggested [ 111: (1) the test sequence must be approximately 100 amino acids long; (2) it must contain cysteine residues at positions homologous to

205 TABLE 1 Structural features of immunoglobulin units A. Folding pattern characteristic of immunoglobulin units is suggested by:

1. conserved cysteines and tryptophan; 2. p-strand motifs; these show a prevalence of replacements, rather than of insertionsideletions and are organized in two p-sheets, such that: a. mid-sheet p-strands are more hydrophobic than edge p-strands; b. side chains pointing toward the opposite p-sheet tend to be hydrophobic. B. V-C distinguishing features: I. core: 1. V units have an extra hairpin loop and two extra p-strands (C' and C ) . The latter are found in a five-stranded p-sheet with strands C, F and G. As a consequence, the number of residues between the disulphide-bonded cysteines is higher in V (-70) than in C (-60) units; 2. p-strand motifs can be defined that are somewhat characteristic of each type of unit [9]; 3. V units pair through the p-sheets composed of strands C, ( C ' , C ) , F, G, whilst C units pair through the p-sheets composed of strands A, B. D, E. Since interface p-sheets are more conserved than solvent-exposed P-sheets, @strands C, F and G are more conserved amongst V units than A, B, D and E, whilst the converse is true amongst C units. 11. loops: V units have a conserved glycine (or less frequently aspartate, i.e. a turn-promoting residue) between p-strands A and B. Furthermore, a salt bridge is usually found between an arginine (or less frequently a lysine) near the carboxyl end of the C/D loop and an aspartate at the carboxyl end of the E/F loop. C units have a conserved proline in each of the loops between strands A/B and C/D.

C. V,-VH distinguishing features: 1. CDRl is longer in VL whereas CDR2 is longer in VF,. 2. The residues conserved in interface @sheets (residues cumulatively present in 3 0 % of sequences at the given position) are: strand

V,

position

V,

vosition

C C'

Val/Ile Leu TrP Trp

37 45 47 103

Phe/Tyr Pro/Phe LeuIGly Phe

36 44 46 98

G

(position number is according to [206]).

D. VH-VLcontacts involve [lo]: 1. hydrogen bonding between glutamines in strands C; 2. herringbone pairs involving hydrophobic residues in strands C, F, G.

E. Unit pairing requires identity of type and is suggested by: 1. lower primary sequence variability in one p-sheet (presumably the interface p-sheet) rather than in the opposite p-sheet (presumably solvent-facing); 2. electrostatic charge compatibility.

206

w IgG FcR

Poly-lQR

a,B-gp

Thy-1

MRCOX-2

NCAM

4 1 , MAGlNCp 3

Fig. 2. Presently known incnibci-s o f the imniuno~lobulinsupcrfamily. 0. immunoglobulin-like V units: units: @. non-imiiiiiiioglohulin-lil\c V units: 0. C units. Interchain disulphide bonds arc not sho\\n. The douhlc line irepresents the plnsina inemhrane to which ‘lliy-1 and a form of NCAM a t e attached via ;I link to ;I phophoinositide. ‘The structure of the TcR ii chain is hypothetical.

0. units distantly related to iminuno~lohiilinV

the cysteines which establish the immunoglobulin intra-unit disulphide bond; (3) structural predictions [ 12-14] must indicate thc potential of P-strand formation at positions homologous to those o f immunoglobulin units; (4) sequence homology to immunoglobulin units must be statistically significant [ 151. Furthermore, if pairing of units is to occur, a further set of criteria is expected to be met (Table I ) . Members of the immunoglobulin superfamily which have been identified at present are sketched i n Fig. 2. In this review, 1 only shall discuss in detail members (other than immunoglobulins) which consist of at least one unit meeting the standard criteria and shall only mention other inore distantly related members in the context of the cvolution o f the whole supcrfamily.

3. T h e T cell receptor Although it has long heen known that T lymphocytes interact with antigen by way of specific receptors, the identification of such receptors has been accomplished only recently (reviewed in [16]). At least two kinds o f receptors are presently known. which are expressed b y T cclls which most likely belong t o different lineages. I shall refer to these as the a/p and the $6 T cell receptors (TcR’s).

207

3. I . The alp T cell receptor. This is the antigen receptor which is expressed by the majority of mature T lymphocytes. Like immunoglobulins, it is highly diverse and its diversity is clonally distributed. However, in contrast to iiiiniiinoglobulins. it only recognizes foreign antigen in t h e context o f a major liistocot7ipatibility complex molecule; this targets recognition t o cell-bound structures. Whilst the a/@TcR's and immunoglobulins have evolved under different selective pressures, they clearly share a common ancestor. as both their structure and genetics are fundamentally organized according to the same principles. 3. I . I . Overall structure Like immunoglobulins, the alp T c R consists of two chains which arc disulphidebonded a n d it is organized into ;I V and ;I C region. There are. however, a number of significant differences from i m in LI n oglo h i t I ins . First , w hi I st i m in unogl o bu I i n s a re hoth cell-bound and soluble receptors. the u l p T c R is o n l y cell-bound. Second, the binding site is monomeric. Third, whilst hoth componcnt chains span the plasma membrane, the C region is much smaller than that of immunoglobulins such that t h e extracellular portion of the tv/P TcR molecule rather resembles an immunoglobulin Fab arm. Fourth. the a/@TcR is closely associated with a set of proteins (the CD3 complcx). which act a s ;I signal transducer for the coupling of antigen binding to cell activation [ 171. In addition. other molecules (CDJ and CD8, see below) may play an important role in binding of the T cell to the antigeniMHC complex.

3.1.2. Strcict~ireof the varirihlr r .' 'c1 ~0 1' 1 This is the domain in which the cwip T c K is most homologous t o immunoglobulins. .4lignmetit o f primary sequences (Fig. 3 ) shows conservation o f @-strand patterns as well of residues which are known t o be critical in V-V packing. Some features of loop architecture are also conserved. This suggests that both folding and pairing of V,, and V, units occur in ;I similxr way to that o f immunoglobulin V domains Variability plots show the existence i n hoth V,, and V, of three hypervariable regions, which in the primary sequences map to positions homologous to the complementarity determining regions ( C D R ' s ) o f immunoglobulins [ 19-25]. This suggests that the antigeniMHC binding site o f the NIP TcR largely results from the juxtaposition of these three CDR's. In these analyses, both V,, and V, display an overall high level of variability and extra hypervariable regions have been proposed to occur in V, [19]. However, this does not necessarily imply an extra binding site. In the first place, the significancc of the extra hypervariable regions in V, has been questioned, since the sequence sample is statistically small and V,'s do not represent a homogeneous population ([26] and see below). Secondly, sequence variability reflects freedom from structural constraints, which only indirectly correlates with antigen binding; for example, a fourth region of hypervariability exists in human V, [27]and this does not form part of the antigen binding site.

(A) V,

QQ

10

~

1 0

~

30

!

: 104

~

5;

~

70

60

80

9OI

~

;I00

U

:

l

Z

O

I

!2

cr:

NVQ QSPESLIVPEGAR TSLNCTF SDSAS******Q YFWWY QHSG KAE'KALM SIFSNGEK********EEGR FTIH LNKAS LHFSLHI RoSQPSD SALYLCAV LYGGSGNK LIFGTGTL L S W

Vp NS KVI QTPRYLVKGQGQK AKMRCIP EKGHP******* W F W Y

QNKN NEFKFLI N*FQNQEVL€Q*IDMTE*KR FSAE CPSNS PC*SLEI QSSEAGD SALYLCAS L*NWSQDT QYFGPGTR L L m

-

V., GHG KLE QPEISISRPRDET AQISCKV FIESFRS****V TIHWY QKPN QGLEFLL YVLATPTHI*'*FLDKEYKK MEAS KNPSA STSILTI YSLEEED EAIYYCSY MSDSSGFH KVFAEGTK LIVIPS V,

DI VMT QSPSSLSVSAGER VTMSCKS SQSLLNSGNQKN FLAWY QKPG QPPKLLI YGASTRESG*******VPDR FTGS **GSG TDFTLTI SSVQAED LAWYCQN

HS****YP LTFGAGTK

VH

*E VKL VESGGGLVQPGGS LRLSCAT S*GFTFSDF*** YME

YYGSTWYF DVWGAGTT VTvSS

QPPG KRLEWIAAS RNKGNKYTTEYSASVKGR FIVS RDTSQ SILYLQM NALRAED TAIYYC

A

B

A

Human C k

CDRl C

C'

B

C

E

D

CDR2

Q P W S

+ Human CBI EDLNKVFPPE Mouse C ---RN-T--I(

P

Human C,

Mouse C,

t

Ph -Y

A

D

E

F

CDRB

G

209 I t has been remarked that both V,, and V, are somewhat more homologous to V, than t o V , in their core structurc. However, this does not imply significant differences of the binding site, since. for example. V, CDR loop lengths are closer to those of V,, than to those of V,; furthermore, V, dimers d o not necessarily pack in a significantly different way from V,.-V,, [ 101. 3.1.3. Genetic basis of diversity

In the germ line both V,, and V,. like immunoglobulin V regions, are encoded in separate segments, each present i n multiple. diverse copies (families). During the somatic assembly process, segments can recombine independently and nucleotides can be inserted and/or deleted from the junctions. In this way. a large repertoire of different V regions can be derived from a much smaller repertoire of germ-line sequences (see Chapters 3 and 4). T h e segmental arrangement of V regions is clearly a critical factor in the generation of diversity. It has been suggested that this arrangement resulted from the insertion of a transposon-like element into a primordial V exon [28]; this element became stably integrated into the germ line. whilst maintaining the ability to be excised during the somatic development o f immunocompetent cells. Sequence variability resulting from imprecision i n the excision process became utilized in the generation of the antigen binding site. Consistent with this, the insertion is found at a position corresponding to ii region in the polypeptide ( C D R 3 ) which is subject to few structural constraints. The ends of the excised D N A sequence have a unique organization [29]: ( a ) each e n d consists of two short conserved sequences (a heptamer and a nonamer) which a r e separated by a spacer; (b) the spacer can span either one o r two turns of t h e D N A helix (one- o r two-turn type); (c) the heptamerinonamer sets at the two ends a r e in inverted orientation with rcspcct to each other; (d) the spacers at the two ends a r e of different type. i.e. one-turn type at o n e end and two-turn type at the opposite e n d . This organization is shared by all V region loci. Heptamerinonamer sequences a r e also conserved across loci. i.c. the same sequences are found in both immunoglobulin and TcR gene segments. This pattern of conservation suggests that these sequences are critical to recombination a n d that the recombination (joining) machinery has similar requirements i n T a n d B cells. Indeed, this has already been shown t o be the case ( [ 3 0 ]and Chapter 4). ~~~

~~

~

~

Fig. 3 . (A.B). Alignment of immunoglobulin and T c e l l receptor V (panel A) and C (panel B) units. P-strand sequences (from [6] and 12051) xi-c boxed and indicated by letters A to G.*, padding chnrCDR’s (7’cell receptor hypervariable regions acters introduced to maximize nlignmentz. I’ancl A : map essentially t o corresponding positions. see text). conserved glycine residue in the loop connecting P-strands A and B in V units; *. kcv residues in V,,-V, packing [ l o ] . Horizontal brackets, salt bridgcs. V, and V l l sequences are from the niwse myeloma MCPC 603 [206] (this V, is unusual in the length of its C D R l [205]):V,, and V, iirc Iron1 the mouse T-helper hybridoma 2B4 [207.208]: V, is the nimt frequently rearranged V at thc miuse TcRyl locus (1771 and see tcxt). Panel B: - , interspccies identity; +. identity to human C,. C‘ i-cgion sequences are from [2Oh] (human Ig CJ. (209l (human T’cR C B l ) .[ Z I O ] (mouse TcR C,, and C,,, arc identical), [ Z I I ] (human TcR Cy,) and [77](mouse TcR Cd).

=.

210

Fig. 3 . Orientation o f immunoglobulin a n d TcR V gene segments relative to joining signals. The symbol '2' indicates ii two-turn type joining signal a n d '1' ii one-turn type joining 4gnal. Within each type of signal. the conserved hcptarners :IIK adincent to the V gene segment. whilst the conserved nonamers point toward each other. Although n o D segment has s o far been shown at the TcR y locus. its existence has not been excluded.

T h e V, locus consists of V a n d J segments, whereas the V, and V,, loci are composed of V , D and J segments. The existence o f D segments has also been recently reported at the V, locus [316]. Differences between these loci are found in the relative orientation of recombination signals and coding segments (Fig. 4); this is most significant when contrasting V, and V,. A V , , region is assembled in two steps of which D-J joining is the first. D-J joining may also occur as an obligatory first event in V, assembly; however, germ-line V, segments can join both to D-J and directly to J . As a consequence D segments are obligatory in IgH V regions. dispensable in TcR V regions [31-331. D - D joining can occur i n V, [13]and may also in principle occur occasionally in Vk,. iis both 21 one-turn and a two-turn recombination signal sharing the nonamer scquence can be found o n the 5' side of some D, segments [34]. Comparison of the germ-line V gene repertoires of immunoglobulins to those of aip TcR's shows significant differences (Table 1). Both V,, and V, segments are more heterogeneous in sequence than immunoglobulin V segments; this is reflected in the larger number of subfamilies, as defined by nucleotide homology below 75% [20-251 and correlates with the high background in variability plots (see above). O n the other hand, T c R a and T c R P V segment subfamilies contain fewer members. D, segment complexity is amplified by the use of multiple reading frames [35]. T h e J,, a n d J, segments are more numerous and more divergent, as well as longer than immunoglobulin J's. Thc J,, segments a r e unusually long and the sequences of their 5' ends show homology t o D, segments; it has therefore been hypothesized that the J,, segments are equivalent to a D-J fusion. possibly as a result of some germ-line D N A rearrangement 1361. The organization of the J, family is unique: in the mouse it is estimated to consist o f more than 50 segments which are distributed over at least 60 kb and are more widely spaced than those of any other J cluster [37]. A similar organization is likely to exist in man [38]. Such a n arrangement has important implications for transcription, since transcripts of a rearranged T c R a locus must span up to 70 kb. T h e somatic recombination which results in V region assembly introduces diversity by three mechanisms: first, different combinations of V, D and J segments; second, variability in the position of strand breaks; third, template-independent

21 1 'I'ABLE 2 Size of the germ-line repertoire o f T c e l l receptor and immunoglobulin V gene segments (including pseudogenes) V

J

D

total

subfamilies

total

subfamilies

total

B human

6X (hO 300

3 0

human mouse

4(NO 300

4 2-4

4 2

3 I

13 14

N

Y

X

H >4 12

9 4

K

4

5

A

human mouse

>9 4

Subfamilies are defined by greater than 75'r homology amongst members. Data f o r V, and human V,, arc extrapolated from the number and frequency of k n o w segments. the value in brackets giving the 95% confidence upper limit [22-25.215]; for inouse V,, the data mainly derive from Southern blotting experiments [20.21]; for V, from Southern hlotting as well as from cloning experiments [75-78.8(kX2]. For J,, the numher or elements known 211 present is given: the total repertoire size is estimated to he larger. For the germ-line repertoire of iiniiiunoglobulin V gene segments scc Chapter 3.

addition of nucleotides to the recombining termini (N segments). T h e first two mechanisms occur at all V loci. the third. which is due to the action of terminal transferase [39], occurs nearly exclusively a t V,, V,, and V, loci. Vl- a n d V, assembly occur at a later stage than V,, and V,. Thus, differences in the occurrence o f N segments may simply reflect different developmental regulation o f terminal transfcrase activity in the T and B lineages. Combinations of V region gem segments. although largely unrestricted. may not be completely random; some bias has bccn ohserved at the IgH locus which is linked to the temporal order of asscnibly (see Chapters 4 and 7). It is not known whether a similar situation exists a t either the TcRa o r at the TcRP locus: however. some bias in J segment usagc ma): exist i n both mouse and man [40,21]. O n average, only a small proportion o f assembled V regions is functional. In immunoglobulins, rescue of non-productive chromosomes may result as a conse-

212 quence of secondary joining or of V segment replacement in non-functional V regions ([39] and Chapter 4). Neither in v, nor in v, segments are the recombination signals found which have been postulated to mediate V segment replacement [41]. However, the larger number of J segments. especially at the T c R a locus. augments the potential for secondary joinings. Furthermore, since D, segments, unlike D,'s, are used in all three translational frames, productive assembly is more likely at the TcRP than at the IgH locus. Assembled V, and V, genes can mutate at a high rate by an as yet unknown mechanism. Mutations are rare in primary B cells, but accumulate during the progression of the immune response and correlate with an increased affinity for antigen (maturation) (see Chapter 7). With one exception [42]. no evidence for a similar mechanism has so far emerged from the analysis of expressed V, or V, regions. Thus, affinity maturation may n o t play any significant role in a/p TcR recognition. This may be due t o ;I bias in the sampling, since most of the T cells analysed may be equivalent to primary B cells. However, it is more likely to reflect different selective pressures: no specificity control mechanism (thymic selection, and/or dependence on regulatory T cells) is operational in peripheral T cells and a/p TcR mutations which might cause loss of self MHC restriction (see below) and/or the generation of self-aggressive specificities must be prevented. In summary, the genetic strategy of the alp TcR is characterised by two main differences from immunoglobulins: first, greater heterogeneity of framework sequences; this may well correlate with diversity at the binding site, for example by dictating different conformations of CDR's, in analogy with immunoglobulin V regions [6]. Second, restriction of somatic diversification to CDR3: this may imply that variability at C D R l and CDR2 is under genetic selection (see below). 3.1.4. Structure of the coristuiit regiori

The constant region of the aij3 TcR consists of a C-like domain (Ccrplus C,) linked by a connecting peptide to the transmembrane and intracytoplasmic tail. Both C, and C, show conservation of p-strand pattern and contain key residues typical of immunoglobulin C units (Fig. 3). Howevcr, in C, the homology unit is only 90 amino acids long and little homology exists at positions corresponding to p-strands C, F and G, which are also poorly conserved across species. Divergence is most evident in the sequence aligning with j3-strand C, which is lacking the tryptophan residue that is an invariant component of the core of the immunoglobulin unit [6]. Thus, whilst conservation of j3-strands A . B, D and E and of few other critical residues suggests that C,, and C, pack in a manner analogous to that of immunoglobulin C domains, it remains to be seen whether C,, folds like a typical C unit. Both (Y and p chains contain one cysteine residue located approximately 20 amino acids from the cell membrane within the connecting peptide. Thus, although C, also contains a conserved unpaired cysteine, the disulphide bond linking the two chains most likely occurs between the connecting peptides. Both chains are known to be N-glycosylated, the extent of glycosylation varying between species, between chains and even within the same chain [43,44]. Glycosylation sites are found in both C units and in the connecting peptides.

213

a

= Cunii

0=

=

0-

100 basepairs

Connecting peptide

= Transmembrane =

0=

lntracytoplasmic

3 untranslated region

m.

Fig. 5 (A.B). ( A ) Genomic organization of humaii TcR C region genes. exons; //, introns. Numbers give approximate intron lengths i n lib. Thc TcR y chain connecting peptide is encoded by a number (1-3) of highly related exons which varies hctwecn individuals (1741 and see Fig. 6). (B) Gcnomic organization of mouse TcR C, genes. Identical tilling pattern indicates sequence homology. Arrows indicate direction of transcription.

Finally, the transmembrane stretch of both a and p chains contains basic residues which have been hypothesized to form salt bridges with acidic residues occurring in equivalent position in the polypeptides of the CD3 complex [18]. Crosslinking studies have shown the TcRP chain to be topographically close to the CD3 y chain at the cell surface [4S]. The genomic organization of TcRa and TcRP C region genes is illustrated in Fig. 5 . Two tandemly arranged TcRP C region genes exist in both mouse and man, resulting from a duplication that also involves the D, and J, segment cluster. Se-

214 quence identity of over 95%, in the coding regions of the two C,’s suggests strong selection. The two TcRP C region genes are equally used by functionally different classes of T cells (helperlcytotoxic). The TcRP C region genes are unusual in that the C, immunoglobulin homology unit is not encoded in a separate exon.

3. I .5. The alp T cell receptor and the major histocompatibility complex It has long been known that most T cells recognize a foreign antigen only in association with a major histocompatibility complex (MHC) molecule of a specific class (either class 1 or class 11) and allotype, a phenomenon known as MHC restriction [46]. There is good evidence that it is the alp TcR that determines all these specificities [47-501. I n contrast, antibody recognition is not generally MHC restricted. What is the basis of this difference? Two pieces of evidence suggest that obligatory MHC restriction is not due to basic structural differences between alp TcR’s and immunoglobulins: first, the alp TcR and immunoglobulin V domains are highly homologous, suggesting a similar molecular architecture. with a single binding site shaped according to identical principles; second, antibodies recognizing foreign antigen in association with an MHC molecule do exist (511. Thus the bias in the aip TcR recognition repertoire must be due to selection. Where does such a selection occur? Several lines of evidence indicate that MHC class and allotype restriction is due to thymic selection of a/p complexes [52]. Thus. in most cases neither the a chain alone nor the p chain (nor any V,,or V, gene segment) is individually sufficient for the recognition of MHC class ([32,53-55] one exception is discussed in [ 5 6 ] ) and allotype [35,57-59]. However, individual a and /3 chains may recognize MHC molecules non-specifically, i.e. independently of class and type. Thus, the same a chain is shared by alp TcR’s specific for: ( 1 ) pigeon cytochrome c and MHC class I1 Ek or Ek”’ [37,58]; (2) ovalbumin and MHC class I1 A” [5Y]; (3) an allotypic MHC class I Dh 1591. Since the three a/p TcR’s listed have been generated by in vitro manipulations (i.e. they have not been selected as a complex in the thymus) and do not crossreact, this particular a chain may impose a bias towards MHC recognition that is independent of class and allotype [59]. Whilst this bias may also be due to selection of individual chains in the thymus, V,, andior V, gene segments are clearly not individually selected [53,55]. These conclusions have two significant implications. First, they support an early hypothesis that there is a germ-line repertoire of V regions that are specific for MHC molecules [60]. However, by making such specificity independent of MHC class and allotype. they dispense with an otherwise problematical requirement for a co-evolution at the population level of V region and MHC genes [61]. Second, they suggest that this MHC bias is carried by germ-line encoded CDR’s (CDRI and 2), whilst determination of MHC class- and allotype-specificity as well as foreign antigen binding must largely be the role of CDR3, which is the only target of somatic variability. Different requirements for germ-line repertoire selection may explain why, despite prima facie wastefulness [62], immunoglobulins and cdp TcR’s have evolved as two completely independent. yet equally specific recognition systems.

215

3.2. The $6 T cell receptor Despite lack of direct evidence for a receptor function, two elements strongly suggest that this molecule constitutes a second type of TcR. First, both the structure and the genetics of at least one of the component chains, the y chain, are closely homologous to those of TcR cy and /3 chains, including features of diversity (see below). Second, the TcRy is expressed at the cell surface in association with the CD3 complex, which is postulated to act a5 a signal transducer in coupling antigen binding and cell activation in a//3 TcR-positive T cells [17]. The CD3 present on T cells expressing TcRy is known to be functional [63,64-671. Whilst early in ontogeny the TcRy is expressed by a large number of thymocytes, in the adult it is found only on a small minority (less than 5 % ) both of thymocytes and of circulating T cells. All such TcRy expressing cells are alp TcRnegative [67,66], express immunoglobulin Fc receptors and can effect antibody dependent as well as direct cytotoxicity. The in vitro killing of target cells, albeit specific, occurs independently both of previous sensitization and of MHC allotype and is not inhibited by antibodies against MHC molecules, showing similar features to natural killer cells [64.65,68]. Thus, the ylS TcR is likely to be non-MHC restricted. Consistent with this, TcRy-positive cells express neither CD4 nor CD8 (these molecules possibly playing an accessory role in MHC recognition by d p TcRpositive cells, see below) and may not be thymus-dependent [67,66,69]. In humans, TcRy's are heterogeneous in structure; it is not clear whether this heterogeneity has functional relevance and/or whether it identifies different classes of T cells. Data from different laboratories are tentatively summarized in Table 3 . On the basis of the presence of interchain disulphide bridges, two forms can be distinguished, the non-disulphide-bonded form probably representing more than TABLE 3 Structural features of TcR's Humaii

Mouse

y chain

40-44 + occasionally 3&41

55-60 (4(k45)h 40

35

31-34

6 chain

43 (invisihlc. ? ) '

38-40 (62)"

45

Interchain disulphide

+

-

+

glycosylated polypeptide backbone

32

Numbers represent range of molecular weights in kDa; for the y chain these are given for both the glycosylated (cell surface) form(s) and the polypeptide hackhone. I and I1 refer to two distinct types of TcR's which have been identified in huinaiis. See text for details. " In the human disulphide-bonded form a 6 chain was directly visualized in six out of a total of nine clones analysed [63-65.681. hypothcsized in one other case [65]and possibly missing in the remaining two [64]. In the non-disulphide-bonded form, thc data in parentheses refer to a polyclonal TcRy-positive cell line from normal peripheral blood ( y chain only. [ h S ] )and t o a thymocyte clone (y/S chains, [h3)).

216 half of the alp TcR-negative, CD3-positive circulating T cells [65]. At least two y polypeptides of molecular weight 31-34000 and 40000 have been found, of which the first may be more common and the second may be restricted to disulphidebonded complexes. Both polypeptides are heavily glycosylated; occasionally, partial glycosylation products may be found in variable amounts on the cell surface [64,65]. Much less is known about the non-y (6) chain for lack of specific probes. However, at least two different forms of this chain exist in non-disulphide-bonded complexes, one of which might be thymus specific [63]. On the basis of its pattern of expression in ontogeny, it has recently been proposed that a newly identified TcR C,-like gene, located approximately 70 kb upstream of C,, might encode C, [216]. Some TcRy’s might be homo-. rather than heterodimers [64]. A single form of $6 TcR has been described in the mouse [70-72]. Like a and p chains, the y chain consists of a V and a C region. There are two isotypic TcR C, region loci in man [73,74]. Three TcRy loci exist in the mouse [75-781: there is no evidence that the TcRy2 and y4 loci (nomenclature according to [77]) can encode functional polypeptides (see below). A fourth locus (TcRy3) is found in the BALBlc mouse, but it is likely to be of little functional relevance, since it contains multiple defects (stop codons in all three reading frames of the single J segment as well as defective splice and polyadenylation sites) and it is not conserved in different strains (75,761. The sequence of all human and mouse y isotypes reveals typical features of an immunoglobulin C homology unit and contains a conserved N-glycosylation site in mouse T c R y l as well as in both human isotypes. The C unit is followed by a connecting peptide, of which the membranedistal segment shows the most dramatic variations amongst mouse and human TcR C, regions (Fig. 6). In humans this is due to different numbers (between 1 and 3) and combinations of homologous 16 amino acid long units, each unit being encoded by a separate exon [73,74]. Three different C, regions originate from the CY2locus probably due to polymorphism in the number and type of such exons rather than to differential splicing. Differences in connecting peptide size may be amplified by the occurrence of extra N-glycosylation sites. The membrane-proxima1 segment of the connecting peptide is conserved between isotypes as well as across species and is encoded by the same exon as the transmembrane and intracytoplasmic tail. In man, an unpaired cysteine is found only in the C,, connecting peptide. Thus, the human disulphide-bonded form of y chain can only correspond to this isotype. The only mouse y polypeptide chain that has been identified is both N-glycosylated and found in a disulphide-bonded dimer [70-721. All mouse y isotypes are predicted to have an unpaired cysteine (in the connecting peptide), but only CYI (in the C domain) and C, (in the connecting peptide) have N-glycosylation sites and only the CYIlocus is substantially transcribed in foetal thymocytes, where the y chain is known to be expressed [77,79]. All y chain transmembrane segments share with a and p chains the presence of a charged basic residue, which is presumably implicated in the association with CD3. The sequence of V, regions shows features typical of immunoglobulin and TcR V,, and V, regions (Fig. 3). An exception is found in p-strand G, where the se-

217 C unit

1

S

N

1

I

s

I

Connecting Peptide

N

I

S

I

N

I

N

5

I

I S

N

s I

I

S

S

I

I

I

I

I

t

N

N N

I

I

N

N

I

I

H U M A N y 2 (FRO 2 2)

1

HUMAN y 2 ( H P B MLT)

1

H U M A N y 2 (PEER)

1

MOUSE y 1

N

l

N N I l

I

l

N l

s

s

I

I s

s I

I

I

s I

N

111

N N I 1

n

s I

NSN

HUMANyi

5

I

I

1

TM + CY

I

1

MOUSEyP

s

I

I

N 1

S 1

I - -

MOUSEy4

Fig. 6. Structure of TcR y chain connecting peptitle\. as deduced from c D N A clone>, Boxm with siniilar filling patterns (i.c. either cross-stripc\ o r shatlcd) are homologous in sequence. Blank spaces are introduced to maxiniizc alignments. Fro 2.1. HPB-MLT and PEER are cell lines established horn dilferent individuals [7JI. The lengths of thc C’ u n i t ( II 0 amino acids) and ol the transmembrane and cytoplasmic segment (approximatcly 53 aniiiio : d s ) is a l s o \hewn 11s a reference. S. potential disulphidc liridge: N. potential N-linked glycosylatioii \ i t c

quence Ah-X-Gly in all V,’s rcplaces the Gly-X-Gly which is found absolutely conserved in all other V units. This sequence is encoded by the J segment and in V, and V,. forms a P-bulge which contributes in a characteristic way to V-V domain packing [XI. Comparison of V, sequences shows hypervariable regions analogous to immunoglobulin CDR’s. The genetic organization of V, regions is similar to that of V, and V,. The germline repertoire of V gene segments is limited (Table 2). Only one V and one J segment are present at mouse TcRy2. y3 and y4 loci and these segments belong to the same subfamily [75,76,78]. However, the complexity of the mouse TcRyl Vsegment family is considerably greater and this is true to an even larger extent of the human family [77,80,81,74,82]. In the latter, four V segments are pseudogenes [go]. These may nonetheless play a functional role, as pseudogenes have been shown to contribute to the somatic generation of diversity at the chicken V, locus (see Chapter 7). No D segment has yet been shown to exist; however, J, segments are similar to J, and may also be possibly equivalent to D-J fusion events. V, region assembly involves limited combinatorial variability in TcRy-positive cells. At the mouse TcRyl locus, the only one which consists of multiple V seg-

218 ments and which is known to encode a functional y chain, equal usage of V segments is observed only in early foetal thymocytes; later in ontogeny as well as in the adult only a single J-distal V segment is found assembled [77.79]. Likewise, there is evidence to suggest that, in adult humans. fewer V, segments are used by TcR y-positive cells than are found in the non-functional rearrangements of TcR ynegative cells [80,65]; the V, pseudogene segments are never assembled [80]. This situation appears to be the reverse of that reported at the IgH locus. where restricted use of J-proximal V segments is found early in ontogeny, whilst the adult repertoire is largely unbiased (see Chapter 4). The restricted combinatorial diversity at TcRy loci might be due to selection for receptor binding specificities: however. it may also result from V segment replacements following non-productive assemblies. Indeed. in contrast to V, and V,, but like V, segments, a sequence corresponding to the putative replacement signal is absolutely conserved in all known V, segments, both in mouse and man [41]. Whilst the two human TcRy loci share V segments (analogously to the IgH and TcRp loci) the three mouse TcRy loci do not. Although mouse y2 and y4 V segments are closely linked [ 7 5 ] ,no instance of 'scrambling' has yet been reported. Thus, in the mouse. the assembly and transcription o f one y locus does not preclude the assembly and transcription of another y isotype. Given the close proximity and opposite orientation, the mouse TcRy2 and y4 genes might well share control elements. Somatic diversification of V, regions does result from junctional variability ( N segments and flexibility in recombination breakpoints), but. similarly to V, and V,, is never observed after assembly. Thus, variability in y chains is likely to be topographically limited to the third CDR loop within a substantially conserved framework.

3.3. Relationship between the expression of the alp rind of the ylS T cell receptors The TcRy-positive cells have often assembled a functional V, region gene on only one chromosome and as a rule contain partially assembled V, regions (D joined to J ) with the a loci in germ-line configuration, thus being aip TcR-negative [64,65,83,84]. Conversely, alp TcR-positive cells contain assembled, mostly nonfunctional V, region genes on both chromosomes and are y TcR-negative [85,86,87,78]. In ontogeny and during intrathymic differentiation, V region assembly begins approximately synchronously at the TcRy and p loci and precedes V,J, joining [83,84]. A developmental program has been proposed (Fig. 7) [72], which resembles that postulated for immunoglobulin loci (see Chapter 4). According to this view, expression of y/S TcR polypeptides prevents assembly and expression at TcRa and p loci and thus identifies a separate lineage of T cells. This model is supported by the finding that, contrary to the alp TcR, the ylS TcR pathway is neither MHC-restricted nor thymus-dependent. and may be functionally more relevant at early stages in ontogeny.

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4. The major histocompatibility complex The members of the immunoglobulin superfamily that are encoded in the major histocompatibility complex (MHC) (Fig. 8) constitute a large family which, on the basis of functional criteria. can be divided into two major sets [SX-911. The first consists of antigen-presenting molecules and includes the classical transplantation antigens (class I, see below) and immune response factors (class 11). The function of the second is presumably in differentiation, since these molecules (which in the mouse include the Qa and TL antigens [92]) are expressed on specific cell types; molecules analogous to this second set V (CD1) are also encoded by a small family .--._--

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220 of MHC-related genes. which map to a different chromosome [93]. Both sets consist of cell-surface molecules. Alongside the genes coding for the identified protein products, a much larger number of genes is found with homologous coding potential. some of which a r e clearly pseudogenes. Here I shall concentrate exclusively on t h e first set, since little is known about the function of the second. Antigen presentation can be viewed as an adaptation of a n older cell-cell recognition function into two separate systems which identify distinct classes of M H C molecules. T h e first system has evolved to monitor a n d ensure the integrity of cell surface structures and consists o f M H C molecules expressed on most cell types and mainly recognized by cytotoxic T cells (class 1 molecules). T h e second system has evolved to mediate cell-cell interactions in the immune response and consists of M H C molecules mostly expressed on macrophages and B cells and mainly recognized by regulatory T cells (class 11 molecules). T h e functional diversification between class 1 and class 11 molecules is reflected in substantial structural differences (see below). T h e antigen-presenting function correlates with a high degree of diversity, resulting from two simultaneous requirements [46]: ( a ) the ability to bind to foreign antigens; (b) the ability to bind to TcR’s. A s a rule. both requirements must be met by the same M H C molecule in order to trigger a T cell response. However. since the effective trigger must be a trimolecular complex of M H C molecule, T c R and foreign antigen, any pairwise interaction between individual components is generally much weaker than that between classical receptor-ligand pairs (e.g. antigen-antibody. hormone-hormone receptor). Each class of M H C antigen-presenting molecule consists of multiple isotypes. Thus. three class I isotypes ( H L A A , -B and -C) and at least three class 11 isotypes ( H L A - D P , -DQ and - DR) are found in man. Each isotype occurs in ;I large number o f diverse forms. T h e diversity of M H C antigen-presenting molccules differs from that of immunoglobulins in two major respects. First, it cxists primarily within the species (polymorphism) and is much more limited within each individual. This polymorphism is unique in ( a ) the large number of alleles at each locus (more than 40 serologically detectable variants are presently known at the H L A - B locus a n d primary sequences show even greater heterogeneity). ( b ) the comparable abundance of each allele and (c) the extent of primary scquence divergence between alleles (this can be more than 8% in amino acid differences at the H L A - B locus). Clearly, a large, balanced polymorphism maximizes heterozygosity [94]and implies that limited M H C diversity confers a selective advantage. However, it is equally clear that the M H C , unlike the immunoglobulin and T c R systems, has not evolved to provide maximum diversity within o n e individual. Thus, within the individual, some M H C diversity is likely to be of selective advantage, but a large diversity may be detrimental. Indeed it may be suggested that the extent of M H C diversity has conflicting effects on the antigen-presenting potential and the T cell repertoire within each individual, since, whilst expanding the former, it reduces the latter as a consequence of the deletion of T cell clones that a r e reactive to self M H C [95]. A second important difference between M H C antigen-presenting molecules and immunoglobulins is in the distribution of the repertoire of diversity within each in-

22 1 dividual. Whilst immunoglobulin and TcR diversity is distributed amongst a set of cell clones, M H C diversity (within each M H C class) is entirely represented in each cell. These patterns of distribution reflect the requirement of an interaction between a target (the M H C molecule) and an effector (the TcR) for the immune function. Clearly, for the cellular immune system to be effective, the target must be present on all cells whilst the eftector must be specific. These requirements also impose different constraints on the amount of diversity which can be achieved by the immunoglobulin and the MHC systems within the individual. 4.1. Overall structure

Although little is known about the three-dimensional structure of M H C molecules, on the basis of primary sequence data as well as of preliminary crystallographic evidence [96] it can be assumed that their general structural plan is not very different from that of TcR’s. Thus, they are composed of two different chains, a heavy ( a ) and a light ( p ) chain, and consist of a membrane-distal domain, to which variability is essentially restricted, a membrane-proximal domain, which is essentially invariant, a transmembrane and an intracytoplasmic tail. Both extracellular domains result from the pairing of two units (Fig. 2); however, in class 1 both V domain units (a1 and a2) are contributed by the same chain ( a ) ,whilst the C domain is contributed by one a chain unit ( a 3 ) and by &-microglobulin (which is not M H C encoded). In class 11, both domains result from one a chain and one p chain unit (the V domain from a1 and p l and the C domain from a2 and p2). Furthermore in class I molecules, but not in class I1 or T cell receptor molecules, the transmembrane and intracytoplasmic segments are not dimeric. 4.2. Structure of the variable regiori

This region consists of the a1 and a2 units in class I and of the a1 and p l units in class I1 molecules. All these units have lengths very close to those of classical immunoglobulin units, but none of their canonical features (see Section 2 in this chapter). Thus, it is likely that both the folding and the packing of the two units of each domain are drastically different from those of immunoglobulin V units. Two elements, however, suggest that MHC V domain units share a basically conserved architecture: first, the variability in primary sequence is clustered, allowing the distinction of framework and hypervariable regions [97,98]; second, features of predicted secondary structure are conserved amongst all units [99,100]. Thus, in class 1 a1 and a2 and class I1 P l units the amino-terminal half would consist of three P-strands in antiparallel orientation, whilst another (possibly two) P-strand would span the carboxy-terminal stretch. The structure of the middle section is less easy to predict, but it is likely that it folds at least partly into an a-helical conformation (Fig. 9). Both class I a2 and class I1 p l domains are likely to have an internal disulphide bond. It has been proposed [loo] that these secondary structure elements fold into an ‘open sandwich’ type of tertiary structure [ l o l l that consists of a single P-sheet covered on one of its sides by a layer of helices and loops; how-

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