Immunobiology
PRINCIPLES OF MEDICAL BIOLOGY A Multi-Volume Work, Volume 6 Editors: E. EDWARD BITTAR, Department of Physiology, University of Wisconsin, Madison NEVILLE BITTAR, Department of Medicine, University of Wisconsin, Madison
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Principles of IVIe
A Multi-Volume Work Edited by E, Edward Bittar, Department of Physiology, University of Wisconsin, Madison and Neville Bittar, Department of Medicine University of Wisconsin, Madison This work provides: * A holistic treatment of the main medical disciplines. The basic sciences including most of the achievements in cell and molecular biology have been blended with pathology and clinical medicine. Thus, a special feature is that departmental barriers have been overcome. * The subject matter covered in preclinical and clinical courses has been reduced by almost one-third without sacrificing any of the essentials of a sound medical education. This information base thus represents an integrated core curriculum. * The movement towards reform in medical teaching calls for the adoption of an integrated core curriculum involving small-group teaching and the recognition of the student as an active learner. * There are increasing indications that the traditional education system in which the teacher plays the role of expert and the student that of a passive learner is undergoing reform in many medical schools. The trend can only grow. * Medical biology as the new profession has the power to simplify the problem of reductionism. * Over 700 internationally acclaimed medical scientists, pathologists, clinical investigators, clinicians and bioethicists are participants in this undertaking.
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Immunobiology Edited by E. EDWARD BITTAR Department of Physiology University of Wisconsin Madison, Wisconsin NEVILLE BITTAR Department of Medicine University of Wisconsin Madison, Wisconsin
( ^ Greenwich, Connecticut
jAI PRESS INC. London, England
Library of Congress Cataloging-in-Publication Data Immunobiology / edited by E. Edward Bittar, Neville Bittar. p. cm.—(Principles of medical biology ; v. 6) Includes index. ISBN 1-55938-811-0 1. Immunology. 2. Molecular immunology. I. Bittar, E. Edward. II. Bittar, Neville. III. Series. [DNLM: 1. Immune System. 2. Immunity. QW 50413223 1996] QR181.I454 1996 616.07'9—dc20 DNLM/DLC 96-35160 for Library of Congress CIP
Copyright © 1996 by JAI PRESS INC. 55 Old Post Road, No. 2 Greenwich, Connecticut 06836 JAI PRESS LTD. 38 Tavistock Street Covent Garden London, England All rights reserved. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, filming, recording, or otherwise, without prior permission in writing from the publisher. ISBN: 1-55938-811-0 Library of Congress Catalog No.: 96-35160 Manufactured in the United States of America
CONTENTS
List of Contributors
ix
Preface £ Edward Bittar and Neville Bittar Chapter 1 The Thymus in Immunity J.FA.P. Miller
xii i
1
Chapter 2 The B-Cell in Immunity David Tarlinton
21
Chapter 3 Cell-to-Cell Interactions in the Immune System William A. Sewell and Ronald Penny
47
Chapter 4 Immunological Tolerance J.FA.P. Miller
63
Chapter 5 The Generation of Diversity in the Immune System E.J. Steele and H.S. Rothenfluh Chapter 6 The Antigen-Antibody Complex: Structure and Recognition P.M. Colman vii
85
107
viii
CONTENTS
Chapter 7 The Major Histocompatibility Complex Brian D.Tait
121
Chapter 8 B and T Cell Signaling at the Molecular Level Tomas Mustelin and Paul Bum
137
Chapter 9 Cytokines in Immunology Andrew J. Hapel and Shaun R. McColl
151
Chapter 10 Activation and Control of the Complement System B.Paul Morgan Chapter 11 Phagocytes in Immunity and Inflammation Philip ISA. Murphy
171
197
Chapter 12 Anaphylaxis Caiman Prussin and Michael Kaliner
231
Chapter 13 Autoimmunity and Autoimmune Disease Sudershan K. Bhatia and Noel R. Rose
239
Chapter 14 Cell Death and the Immune System R.M. Kluck and].W. Halliday
265
Chapter 15 Designer Antibodies Andy Minn and Jose Quintans
281
Chapter 16 Psychoneuroimmunology Ruth M. Benca
303
INDEX
315
LIST OF CONTRIBUTORS Ruth M, Benca
Department of Psychiatry University of Wisconsin Madison, Wisconsin
Sudershan K. Bhatia
Department of Immunology and Infectious Diseases The John Hopkins University School of Hygiene and Public Health Baltimore, Maryland
Paul Bum
Department of Biology Hoffmann-La Roche Inc. Nutley, Ne Jersey
P.M. Colman
CSIRO Division of Biomolecular Engineering Parkville, Victoria, Australia
J.W. Halliday
Liver Unit Queensland Institute for Medical Research Queensland, Australia
Andrew J. Hapel
Experimental Haematology Group John Curtin School of Medical Research Australian National University Canberra, Australian Capital Territory, Australia
Michael
Institute for Asthma and Allergy Washington, D.C.
Kaliner
R.M. Kluck
Liver Unit Queensland Institute for Medical Research Queensland, Australia IX
LIST OF CONTRIBUTORS J.FA,P. Miller
The Walter and Eliza Hall Institute of Medical Research Royal Melbourne Hospital Melbourne, Victoria, Australia
Andy Minn
Department of Pathology The University of Chicago Chicago, Illinois
B. Paul Morgan
Department of Medical Biochemistry University of Wales College of Medicine Heath Park, Cardiff, Wales
Philip M. Murphy
The Laboratory of Host Defenses National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland
Tomas Mustelin
La Jolla Institute for Allergy and Immunology La Jolla, California
Ronald Penny
Centre for Immunology St. Vincent's Hospital and University of New South Wales Sydney, New South Wales, Australia
Caiman Prussin
National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland
Jose Quintans
Department of Pathology The University of Chicago Chicago, Illinois
Noel R. Rose
Department of Immunology and Infectious Diseases The Johns Hopkins University School of Hygiene and Public Health Baltimore, Maryland
List of Contributors
XI
H.S. Rothenfluh
Division of Immunology and Cell Biology The John Curtin School of Medical Research Australian National University Canberra, Australia
William A. Sewell
Centre for Immunology St. Vincent's Hospital and University of New South Wales Sydney, New South Wales, Australia
E.I. Steele
Department of Biological Sciences University of Wollongong Wollongong, New South Wales, Australia
David
The Walter and Eliza Hall Institute of Medial Research Royal Melbourne Hospital Melbourne, Victoria, Australia
Tarlinton
Brian D. Tail
Tissue Typing Laboratories Royal Melbourne Hospital Parkville, Melbourne, Australia
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PREFACE
As this volume demonstrates, immunobiology is a young science which is undergoing explosive growth. Judged by results, it is already an elaborate discipline which cuts across every other area in biomedical research and even has its own vocabulary (e.g., the “veto” effect). Rather than inculcate the habit of superficial learning by having the student go through a maze of details, we have sought to gather together sixteen essays that range from T-cells to psychoneuroimmunology. This is in keeping with the growing understanding that the student is expected to read and think far more for herselfhimself. Next to nothing is known about innate immunity. However, recent evidence suggests that collectins might bridge the gap between innate immunity and specific clonal immune responses. Collectins are soluble effector proteins that include serum mannose-binding protein, and lung surfactants A and D. They are considered to be ante-antibodies. Our most grateful thanks are due to the contributors who have made this volume possible. They are also due to Ms. Lauren Manjoney and the production staff of JAI Press for their skill and courtesy.
E. EDWARD BITTAR NEVILLE BITTAR
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Chapter 1
The Thymus in Immunity J.F.A.P. MILLER
Introduction Historical Background Antigen Recognition and the Major Histocompatibility Complex (MHC) Peripheral T Cell Subsets T Cell Migration Recirculation of Naive T Cells Tissue-Selective Homing of Activated and Memory T Cells Intrathymic Events The Thymus in Disease States Summary Recommended Readings
Principles of Medical Biology, Volume 6 Immunobiology, pages 1-20. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-811-0
2 4 7 10 12 13 14 15 17 18 19
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J.F.A.P. MILLER
INTRODUCTION The cells in the immune system responsible for specifically targeting and causing the removal of foreign material or antigen are known as lymphocytes. They circulate in blood and lymph and populate areas of the body known as lymphoid tissues which include the spleen, lymph nodes, thymus, tonsils, adenoids, and Peyer's patches, the last three being located along the alimentary tract. The thymus in mammals is situated in the upper part of the thoracic cavity where it overlies the heart and some of the major blood vessels (Figure 1). It is unique among lymphoid tissues, both as regards structure and function. Relatively large in the infant, its maximum size is reached at the time of puberty, after which it regresses slowly, becoming reduced to little more than a vestigial structure in old age. It is divided into lobules each with a central part or medulla and a peripheral part or cortex (Figure 2). The main types of cells are the lymphocytes and the so-called stromal cells which include the cells of the epithelial framework and of the dendritic-macrophage lineages. T cell precursors (derived from fetal liver or later from bone marrow) enter from vessels at the cortico-meduUary junction and first associate with macrophages. Two or three days later they are found m the subcapsular cortex. They eventually give rise to more differentiated thymus lymphocytes. The cell composition of the thymus may be divided into three distinct layers. (1) In the outer cortex, beneath the capsule, is a layer of dividing primitive lymphocytes (lymphoblasts), which constitute 5 to 15% of the total thymic lymphocyte population. Some lymphoblasts interact with specialized epithelial cells, the "nurse cells," which promote their proliferation and differentiation to more mature smaller forms. (2) The newly derived lymphocytes migrate from the cortex towards the medulla. In the deep cortex are three major classes of cells: small lymphocytes, dendritic cortical epithelial cells and macrophages. The lymphocytes have a thin rim of cytoplasm, make up about 80 to 85% of the thymic lymphocyte population and are
Thyroid
Figure 1. The location of the thymus in the chest.
The Thymus in Immunity capsule subcapsular blasts - c O .
nurse cells small cortical thymus lymphocytes
cortical dendritic epithelial cell
medullary epithelial cells
Q
MEDULLA
O
interdigitating dendritic cells
medullary thymus lymphocytes
Figure 2. Structure of the thymus. Diagram to show cellular architecture of the thymus (see text).
in intimate contact with the dendritic epithelial cells. These have long processes and are connected to one another by junctions known as desmosomes. They may be involved in selecting the T cell repertoire (see later and Figure 11). Interspersed among the network of dendritic epithelial cells are macrophages which engulf the many lymphocytes that have died or are destined to die. On the medullary side of the cortico-meduUary junction lie structures called Hassall's corpuscles which constitute the final graveyards for the massive numbers of dying lymphocytes. (3) The medulla contains medium sized thymic lymphocytes, macrophages, spatulate medullary epithelial cells and bone-marrow derived interdigitating dendritic cells. The latter are most conspicuous near the cortico-meduUary junction and are involved in negative selection of those lymphocytes which have the potential to inflict damage on the body's own tissues, the so-called self-reactive lymphocytes (see below). Some medullary mature T cells may be derived partly from the intrathymic maturation process and partly from extrathymic circulating T cells. The proportion of lymphocytes undergoing cell division (mitosis) is much higher in the thymus than in any other lymphoid tissues throughout the life of the individual. Furthermore, thymus lymphocyte mitotic activity, unlike similar activity elsewhere, is not dependent on antigenic stimulation but is preprogrammed and hence controlled intrinsically (from within the thymus). During development, the thymus, unlike other lymphoid tissues, is a purely epithelial organ. Lymphocytes first appear in the epithelial network at about 10
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J.F.A.P. MILLER
weeks of gestation in the human and 12 days in the mouse. They are derived by differentiation of hemopoietic ancestral or stem cells which enter from the blood stream. It is only much later that lymphocytes make their appearance in other lymphoid organs. The thymus is thus often referred to as a primary or central lymphoid organ and the other lymphoid tissues as secondary or peripheral. When animals are immunized by antigen, characteristic cellular changes occur in lymph nodes and spleen. For example, small lymphocytes enlarge to larger "blast" cells which stain with a particular RNA-staining dye (methyl green pyronin). These undergo mitosis and antibody forming "plasma cells" accumulate in certain areas. None of these antigen-induced changes have ever been found in the intact thymus of immunized animals under normal conditions. These findings raised questions as to whether the thymus played any role in immunity.
HISTORICAL BACKGROUND Prior to I960, the functions of the thymus and its lymphocytes were obscure. By contrast, the circulating small lymphocytes, as found in blood, lymph and lymphoid tissues, were proven to be immunologically competent by the work that Gowans and his collaborators performed in the late fifties and early sixties (Gowans, 1961). Yet although the thymus was known to be a lymphocyte-producing organ, immunologists did not consider it to have any immunological function. This may have been because some investigators, for example. Good and his collaborators (MacLean et al., 1957), concluded from experiments, in which the thymus was removed from adult rabbits, that they had obtained "evidence that the thymus gland does not participate in the control of the immune response." In the early sixties, Medawar (1963) even suggested that "we shall come to regard the presence of lymphocytes in the thymus as an evolutionary accident of no very great significance." What then was responsible for reversing the tide? In the late fifties and early sixties, Miller, then working with a leukemogenic virus of mice, surgically removed the thymus (thymectomized) of newborn (neonatal) mice to determine whether the virus, when introduced at birth, had first to multiply in thymus tissue. He found that neonatally thymectomized mice died prematurely from causes unrelated to leukemia induction and suggested "that the thymus at birth may be essential to life" (Miller, 1961a). Further experiments showed clearly that mice thymectomized at one day of age, but not later, were highly susceptible to infections, had a marked deficiency of lymphocytes in the circulation and in lymphoid tissues and were unable to reject skin grafts taken from incompatible mice of other strains (Miller, 196 lb). These results led to the hypothesis that "during embryogenesis the thymus would produce the originators of immunologically competent cells many of which would have migrated to other sites at about the time of birth. This would suggest that lymphocytes leaving the thymus are specially selected cells" (Miller, 1961b). In adult mice, thymectomy had for long been known not to have any untoward effects. Miller (1962a), however.
The Thymus in Immunity exposed adult thymectomized mice to total body irradiation which partially destroyed the lymphoid system and was able to show that the recovery of lymphoid and immune functions was thymus-dependent. Implanting thymus tissue into neonatally thymectomized or adult thymectomized and irradiated mice allowed a normal immune system to develop. When the thymus graft came from a foreign strain, the neonatally thymectomized recipients failed to reject skin from mice of the strain that had donated the thymus, although they could reject skin graft from other incompatible strains. This led to the suggestion that "when one is inducing a state of immunological tolerance in a newly born animal," for example by the classical technique of injecting foreign bone marrow cells at birth (see Chapter 4), "one is in effect performing a selective or immunological thymectomy" (Miller, 1962b). Thus, lymphocytes developing in the thymus in the presence of foreign cells would be deleted, implying that the thymus should be the seat where tolerance to the body's own tissues (self tolerance) is imposed. Some of these findings were soon confirmed by groups working independently, notably those headed by Waksman and by Good (Amason et al., 1962; Martinez et al., 1962). In the late fifties and early sixties, only a single variety of lymphocyte was believed to be involved in performing all types of immune responses in mammalian species. In birds, however, it seemed that two distinct subsets of lymphocytes performed those immune responses mediated by antibody (the "humoral" immune responses) and those in which cells, but not antibody, were involved (the "cellmediated" immune responses). The latter include transplant rejection, delayedhypersensitivity reactions such as tuberculin sensitivity, and killing or "lysis" of target cells. The finding of a division of labor among avian lymphocytes was first reported by Szenberg and Warner (1962) using newly hatched chicks: surgical removal of the bursa (an organ found only in birds and analogous to the thymus but situated near the cloaca) soon after hatching was associated with defects in antibody formation and early thymectomy with defects in cellular immune responses. Since mice do not have a bursa and since neonatal thymectomy in that species prevented both cellular and most humoral immune responses, it was widely believed that the mammalian thymus fulfilled the fimctions of both the avian thymus and bursa. A hint that two distinct lymphocyte subsets may indeed be involved in immune responses in mice, however, came from the experiments of Claman and his colleagues in 1966. They showed that irradiated mice receiving a mixed population of marrow and thymus cells produced far more antibody than when given either cell source alone. Having no genetic markers on their cells, they could not, however, determine whether the antibody-forming cells were derived from the thymus or the marrow. In independent investigations, (Miller and Mitchell, 1967,1968; Mitchell and Miller, 1968) introduced genetically marked cells into neonatally thymectomized or thymectomized irradiated hosts and established beyond doubt and for the first time that antibody-forming cell precursors (subsequently known as B cells)
5
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J.F.A.P. MILLER
were derived from bone marrow, and that thymus-derived cells (now called T cells) were essential to help B cells to respond to antigen by producing antibody. The existence of two distinct lymphocyte subsets, T and B cells, was not only confirmed but led to a re-investigation of numerous immunological phenomena including memory, tolerance, autoimmunity, and genetically determined unresponsive states. T cells were clearly responsible for the "cell-mediated" immunities, and T cells were themselves soon subdivided into subsets based on function, cell surface markers and secreted products or "lymphokines." In 1957, prior to the discovery of T and B cells, Burnet postulated that lymphocytes had predetermined reactivities. A cell with a receptor that best fitted a given antigenic determinant is selected by that antigen and activated to divide producing a clone of daughter cells, all with the same specificity (Figure 3). The antigen receptor on the membrane of these progeny cells would be identical in its binding site to the antibody eventually secreted by members of the clone. The theory has stood the test of time and for B cells, it was clear that the antigen recognition unit or receptor was an accurate sample of the antibody or immunoglobulin (Ig) which that cell would produce after successfiil antigenic stimulation. It was also found that a small proportion of naive B lymphocytes could specifically bind labeled antigen and that this binding could be blocked by antibody directed against the immunoglobulin receptor itself Yet T cells could never be shown to bind antigen Clonal Selection lymphocytes
^^R
antigen
^A
^^
lymphocyte-antigen interaction lymphocyte proliferation and differentiation clone
antibody
k 1 i 'A^ '
Jli\
Figure 3. Burnet's clonal selection theory. The antigen-specific receptor is unique on mature lymphocytes. A cell with a receptor into which a given antigenic determinant can be accommodated is selected by the antigen to divide and produce a clone of daughter cells, each with the same antigen specificity. In the case of B cells, as shown in this diagram, the membrane receptor is identical in its binding site to the antibody which members of the stimulated clone will eventually secrete.
The Thymus in Immunity
7
and great controversy raged for many years over the nature of the antigen receptor on T cells.
ANTIGEN RECOGNITION AND THE MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) Unlike B cells, T cells perceive, not naked antigen, but antigen presented on the surface of other cells. Highly visible to T cells are molecules encoded by the major histocompatibility complex (MHC), a series of genes which code for molecules on the surface of a variety of cell types (see Chapter 7). They provoke violent rejection reactions on the part of responding T cells and are perfect targets for killer or cytotoxic T cells. Following virus infection and virus entry into cells, as first shown by Zinkemagel and Doherty in 1974, T cells recognize not just the virus derived antigenic determinants, but these in association with MHC-encoded molecules on CLASS peptide groove
CLASS II peptide groove
^2^
Figure 4, Class I and class II MHC molecules. The class I molecule is composed of two polypeptide chains. The heavy chain has 3 external domains, a l , a2 and a3, a transmembrane portion (TM) and a cytoplasmic tail (CY). It is associated in its extracellular portion with the light chain, p2-microglobulin (p2m), a molecule not encoded by the MHC gene locus. The polymorphic regions of the class 1 heavy chain are those where the amino acid sequences of the polypeptide chain differ among unrelated individuals. They are situated in the a1 and a2 domains which form a groove that can accommodate peptide fragments derived from the processing of proteins synthesized within the cell (e.g., self proteins or virus-derived proteins in virus infected cells). The class II molecules are composed of two polypeptide chains, a and p, both encoded by the MHC gene locus. Each chain spans the membrane and hence has a transmembrane region, a cytoplasmic tail and an extracellular portion. Both the a and the p chains have two external domains, a1, a2, and p i , p2. The polymorphic regions lie in the a1 and pi portions which also form a groove into which can be accommodated peptides generally derived from proteins taken up by the cell from the external milieu (see also Figure 5). A separate class of antigens known as "superantigens'' (e.g., certain bacterial toxins) bind not to the groove of the class II molecules but to the external face of the domains and to the p chain of the TCR.
J.F.A.P. MILLER
8
the cell surface. This phenomenon became known as MHC restriction and the MHC molecules involved as restriction elements. The MHC molecules that serve as targets of T cell responses occur in two major forms, termed class I and II (Figure 4). The former are found on most tissue cells and are composed of two noncovalently linked polypeptide chains—SL heavy one (molecular weight 45 kD) spanning the cell membrane and having three extracellular portions or domains ( a l , a2 and a3), and a lighter chain termed p2-microglobulin. This does not span the membrane and is encoded by a gene distinct from the MHC genes. The class II molecules consist of two noncovalently linked a (28 kD) and P (34 kD) chains, both encoded by the MHC and both having two extracellular domains. The distribution of class II molecules is restricted mostly to B cells, macrophages, and dendritic cells. Both class I and II molecules exhibit a striking degree of structural variation or polymorphism within individuals of the same species. The polymorphic regions of the molecules, where there are differEndogenous pathway
r
peptides O
degradation
class I
I
proteins
\ \
endogenous synthesis
Exogenous pathway
JL
r t
[o]*-(cO**—O exogenous antigen endocytosis
class 11
Figure 5. Antigen-presenting cell (APC) and processing pathways. Professional ARC present processed antigen in association with MHC class I and ii molecules. Two pathways of antigen processing operate: they are referred to as endogenous and exogenous. (1) Some proteins synthesized by the APC are chopped into fragments (degraded into peptides) by cellular enzymes. Most newly synthesized class I molecules are unstable unless peptide is associated with them. The binding of peptides to M H C class I molecules occurs in an intracellular compartment known as the endoplasmic reticulum and the peptide-MHC complex can then be transported to the surface. This particular route is known as the endogenous pathway. (2) External antigens taken up by the APC ("endocytosis") are degraded In compartments known as endocytic vesicles which fuse with other vesicles that contain class ii but not class I molecules. This type of transport is referred to as the exogenous pathway.
The Thymus in Immunity
9
ences in amino acid sequences among unrelated individuals, are situated in the a l and a2 domains of the class I molecules and the a l and pi domains of the class II molecules. These domains form a groove or pocket capable of binding fragments derived from the enzymatic degradation or processing of self or foreign components (Bjorkman et al., 1987; Brown et al., 1993). Such fragments derived from protein antigens are known as peptides and are made up of short sequences of amino acids with a carboxyl end or "terminus" and an amino terminus. Cells which perform the processing task and transport the peptide-MHC complex to their cell membrane where T lymphocytes can examine them, are termed professional antigen-presenting cells (APC) (Figure 5). The antigen-specific receptor on T cells (the "TCR") has specificity for both the peptide and the external surface of the MHC molecule which accommodates the peptide (Davis and Bjorkman, 1988). Most TCRs are composed of two disulfidelinked polypeptide chains, a and p (Figure 6), although less common TCR use other chains termed y and 5. Each chain has a constant amino acid sequence in its carboxyl terminus (C) and a variable sequence in its amino terminus (V). Other molecules intimately associated with the TCR are the CD3 complex composed of three polypeptide chains (y, 5, and s) and the so-called q-q "homodimer" composed of a pair of identical polypeptide chains. The CDS and q-q molecules are essential for
TCR
extracellular membrane cytoplasm Figure 6. The antigen-specific T cell receptor (TCR) and associated CD3 and q-q complexes. The antigen-specific TCR is composed of two disulfide-linked polypeptide chains, a and |3. Each chain has a constant amino acid sequence in its carboxyl terminus (C) and a variable sequence in its amino terminus (V). The CD3 complex, composed of three polypeptide chains y, 6, and 8, and the homodimer q-q are intimately associated with the TCR and are involved in TCR assembly and signal transduction when the TCR has bound a peptide-MHC complex.
J.F.A.P. MILLER
10
the assembly and transport to the cell surface of the TCR and play a role in transducing signals after occupation of the TCR by a peptide-MHC complex. The TCR chains are encoded by several genes which rearrange during T cell development and contribute to the great diversity of specificities associated with TCRs (Davis and Bjorkman, 1989). This is described in detail in Chapter 5. Briefly, individuals inherit from their parents sets of "germline genes" which code for the combining site of antigen-specific receptors on both T and B cells. A variety of mechanisms then operate during T and B cell differentiation to rearrange and join together the germline elements and eventually give rise to the active gene which is a mosaic of these units. Hence, an enormous diversity can be generated and a great variety of antigen-specific receptors is made available to ensure that lymphocytes can recognize an infinite number of antigenic determinants.
PERIPHERAL T CELL SUBSETS Although almost all T cells bear the Thy-1 marker, they are heterogeneous with respect to function and other cell surface markers. The two major subsets of T cells are termed CD4'" and CD8"^ T cells (Figure 7). The former are characterized by the presence on the membrane of the CD4 molecule and act as "helper" cells by assistmg B cells in producing certam types of antibody. The collaboration between T and B cells is described in Chapter 3, The CDS"^ T cells have CDS molecules on
Figure 7. The CD4 and CDS co-receptor molecules on T cells. The CD4 and CD8 molecules characterize mature peripheral T cells which recognize peptides m association with MHC class II and class I molecules, respectively. The CD8 molecule has an affinity for specific sites on the a3 domain of the class I molecule and the CD4 for some sequences on the nonpolymorphic portion of the class 11 molecule.
The Thymus in Immunity
11
their surface and, after direct contact with their target cells, act as killer or cytotoxic cells destroying foreign cells and cells infected by viruses. In some situations CDS"^ T cells require help from 004"" T cells for cytotoxic activity. The CD4 and CDS molecules are "coreceptors" as they act m concert with the TCR. The CD4 co-receptor has a binding site specific for a portion of the MHC class II molecule and the CDS co-receptor has one specific for a part of the a3 region of the class I molecule. Co-aggregation of CD4 or CDS molecules with the CD3-(;-c; complex and the TCR, once bound to its specific peptide-MHC complex, initiates a signaling cascade to "activate" the T cell, turning on its functional and lymphokine-secretion machinery (Janeway, 1992). T cell-derived lymphokines control the differentiation of a wide variety of cells of the hemopoietic and lymphoid systems and are active in initiating inflammatory responses such as delayed-type hypersensitivity. CD4'^ T helper (Th) cells are themselves heterogeneous in terms of their lymphokine release pattern. Although the naive CD4 cell can synthesize a variety of lymphokines immediately after activation, the way in which antigen is presented by different cells eventually restricts the secretion pattern. Thus Th cells can be divided into ThO (which can secrete various lymphokines) and into further differentiated forms known as Thl and Th2 cells (Mosmann and Coffman, 1989). Although both these cells can secrete the lymphokines IL-3, GM-CSF and TNF-a, they differ in their pattern of release of other lymphokines. Thus, Thl cells produce interferon-y and interleukin-2 while Th2 cells secrete interleukin-4, 5, 6, 10, and 13. Thl and Th2 can antagonize each other thus playing a role in immunoregulation (see also Chapters 4 and 9). Under certain conditions, T cells can suppress the responsiveness of other lymphocytes. Whether such "suppressor T cells" exist as a distinct subset or reflect the production of inhibitory cytokines, such as TGF-(3 (A. Miller et al., 1992), has yet to be established. T cells can also be subdivided according to their previous antigenic experience. Those which have not met antigen are termed "naive" or "virgin" cells and are characterized by the presence of distinct molecules on their surface. For example, they express the high molecular weight forms of the CD45 molecule (notably CD45RA), low levels of the molecule known as CD44 and high levels of L-selectin (also called MEL-14). Those T cells which have been stimulated by antigen are the progeny of naive T cells and are large "blasts" known as effector or activated T cells. They may become small "memory" cells whose existence may depend on continuous antigenic stimulation. Both activated and memory T cells exhibit on their cell membrane the CD45RO molecules, high levels of CD44, low levels of L-selectin and various adhesion molecules such as LFA-1 and CD2. All these molecules are involved in various T cell functions including intracellular signaling, adhesion to APC or to cells lining blood vessels ("endothelial cells") (Mackay, 1993; Sprent, 1993). Some of the molecular interactions occurring between specific T cells and professional APC are shown in Figure S.
J.F.A.P. MILLER
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APC or target cell
Figure 8. Adhesion and co-stimulatory molecules involved during the interaction of T cells with TCR specific for an MHC-peptide complex presented by a professional APC. Cell surface molecules expressed on T cells may play a role in immune responses by functioning as receptors for cell surface molecules expressed on APC. The interaction of such molecules may strengthen the binding between the T cell and the APC and may be Involved in transmembrane signals initiated by TCR occupancy or independent of the TCR. The B7 molecule is characteristically expressed by professional APCs and its binding to the T cell's CD28 molecule produces a powerful co-stimulator signal to ensure that the T cell becomes fully activated following the binding of its own TCR to the MHC-peptide complex presented by the APC.
As stated above, most T cells utilize TCR a and P genes but a smaller subset use the genes y and 6. Some 76 cells exist in certain epithelial environments and, unlike aP T cells, exhibit a highly restricted TCR specificity. They may thus express invariant TCRs and perform totally different tasks. Much remains to be learned about their functions.
T CELL MIGRATION The total number of cells released from the thymus is small being of the order of 1 to 2 million per day in young mice. Output is maximal at an early age and declines when thymic atrophy sets in, reaching very low levels in old age. The cells leaving the thymus are typical CD4^ and CDS"*" T cells which may have to undergo a further period of maturation during several days. The emigrants migrate non-randomly along well defined routes, the actual pathway depending on whether the T cells are naive or activated.
The Thymus in Immunity
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Recirculation of Naive T Cells
Naive small T cells recirculate from blood through lymphoid tissues and back to blood directly or via the lymph. They have a long lifespan and do not divide unless stimulated by antigen. Recirculation allows naive T cells to patrol the body and home in on sites in lymph nodes and spleen which have trapped antigens and invading micro-organisms. Naive T cells have specialized receptors ("L-selectin") which allow them to bind to distinct molecules on the surface of endothelial cells lining specialized venules in the lymph nodes known as "post-capillary" or "high endothelial" venules (HEV) (Figure 9). They then enter lymph nodes through a region known as the paracortex which contains a network of specialized APCs including the so-called dendritic cells. This area of the node is known as the T-cell dependent area and antigens which provoke cellular immunity produce histological changes in this area, the small T cells enlarging to large blasts which divide. Other areas of the lymph nodes, including the follicles are known as the B-cell dependent areas (see Chapter 2). After traversing the paracortex, the recirculating T cells enter the medulla of the nodes and leave by efferent lymphatics which drain into other lymph nodes or end up in the thoracic duct. This large vessel empties into a major blood vessel in the neck. The spleen is divided into red and white pulps (Figure 10), the former containing many hemopoietic cells such as red blood cells, the latter being populated by lymphocytes. The spleen does not have significant lymphatics and T cells enter via the splenic artery which terminates in a loose network of vessels in the red pulp. The T cells migrate to the area of the white pulp around the arterioles (the "periarteriolar lymphocyte sheath" or PALS), a T-cell dependent area rich in APCs
afferent lymphatic subcapsular sinus primary follicle (B area)
cortex
medulla
medullary cord (B area) medullary sinus efferent lymphatic Figure 9, Microanatomy of a lymph node (see text).
I.F.A.P. MILLER
14
red pulp
marginal sinus central arteriole
t
primary
follicle
(B area) marginal zone (B area)
Figure 10. Microanatomy of a section of the spleen (see text).
including dendritic cells, and the B cells migrate to the follicles in the white pulp. T cells leave the spleen by going to the red pulp and entering tributaries of the splenic vein. If recirculating T cells encounter antigen presented by APCs in the T-cell dependent areas of lymph nodes or spleen, those T cells with TCR specific for the antigen are sequestered from the circulating pool and activated to proliferate and to produce effector cells which perform the cell-mediated immune responses (Sprent et al., 1971). Tissue-Selective Homing of Activated and Memory T Cells
T cells homing to the alimentary canal ("gut-tropic cells") are either activated blasts or smaller CD45RO"^ memory-type cells. After antigen activation, T cells downregulate the expression of L-selectin which was present on naive T cells and instead express the a4(37 adhesion molecule ("integrin"). This allows them to bind to specific molecules on endothelial cells found in gut mucosa and gut-associated lymphoid tissues and thus to enter such tissues. The skin represents a major entry point for microorganisms. T cells that home to the skin are almost exclusively of the memory type and express the a4pi integrin. This serves as an adhesion receptor for the molecule E-selectin which is found in inflamed skin.
The Thymus in Immunity
15
Memory-type T cells predominantly localize to inflamed sites. The inflammatory response can also affect lymph nodes and antigen stimulation induces the expression of defined vascular adhesion molecules on the endothelial cells of HEV. This in turn allows a marked increase in the migration of memory-type T cells to the node. The different migration behavior of naive and effector or memory T cells ensures that the immune system provides a most economical way of displaying its resources to fight against dangerous intruders.
INTRATHYMIC EVENTS Most T cells arise in the thymus as a result of the programmed differentiation of incoming stem cells. There is little evidence for extra-thymic production of T cells. Since T cells are specialized to recognize antigenic determinants in association with self MHC molecules, the thymus must provide a repertoire of T cells by selecting those cells with TCR that have some degree of specificity for these MHC molecules. But because TCR specificities are randomly generated and there is extensive MHC polymorphism among individuals of a particular species, the specificities of T cells in the preselected pool of differentiating thymus lymphocytes must by chance be directed to all the MHC molecules expressed in the species. Most thymocytes will therefore lack the correct specificity and hence will be unsuitable. This presumably accounts for the vast numbers of thymocytes generated each day (about 10^ in mice), the massive rate of cell death and the small number exported to the periphery (about 10^ per day). The earliest T cell precursor derived from stem cells entering the thymus is characterized by the surface expression of the molecule CD44 and very low levels of CD4. Soon after, these early cells lose the CD4 marker and transiently express, in addition to CD44, the CD25 molecule (which is a receptor for the interleukin, IL-2). At this stage, the cells are referred to as "double negative (DN) cells" because they lack expression of both CD4 and CDS which characterize mature T cells. The DN cells then lose CD44 expression and begin to rearrange and express the (3 chain genes of the TCR. This is followed by rearrangement of the TCR a locus, expression of low levels of the aP TCR on the surface and loss of CD25. A separate subset of DN cells rearrange the y and 8 locus of the TCR to express a y8 TCR. The early thymocytes account for less than 3% of the cells in a mouse thymus. They proliferate extensively in the subcapsular cortical zone presumably as a result of interacting with cortical epithelial cells which produce a number of factors or "cytokines" influencing cell proliferation. The DN cells then migrate to the deeper cortex. A few can go out to the periphery without expressing the CD4 or CDS coreceptor molecules, but most give rise to cells that express both CD4 and CDS (termed double positive cells) and low to intermediate levels of the TCR. They are now subjected to stringent selection tests (Figure 11).
16
J.F.A.P. MILLER
Random expression of ap TCR genes
Positive selection by MHC molecules Negative selection towa/S TCR expression
high a/S TCR expression
cortical
low TCR a/?
macr^hages I medullary epithefium
^
(7)
JO
m 30
Pre-B
Virgin B
O © Q DtoJH
VHtoDJH VLtoJL
igM
Mature B
0
1 ^ IgM IgD
Activated B
Q„
Memory B
0 IgGor IgA
Plasma cell
{
(§5 IgM, IgQ or IgA secretion |
DR CD10 CD19 CD20 CD23 CD38 CD40 B7
Figure 4. The cell surface proteins expressed by B cells change with development. As a B cell matures it loses expression of some markers, such as CD10, and gains others, such as CD23. These changes define developmental stages and often correlate with changes occurring at the immunoglobulin gene locus with respect to gene rearrangement or isotype switching. Plasma cells, which specialize in antibody secretion, remove practically all cell surface proteins including the B lineage specific markers. They are however easily identifiable by their shape and the presence of immunoglobulin in the cytoplasm. A similar scheme can be drawn for mouse development with some minor differences.
28
DAVID TARLINTON
positive signal is delivered to a pre-B cell through the surface expression of the Ig heavy chain in association with two proteins called collectively the pseudo light chain complex, and individually VpreB and 0:^5. Only those pre-B cells with a productive IgH gene rearrangement can express such a molecule and thus receive the growth stimulus necessary to remain viable and to initiate light chain gene rearrangement. Whatever the mechanism, cells with two nonproductive IgH gene rearrangements are efficiently removed from the B cell population since they are not seen beyond the pro-B cell stage and they do not appear to accumulate in the pre-B compartment. Immature B cells represent the first time that IgM is expressed on the cell surface. This is a delicate time in B cell development since at this point any autoreactivity in the B cell repertoire will be exposed. The way in which the immune system deals with self-reactive cells is the study of tolerance and is discussed in greater detail in Chapter 4. While it is difficult (if not impossible) to follow the fate of self-reactive B cells which develop in a normal individual because of the heterogeneity of the population, a number of animal models have shed light on the fate of such cells. In these models, mice have been made transgenic for the genes necessary to produce an immunoglobulin molecule of a known specificity. That is, all the B cells developing in these mice will express identical immunoglobulin molecules with identical specificity. These Ig-transgenic mice can then be crossed with mice which express the antigen recognized by the transgene derived Ig. In such a way, every B cell which develops in the doubly positive progeny will be autoreactive, and the fate of individual B cells is now reflected in the fate of the population. Furthermore, the nature or context of the autoantigen can be altered. In two well characterized examples such pseudo-self antigens have been expressed as integral membrane proteins in either the bone marrow (Hartley et al., 1991) or the periphery, specifically the liver (Russell et al., 1991). In both cases self-reactive B cell were killed either as soon as they were formed in the bone marrow or upon entry into the periphery. Finally, the effect of the self-antigen being soluble (i.e., a serum component) has been examined (Goodnow et al., 1988). In this case, the autoreactive B cells were not deleted but rather rendered incapable of responding to the antigen. This state is referred to as B cell anergy and in conjunction with clonal deletion (outlined above) constitutes the major form of purging the B cell repertoire of self-reactivity. While it should be borne in mind that these transgenic systems are model systems and are by necessity somewhat contrived, they adequately demonstrate the potential fate of self-reactive B cells. Provided an immature B cell is not deleted because of its self-reactivity, it then leaves the bone marrow and enters the periphery. Whether the newly emigrant immature B cell gains entry to the recirculating B cell pool appears to depend on a number of factors, one of them being self-reactivity as outlined above. In summary, there appears to be a period in the development of a B cell in which stimulation by antigen leads to cell death rather than activation. The reasons why the same stimulus
The B-Cell in Immunity
29
should have such different outcomes at different stages in B cell development are as yet unknown. Approximately fifty million B cells leave the bone marrow of an adult mouse and enter the periphery every day, sufficient to replace the entire peripheral B cell pool in about five days (Osmond, 1986). Lifespan studies, however, indicate that this is not the case. In fact, in adult rats (these studies have been best performed in rodents) only around five percent of emigrant B cells gain access to the peripheral pool. This low level of replacement suggests that the majority of peripheral B cells are long lived, a fact that has recently been directly demonstrated in a number of experimental systems. Entry into the recirculating B cell population is also accompanied with a further change in cell surface markers. The most obvious is the expression of IgD in addition to IgM on the cell surface (Figure 4). On a given B cell, both the IgM and IgD molecules have the same antigen specificity since these heavy chains are generated by the alternative splicing of a single variable region unit onto the two different constant regions. It was at one time maintained that since the onset of IgD expression correlated with the end of the period during which deletion of B cells could be induced by antigen, its expression was somehow related to this event. Recent analysis of mice unable to express IgD, however, suggests that this is either wrong or an oversimplification. Indeed, the analysis of these IgD""" mice has yet to reveal any clear-cut function for IgD. In addition to IgD, recirculating B cells differ from their precursors by expressing CD23 (the low affinity receptor for IgE) and higher levels of MHC class II. The recirculating B cell population is also uniformly positive for expression of CD40, a molecule which is critical for the activation of B cells by T cells. Prior to this stage, not all B lineage cells express CD40 (Figure 4). In principle, access to the recirculating B cell pool may be by either of two processes: entry is either random or selective. While current experimental evidence is not conclusive, it tends to favor the selective recruitment of B cells into the recirculating pool. The most suggestive evidence comes from analyzing B cell populations for changes in Ig variable region gene usage. On two levels there is a shift in VH gene usage associated with becoming a long lived B cell. The best characterized example is that in which the VH gene families located closest the DH and JH elements are used preferentially in pre-B cells, while peripheral B cells utilize VH families according to their number of members (Malynn et al, 1990). The importance of this observation is that since pre-B cells do not express complete Ig on the surface, their repertoire is probably unselected. If entry into the peripheral pool were stochastic, then the peripheral repertoire would be expected to be the same as that of the pre-B cells. The fact that these two repertoires differ indicates selection on the basis of the expressed Ig. This selection could be either positive, negative or a mixture of both. That is, certain B cells could be either recruited because of some benefit their antigenic specificity endowed to the animal or, alternatively, immature B cells could be deleted because of self reactivity with the
30
DAVID TARLINTON
few survivors becoming long lived B cells. The correct answer will probably be a mixture of both.
B CELL MIGRATION In order to appreciate how B cells function in the immune system, it is necessary to understand a little of how they are distributed in the body and how they migrate from place to place. As has been detailed above, once generated in the bone marrow B cells then leave and enter the blood as immature B cells. These cells then enter the spleen at the periarteriolar lymphocytic sheath (PALS), an area rich in T cells and another specialized cell type called interdigitating dendritic cells (IDCs). One view of B cell maturation holds that the immature B cells are exposed to antigen at these locations and are thereby selected for entry into the peripheral pool (Gray and MacLennan, 1988). It certainly appears that there is a bifurcation in B cell circulation at this point, since beyond it only a small fraction of the newly produced B cells survive. This small fraction of the emigrant B cell population migrates from the PALS into adjacent B cell rich areas called follicles. Follicles are organized structures in the secondary lymphoid organs with a reproducible cell distribution and a critical role to play in immune responses. The distribution of follicles in spleen and a typical follicle are shown in Figure 5. Within the follicle greater than 90% of the cells are B cells with the phenotype of recirculating B cells, namely IgM"^IgD^'. The remaining cells are CD4"*" T cells, a specialized kind of antigen presenting cell called a follicular dendritic cell (FDC) and macrophages. These various cell types cooperate in generating germinal centers in response to antigenic challenge. Surrounding the follicle is the marginal sinus and surrounding this the marginal zone. Recirculating B cells enter the secondary lymphoid organs via high endothelial venules in the marginal zone, and then migrate to the follicles. The B cells traverse the follicle and if they do not encounter their cognate antigen, they exit through the marginal sinus, which represents the efferent lymph, and repeat the whole process. The marginal zone is typically considered to be part of the so-called white pulp in the spleen, meaning that it is part of the lymphocyte area. It does however form the junction between the red and white pulps and may be involved in the trafficking of lymphocytes between the two areas. B cells which reside in the marginal zone (as opposed to traversing it) have a phenotype which is distinct from that of the follicular B cells. MZ B cells express high levels of IgM, low levels of IgD and are negative for CD23, the inverse of follicular B cells. The exact role of the marginal zone B cells is not precisely known, but some experimental evidence suggests that these B cells may be involved in immune responses which are independent of T cell help and in transporting immune complexes into the follicles. The basic organization of a follicle is quite well conserved in evolution. While present in essentially all secondary lymphoid organs it may have evolved from the organ in which the early events of B cell development occurred in more primitive
The B'Cell in
Immunity
31
>.^^. ^
%l D
Figure 5, Histological staining of B cell structures in secondary lymphoid organs. {A) a transverse section of a mouse spleen, stained with an antibody specific for B cells. Note how the B cells are organized Into horseshoe-shaped follicles, the center of which Is the T-cell rich area. (B) a close up of one follicle. The lightly staining cluster of cells Indicated by the arrow is a germinal center formed within the follicle. (Q cells of a germinal center revealed more clearly with a specific stain and (D) a focus revealed by an antl-immunoglobulln stain. Note the high level of staining which Indicates a high level of immunoglobulin production by the B cells in the focus. (Courtesy of B. Pulendran, The Walter & Eliza Hall Institute.)
animals. Indeed, in chickens and sheep the antigen independent phase of B cell development occurs in follicles structurally similar to those described above. In chickens these follicles are localized to an organ called the bursa of Fabricius (hence the B in B cell).
B CELL RESPONSES TO ANTIGEN At this point the development of B cells has been chartered from stem cells up to recirculating B cells in the periphery. The various selective forces which operate on B cell development and which shape the repertoire have been described. What now needs to be considered is what happens when B cells encounter antigen in the periphery. That is, how do B cells participate in an immune response.
DAVID TARLINTON
32
GERMINAL CENTER
3
s
©
I
immune complexes
O -*(©-"•"
0
• O —'(£)3
. IgG
4
DAYS POST-IMMUNIZATION
Figure 6. The early stages of a primary T-cell dependent immune response. Antigen complexes are localized on the surface of interdigitating dendritic cells (IDCs) outside the follicle. B and T cells are stimulated by the antigen and begin the response. In the extrafollicular pathway, B cells proliferate and differentiate into plasma cell in a focus. In the intra-follicular pathway, B cells migrate into the follicle, proliferate and form a germinal center. Immune complexes become localized on the follicular dendritic cells (FDCs) and are important for B cell selection during affinity maturation.
When antigen is introduced into an animal it initiates a cascade of events that eventually lead to its clearance and to the production of cells which will more efficiently respond to the same antigen upon re-exposure. An outline of the early events in an immune response is shown in Figure 6. Not all antigens, however, elicit the same kind of response. Protein antigens generally elicit the participation of T cells in the production and secretion of antigen specific immunoglobulin while non-protein antigens do not. This basic distinction of T-dependent/T-independent responses has several important ramifications. Since most antigens encountered are T-dependent, these shall be considered first. The basic components of a T-depend-
The B'Cell in Immunity
33
ent response are the production of antigen specific plasma cells, the germinal center reaction and the production of memory B cells.
T CELL DEPENDENT RESPONSES The first thing to appreciate about immune responses is that they are actually quite difficult to initiate. The antigen has to be of a particular nature in order to elicit a response, it must either be given in the form of an aggregate or it must aggregate soon after introduction. The reason for this is that the cells of the immune system basically will not recognize soluble antigen. Indeed, antigen given in such a form is usually tolerogenic rather than immunogenic. Soluble antigen can be aggregated by immunoglobulin already circulating in the animal. Although this immunoglobulin will bind poorly to the antigen because of its low affinity, this can be compensated for by the increased avidity of binding that comes from being IgM. Secreted IgM is a pentameric molecule and therefore has ten antigen binding sites rather than the usual two. These multiple binding sites help to stabilize low affinity interactions. While in general, immune complexes are removed from the system by phagocytic cells such as macrophages, a fraction is transported to secondary lymphoid organs where it acts to initiate a humoral immune response. This transportation is carried out by specialized dendritic cells in the periphery called Langerhan's cells. These cells migrate with antigen to the secondary lymphoid organs where they differentiate into extrafoUicular or interdigitating dendritic cells (IDCs). These dendritic cells are efficient in priming T-dependent responses because they express at high levels receptor molecules necessary for binding immune complexes (receptors for immunoglobulin Fc and complement component C3b), high levels of MHC class II antigens and they constitutively express T cell co-stimulatory molecules such as B7 (these are described in greater detail in Chapter 3). Both the class II and B7 molecules are important for priming helper T cells. As indicated earlier, the dendritic cells are visited both by recirculating B cells and newly generated virgin B cells and they exist in areas rich in T cells. If B cells from either of these sub-populations recognize the antigen on the surface of the dendritic cells in the presence of antigen primed T cells, they then follow one of two pathways; they either stay in the extrafoUicular PALS area and differentiate into an antibody producing plasma cell or they migrate to the follicle and initiate the formation of a germinal center (Figure 6). The direct differentiation of antigen specific cells into plasma cells in the PALS forms the earliest B cell response to antigen. These antigen specific plasma cells are localized in a histologically identifiable structure called a focus, shown in Figure 5. The observation that each focus is adjacent to a germinal center has led to speculation about the relationship between the B cells in the focus and those in the germinal center. Are they, for example, clonally related or unrelated? Do B cells migrate from the germinal centers to the focus and there begin antibody secretion? While the answers to these questions are not yet known, they do provide for some
34
DAVID TARLINTON
interesting speculations, some of which are outlined below. Foci persist for about one week, after which they dissipate. Although antibody production continues essentially unabated after this time, it presumably derives from plasma cells located in either the red pulp of the spleen or other organs entirely. Indeed, significant numbers of plasma cells are located in the bone marrow and the lamina propria, especially during a secondary response. An additional feature of B cell differentiation is also frequently visible in the plasma cells of the foci, namely immunoglobulin isotype switching. Isotype switching is the name given to the process of genetic recombination whereby the constant region of an immunoglobulin molecule is replaced with another. All B cells start out life expressing IgM. They then co-express IgD, although this is not by isotype switching since it does not involve recombinatorial deletion of the intervening DNA. Upon stimulation by antigen, however, some fraction of the stimulated B cells will switch isotype at the heavy chain locus, transposing the VHDHJH rearrangement from upstream of IgM to being upstream of one of the other constant regions. For example, a cell may switchfi-omIgM to IgG, IgA, or IgE. The choice is not random, quite the opposite with the downstream isotype selected being determined by the stimulus the B cell receives. Certain T cell derived lymphokines act to dictate the isotype selected. Immune responses which elicit helper T cells of the so-called T^l subset, for example, are dominated by immunoglobulin of the IgG4 (human) or IgGl (mouse) isotype. The reason for this is that TH2 cells secrete the lymphokine IL4 which acts as a switch commitment factor to IgG4 and IgGl (and to IgE at higher concentrations). By itself IL4 cannot induce switching, but it does insure that if switching occurs, then it is directed to one isotype. There is a certain symmetry to this system in that the nature of the antigen determines the nature of the response. That is, different antigens elicit TH subsets which produce different sets of lymphokines which, in turn and among other things, favor different immunoglobulin isotype switch outcomes. The different isotypes have different effector functions, and thus the response is tailored to the antigen. IgM and IgA, for example, are efficiently and specifically transported across epithelia and into the mucosa and one finds extensive switching to IgA in the gut associated lymphoid tissues (GALT). Although exactly how different antigens elicit TH cells secrefing different lymphokine profiles is currently unknown. A small number of the B cells stimulated by antigen on the extrafollicular dendritic cells move into the follicle where they form a germinal center. Germinal centers derive from the extensive proliferation of these B cell clones and form distinct histological structures examples of which appear in Figure 5. The composition of germinal centers is the same as that of the follicle in which they develop, namely B cells (90%), CD4'' TH cells (5%), follicular dendrific cells (2%) and macrophages (3%). These macrophages are unique in that they contain small dense bodies called Tingible bodies which represent the nuclei of dead lymphocytes that
The B-Cell in Immunity
35
have been phagocytozed. The requirement for such a housekeeping cell in the germinal center will become apparent shortly. Germinal centers are usually first detectable about five days after the introduction of the antigen, although the day prior to their appearance rapidly dividing centroblasts can be seen in the primary follicle. Indeed, centroblasts have been estimated to have a doubling time of 6 hours! During the period seven to fourteen days post-immunization, the full architecture of the GC is resolved with the so-called light and dark zones becoming apparent. The dark zone is full of dividing centroblasts which are surface Ig negative while the light zone contains non-dividing centrocytes and is rich in follicular dendritic cells. The germinal center reaction can persist for several weeks with the average being four to five weeks. Finally, it appears that all of the cell types found in the germinal center are essential for its continued functioning. If the CD4"^ TH cells are removed by antibody treatment, for example, then the reaction immediately stops. The germinal center is not only a site of extensive and rapid B cell proliferation but also a site of cell death. Indeed, the whole reaction appears to be in a sort of equilibrium in that despite the proliferation, the germinal centers reach their maximum size at about day 14. What then happens to the majority of the cells being generated in the germinal center? While some presumably exit and become plasma cells or memory cells during the course of the reaction, it appears that most die. The reason for this extensive death is intimately linked with the processes of affinity maturation occurring in the germinal center. Affinity maturation is the name given to the phenomenon whereby the average affinity of antibody for antigen increases during the course of a response. It is the result of two processes. One is the preferential growth of B cell clones whose Ig molecules have an intrinsically higher affinity for antigen. Such clones will be favored as the response continues and antigen becomes limiting. The other is the process of somatic mutation (or hypermutation) whereby higher affinity variants are created by deliberately introducing point mutations into the variable region gene segments of the immunoglobulin heavy and light chain genes. Although the mutational process is not random in that some nucleotide changes are more likely than others, and some positions are more likely to be mutated than others, it is not very selective. Thus, while some mutations will improve antigen binding, others will have either no effect or be deleterious. Some mutations may in fact destroy the ability of the immunoglobulin molecule either to bind antigen or even to be made at all. In order to function properly the system requires an efficient method of selecting B cells with improved affinity for antigen and deleting the remainder. This selection occurs in the germinal center. A current model for the functioning of a germinal center is as follows. Extensive clonal expansion of a B cell occurs in the dark zone of the germinal center. These centroblasts have down regulated surface Ig expression and are actively undergoing somatic mutation. After each round of mutation, which may be after every round
36
DAVID TARLINTON
of division, the cells migrate from the dark zone to the light zone. The light zone is rich in follicular dendritic cells which are covered in immune complexes and which express molecules important for B cell proliferation and differentiation. In the light zone, the centroblast has now become a centrocyte and again expresses surface Ig, presumably the most recently mutated form. In order to survive, this B cell has to gain access to the antigen on the surface of the FDC. This can only happen if the newly mutated surface Ig can displace the antibody already coating the antigen on the surface of the FDC. If the mutations improve affinity, then the B cell has a good chance of displacing lower affinity antibody. If the mutations are deleterious to antigen binding, then the B cell will not gain access to the antigen on the FDC and will rapidly die. These dead B cells are removed by the resident macrophages. Selected B cells on the other hand, probably re-enter the dark zone for further rounds of mutation and selection. There are two lines of evidence to support this model. First, in somatically mutated high affinity B cells, the mutations are not distributed randomly but are concentrated in regions of the genes which encode the antigen binding segments of the protein called complementarity determining regions (CDRs). Second, members of single B cell clones have been recovered which show hierarchical distributions of mutations (Jacob et al., 1991). Such a scheme is depicted in Figure 7. The simplest explanation for the concentration of mutations in the CDRs and the occurrence of mutational trees is for rounds of mutation to be punctuated by selection. Not all somatic mutants re-enter the mutational process. Some apparently exit from the germinal center and become antibody secreting cells in the extrafollicular areas. Thus, as the response continues the average affinity of the antibody in circulation improves. The means by which the choice between continued mutation or antibody secretion is made are unknown. One possibility is that it depends on the availability of unbound antigen on the surface of the FDCs, the more free antigen the more likely that the cell will become a plasma cell. It has in fact been proposed that the germinal center reaction itself is terminated when there is no free antigen on the surface of the FDCs. The final cell type to emerge from the germinal center is the memory B cell. This cell is isotype switched and its V genes are extensively mutated and selected. That is, the immunoglobulin expressed by this cell is close to the optimum possible for the antigen. It is not clear whether memory B cells are generated throughout the course of the germinal center reaction or only at the end when the reaction ceases. However it happens, the end result is a population of cells with high affinity for antigen. These cells then enter the circulation and persist for many months, even years. Recent experiments have indicated both that antigen is necessary for the persistence of memory B cells (Gray and Skarvall, 1988) and that memory B cells are not dividing (Schittek and Rajewsky, 1990). These are not necessarily contradictory statements, since they imply that memory B cells need to be regularly exposed to antigen but apparently not in a form which induces a response. It has
The B-Cell in
e
Immunity
DIVISION
/
/
/
\
\
37
/
\
\
Figure 7, A schematic representation of somatic mutation and selection in a single B cell clone. As the daughter cells divide, their progeny incorporate different mutations which may or may not be selected for. If they are, the cell continues to divide; if not, the cell dies. The B cells at each generation possess the mutations of their antecedents v^/hich aHou/s for the order in which the mutations were introduced to be determined.
been known for some time that immune complexes remain on the surface of FDCs and IDCs for many months if not years, and this may be the source of the continual antigenic exposure necessary to maintain the memory B cell population. One final thing to say about T-dependent responses concerns the relationship between the cells of the foci and those of the germinal center. Two ideas have been proposed for the formation of these structures. In one the B cells which populate the GC are derived from a different "lineage" than those which enter the foci (Linton et al., 1988). That is, entry into the GC or the focus is intrinsic to the B cell and is determined before the exposure to antigen. The alternative view holds that the choice is made at the point of exposure to antigen, and that in fact progeny from the one B cell clone may populate both the GC and its adjacent focus (Jacob and Kelsoe, 1992). Once again, there is experimental evidence supporting both views.
A SECONDARY RESPONSE When an animal is re-exposed to an antigen, immune complexes are again necessary for triggering the secondary or memory response. In this case there is often antigen specific immunoglobulin still circulating from the primary response. These
38
DAVID TARLINTON
complexes are again localized on extra-follicular dendritic cells in the secondary lymphoid organs where they are exposed to circulating memory B cells. When memory B cells encounter antigen, they rapidly begin to proliferate and differentiate into antibody secreting plasma cells. These plasma cells migrate to the bone marrow where they remain and continue to secrete immunoglobulin for a considerable time. Since the memory B cell population is already isotype switched, somatically mutated and affinity selected, the secondary response is both more rapid and of higher initial affinity than the primary response. In fact, the peak antibody titer is reached three to five days after secondary antigen exposure compared to the two weeks required in the primary response. Whether or not germinal centers arise as part of the secondary response is not completely clear. Some investigators have found that they do, others that they do not. The difference appears to reflect differences in immunization protocols, such as the length of time between primary and secondary challenges.
T CELL INDEPENDENT RESPONSES Some antigens do not require T cell participation in order to elicit an antibody response. This is graphically demonstrated by the ability of congenitally athymic nude mice (which lack all thymus derived lymphocytes) to respond, albeit weakly, to certain antigens. The common feature of T-independent antigens is that they lack protein epitopes and are therefore unable to be presented in a recognizable form to T cells. Antigens such as polysaccharides and lipids are examples of T-independent antigens. T-independent antigens are further divided into Type 1 and Type 2. Type 1 antigens have intrinsic polyclonal B cell activating properties, in that they are themselves mitogenic. Lipopolysaccharide (LPS) is an example of a murine Type 1 antigen. Type 2 antigens are defined by the inability of mice bearing the X-linked immunodeficiency (xid, see below) mutation to respond to them. An example of a Type 2 antigen is the polysaccharide of pneumococcus. There are a number of important differences between the response induced by T-dependent and independent antigens. Foremost among them is that T-independent antigens do not result in memory B cells or affinity maturation through V gene somatic mutation. Indeed, T-independent responses are generally composed of IgM antibodies, are of short duration and low affinity. Some isotype switching may occur, typically to IgG3 and IgG2a in the mouse and IgG2 in humans. The significance of T-independent antigens comes from the fact that a number of pathogens elicit only this kind of response. Since these responses do not produce memory B cells, these antigens are difficult to vaccinate against. Finding a way of circumventing this is a major goal of current immunological research.
The B'Cell in Immunity
39
OTHER TYPES OF B CELLS A number of recent studies have suggested that not all B cells in the periphery are the same. Sub-populations have been identified by their cell surface phenotype and, in some cases, by functional and developmental differences from the majority of B cells. One well characterized sub-population is that of the Ly-1 B cell. These B cells express the pan-T cell antigen CD5 (which, in the mouse, used to be called Ly-1, hence the name). Additionally, Ly-1 B cells express low levels of IgD, high levels of MHC class II and lack CD23. They are most frequent in the neonatal period and, in mice, are localized to peritoneal and pleural cavities. The equivalent B cell population expressing CD5 exists in humans, but it differs somewhat in its location, being absent from the peritoneum and mainly restricted to the blood. The developmental differences between Ly-1 B cells and follicular B cells are so numerous that a number of investigators have proposed that these B cells represent a different hematopoietic lineage. Needless to say, this is quite a contentious issue and the evidence is not yet conclusive. At present the significance of the CD5+ B cell population is difficult to assess. These B cells have been implicated in the production of a number of autoantibodies in both humans and mice, and as being the population from which the vast majority of B chronic lymphocytic leukemias (B-CLLs) develop (greater than 90% of human B-CLL are CD5+). It is postulated that the inherent characteristics of CD5 B cells predispose them to becoming CLLs in that these cells are thought to be self-renewing IgM positive cells, thereby providing a greater opportunity for oncogenic transformation.
B CELL DEFICIENCIES A number of immunodeficiencies involving different aspects of B cell immunology are known. These are listed in Figure 8, giving the human and mouse forms and the underlying genetic lesion where known. In fact, in the very recent past, the exact molecular nature of the defects in both X-linked agammaglobulinemia (XLA) and XLA with hyper-IgM were discovered. These findings represent the culmination of years of basic research in immunology and offer great hope of successful intervention in these diseases. X-Linked Agammaglobulinemia (X-LA)
This condition, first described by Bruton in 1952, manifests as an almost complete absence of circulating immunoglobulin with peripheral B cell numbers also being profoundly decreased (less than 1% of normal). During early life affected individuals are protected by maternal IgG passed through the placenta, but as this declines, they become susceptible to recurrent infections, particularly of the respiratory tract. They can, however, be kept healthy by frequent infusions of gam-
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:r 03 -g3_2 13 .E 7 ^
•i < •§ ^ u ^ . a
•2 >< < § t3 '^ _j ci
ro c X 03 tn CD .to CD p CD CD
•y c §^ _^ ' _^ a; c/) CD o -2 c
u •-
CD O) a; -^ u ^,^ -o i^
CD
.i;;
E o
i.l
DC C
C DO " • _ ^ CD 03 a> n DC I/)
C
C to +CD (D 03
300 >12 4 Rare
200-250 0 4
100 0 50
+
+
-
25-50 2 12 Common
+
+
+ +
+
-
+
+
-
?
?
Source: Adapted from Steele, E.j. et al., 1993.
The situation is not as simple as it first appears, as many of the VH/VL [ and Va/Vp ] combinations are likely to be non-viable (Cohn, 1968). Data on this point for TcRs is not yet available but it is known that preferential association of polypeptide chains appears to be a feature of Ig assembly from H and L polypeptide chains (called the Mannik Phenomenon, see Cohn, 1968). Junctional Diversity
Additional diversity can be created during the rearrangement process. During Ig heavy chain and TcR (3 chain rearrangement, two D genes can be rearranged giving rise to a V-D-D-J variable region which may be fully functional (see Table 2). Such a rearrangement was recently found in an antibody heavy chain isolated from a systemic lupus erythematosus patient (Davidson et al., 1990). When the various genetic elements are lined up for joining via the heptamer/nonamer recognition sequences, an exonuclease may remove nucleotides from the ends of the genetic elements involved to create a novel and unique join (Figure 7). Many Ig H chain and TcR a and (3 chain V regions have been found to possess varying numbers of non-templated nucleotides at the joins i.e., nucleotides not coded for by the genetic elements being rearranged. The additional sequences are known as N regions and they are inserted by the enzyme terminal deoxynucleotidyl transferase (Desiderio et al., 1984, Figure 7). N region additions are not evident in fetal and early neonatal Igs but are prominent features of rearranged V[D] J genes in adults (Feeney, 1990). In many V regions there is evidence of both removal of nucleotides from the joins and N region additions. No such deletions or N regions have yet been reported for antibody light chains; however, they too are capable of producing extensive junctional diversity during rearrangement. During
98
E.I. STEELE and H.S. ROTHENFLUH
Exonuclease ID
I
I
A. l7l l9l
J I
inw
D-J ends held together by a putative joining enzyme. Signal sequences are joined
m
TZA
An exonuclease removes nucleotidyltransferase
bases. Terminal deoxyadds bases.
"GTGAACCG -CACTTGGC
i L
N-Region Figure 7. Nucleotide removal and N region addition during the D-J rearrangement of an IgH chain. The heptamer (7)/nonamer (9) signal sequences are involved in lining up the genetic elements involved. The same mechanism is also involved in the V-DJ rearrangement step which follov^s the D-J rearrangement. Often the ends of the genetic elements involved are removed by the exonuclease, indeed it is not unusual for the D gene to be almost completely removed and replaced by a N region. (Adapted from A l t , F. W., and Baltimore, D., 1982).
Ig L chain rearrangement the joining sites of the V and J elements involved can vary from one rearrangement to the next, thus resulting in many unique rearrangements even v^hen the same two genetic elements are involved. However, the immune system pays a heavy price for being able to generate so much junctional diversity: many if not most of these nucleotide deletions, N regions and imprecise joins will result in a non-functional protein due to loss of the reading frame. All the cells with non-functional receptors must be deleted from the repertoire, and hence, the high rate of lymphocyte precursor production in the bone marrow is coupled to a high rate of cell death.
99
The Generation of Diversity in the Immune System Secondary Rearrangements
A supplementary somatic process has been identified termed a "secondary rearrangement." Here the V segment of a rearranged V[D]J is replaced by a DNA recombination event which inserts a different upstream V segment (Reth et al., 1986). Heptamer recognition sequences (palindromes) embedded within the 3' ends of V segments and possibly related to heptamers immediately 5' and 3' of D and J elements are thought to direct the specificity of the secondary recombinase enzymes. The significance of variable region replacement is still not clear. Recent developments on the mechanism of self tolerance induction in B cells suggest it may be the genetic mechanism underlying the process called "receptor editing." If a developing B cell encounters a self epitope, the self reactive cell has a 'second chance' to change the specificity of its Ig receptor by V gene replacement (reviewed in Nossal, 1993). T cells developing in the thymus may also employ a similar V replacement strategy (Petrie et al., 1993).
o •a
-1000
1000 2000 3000 Nucleotides from cap site
PcL
VDJHI J H 2 J H 3
4000
JH4
Figure 8. Distribution of somatic mutations around a putative V-D-JHI rearrangement. The distribution of somatic mutations is asymmetric and the upstream boundary for the somatic hypermutation process lies around the promoter (P)/transcription start site (c) region. The downstream boundary seems to lie around the enhancer region (E). (Adapted from Rothenfluh, H. S. et al., 1993).
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E.J. STEELE and H.S. ROTHENFLUH
Somatic Hypermutation of Rearranged V(D)J Genes Additional somatic diversity can be generated as a consequence of antigen-stimulated somatic hypermutation, a process that introduces nucleotide changes (mainly point mutations) into the DNA sequence within and adjacent to rearranged V genes. Antigenic stimulation is obligatory to activate the process and it is entirely dependent on T cell help. T-independent antigens, such as lipopolysaccharide, do not activate the mutator. Whilst the detailed molecular mechanism is still unknown, many molecular and cellular features of the process are now clear (reviewed in Steele, 1991). In mice it is established that somatic point mutations are introduced mainly into the transcribed region of the genomic DNA of rearranged V[D]J regions (Steele et al., 1992; Rothenfluh et al, 1993). A comparison of many somatically mutated genes shows an asymmetrical distribution of mutations around rearranged V genes with a single major mode centered on the V[D]J region and a positively skewed tail into the non-translated J-C intron (Figure 8). A subset of mature B cells once specifically activated in a T-dependent manner migrate to primary lymphoid follicles to form a germinal center (Figure 3). In these sites the selected 'founder' B cell mutates its productively rearranged V[D] J genes at a very high rate, estimated to be 10'^ to 10""^ per base pair per replication event (mutation rates in other eukaryotic genes are 10"^ to 10'^'). The founder B cell undergoes clonal growth and rapid point mutation targeted to V[D]J within the germinal center (Jacob et al., 1991; MacLennan, 1991). A stringent Ag mediated selection process ensures the emergence of higher affinity mutants only (Figure 3). Ag-Ab complexes consisting of Abs of lower affinity produced in the initial phase of the primary response present selecting epitopes from the surface of interdigitating follicular dendritic cells (FDCs). Mutated B cell progeny ('centrocytes') which no longer bind Ag (or bind at a much lower affinity) do not receive an activating cross-linking signal and 'commit suicide' via a process called apoptosis, or programmed cell death. However, those progeny with mutant Ig receptors of higher affinity compete to dislodge the lower affinity antibodies in the Ag-Ab complex, and are 'selected' to survive as long lived memory cells. It can be seen that we have a process of rapid antigen-mediated Darwinian positive selection or 'evolution in microcosm' (Cunningham, 1977), supplying the host organism with high affinity protective antibodies specific for native conformational epitopes. Thus, the somatic hypermutation and selection process within a germinal center basically results in the fine tuning of the specificity repertoire during an immune response (Goverman et al., 1986). It only contributes to the diversification process with respect to cross reactive specificities. However, the target area of mutation extends into the flanking regions, particularly into the 3' J-C intron Figure 8 and the resultant concentration of mutational differences to the CDRs or hypervariable domains (Wu and Kabat, 1970) are clearly the result of antigen binding selection for functional antibodies (Weigert et al., 1970) i.e., amino acid replacement
The Generation of Diversity in ttie Immune System LVj
LV„
LV2
Di.„
101
Ji^
Cj,
-ii«r[HZ:i''-l»-^HHHF^^^H}{}ff-^HlHlHllBL VDJ
i
Cfi
Productive VDJ rearrangement in a mature B cell 1 ^ Antigenic stimulation Somatic hypermutation of rearranged V region in germinal center L
VDJ
Cil DNA
L VDJ cap-||»**»|«
C^i AAAAA mature mRNA
Further antigenic selection within germinal center
-IH
m
m i:
CDRl CDR2 CDR3 cap-H
iA
m
AAAAA
Figure 9. The sequence of events resulting in the selection for somatic mutations in the CDRs (Wu-Kabat structures). When a mature B cell expressing a functionally rearranged Ag specific Ig comes in contact with the Ag it may become an unmutated plasma B cell or it may undergo the somatic hypermutation pathv^ay. In the latter case, the Ag stimulated B cell will enter a germinal center in the spleen or in a lymph node. Mutations in the cartoon (represented by the concentrations of black dots) are introduced into the VDJ and its flanking regions. The mutation mechanism does not extend into the constant region exons. During antigenic selection within the germinal center, B cells bearing surface Ig with mutated CDRs but conserved FRs are selected for providing the mutated Ag binding site has a higher affinity for Ag. Thus B cells leaving germinal centers have accumulated changes in their CDRs but few if any in their FRs. CDR3 is crosshatched to simply indicate that most of its diversity results from junctional diversity rather than somatic mutations, cap = cap structure at 5' end of processed RNA. AAAAA = poly-A tail added at 3' end of processed RNA. Note, in the DNA molecule of the lower diagram only the first exon of C^ is shown.
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E.J. STEELE and H.S. ROTHENFLUH
mutations in the CDRs and conserved (silent) nucleotide changes in the FR regions. The sequence of events resulting in the emergence of somatically fashioned 'Wu-Kabat' structures within V[D]Js (Steele et al., 1993) is shown in Fig. 9. It should be noted at this point that of the three CDRs in a rearranged variable region the CDR3 is almost solely a somatic construction in that it spans the V-J join in IgL chains or the V-D and D-J joins in IgH chains. Thus, most of the diversity found in CDR3 is due to imprecise joining, nucleotide deletions and/or N region addition rather than somatic hypermutation. The position of the other two CDRs, CDRl and CDR2 are abeady embedded in the germline encoded V segment (below). The somatic mutator is clearly regulated in both space and time. This is necessary given the potential to generate lethal genetic errors in somatic cells. The possibility that autoimmune B cells may be generated cannot be excluded in this schema and such a danger exists for all hyperimmune memory B cell responses. However, this would be limited by the relative lack of T cell help as mature post-thymic T cells themselves are unlikely to undergo antigen driven somatic mutation of their TcRs (Steele et al., 1993) i.e., autoreactive Th cells would be clonally deleted in the thymus. We will now turn to a new concept that is emerging from work in our laboratory on how the germline V gene repertoire has evolved (Rothenfluh and Steele, 1993a).
"DIRECTED MUTAGENESIS"—EVOLUTION OF THE IMMUNE SYSTEM? A moments' reflection on the facts of somatic hypermutation shows that the mammalian immune system has evolved a tightly regulated process of "directed mutation" (Steele, 1989). It is also clear that the process depends very much on positive Darwinian selection but now occurring within a somatic cell population of a multicellular organism. Indeed, it was this aspect which proved decisive in the acceptance of Burnet's Clonal Selection Theory over its more instructionalist forerunners (Steele, 1991b). Rapid somatic mutation and antigen-binding selection results in a brisk affinity maturation of the memory antibody response. It is important to re-emphasize that this is not old fashioned 'directed mutation' in the sense of the environment instructing genetic change directly on the DNA. A key evolutionary question can therefore be posed: Does this acquired and clearly beneficial somatic adaptation die with the individual or can acquired somatic mutations in V genes be inherited through the germline? (Steele, 1979; Rothenfluh and Steele, 1993a,b). Current research in our laboratory on DNA sequences of a large number of germline V segments and their non-transcribed and non-translated 5' flanking regions (from the genomic DNA of inbred mice) lead us to deduce that the germline V genes bear all the hallmarks of powerful somatic selection pressure namely, positive Darwinian selection occurring first in the soma coupled to physical transfer and integration of these somatically fashioned V genes into homologous target sequences in the germline DNA (Rothenfluh et al., 1995).
The Generation of Diversity in the Immune System
103
SUMMARY The mammalian immune system has evolved sophisticated germline and somatic strategies for the generation of an immense repertoire of antigen-specific lymphocytes. The key evolutionary selective forces have been the need to protect the individual against 'unexpected' infections and to avoid autoimmune disease. The germline is composed of a large tandem array of V segments located upstream of joining elements (D,J) which are themselves proximal 5' to the constant region coding exons at Ig and TcR genetic loci. During lymphocyte development V genes rearrange to produce complete V[D]J variable regions which are transcribed, translated and the protein chains assembled into functional antigen-specific Ig and TcR receptors. Such receptors are clonally distributed such that any mature B cell or most T cells express only one antigen-specific receptor on their surface membrane. Combinatorial DNA recombination of the germline encoded elements (V-to-[D]to-J) together with combinatorial association and assembly of complete polypeptide chains can by itself generate a potentially very large recognition repertoire (>10'^), although many of these 'random' combinations may not be functional receptors. Additional somatic diversification processes include V to [D] J junctional diversity, nucleotide deletions, N region additions and 'secondary rearrangements' that can lead to receptor replacement and therefore a complete change of clonal specificity. Finally, there is the tightly regulated antigen-driven process of somatic hypermutation of rearranged IgV genes. It is confined to a subset of mature B cells during differentiation to memory cells in specialized post-antigenic lymphoid structures called germinal centers. Memory B cells arising from germinal centers express and secrete mutated high affinity antibodies. In this way the specificity of the antibodies is fine tuned during an immune response. It is not known whether a similar process occurs in T cells but if it does it would have to occur during T cell development in the thymus to ensure clonal deletion of autoreactive cells. Positive Darwinian selection drives the development and evolution of both the germline and somatic variable gene repertoires. Indeed, there is emerging evidence from the structure and pattern of germline V gene sequences that acquired somatic mutations in V[D]J genes may be inherited in the germline DNA.
REFERENCES Alt, F.W., Oltz, E.M., Young, F., Gorman, J., Taccioli, G., & Chen, J. (1992). V[D]J recombination. Immunol. Today. 13, 306-314. Alt, F.W., & Baltimore, D. (1982). Joining of immunoglobulin heavy chain gene segments: Implications from a chromosome with evidence of three D-JH fusions. Proc. Natl. Acad. Sci. USA 79, 4118-4122. Berek, C , & Milstein, C. (1987). Mutation drift and repertoire shift in the maturation of the immune response. Immunol. Rev. 96, 23-41.
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Berek, C, & Milstein, C. (1988). The dynamic nature of the antibody repertoire. Immunol. Rev. 105, 5-26. Blanden, R.V., Hodgkin, P.D., Hill, A., Sinickas, V.G., & Mullbacher, A. (1986). Quantitative considerations of T-cell activation and self tolerance. Immunol. Rev. 98, 75-93. Bothwell, A.L.M. (1984). The genes encoding anti-NP antibodies in inbred strains of mice. In: The Biology of Idiotypes. (Greene, M.I., & Nisonoff, A., Eds.) pp. 19-43. Plenum Publishing Corp., NY. Burnet, F.M. (1959). The Clonal Selection Theory of Acquired Immunity. Cambridge University Press, London. Cohn, M. (1968). The molecular biology of expectation. In: Nucleic Acids in Immunology. (Plescia, O.J., & Braun, W., Eds.) pp. 671-715. Springer-Verlag, NY. Coleman, R.M., Lombard, M.F., & Sicard, R.E. (1992). Fundamental Immunology. Wm. C. Brown Publishers, Dubuque, lA. Cunningham, A.J. (1977). Evolution in microcosm: The rapid somatic diversification of lymphocytes. Cold Spring Harbor Symposia on Quant. Biol. 41, 761-770. Davidson, A., Manheimer-Lory, A., Aranow, C , Peterson, R., Hannigan, N., & Diamond, B. (1990). Molecular characterisation of a somatically mutated anti-DNA antibody bearing two systemic lupus erythematosus-related idiotypes. J. Clin. Invest. 85, 1401-1409. Davis, M.M., & Bjorkman, P.J. (1988). T-cell antigen receptor genes and T-cell recognition. Nature 334, 395-402. Davis, M.M., & Chien, Y-H. (1993). Topology and affinity of T-cell receptor mediated recognition of peptide - MHC complexes. Curr. Opin, Immunol. 5, 45-49. Desiderio, S., Yancopoulos, G., Rosa, M., & Baltimore, D. (1984). Insertion of N-regions into heavy-chain genes is correlated with expression of terminal deoxynucleotidyl transferase in B-cells. Nature 311:752-755. Edelman, G.M., & Gaily, J.A. (1970). Arrangement and evolution of eukaryotic genes. In: The Neurosciences. 2nd Study Program. (Schmitt, F.O., Ed.) pp. 962-972. Rockfeller University Press, NY. Eisen, H.N. (1986). Why affinity progression of antibodies during immune responses is probably not accompanied by parallel changes in the immunoglobulin-like antigen specific receptor on T cells. BioEssays 4, 269-272. Feeney, A.J. (1990). Lack of N regions in fetal and neonatal mouse immunoglobulin V-D-J junctional sequences. J. Exp. Med. 172, 1377-1390. Goverman, J., Hunkapiller, T., & Hood, L.E. (1986). A speculative view of the multicomponent nature of T cell antigen recognition. Cell 45, 475-484. Hood, L.E., Weissman, I.L., Wood, W.B., & Wilson, J.H. (1984). Immunology. 2nd ed. The Benjamin/Cummings Publishing Co., Menlo Park, CA. Honjo. T. (1983). Immunoglobulin genes. Ann. Rev. Immunol. 1, 499-528. Honjo, T., Alt, F.W., & Rabbitts, T.H. (1989a). Immunoglobulin Genes. Academic Press, NY. Honjo, T., Shimizu, A., & Yaoita, Y. (1989b). Constant-region genes of the immunoglobulin heavy chain and the molecular mechanism of class switching. In: Imunoglobulin Genes (Honjo, T., Alt, F.W., & Rabbitts, T.H., Eds.) pp. 123-149. Academic Press, NY. Jacob, J., Kelsoe, G., Rajewsky, K., & Weiss, U. (1991). Intraclonal generation of antibody mutants in germinal centres. Nature 354, 389-392. Kagi, D., Ledermann, B., Burki, K. et al. (1993). Abstract: Function of CD8+ T lymphocytes and NK cells in perforin deficient mice. EMBO Workshop on Cell Mediated Cytotoxicity. Weismann Institute of Science, Rehovot, Israel, 29th August-lst Sept., 1993. Kees, U., & Blanden, R.V. (1976). Single genetic elements in H-2K affects mouse T cell anti-viral function in poxvirus infection. J. Exp. Med. 143, 450-456. Langman, R.E., & Cohn, M. (1987). The E - T (Elephant - Tadpole) paradox necessitates the concept of a unit B cell function: the protection. Mol. Immunol. 24, 675-697.
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Lanzavecchia, A. (1985). Antigen-specific interaction between T and B cells. Nature 314, 537-539. Lanzavecchia, A., Roosnek, E., Gregory, T., Nerman, P., Abrignani, S. (1988). T cells can present antigen such as HIV gpl20 targeted to their own surface molecules. Nature 334, 530-532. MacLennan, I. (1991). The centre of hypermutation. Nature 354, 352-353. Manser, T., Huang, S.-Y., & Gefter, M.L. (1984). Influence of clonal selection on the expression of immunoglobulin variable region genes. Science 226, 1283—1288. Nossal, G.J.V. (1993). A second chance for bad B cells. Curr. Biol. 3,460-462. Ohno, S. (1970). Evolution by Gene Duplication. Springer-Verlag, Berlin. Padovan, E., Casorati, G., Dellabona, P., Meyer, S., Brockhaus, M., & Lanzavecchia, A. (1993). Expression of two T-cell receptor a chains: Dual receptor T cells. Science 262, 422-424. Petrie, H.T., Livak, F., Schatz, D.G., Strasser, A., Crispe, I.N., & Shortman, K. (1993). Multiple rearrangements in T cell receptor a chain genes maximize the production of useful thymocytes. J. Exp. Med. 178,615-622. Rathbun, G., Berman, G., Yancopoulos, G., & Alt, F.W. (1989). Organization and expression of the mammalian heavy-chain variable-region locus In: Imunoglobulin Genes. (Honjo, T., Alt, F.W., & Rabbitts, T.H., Eds.) pp. 63-90. Academic Press, NY. Reth, M., Gehrmann, P., Petrac, E., & Weise, P. (1986). A novel VH to VHDJH joining mechanism in heavy-chain-negative (null) pre-B cells results in heavy-chain production. Nature 322, 840-846. Roes, J., & Rajewsky, K. (1993). Immunoglobulin D (IgD)-deficient mice reveal an auxiliary receptor function for IgD in antigen-mediated recruitment of B cells. J. Exp. Med. 177, 45-55. Rothenfluh, H., & Steele, T. (1993a). Lamarck, Darwin and the immune system. Today's Life Science 5, 8-15 and 16-22. Rothenfluh, H.S., & Steele, E.J. (1993b). Origin and maintenance of germline V-genes. Immunol. Cell Biol. 71,227-232. Rothenfluh, H.S., Taylor, L., Bothwell, A.L.M., Both, G.W., & Steele, E.J. (1993). Somatic hypermutation in 5' flanking regions of heavy chain antibody variable regions. Eur. J. Immunol. 23, 2152-2159. Rothenfluh, H.S., Blanden, R.V., & Steele, E.J. (1995). Evolution of V genes: DNA sequence structure of functional germline genes and pseudogenes. Immunogenetics 42, 159-171. Sinha, A. A., Lopez, M.T., & Devitt, H.O. (1990). Autoimmune diseases: The failure of self tolerance. Science 248, 1380-1386. Steele, E.J., Rothenfluh, H.S., & Both, G.W. (1992). Defining the nucleic acid substrate for somatic hypermutation. Immunol. Cell Biol. 70, 129-144. Steele, E.J. (1979). Somatic Selection and Adaptive Evolution: On the inheritance of acquired characters. 1st Edn. Williams-Wallace, Toronto; 2nd Edn. 1981 University of Chicago Press, Chicago. Steele, E.J. (1989). Mechanism of directional mutations? Mol. Rep. Dev. 25, 231-232. Steele, E.J. (ed.) (1991a). Somatic hypermutation in V-regions. CRC Press, Boca Raton, FL. Steele, E.J. (1991 b). Somatic mutation: past, present and future In: Somatic Hypermutation in V-regions. (Steele, E.J. Ed.) CRC press, Boca Raton, FL. pp. 1-9. Steele, E.J., Rothenfluh, H.S., Ada, G.L., Blanden, R.V. (1993). Affinity maturation of lymphocyte receptors and positive selection of T cells in the thymus. Immunol. Rev. 135, 1-7. Steinman, R.M., Gutchinov, B., Witmer, M.D., & Nussenzweig, M.C. (1983). Dendritic cells are the principal stimulators of the primary mixed leukocyte reaction in mice. J. Exp. Med. 157,613-627. Tonegawa, S. (1983). Somatic generation of antibody diversity. Nature 302, 575-581. Unanue, R.R. (1984). Antigen-presenting function of the macrophage. Ann. Rev. Immunol. 2,395-428. Wilson, R.K., Lai, E., Concannon, P., Barth, R.K., & Hood, L.E. (1988). Structure, organization, and polymorphism of murine and human T-cell receptor a and p chain gene families. Immun. Rev. 101, 149-172. Weigert, M.G., Cesari, I.M., Yonkovich, S.J., & Cohn, M. (1970). Variability in the Lambda light chain sequences of mouse antibody. Nature 228, 1045-1047.
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Wu, '^ T., & Kabat, E.A. (1970). An analysis of the sequences of the variable regions of Bence Jones p'oteins and myeloma light chains and their implications for antibody complementarity. J. Exp. Med. 132,211-250.
RECOMMENDED READINGS Ada, G.L., & Nossal, G. (1987). The clonal selection theory. Sci. Amer. 255, 62-69. Moller, G. (Ed.) (1987). The role of somatic mutation in the generation of lymphocyte diversity. Immunol. Rev. Vol. 96, Munksgaard, Copenhagen. Moller, G. (Ed.) (1992). Germinal centers in the immune response. Immunol. Rev. Vol. 126. Munksgaard, Copenhagen. Tonegawa, S. (1985). The molecules of the immune system. Sci. Amer. 253, 104-113.
Chapter 6
The Antigen-Antibody Complex: STRUCTURE AND RECOGNITION
P.M. COLMAN
Introduction Antibody Structure Antigen Structure Antibody-Antigen-Complexes Summary Recommended Readings
107 108 113 116 119 120
INTRODUCTION Biological processes generally are controlled by interactions between molecules. Mostly these interactions are the result of evolutionary refinement and optimization of the interacting molecular species. Specific immune responses to an antigen call Principles of Medical Biology, Volume 6 Immunobiology, pages 107-120. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-811-0
107
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P.M.COLMAN
for an interaction between immune receptors and that antigen, but in this case the antigen may not necessarily have been encountered during either the evolutionary history or the lifetime of the animal. Survival of the animal depends on a system for generating an ensemble of diverse molecules, one or more of which is capable of attachment to a particular antigen. The amino acid sequence variability among antibodies which results from gene segment rearrangement and somatic mutation is a major source of diversity of antibody specificities. The purpose of this chapter is to examine the structural basis of antigen binding by antibody, and antibody variability is a large part of the story. However, beyond that there are strictly conserved structural features of antibodies whose role in binding of antigen is quite fundamental and we shall discuss also those special architectural features of antibody molecules which suit them well for their task.
ANTIBODY STRUCTURE We consider here immunoglobulin type yl (IgGl) although the general principles extend to all types. IgG is a four chain structure, a dimer of heavy (H) and light (L) chains which are covalently joined by disulfide bonds. These polypeptides are arranged into a -Y- shaped structure (Figure 1). Each arm of the -Y- contains one
Figure 1. Quaternaryarrangementof immunoglobulin domains in an IgGl molecule. Variable (V) and constant (C) domains have the structure shown in Figure 2.
109
The Antigen-Antibody Complex
light chain and the amino terminal half of the heavy chain. The C-terminal halves of the two heavy chains constitute the stem of the molecule. Proteolysis of many antibodies results in three fragments being produced, two Fab (fragment antigen binding) being the arms and one Fc (fragment crystalline) being the stem. Heavy and light chains are composed of multiple copies of a single structural domain. This domain of circa 100 amino acids is the building block of many molecules of the immune system and indeed of some non-immune system molecules. Heavy chains contain four domains (two in the Fab and two in the Fc) and light chains contain two domains. In both heavy and light chains, the N-terminal domains are highly variable across antibodies of different specificity, whereas the remaining domains display conserved amino acid sequences amongst different molecules. To reflect this pattern of variable and constant chemical structure, the domains on the two polypeptides are referred to as VH, CHI, ^m ^^^ ^HS, and VL and CL, reading in each case from the N-terminus (Figure 1). The basic domain structure is illustrated in Figure 2. It is a p-sheet sandwich composed of seven P-strands labelled A through G. Strands ABED form one sheet and CFG the other. The two sheets are covalently connected through cysteine residues on strands B and F. The strand orientations in the two sheets are almost parallel to each other and in this respect the domain structure is typical of many other P-sheet structures in proteins which display a similar 'aligned' packing of CDR1
CDR 2
Figure 2. Immunoglobulin domain structure. Constant domains comprise seven strands, A through G. Variable domains have two additional strands C and C\ Complementarity determining regions of variable domains are labeled CDR1 through CDR3.
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P.M.COLMAN
p-sheets. Variable and constant domains are distinguished from each other structurally by the inclusion in variable domains of two additional P-strands between strands C and D. These are labelled C and C Two aspects of this elaboration of the seven-strand structure are important for the special function of variable domains. The C'C" loop forms part of the antigen binding site and the extended P-sheet C'CFG plays a crucial role in determining the pairing of VH and VL domains (see below). Sequence variation between antibody molecules is not uniformly distributed throughout the V^ and VH domains. Rathermore, in each case, it is concentrated in three places in loops between the p-strands. These hypervariable loops, BC, C'C" and FG, are associated directly with the specificity of a particular antibody and are referred to as the three Complementarity Determining Regions or CDRs. They are all located at the same end of the domain structure. The three CDRs themselves are not equally variable. Most variation occurs in the third CDR of VH ( C D R H 3 ) followed by CDR L3 and, as illustrated below, these two CDRs are centrally located in the antigen binding site. CDR H3 is also most variable with respect to length and to the conformation of its polypeptide backbone. Immunoglobulin variable domains have a segmented gene structure. VL domains are encoded by a V gene and a J gene which correspond respectively to strands A through F (and including most of CDR L3) and strand G. VH domains have an additional level of complexity in their gene structure. The V gene in that case does not include CDR H3. That loop is encoded by a separate element known as D (for diversity), and thus explains the supravariability of CDR H3 compared with the other five CDRs. The J gene structure for VH domains is similar to that for VL domains, i.e., it encodes strand G of the p-sandwich structure. In man, heavy chain variable domains derive from combinatorial association of one each of 500 V genes, 15 D genes and 4 J genes, giving rise to some 30,000 possible VH domains. For light chains, there are 200 V genes and 4 J genes, and 800 possible VL domains. Different pairwise association of VL and VH domains potentially produces over 20 million different antigen binding sites. Other aspects of the recombination process, such as variability in the joining sites of the genes, further expands the repertoire of different structures. The organization of the domains into the -Y- shaped molecule is illustrated in Figure 1. Domains associate laterally along the length of the molecule through extensive noncovalent interfaces. CHI and CL domains form a dimer through association of the ABED face of each domain. CH3 domains dimerize in a very similar manner. In both cases the P-strand orientation of one of the ABED faces in the interface is approximately 90° to that on the partner ABED face. Like the aligned packing described for the domain structure itself, orthogonal packing of P-sheets is also a very common structural motif in protein molecules. Association of CH2 domains is unusual because of the role of carbohydrate in covering the ABED face there.
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In contrast to constant domains, VL and VH domains dimerize through their C'CFG faces, and they do so in a way which is quite unusual compared to other known protein structures. The packing of the two C'CFG P-sheets conforms to neither the ahgned nor orthogonal classes. Rather, the strand orientations in this case are inclined at approximately 60° to each other (Figure 3). The basis for this unusual interaction is two-fold. First, there are some very characteristic and conserved amino acid sequences in the C and G strands which introduce bulges into the regular p structure of these strands and give the C'CFG face a strong curvature (Figure 3). Secondly, conserved amino acids projecting outwards from the C'CFG face contribute to the complementary surfaces of the VL and VH domains at this interface. These two features of variable domain structure are both special to and conserved in variable domain structures. The VL-VH association brings into close spatial proximity the six CDRs of the two domains (Figure 3), CDRs HI, H2, LI and L2 being peripheral and CDRs H3 and L3 central. This arrangement is important in view of the fact that H3 and L3 are more variable than the other four CDRs. Antigens do not always interact with all six CDRs but in all cases studied so far CDRs H3 and L3 are part of the interaction. Together, the six CDRs form the entire surface of the extremities of the Fab arms. It is generally very common to find the 'active sites' of protein molecules located either at the subunit boundaries of oligomeric proteins or at structural domain interfaces. There is an approximate two fold symmetry relationship between the VL and VH domains within the heterodimer and Figure 3 is a view down this pseudo symmetry axis. In that figure, close to the viewer, the CDRs are seen to participate in interactions across the dimer interface. Most of the CDR contacts in this interface involve H3 and L3. The variability in CDR sequences and structures from molecule to molecule result in small perturbations of the geometry of pairing of VL and VH domains between different antibodies. It appears that any given antibody molecule has a well defined and preferred pairing geometry for the variable domains, but variation among molecules can be as large as 15°, i.e., after alignment of, say, the VH domain of two different antibodies, their VL domains may differ in alignment by up to 15°. Thus, despite the conserved structures in this interface (to the rear of the view in Figure 3), the variable CDR structures modulate the interacfion. The VL-VH interface structure can also be modified during encounter with an antigen (see below). Fv fragments are VL-VH domain pairs which can sometimes be generated by proteolytic digestion of antibodies. They are now readily producible by genetically engineering bacteria to over-express VL and VH genes. Fv can be made this way either as a two chain structure which forms a heterodimer VL-VH pair or as a single chain entity in which the two domains on a single polypeptide are joined by an appropriate linker. Linkers of 15 amino acids suffice to allow the C terminus of one V domain to be joined to the N terminus of the other without distorting the Fv quaternary structure. The three dimensional structures of Fv fragments, either of
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Figure 3, V L - V H pairing to form the antigen binding surface. This view is down the approximate two fold symmetry axis relating V H (above) and VL (below) with the six CDRs towards the reader. The C',C,F, and G strands VL V H on the two domains are shaded. In each case strand C is farthest from the reader and strand G is closest.
the two chain or single chain type, have shown that VL-VH pairing is largely unaffected by the removal of constant domains. This result was anticipated during the 1970s when studies with Bence-Jones proteins (dimers of light chains) showed that the presence of the CL domains did not influence the way in which VL domains dimerized. It is possible that some CDR sequences are not very well accommodated at the VL-VH interface and in those cases the presence of constant domains may be important in maintaining the structure of the antigen binding site by stabilizing the interaction between the heavy and light chains. The linkages between domains along the length of the polypeptide chains are less extensive than the lateral contacts described above and are of varying flexibility.
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The VL-CL and VH-CHI links are loose and allow an elbow movement within the Fab arm of the molecule. The CHI-CH2 link is through a cysteine rich peptide known as the hinge which allows large movements of the Fab arms with respect both to each other and to the Fc stem. The CH2-CH3 link is less flexible. The antigen binding site is distant from and loosely held to the sites of effector functions in the Fc region. Such a structure is consistent with the idea that these functions are not fired by a specific conformational trigger but rather by aggregation of bivalent antibody by multivalent antigen. Amino acid sequences of T cell receptors suggest features common to Fab fragments of antibodies, including the special architecture at the interface of the variable domains of the a and p chains. However, no direct three dimensional structures are yet available either of the receptor or of its complex with MHC antigen and peptide.
ANTIGEN STRUCTURE The definition of the antigen binding site of an antibody can be done with some precision by a study of antibody structure and the comparison of molecules with different specificities. In contrast, the definition of an antibody binding site on an antigen is not only difficult a/^r/on but also has meaning only in the context of a specific antibody molecule. Antigenic molecules can be proteins, polysaccharides, nucleic acids (or the oligomeric units of any of these), or naturally occurring or man made organic compounds (usually conjugated with larger carrier molecules). Most of the attention here will be on proteins and peptides because (i) more is known in these cases about the structures of complexes, (ii) there is an extensive database of protein-protein interactions against which to compare antibody-antigen complexes, and (iii) protein antigens are generally important in the development of protective immunity to pathogens. We consider here one example of a well-characterized antigen which addresses some of the practical issues. What are the physico-chemical determinants of antibody binding to antigen? What determines cross-reactivity of anti-sera to two antigens? What minimal changes to antigen can abolish binding by a monoclonal antibody and conversely what antigenic changes can be tolerated by a monoclonal antibody? Influenza viruses undergo continuous antigenic variation. The selection pressure of antibodies to strains of the virus currently and previously infecting man ensures a survival advantage to virus variants to which these antibodies cannot bind The virus has two different glycoproteins displayed on its envelope, a neuraminidase and a hemagglutinin, and both of these are subject to two types of antigenic variation. On the one hand, single amino acid sequence changes in the antigens accumulate continuously and can lead to a variant capable of reinfecting an individual. On the other hand, there occur occasional and sudden dramatic changes in antigenic structure caused by reassortment of the segmented viral genome and
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resulting in a new neuraminidase or hemagglutinin molecule with only 50% amino acid sequence identity to the antigens of previously circulating strains. Such events characterize new subtypes of the antigens, defined experimentally by the lack of cross-reactivity between antisera to antigens of different subtype. Cross- reacting anti-sera characterize variants within a subtype, where amino acid sequence similarities are usually higher than about 80%. Studies of the three dimensional structure of neuraminidase of different subtypes show, as expected, identical three dimensional structures at the level of the fold of the polypeptide chain. However, a comparison of the surface structures of two different neuraminidase subtypes (Figure 4) reveals that conserved structures are dispersed around the surface and do not segregate into any single, large patch. It is known (see below) that antibodies need to attach themselves to a large surface on the antigen (-700A^ or more and involving 15 or more amino acids) in order to bind effectively and the absence of such large conserved surface patches between antigens of different subtypes is the likely cause of the failure of antisera to cross-react. The largest conserved surface structural feature across neuraminidase subtypes is the enzyme active site, about 600A^ in area (Figure 4). Monoclonal antibodies have been used in many virus systems to suppress wild-type virus growth and to select variants which are able to grow, presumably because of the failure of the antibody to bind to them. These variants typically differ by a single amino acid from wild-type and three dimensional structure analysis of such variants of both influenza virus hemagglutinin and neuraminidase show that the structural consequences of these amino acid substitutions are usually very localized to the site of mutation. Furthermore, sometimes the single change in amino acid which abolishes the antibody binding is a change which is considered structurally conservative, e.g., alanine to valine, or asparagine to aspartic acid (or vice versa). Superficially, these observations sit uncomfortably with the requirement for large surfaces of interaction. Why should one amino acid sequence change out of 15 or more in the binding site to antibody make such a large difference? One answer is that in some cases it does not. Examples are known in which the substitution of one amino acid within the interface by another, even of dissimilar physico-chemical properties, has only a small effect on the binding affinity and only a local structural effect on the antibody-antigen interface. In other cases, the contribution to the total binding energy by a single amino acid can be sufficiently large that its substitution by another residue effectively abolishes binding. Note that this does not imply a particularly strong contribution for any one amino acid in the interaction. An antibody-antigen complex with a dissociation constant Kd~10^^M will be severely compromised by the loss of a single tight hydrogen bond (4kcal/mole) which will raise K^ by a factor of nearly 1000. Thus, a change as subtle as a single serine to alanine, and the concomitant loss of the hydroxyl group, can produce a variant antigen to which a monoclonal antibody no longer binds. It is not possible to formulate rules about the effects of general or specific amino acid
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sequence changes in an antigen on the binding of a particular antibody. In some cases, subtle changes can have large effects and in other cases more substantial changes have little effect. A particular outcome depends on the structural context of the mutated amino acid within the antibody-antigen interface.
ANTIBODY-ANTIGEN-COMPLEXES X-ray crystallography of antibody-antigen complexes has allowed a direct examination of the type and extent of chemical interactions which form. In several cases, it has also enabled a comparison of the bound and free forms of the antibody. The diverse nature of antigen and the capacity of antibody to accommodate it suggests that conclusions from a handful of structures will not paint a complete picture. Antibody-antigen interfaces are extensive (Figure 5). The lower limit of buried surface area between two interacting protein molecules in a biological complex stands at around 1200A^, i.e., ~600A^ from each partner. Buried surface areas in antibody-protein interfaces are typically somewhat higher than this, but the figure may be a little less for antibody-peptide interactions. The physico-chemical nature of the interaction can include hydrophobic interactions, hydrogen bonding and ion pair formation. In the handful of protein antigen-antibody complexes studied in three dimensions at this time, there are between ten and fifteen hydrogen bonds, and up to three ion pairs within the interfaces. There is an unusually high proportion of aromatic residues within the CDRs of the VL and VH domains. In comparison, aliphatic amino acids contribute less to the binding site. This may relate to the fact that antigen binding to antibody will reduce the conformational entropy of side chains within the binding site by virtue of restricting rotational freedom around side chain bonds. Aromatic amino acids can contribute large buried surface areas to the interface with minimal unfavorable entropic consequences. Not only do the surfaces display a measure of chemical complementarity, but their shapes are also complementary (Figure 5). This means that water molecules are generally excluded from the buried interior of the interface. This exclusion is not absolute and, in addition, water molecules can bridge the antibody and antigen around the solvent exposed perimeter of the interface. The antibody binding surface of protein antigens is usually comprised of several discontinuous peptide segments (2-5 in currently available structures). The antibody does not always engage all six of its CDRs in forming the complex. In cases studied so far, CDRs H3 and L3 are always participating in binding antigen, and at least two other CDRs also contribute. Sometimes an amino acid outside of the CDRs, but structurally adjacent to them, forms part of the contact surface with the antigen. Anti-peptide antibody complexes with peptide have been observed to bind peptide either in extended or folded conformations. Buried surface areas are somewhat less than the 1200A^ lower limit of protein-protein interfaces and the number of amino acid residues of the peptides seen to interact specifically with the antibody is about half the usual 15 or so seen in protein antigen-antibody com-
The Antigen-Antibody Complex
117
Figures, Antibody-antigen interfaces are large surfaces of complementary shape and chemistry. Here the influenza virus neuraminidase is shown on the left with the variable domains of an antibody on the right. The right hand view has separated the antigen and antibody by 8A to illustrate the shape complementarity of the interfaces. In this example, antigen and antibody both bury 900 A^ of their surface in the interface.
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plexes. In the case of antibody-peptide interactions, the binding site on the antibody is not so much the surface of the six CDRs as a groove running between the V^ and VH domains. For low molecular weight haptens, the binding site is usually a pocket near the center of the CDR surface. The interactions between antibodies and small antigens are more reminiscent of enzyme-substrate interactions than of complexes between macromolecules. In regard to the chemical and physical complementarity of the interfaces there is little to distinguish an antibody-antigen complex from other known protein-protein or protein-ligand complexes. During secondary immune responses, somatic hypermutation introduces additional diversity into the antibody repertoire, and from these somatic variants antibodies with higher affinity to the antigen are selectively produced. The sites of amino acid sequence changes in the antibody introduced in this way are sometimes within the CDRs and sometimes within framework regions of the variable domains. In the case of changes in the CDRs it is likely that an antibody-antigen interface residue has been altered. It is also likely that the cause of the increased affinity is a local improvement in physical and/or chemical fit around the site of the mutated residue, rather than any gross rearrangement of the antibody on the antigen. Where the somatic changes are not in CDRs it may be plausible to argue an indirect effect on affinity for antigen. One example of an indirect effect on CDR structure is in VH domains, where the size of a residue in the DE comer of the domain influences the conformation of CDR H2. Similar 'knock-on' effects have been observed in the structures of dimers of VL domains where sequence changes in CDR L3 affect the structure of CDR L2. Binding studies reveal that the affinity of Fv fragments for antigen may be several fold weaker than of the parent Fabfi-agment.Fv fragments have been crystallized in complex with antigen, and studies of these structures show that they bind to antigen in the same way as the Fab fragment from which they derive. Genetically engineered Fv fragments can now be used to probe many aspects of antibody-antigen binding and recognition, including the contributions of individual amino acids to the binding energy. In protein-ligand complexes generally it is observed that sometimes the complementarity of the partners pre-exists quite precisely prior to engagement. In other cases, conformational changes in the protein are required to achieve complementarity. These conformational changes frequently involve movements of side chains on the surface of the protein and sometimes also include changes in the conformation of the polypeptide backbone. Both types of conformational change have been seen to occur in the CDRs of antibodies as a consequence of antigen binding. However, there is an additional and unique dimension to the capacity of antibody to adapt its shape to an antigen. In some cases it has been observed that the VL-VH interface is perturbed by binding antigen. Thus, in addition to local structural effects within the CDRs, the three VH CDRs are able to be moved en bloc with respect to the three VL CDRs. This movement in some cases is as much as a few A, i.e,
The Antigen-Antibody Complex
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approaching the distance between adjacent amino acids on a polypeptide chain (3.8A). Since the purpose of such a rearrangement of the VL-VH interface is to bind antigen, it is not surprising that the magnitude and direction of the movement is different in different complexes, and in some cases may be very small or zero. The special design features of the VL-VH interface described earlier appear to allow it to function as an adaptor whose purpose is to improve the binding of poorly fitting structures. When ligands bind to proteins at subunit or domain interfaces, it is commonly observed that some rearrangement of the subunit or domain structures occurs. Such quaternary structure changes are a special feature of allosteric proteins where the altered arrangement of subunits usually affects the affinity of the protein for ligand binding at remote sites. Hemoglobin is the best studied structural example of this phenomenon. There is no evidence to suggest that the antibody adaptor is functioning in this way. Unlike allosteric proteins where a single ligand induces a specific quaternary structure change, antibodies utilize the adaptor to maximize interaction with an antigen, resulting in quaternary structure changes which are antigen and antibody dependent. As important as this adaptor may be for binding particular antigen, it should not, and apparently does not, seriously compromise the specificity of the immune response. However, there are some situations in which one antibody is able to bind two quite different antigens. Antiidiotypy is an example of this. If antibody 1 (Abl) is raised against antigen (Ag) and Ab2 against the idiotype of Abl, it might be expected that Ab2 would resemble Ag, since both are bound by Abl. Such resemblances are rare although some are known. They require not only that Ab 1 is rigid, at least in the sense that its structure is identical in complex both with Ag and Ab2, but also that all of the amino acids of Ab 1 in the interface with Ag or Ab2 are behaving in the same way, e.g. an asparagine residue should in both cases donate hydrogen bonds to the interface. More common is the observation that Ag and Ab2 are structurally unrelated. Structural differences of Ab 1 in the two complexes is one possible explanation for this, and different physico-chemical utilization of particular amino acids of Abl in the two complexes is another.
SUMMARY The antigen binding site of antibodies is a chemically and structurally variable surface of amino acids located at the extremities of the arms of the -Y- shaped molecule. Six hypervariable loops (Complementarily Determining Regions) contribute to this surface and determine the specificity of the antibody. Three of these CDRs are on the VH domain and three are on the V^ domain, and their relative positions in space are determined by the interactions at the VL-VH dimer interface. That interface has unusual architectural features when compared with the database of protein structures.
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Antibodies behave like other protein molecules when they associate with a ligand. They may change their structure, at the level either of the peptide backbone or side chain conformation, to achieve improved fit with the antigen. In addition, the VL and VH domains themselves may undergo some rearrangement across the VL-VH interface upon binding an antigen. The VL-VH interface plays the role of an adaptor, permitting movement of VL CDRS relative to VH CDRS SO that shape complementarity of the entire CDR surface to the antigen can be optimized. The diversity of CDR sequences, coupled with the ability to adopt a number of related conformations, results in a formidable armada of antibody specificities.
RECOMMENDED READINGS Colman, P.M. (1988). Structure of antibody-antigen complexes: Implications for immune recognition. Adv. Immunol. 43, 99-132. Colman, P.M. (1989). Neuraminidase enzyme and antigen. In: The Influenza Viruses (Krug, R.M., Ed.) pp. 175-218. Plenum N.Y. Colman, P.M. (1991). Antigen-antigen receptor interactions. Curr. Opin. Struct. Biol. 1, 232-236. Davies D.R., & Chacko, S. (1993). Antibody Structure. Ace. Chem. Res., 26, 421^27. Herron, J.N., He, X.M., Ballard, D.W., Blier, P.R., Pace, P.E., Bothwell, A.L.M., Voss, E.W., Jr., & Edmundson, A.B. (1991). An autoantibody to single-stranded DNA: Comparison of the threedimensional structures of the unliganded Fab and a deoxynucleotide-Fab complex. Proteins: Structure, Function, and Genetics 11, 159-175. Stanfield, R.L., Takimoto-Kamimura, M., Rini, J.M., Profy, A.T., & Wilson, I.A. (1993). Major antigen-induced domain rearrangements in an antibody. Structure 1, 83-93. Tulip, W.R., Varghese, J.N., Baker, A.T., van Donkelaar, A., Laver, W.G., Webster, R.G., & Colman, P.M. (1991). Refmed atomic structures of N9 subtype influenza virus neuraminidase and escape mutants. J. Mol. Biol. 221, 487-497. Tulip, W.R., Varghese, J.N., Laver, W.G., Webster, R.G, & Colman, P.M. (1992). Refmed crystal structure of the influenza virus neuraminidase-NC41 Fab complex. J. Mol. Biol. 227, 122-148. Wharton, S.A., Weis, W., Skehel, J.J., & Wiley, D.C. (1989). Structure, function and antigenicity of the haemagglutinin of influenza virus. In: The Influenza Viruses (Krug, R.M., Ed.), pp. 153-174. Plenum, New York.
Chapter 7
The Major Histocompatibility Complex BRIAN D. TAIT
Introduction Basic Genetic Structure of the MHC Class 1 Region Class 11 Region Class III Region Protein Structure of MHC Molecules Class I Molecules Class II Molecules MHC Polymorphism and Nomenclature Class I Class II Techniques for Detecting MHC Polymorphism Serology Molecular Techniques Function of Class I and Class II Molecules HLA in Transplantation Matching
Principles of Medical Biology, Volume 6 Immunobiology, pages 121-136. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-811-0
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Sensitization HLA and Disease Associations Molecular Mimicry Restriction of Antigen Presentation Class III Association Summary
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INTRODUCTION The major histocompatibility complex (MHC) is found in all vertebrate species and its fundamental biological function is the recognition of self from non-self As such, the MHC plays a pivotal role as a regulator of immune function. This role is achieved by a variety of molecules that are the products of a gene cassette with varying but coordinated functions. In the human, the MHC gene cluster is found on the short arm of human chromosome 6. The majority of the genes which comprise the human MHC are polymorphic, the degree of polymorphism varying between loci. The clinical relevance of MHC polymorphism is threefold. First, the study of MHC polymorphism and its effect on the functioning of the various products of the MHC gives insights into how these molecules operate at the molecular level in normal immune responses. Secondly, many human diseases, particularly those of autoimmune nature, show associations with particular MHC alleles; the study of these associations can assist in the understanding of these disease processes and the role MHC products play. Thirdly, some of the MHC gene products are powerful stimulators of alloresponsiveness in the clinical transplant situation. This alloresponsiveness takes the form of both antibodies and cytotoxic T cells directed at polymorphic regions of certain MHC molecules. These various facets of the MHC will be discussed in this chapter.
BASIC GENETIC STRUCTURE OF THE MHC The MHC consists of 4,000 Kb of DNA on the short arm of human chromosome 6 and is divided into three classes of genes based on structure and function (Figure 1). The genes within the MHC are mherited in a co-dominant fashion, i.e., alleles on both chromosomes code for a protein product. The combination of MHC genes on one chromosome is termed a haplotype. Class I Region
HLA (originally termed histocompatibility locus -A) -A,B,C are three highly polymorphic genes within the class I region. The HLA-A gene was the first gene discovered in the human MHC, one allele of which was shown to code for the
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emerged. Despite this long history, new discoveries continue to be made and this superficially simple system continues to surprise. In this chapter I will provide a concise but up-to-date summary of the complement system, focussing on the activation of the system, its regulation, its biologically active products and its involvement in disease. Complement plays a central role in innate immune defense. The primary purpose of complement is to kill invading microorganisms but it is also important in the mediation of inflammatory reactions and the clearance of immune complexes. Complement, via interactions with other components of the immune system, also plays a role in the development of immunity. The complement system consists of a group of eleven soluble plasma proteins which interact with one another in two distinct enzymatic activation cascades (the classical and alternative pathways) and in the non-enzymatic assembly of a cytolytic complex (the membrane attack pathway) (Figure 1). Control of these enzymatic cascades is essential to prevent the rapid consumption of complement in response to trivial stimuli and is provided by ten or more plasma and membranebound inhibitory proteins which act at multiple stages of the system to regulate activation (Table 1). In the first section of this chapter I will describe the activation of the system, the components of the activation and membrane attack pathways and their interactions. In the second section I will discuss the control of the pathway and the regulatory proteins involved. In the third section I will outline the physiological importance of complement, its role in host defence and the biological activities of the products of activation. In the final section I will focus on pathological processes involving complement.
ACTIVATION OF COMPLEMENT The Classical Activation Pathway Activation of CI
The classical pathway is so called because it was the first activation pathway to be recognized. The pathway is initiated by the binding of CI to an activator (Hughes-Jones, 1986; Loos, 1988). Antibody bound to antigen is the best known activator of the classical pathway but many other substances can also bind CI and initiate the classical pathway and these non-antibody activators may be of considerable physiological and pathological significance (Taylor, 1993). CI is a large heterooligomeric complex (molecular weight approx. 800 kD) consisting of a single molecule of Clq and two molecules each of Clr and Cls (Ziccardi, 1983; Arlaud et al., 1990). These components are held together noncovalently in a Ca^^-dependent complex (Figure 2). Clq is itself composed of six copies of each of three distinct polypeptide chains (A, B, and C). One copy each of the A, B and
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Bound C1
Figure 2. Structure and activation of C I . The fluid-phase CI complex, consisting of one molecule of C1 q and two molecules each of CI r and C1 s, binds via at least two of its six 'heads' to immunoglobulin. (1) binding causes conformational changes in C l q , resulting in activation; (2) this causes conformational changes and activation of the first CI r molecule which (3) cleaves and activates the second CI r which in turn (4) cleaves and activates CI s. Reproduced from Morgan (1990). Complement: Clinical aspects and relevance to disease. Academic Press, London, with permission.
C chains are wound around each other in a triple helix containing an extended, collagen-like tail and a large globular head (Reid and Porter, 1981). In the intact Clq molecule the collagen tails of the six triple helices are tightly associated along the amino terminal half but then diverge to form six connecting strands, each bearing a globular head. This complicated molecule is encoded by three closely linked genes present on chromosome Iq in the order A-C-B (Sellar et al., 1991). Clq binds via its globular heads to the Fc portion of IgG. Activation requires the binding of multiple heads of a C1 q molecule by aggregates of IgG. This multivalent interaction greatly increases the strength of binding and triggers conformational changes within Clq which induce activation. IgM is a multivalent molecule and can thus activate Clq efficiently and without the need for aggregate formation. Among the human IgG subclasses the ranking of efficiency for activation of CI is IgG3 > IgGl > IgG2; IgG4 does not activate CI. Clr and C Is are homologous, single chain molecules of molecular weight 80 kD which, in the presence of Ca^"^, form an elongated Clr2Cls2 complex which sits
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between the globular heads of Clq (Figure 2) (Reid, 1986; Sim and Reid, 1991). They are encoded by closely linked genes on chromosome 12. Conformational changes within Clq upon binding antibody allow the auto-activation of the proenzyme Clr, a process which involves cleavage at a single site within the molecule, thereby revealing the active site. Clr then activates Cls in the complex, again by cleaving at a single site in the molecule (Sim and Reid, 1991). Of the non-antibody activators of CI, mannose binding protein (MBP) is of particular importance. MBP is a high molecular weight serum lectin made up of many copies of a single 32 kD chain (Taylor et al., 1989). Its role in vivo is to bind mannose and N-acetyl glucosamine residues in bacterial cell walls. Once bound, MBP can develop the capacity to activate the classical pathway of complement either by binding Clr and Cls to form a MBP-Clr2-Cls2 complex or by binding a novel protein termed MBP-associated serine protease or MASP (Matsuhita and Fujita, 1992). MBP thus provides a rapid, antibody-independent means of activating the classical pathway on bacteria and deficiency is associated with defective bacterial opsonization and repeated infections (Turner, 1991). Other lectins have also been shown to activate complement, leading to the proposal that they represent a distinct complement activation pathway (Holmskov et al., 1994). Binding and Cleavage of C4 The next component in the classical pathway is C4, a large plasma protein (200 kD) which consists of three disulfide-bonded chains (a, (3 and y) (Shreiber and Muller-Eberhard, 1974; Janatova and Tack, 1981). C4 is synthesized as a singlechain precursor which is processed to give the three-chain molecule prior to secretion. The gene for C4 is in the class III region of the major histocompatibility complex (MHC) on the short arm of chromosome 6. In fact, C4 is encoded by two closely linked genes which give rise to the two isotypic variants, C4A and C4B (Campbell et al., 1990). These variants differ by only six amino acids but this small difference causes significant changes in function, C4A binding preferentially to amino groups after cleavage and C4B to hydroxyl groups (see below). C4B is also more efficient in propagating continued activation of complement. C1 s cleaves fluid-phase C4 at a single site near the amino-terminus of the a chain, releasing a small fragment, C4a and in the process exposing a reactive thiolester group in the a chain of the large fragment, C4b. Exposure of this reactive group bestows upon C4b the capacity to bind to membranes or other surfaces (Law et al., 1980; Law and Dodds, 1990). The thiolester group forms covalent amide or ester bonds with exposed amino or hydroxyl groups respectively on the activating surface (Figure 3). The thiolester is extremely short-lived due to its susceptibility to inactivation by hydrolysis. As a consequence, C4b binding to surfaces is a very inefficient process, most of the C4b formed decaying in the fluid phase and C4b binding being restricted to the immediate vicinity of the activating CI complex.
178 ^
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Figure 3. Activation and decay of C3. A. Activation of C3 involves a single cleavage in the a-chain mediated by the C3-convertase of the classical or alternative pathway. A small fragment, C3a, is released and a thiolester group is exposed in the large fragment C3b. Decay of C3b is mediated by factor I which, in the presence of an appropriate cofactor (factor H, CR1 or MCP), cuts at 2 sites in the a'-chain, releasing a 2 kD fragment, C3f, and yielding iC3b (i = inactive). A further cleavage by factor I releases C3c, leaving the fragment C3dg (35 kD) attached to the activator surface. Finally, serum proteases cleave a small fragment (C3g, 5 kD) from C3dg. B. Detail of the thiolester group formed by residues 988-992 in the C3d region of the a-chain of C3. A similar thiolester moiety is also found in the structurally related proteins C4 and a2-macroglobulin.
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Binding and Activation of C2
The next component of the classical pathway is C2, a single chain plasma protein of molecular weight 102 kD which, like C4, is encoded by a gene in the class III region of the MHC on chromosome 6. The gene for C2 is closely linked to that for factor B, its functional homologue in the altemative pathway (see below). Membranebound C4b expresses a binding site which, in the presence of Mg^^ ions, can bind C2 near its amino terminus and present it for cleavage by an adjacent CI complex to yield a 30 kD amino-terminal fragment, C2b, and a 70 kD carboxy-terminal fragment, C2a (Nagasawa and Stroud, 1977). The C2b fragment may be released or remain loosely attached to C4a but is not required for enzymatic activity. The C2a fragment binds to C4b through a newly exposed site near its new amino terminus to form the C4b2a complex, the next enzyme in the classical pathway. Binding and Cleavage of C3
The penultimate component of the classical pathway, C3, is the most abundant of the complement components (1-2 mg/ml in serum). It is a large (185 kD) molecule composed of two chains (a, 110 kD and P, 75 kD) held together by disulfide bonds (Lambris, 1988). Like C4, with which it shares many structural features, C3 is synthesized as a single chain precursor molecule and cleaved intracellularly prior to secretion. The gene for C3 is on chromosome 19. C2a in the C4b2a complex cleaves C3 in the fluid phase at a single site in the a chain, releasing a small fragment from the amino-terminus (C3a, 9 kD) and exposing in the large fragment, C3b, a thioester group and binding sites for several complement receptors and regulatory proteins (see below). The labile thioester group confers upon C3b the capacity to bind covalently to the activating surface as described above for C4b (Figure 3). Binding is inefficient and the bulk of the C3b formed decays in the fluid phase to form C3bi ('i' = inactive). Only C3b bound to the activating surface in close proximity to the C4b2a complex takes any further part in activation. Binding and Cleavage of C5
The final component of the classical pathway, and also the first component of the membrane attack pathway, is C5. C5 is a two-chain plasma protein of 190 kD molecular weight, encoded on chromosome 9, which is structurally related to C3 and C4. Like these molecules it is synthesized as a single-chain precursor and cleaved prior to secretion, however, C5 does not contain a thioester group and thus cannot itself bind covalently to surfaces (Law and Dodds, 1990). C3b attached to the membrane binds C5 and presents it for cleavage by C2a in an adjacent C4b2a complex. The C4b2a complex can only cleave C5 bound to C3b and hence the C5 cleaving enzyme (C5 convertase) of the classical pathway is termed C4b2a3b. Cleavage occurs at a single site in the a chain of C5, releasing a small amino-
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terminal fragment, C5a (approx. 10 kD), and exposing on the larger fragment, C5b, a labile hydrophobic surface binding site and a site for binding C6, neither of which are expressed by uncleaved C5 (Hughes-Jones, 1986; Loos, 1988). The Alternative Activation Pathway Initiation of the Alternative Pathway
The alternative pathway provides a rapid, antibody independent route for complement activation and amplification on invading microorganisms and other foreign surfaces. C3 is the key component but two other proteins, unique to the alternative pathway, are also required. Factor B is a single-chain 93 kD plasma protein, structurally and functionally homologous with C2 which, once activated, can cleave C3 at a site identical to that used by the C4b2a enzyme. C2 and factor B have 40% protein sequence identity and are encoded by closely linked genes in the MHC on chromosome 6. Activation of factor B is accomplished by factor D, a 26 kD serine protease enzyme present in plasma in its active form, which cleaves factor B at a single site, releasing a 30 kD fragment, Ba, and exposing a serine protease domain on the large (60 kD) fragment, Bb (Gotze, 1986). Factor D can only cleave factor B bound to C3b. Initiation of the alternative pathway on a surface therefore requires that C3b be deposited in a conformation which allows factor B to bind and be cleaved by factor D. How then is the initiating C3b formed? In many circumstances some degree of classical pathway activation may have occurred, depositing sufficient C3b to trigger the alternative pathway. Nevertheless, it appears that the alternative pathway can be initiated independent of the classical pathway. In biological fluids C3 is continuously hydrolysed at a slow rate to form a metastable C3 (H2O) molecule which, in the presence of Mg^"^ ions, can bind factor B in solution and render it susceptible to cleavage by factor D. Bb in the fluid phase C3 convertase thus formed (C3(H20)Bb) cleaves C3, releasing C3a and exposing the thioester group in C3b which can then bind to adjacent surfaces (Lachmann and Hughes-Jones, 1984; Law and Dodds, 1990). As a result of this "tickover" phenomenon C3b is continuously deposited in small amounts on all cells in the body. Amplification on Activator Surfaces
Deposition of C3b on host cells does not result in continued activation because the surface features do not favor a binding of factor B (non-activator surfaces) and bound C3b decays, a process catalyzed by fluid-phase and membrane inhibitors (see below). In contrast, the surface features of many microorganisms and foreign cells favor amplification (activator surfaces). C3b binds factor B (Mg^'^-dependent) and presents it for cleavage by fluid-phase factor D, thus forming the alternative pathway C3 convertase C3bBb which cleaves more C3. Activating surfaces thus rapidly become coated with C3b molecules, each one of which can itself recruit
Activation and Control of the Complement System
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factor B to form more convertases and further amplify activation. The precise nature of the surface features which determine whether activation will occur are still not clear; however, surface carbohydrates, particularly sialic acid, appear to be important (Pangbum and Muller-Eberhard, 1984; Pangburn, 1986). Cleavage of C5 As in the classical pathway, bound C3b acts as a receptor for C5, enabling the cleavage of C5 by Bb in an adjacent C3bBb complex. The site of cleavage is identical to that caused by C2a in the classical pathway convertase (C4b2a3b). C5a is released and labile membrane- and C6-binding sites are exposed on C5b as described above for the classical pathway. The Membrane Attack Pathway
Cleavage of C5 by the C5 convertase of either the classical or alternative pathways is the final enzymatic step in the complement cascade. The membrane attack pathway involves the non-covalent association of C5b with the four terminal complement components, all of which are hydrophilic plasma proteins, to form an amphipathic membrane-inserted complex. While still attached to C3b in the convertase C5b binds C6, a large (120 kD) single chain plasma protein. Binding of C6 stabilizes the membrane binding site in C5b and exposes a site for C7, a 110 kD single chain plasma protein which is homologous to C6 (see below). Attachment of C7 causes conformational changes in the complex which result in its release from the convertase to the fluid phase and the transient expression of a hydrophobic membrane binding site. If the C5b67 complex encounters a membrane during the brief lifetime of this site, it binds tightly. However, the complex does not penetrate deeply into the membrane and does not disturb the integrity of the lipid bilayer. In addition to the natural decay of the membrane binding site, attachment of C5b67 is further limited by the tendency of the complex to aggregate and by the presence of multiple fluid-phase inhibitors (see below). Deposition of C5b67 is thus limited to the target cell. A small proportion of complexes may attach to the membranes of closely apposed host cells and cause damage or lysis, so-called innocent bystander lysis (Podack and Muller-Eberhard, 1978). The penultimate component of the membrane attack pathway, C8, is a complex molecule made up of three chains, a, p and y (molecular weights 65 kD, 65 kD and 22 kD respectively), encoded by separate genes, a and p closely linked on chromosome 1, y on chromosome 9. The a and p chains are homologous with each other and with C6 and C7 whereas C8y displays no homology. The a and y chains are covalently linked whereas the P chain is non-covalently associated in the complex. The P chain in C8 binds C7 in the C5b67 complex and the resulting complex, C5b-8, becomes more deeply buried in the membrane and forms small pores, causing the cell to become slightly leaky.
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The final component in the pathway, C9, is a single chain plasma protein (molecular weight 69 kD) which is homologous with C6, C7, C8a, and C8(3. The first C9 molecule to enter the C5b-8 complex binds to C8a and undergoes a major conformational change from a globular, hydrophilic form to an elongated, amphipathic form which traverses the membrane and exacerbates membrane leakiness. Unfolding of C9 also exposes binding sites which enable additional C9 molecules to bind, unfold and insert in the membrane. The pore thus grows with the recruitment of additional C9 molecules, individual complexes (membrane attack complexes, MACs) containing anything up to 18 C9 molecules. MACs containing multiple C9 molecules can be visualized in the electron microscope as ring-like structures enclosing a 10 nm pore. Evidence that the pore is formed mainly from C9 molecules is provided by the observation that C9 incubated in vitro in the presence of Zn^"^ ions forms identical ring structures (Podack, 1988; Esser, 1990). The exact mechanism by which the MAC causes lysis is still the subject of debate between those who state that the MAC ring surrounds a rigid, transmembrane pore and those who consider that the MAC induces areas of lipid perturbation (leaky patches) in the membrane (Bhakdi and Tranum-Jensen, 1991; Esser, 1991). Whatever the exact mechanism, the MAC forms functional pores in cell membranes through which ions and small molecules pass, bringing about osmotic lysis of the cell. This strategy of pore formation as a means of killing targets is widely used in nature, notably by bacterial toxins and by mammalian lymphocytes (Peitsch and Tschopp, 1991). The MAC component proteins C6, C7, C8a, C8p and C9 are all highly homologous and have clearly arisen from a common ancestor. C6 and CI are closely linked on chromosome 5 with C9 more distant on the same chromosome (Rogne et al., 1991). C8a and C8p are closely linked on chromosome I. These duplication events have probably arisen to increase the efficiency of targeting and of cytolysis by the MAC.
CONTROL OF THE COMPLEMENT SYSTEM As a consequence of the proteolytic cascade nature of the complement system, small initiating events could result in catastrophic activation of the system. In order to prevent this each part of the complement system is tightly controlled. This control is mediated in part by the inherent instability of the activation pathway enzymes but is also provided by a number offluid-phaseand membrane-associated inhibitors (Figure 4). Control in the Classical Pathway The first step of the classical pathway is regulated by CI-inhibitor (Clinh), a 90 kD plasma protein, a member of the serine protease inhibitor (Serpin) family, which binds to CI and prevents its activation. Clinh also binds activated CI, physically removing Clr and Cls and thus causing disruption of the multimolecular CI
Activation
and Control of the Complement Classical pathway
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183
Alternative pathway
Ct/^----.cT fH C4bp ladder on electrophoresis - commonly buds - decreases - condensation due to extrusion of water - well preserved
- convolutes and buds - loss of microvilli - rounded apoptotic bodies
engulfed by monocytic macrophages without inflammation
Necrosis -scattered mild clumping - random degradation => smear on electrophoresis - never buds - increases - swelling as membrane pumps fail - swelling of endoplasmic reticulum and mitochondria - ribosomes disappear -cell lysis - cell debris inititiates acute inflammatory response
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R.M. KLUCK and J.W. HALLIDAY
contents which initiate further cell damage and inflammation, with eventual clearance by macrophages (see Table 1). Biochemistry of Apoptosis The biochemistry of apoptosis needs to be considered from two perspectives, (1) the molecular changes that either predispose a cell to or protect it from apoptosis and (2) the molecular changes that occur in the cell when it undergoes apoptosis. Distinguishing between these two areas is difficult because the biochemical stage at which an individual cell is irreversibly committed to undergo apoptosis is unknown. Arends and Wyllie (1991) conceptualize cells "primed" for apoptosis as those that have a molecular profile which makes them susceptible to the irreversible "triggering" of the apoptotic pathway. In 1980, Wyllie observed that DNA extracted from apoptotic thymocytes was cleaved at linker regions between nucleosomes, possibly by a Ca^'*"-dependent endonuclease. This cleavage results in a characteristic ladder pattern on gel electrophoresis (Figure 2) and contrasts with the random DNA degradation seen following necrosis (Figure 2). More recently, it has been shown that DNA cleavage to large fragments of 50 and 300 Kb in size is an earlier and more universal feature of apoptosis. This cleavage of DNA in the dying cell provides a means of deleting potentially dangerous genetic material. Inhibitors of protein synthesis inhibit, or delay, apoptosis in some cells encouraging the concept of apoptosis as an active, and programmed, process. However, an essential role for any specific newly synthesised protein has not been verified (Schwartz and Osborne, 1993). The susceptibility (priming) of cells to apoptosis has been shown recently to be influenced by the activity of several proteins in the cell. Three of these proteins are products of cellular oncogenes c-myc, bcl-2, andp55 found in all cell types and are involved in intracellular biochemical pathways. Activity of these proteins is determined by both the rate of gene expression and the rate of degradation, with genetic mutations commonly affecting activity. Susceptibility to apoptosis has been associated with high c-Myc, high p53 and low Bcl-2 protein levels; current investigation of the biochemical properties of these and other proteins suggests great potential for manipulating the apoptotic process. With respect to immune cell development, survival of immature B and T cells seems to require increased bcl-2 expression. The expression of the tumor suppressor gene p5 3 increases in response to DNA damage with a resulting increase in cell death by apoptosis. In mice in which both copies of the p53 gene have been deleted, apoptosis following DNA damaging agents does not occur. Somatic mutations leading to increased c-Myc, increased Bcl-2, and to mutant p53, have been implicated in the etiology of cancer, possibly consequent upon apoptosis failure. Thus a complex network of gene expression is likely to be involved in priming cells for apoptosis. In addition to the expression of intracellular proteins, the expression of receptor proteins on the cell surface of immune cells and their target cells plays a crucial
269
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10^. However, a phage library can be much larger than this, potentially yielding antibodies with higher affinities simply because the affinities of antibodies isolated from a library is thought to be proportional to the size of the library. Murine Expression Systems Although the bacterial expression system is an excellent tool to express engineered genes, it cannot surpass natural immunization for the ease and efficiency of obtaining monoclonal antibodies because of the laborious screening and manipulation process. One could avoid this problem if it were possible to endow a mouse with a human immune repertoire, thereby obtaining human monoclonal antibodies directly. For this approach to work, the mouse needs to tolerate the human proteins
Designer Antibodies
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in order to prevent rejection. Mice that are immunocompromised or mice carrying the human antigens during development are capable of tolerating an artificially endowed human immune repertoire. Using SCID Mice and Transgenic Mice Severe combined immune deficiency (SCID) mice have a defect in immunoglobulin gene rearrangement, resulting in mice without T or B cells. These mice have recently been populated with human peripheral blood leukocytes (PBL) from donors that have not been boosted with tetanus toxoid (TT) antigen for 17 years (Duchosal et al., 1992). Immunization of the human PBL-populated SCID mice (hu-PBL-SCID mice) with a tetanus toxoid booster shot resulted in a significant rise in serum IgG anti-TT levels. B cell RNA was extracted from these mice and used to make a combinatorial library. Screening of the Fab library demonstrated Fab fragments with affinities equal to those antibodies derived directly from boosted individuals. This method of generating high affinity antibody fragments for hu-PBL-SCID mice could be useful in the production of human monoclonal antibodies for therapy. For example, expanded repertoires of anti-HIV antibodies against specific epitopes could be generated by peptide stimulation of SCID mice populated with PBL from seropositive donors, or human autoantibodies could be obtained using appropriate autoimmune donors. Transgenic mice are another valuable tool to molecular biology that could help in the production of human monoclonal antibodies. Unrearranged human antibody genes can be injected into fertilized mice ova. The resulting transgenic mice can then be immunized to produce human antibodies. The spleen cells can be fused to mouse myeloma cells to generate hybridomas that secrete human antibodies. One potential drawback of this approach is that the size of the human Ig gene repertoire in the transgenic mice may be limited by the amount of new DNA that can be carried by the transgenic animal.
DESIGNER ANTIBODY-TARGETED EFFECTOR FUNCTIONS Another rapidly developing field in designer antibodies is the use of antibodies to target an effector molecule to a specific cellular antigen. Our immune system does this naturally in the form of antibody-dependent cell-mediated cytotoxicity and complement-mediated lysis. With the creation of fiision proteins between antibodies and effector molecules such as toxins, radioisotopes, cytokines, enzymes, and other antibodies, novel effector functions can be created. Recent advances have even allowed for targeting antibody molecules to specific intracellular locations. Immunotoxins An immunotoxin is an antibody chemically or genetically conjugated to a toxin for the purpose of delivering a "lethal hif to a specific cell type (Figure 1, bottom).
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ANDY MINN and JOSE QUINTANS
Generally these toxins are of plant or bacterial origin. Ricin is derived from the beans of the Ricinus communis plant, and is capable of inhibiting protein synthesis by decreasing the affinity of 28S ribosomal RNA for elongation factor-2, rendering the ribosome nonfunctional. Pseudomonas exotoxin and diptheria toxin are two bacterial toxins that also prevent protein synthesis by inactivating elongation factor-2 through ADP-ribosylation. When conjugated with an antibody, all of these toxins can be specifically targeted to cells through antibody binding and endocytosis. Often the toxin must be modified to reduce non-specific targeting. For example, sugar moieties on the toxins can bind to receptors on hepatocytes and cells of the reticuloendothelial system; therefore, these sugar residues must be removed. Another problem that is not solved so easily is that the toxin or the antibody used can be immunogenic. However, humanization of immunotoxins has been described (Rybak et al., 1992). A chimeric antibody to the human transferrin receptor was fused to angiogenin, a human homologue of pancreatic RNase, and was effective at inhibiting protein synthesis in cells specifically expressing the transferrin receptor (This receptor is found in high numbers on rapidly proliferating cells). Since this immunotoxin is a chimeric antibody fused to a human protein, it may bear importantly in designing human therapeutic strategies for reducing potential immunogenicity in traditional immunotoxins that incorporate plant or bacterial toxins. Complete tumor regression by immunotoxins has been observed in some animal studies; however, overall, immunotoxin therapy seems to work best with tumors such as leukemias, that are readily accessible to the circulation. Immunotoxins have been rather ineffective in killing solid tumor masses. Additionally, the side effects of immunotoxins remain a significant impediment to their use in human therapy. Indeed, unconjugated antibodies are sometimes better suited than immunotoxins as therapeutic agents. Other Antibody-Effector Molecule Conjugates Radioisotopes can also be effector molecules. In a recent phase I study, a radioiodinated B cell specific antibody was successfully used in 10 patients with non-Hodgkin's lymphomas unresponsive to primary chemotherapy (Kaminski et al., 1993). Four of the treated patients had complete remissions, with no evidence of myelosuppresion. This type of treatment holds promise for improving standard chemotherapy by decreasing toxic side affects and increasing efficacy. As an attempt to reduce the risk of non-specific toxicity of immunotoxins, antibodies have been conjugated to enzymes that convert a prodrug into its active form. In this way, the antibody is first injected and given enough time to find its target and for the excess antibody to clear. The prodrug is then injected and converted to its active form only at the sites where the antibody is bound. Such antibody targeted prodrugs have been shown to be more effective in eliminating tumors than the chemotherapeutic drugs alone.
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Although some cancers may have tumor-specific antigens, the immune system might not be effective in rejecting the tumor because the antigens do not elicit strong immune responses. Recently, Tao and Levy (1993) demonstrated a promising way to improve the potency of the immune system against weak tumor immunogens. They fused the gene for GM-CSF, a cytokine thought to augment antigen presentation in a variety of cells, to the gene for an antibody specific for the variable regions of the immunoglobulin molecules expressed on malignant B cells in order to increase the immunogenicity of the tumor. The fusion protein elicited a strong antibody response and protected mice from subsequent challenge with tumor cells bearing the tumor antigen. These results could have important implications in the design of cancer vaccines. Bispecific Antibodies
A bispecific antibody has two specificities, resulting from the fusion of two hybridomas, or chemical conjugation of two antibodies with different specificities (Figure 1, bottom). Bispecific antibodies can have useful therapeutic applications. For example, having one antigen binding site that is specific for a toxin, drug, enzyme, or radioisotope, can provide an alternative to chemical conjugation of effector molecules. A less obvious use is recruiting effector cells to cellular targets. One antigen binding site can be used to locate a tumor cell, while the other antigen binding site can be used to recruit a cytotoxic T cell or NK cell by recognizing a cell surface protein on the cytotoxic effector cell. Bispecific antibodies may enable the killing of target cells by cytotoxic T cells in an MHC independent manner if they engage a common cell surface antigen such as CD3 (a molecular complex involved in T cell signal transduction). This approach could prove useful in killing tumor cells that have lost MHC expression and as a result are capable of escaping CTL surveillance. The advantages of antibody gene fragments ranging from the ability to use a bacterial expression system to the elimination of Fc reactivity, are also available for use with bispecific antibodies. Winter's group has produced bivalent and bispecific antibody fragments called "diabodies" (Holliger et al., 1993). Diabodies are made by linking a VH and VL domain from different antibodies by a short polypeptide chain (similar to scFv fragments) that is too short to allow pairing of the two V domains (Figure 1, bottom). The two domains are then forced to associate with a complementary Vn-short linker-VL chain that is coexpressed in a bacterial expression system. Winter's group made a double construct: they combined a VH domain gene segment from a monoclonal antibody specific for phenyloxazolone with the gene for the VL domain from a monoclonal antibody specific for hen egg lysozyme, and reciprocally, combined the VL domain gene of the phenyloxazolone antibody and linked it to the VH domain gene of the hen egg lysozyme antibody. The two scFv associated and were shown to bind specifically to phenyloxazolone and/or hen egg lysozyme.
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ANDY MINN and JOSE QUINTANS Intracellular Antibodies
Normally, antibodies are secreted and bind to antigens in the extracellular milieu. The idea of using an antibody to bind an intracellular target has been known for more than a decade; however, it was not until recently that advances in antibody engineering made the use of intracellular antigens much more feasible and promising. Marasco et al. (1993) have illustrated the use of intracellular antibodies by designing an antibody that acts intracellularly to prevent the processing of the HIV envelope protein gpl20, a crucial protein the HIV virus uses to attach and infect target cells. Marasco accomplished this by designing a single chain Fv fragment from an antibody known to bind gpl20 and linking it to a leader sequence so as to target it to the endoplasmic reticulum, the site where gpl20 is processed from its precursor, gpl60. Once expressed in the endoplasmic reticulum of a cell expressing high levels of the envelope protein, the single-chain antibody bound gpl60, prevented its cleavage to gpl20, and inhibited gpl20 mediated syncytial formation by 80-90% (some researchers think syncytial formation is at least partly responsible for the loss of immune cells in AIDS). When the intracellular antibody was expressed in cells that were infected with the entire HIV genome, the virus particles released from the cells were more than 1000 times less infectious than ordinary HIV. Also encouraging was the fact that the intracellular antibody appeared to have no toxic effects on cells. Work on designer antibodies such as this illustrates the prospect of using antibodies in gene therapy. Besides the possible approach to AIDS therapy, intracellular antibodies may be beneficial in cancer therapy as many oncoproteins traverse through the ER. Intracellular antibodies that target other intracellular locations besides the ER are also available and may be exploited. One day, antibodies may prove to be important in both humoral and cellular immunity.
CONCLUSION Paul Ehrlich, who was one of the first to appreciate the specificity of antibodies, became particularly intrigued with using antibodies as weapons against cancer and dubbed them "magic bullets." Although few would dismiss Ehrlich's early visions as unrealistic, attaining the vision has not been without formidable obstacles. Fortunately, recent research has brought a plethora of new tools and ideas that are making the effective therapeutic use of monoclonal antibodies much more of a reality. Antibody molecules are well suited for genetic engineering. Their multichain, domain structure, as well as the fact that the antigen binding sites are loops that rest on top of a scaffold of beta pleated sheets, give them modularity. This modularity was first exploited in the forms of chimeric and humanized antibodies, with the goal of ameliorating such side effects as a HAMA response. The expression of engineered antibody genes in prokaryotes is a development that has the potential to bypass animal immunization altogether and allow us to mimic the generation of
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diversity, selection, and affinity maturation. With these tools, antibodies can be manipulated to change their antigenicity, effector functions, pharmacokinetics, localization, and expression. Basic science is providing knowledge of biological mechanisms giving us insight into how to design antibodies to enhance or interfere with these mechanisms to produce desired results. It would seem that Paul Ehrlich's "magic bullets" are closing in on the bull's eye.
ACKNOWLEDGMENTS We would like to thank Phillip Funk for a critical reading of this manuscript.
REFERENCES Duchosal, M.A., Eming, S.A., Fischer, P., Leturcq, D., Barbas, C.F., McConahey, P.J., Caothien, R.H., Thorton, G.B., Dixon, F.J., & Burton, D.R. (1992). Immunization of hu-PBL-SCID mice and the rescue of human monoclonal Fab fragments through combinatorial libraries. Nature (Lond.) 355, 258-262. Holliger, P., Prospero, T., & Winter, G. (1993). "Diabodies": Small bivalent and bispecfic antibody fragments. Proc. Natl. Acad. Sci. USA 90, 6444-6448. Hoogenboom, H.R., & Winter, G. (1992). Bypassing immunisation: Human antibodies from synthetic repertoires of germline V^ gene segments rearranged in vitro. J. Mol. Biol. 227, 381-388. Jones, P.T., Dear, P.H., Foote, J., Neuberger, M.S., & Winter, G. (1986). Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature (Lond.) 321, 522-525. Kaminksi, M.S., Zasadny, K.R., Francis, I.R., Milik, A.W., Ross, C.W., Moon, S.D., Crawford, S.M., Burgess, J.M., Petry, N.A., Butchiko, G.M., Glenn, S.D., & Wahl, R.L. (1993). Radioimmunotherapy of B-cell lymphoma with ['^^I]anti-Bl (anti-CD20) antibody. The New England J. of Medicine 329, 459-465. Kohler, G., & Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256,495-497. Marasco, W.A., Haseltine, W.A., & Chen, S.Y. (1993). Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gpl20 single-chain antibody. Proc. Natl. Acad. Sci. USA 90, 7889-7893. Marks, J.D., Griffiths, A.D., Malmqvist, M., Clackson, T., Bye, J.M., & Winter, G. (1992). By-passing immunization: Building high affinity human antibodies by chain shuffling. Biotechnology 10, 779-783. Marks, J.D., Hoogenboom, H.R., Bonnert, T.P., McCafferty, J., Griffiths, A.D., & Winter, G. (1991). By-passing immunization: Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222, 581-597. Morrison, S.L., Johnson, M.J., Herzenberg, L.A., & Oi, V.T. (1984). Chimeric human antibody molecules: Mouse antigen-binding domains with human constant region domains. Proc. Natl. Acad. Sci. USA 81, 6851-6855. Rybak, S.M., Hoogenboom, H.R., Meade, H.M., Raus, J.C.M., Schwartz, D., & Youle, R.J. (1992). Humanization of immunotoxins. Proc. Natl. Acad. Sci. USA 89, 3165-3169. Tao, M., & Levy, R. (1993). Idiotype/granulocyte-macrophage colony-stimulating factor fusion protein as a vaccine for B-cell lymphoma. Nature (Lond.) 362, 755-757. Williamson, R.A., Burioni, R., Sanna, P.P., Partidge, L.J., Barbas, C.F., & Burton, DR. (1993). Human monoclonal antibodies against a plethora of viral pathogens from single combinatorial libraries. Proc. Natl. Acad. Sci. USA 90, 4141-4145.
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RECOMMENDED READINGS Mayforth, R.D. (1993). Designer Antibodies. Academic Press, Inc., San Diego, California. Mayforth, R.D., & Quintans, J.Q. (1991). Designer and Catalytic Antibodies. New England J. Medicine 323, 173-178. Riethmuller, G., Scheider-Gadicke, E., & Johnson, J.P. (1993). Monoclonal antibodies in cancer therapy. Curr. Opinion in Immunology 5, 732-739. Waldmann, T.A. (1991). Monoclonal antibodies in diagnosis and therapy. Science 252, 1657-1662.
Chapter 16
Psychoneuroimmunology RUTH M. BENCA
Introduction Evidence for Immune System-Nervous System Interactions Conditioned Immune Responses Stress and Immune Function Stress Effects on Humans Conclusion Recommended Readings
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INTRODUCTION The nature and extent of the relationship between the mind and body has been a central issue for the practice of medicine since its beginnings. The early Greeks believed that emotions could influence the course of physical illnesses, and emphasized the importance of an holistic approach. However, from the 17th to 19th Principles of Medical Biology, Volume 6 Immunobiology, pages 303-313. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-811-0
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century, a variety of influences led to the view that disease was exclusively a physical disorder. The philosopher Descartes proposed that mind and body were separate and distinct entities; the impact of Cartesian dualism continues to this day. The major medical discoveries of this period—^including Hook's discovery of the cell itself, the connection between diseased cells and bodily illness described by Virchow, the identification of pathogens responsible for disease by Pasteur and others, and the development of vaccination by Jenner—resulted in an increasing disregard for the idea that psychological processes might play a role in disease states. The modem era of psychosomatic medicine began in the early 20th century with the theories of Freud and others which postulated that psychological conflict could be expressed as physical dysfunction, as in the case of conversion disorders. In the 1930s, Alexander hypothesized that certain medical illnesses were the result of unconscious psychological conflicts, and that specific emotional states resulted in specific physical disorders. The impact of Alexander's work had a major influence on the modern practice of psychiatry; the standard psychiatric nosology included a listing of psychosomafic disorders, "characterized by physical symptoms that are caused by emotional factors and involve a single organ system" as recently as 1968. Although the failure to provide empirical support for the specificity hypothesis of psychosomatic illness gradually led to an abandonment of that theory, evidence for the relationship between psychological factors and illness has continued to accumulate. One of the first mechanisfic explanations relating psychological factors to illness was provided by Selye in the 1940s. He described a general adaptation syndrome to stress which involved activation of the hypothalamic-pituitary-adrenal cortical axis and proposed that excessive stress could result in physical illness. Subsequently, a number of studies have demonstrated significant correlations between stressful life events and increased risk for illness. "Psychosomatic medicine" has gradually been replaced by the biopsychosocial model of illness, first proposed by Engel in the 1950s, which emphasizes the importance of psychological and cultural factors in influencing the onset and course of disease states. Disease has muUifactorial etiologies, including causative and contributory factors, all of which need to be addressed in clinical treatment. Although there has been an exponential increase in our understanding of the molecular basis for disease over the past several decades, far less is known about the mechanisms linking psychological factors with illness. The relatively new field of psychoneuroimmunology represents an attempt to explain one possible route by which emotional states may influence disease, and is based on evidence that the immune and nervous systems may regulate each other.
EVIDENCE FOR IMMUNE SYSTEM-NERVOUS SYSTEM INTERACTIONS The concept of nervous system control over bodily fimctions has long been accepted, with the major exception of the immune system. Unlike most other
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physiological systems which consist of sessile organs hard-wired to the brain, the immune system is comprised of lymphocytes which perform their surveillance functions by wandering throughout the body and communicating via secretory substances or cytokines. Furthermore, lymphocytes can carry out their various functions adequately when removed from the body and placed in tissue culture dishes, suggesting that the nervous system is unnecessary for a functional immune system. However, recent work has elucidated potential pathways for communication between the nervous and immune systems. First, the immune system has the capacity to receive direction from the nervous system. Although lymphocytes themselves do not receive direct neural input, the vasculature of lymphoid organs is extensively innervated. Noradrenergic sympathetic fibers are found in all primary and secondary lymphoid organs. The thymus, which is the primary organ responsible for T cell production, and the secondary lymphoid organs, lymph nodes and spleen, also appear to receive input from a variety of peptidergic fibers, including vasoactive intestinal peptide (VIP), substance P (SP), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP). Cholinergic fibers may also be present in the thymus, lymph nodes, and spleen. The immunologic significance of lymphoid innervation is not known definitively, but it is possible that neural input may affect lymphocyte traffic through effects on vasculature, and thus impact on immune function. This idea is supported by sympathetic denervation studies, which have shown variable effects on immune responses in rodents. In addition to the demonstration of nerve input to lymphatic organs, lymphocytes themselves have receptors for and/or respond to a variety of neurotransmitters, neuropeptides, and neuroendocrine factors. Beta-adrenoreceptors are found on lymphocytes, including T cells, B cells, monocytes, and macrophages. Norepinephrine has been reported to exert enhancing effects on the inductive phase of immune responses, but has significant immunosuppressive effects on the effector phase of mitogen responses, cytotoxic responses, antibody production, interleukin2 (IL-2) induced proliferation, and natural killer cell (NK) activity. It is thought that norepinephrine exerts its effects on lymphocytes by increasing cAMP activity and thus decreasing responses to IL-2; conversely, alpha-adrenergic agonists and beta-endorphins, which decrease cAMP within cells and enhance their responses to IL-2, have more consistent stimulatory effects on immune responses. The variable effects of adrenergic compounds may also be related to the unequal distribution of receptors on lymphocyte subpopulations. The neuropeptides SP and VIP also influence lymphocyte function. SP binds to tachykinin receptors on lymphocytes and monocytes. It induces chemotaxis in monocytes, as well as stimulating production of the cytokines IL-1, IL-6, and tumor necrosis factor (TNF-alpha). SP stimulates T cell proliferation and increases antibody production by B cells, probably via IL-6. VIP also appears to bind to most lymphocytes, including T and B cells and monocytes, and tends to show immuno-
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suppressive effects on T cell mitogen responses, mixed lymphocyte responses, and NK activity. It may also inhibit homing of lymphocytes into and prevent their migration out of lymph nodes. It should be less surprising that lymphocytes have hormone receptors since this is a characteristic of most cells in the body. Some hormones released as part of the stress response appear to have specific effects on the immune system; the effects of glucocorticoids on immune responses have been studied the most extensively. Glucocorticoids are used widely in clinical practice for their powerful immunosuppressive and anti-inflammatory properties. They have been shown to inhibit monocyte and macrophage activities, including production and secretion of the cytokines IL-1, IL-2, TNF, and gamma-interferon. Conversely, systemic glucocorticoid depletion through adrenalectomy or Addison's disease results in lymphocytosis and thymic hypertrophy. It has been suggested that one of the primary functions of glucocorticoids during stress responses is to prevent the self-reactivity which might occur if the immune system became hyperactivated. On the other hand, prolactin, which is also released during an acute stress response, appears to have immunoenhancing effects. Increased levels of prolactin have been associated with increased mitogen responses. Conversely, suppression of prolactin with administration of bromocriptine suppresses the ability of rodents to combat infections and decreases T and B cell responses. Other hormones may also have effects on the immune system. Hypophysectomy results in thymic atrophy, decreased antibody function, cytoxic T cell activity, and NK activity, all of which can be reversed at least in part with administration of growth hormone. Not only does the nervous system have the capacity to influence the immune system, but the reverse also appears to be possible. Lymphocytes synthesize a number of compounds similar if not identical to those produced by neurons, including endorphins, enkephalins, SP, VIP, CRH, and ACTH. The nervous system also responds to cytokines produced by lymphocytes and macrophages, particularly IL-1, tumor necrosis factor (TNF), and IL-6. IL-1 stimulates ACTH secretion by the pituitary and thus can affect the functioning of the hypothalamic-pituitary-adrenal axis. Glial cells can produce IL-1, and receptors for IL-1 have been demonstrated in the nervous system. Cytokines also have significant effects on temperature and behavioral state. Both IL-1 and tumor necrosis factor (TNF) can act as endogenous pyrogens. IL-1 administered intracerebroventricularly increases non-rapid eye movement (NREM) sleep in various mammalian species. These data have been interpreted to suggest that an infectious process resulting in a significant immune response sends a signal to the brain to increase sleep amount, perhaps a homeostatic response to infection. In summary, the immune system can be regulated by a number of neurotransmitters, neuropeptides, and hormones as indicated by the presence of receptors on
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lymphocytes, as well as their ability to respond to these substances. The nervous system, in turn, has the ability to receive messages from the immune system through receptors for cytokines. Afferent and efferent nerve pathways between the brain and lymphoid organs have also been identified. The data presented so far, however, only demonstrate the potential for communication between the immune and nervous systems. The existence of physiological regulatory connections between the two systems has been more difficult to establish. Most studies to date have focused primarily on nervous system control of immune function through conditioning or via stress effects.
CONDITIONED IMMUNE RESPONSES Classical conditioning is an example of associative learning and involves the attachment of a behavioral response to a stimulus previously unrelated to that response. A neutral, conditioned stimulus (CS) is presented immediately prior to an unconditioned stimulus (US), which is known to elicit the desired behavior. After repeated, paired presentation of the CS and US, however, presentation of the CS alone will elicit the behavior as the organism learns to predict the relationship between the CS and US. At a cellular level conditioning is known to depend on presynaptic facilitation of sensory neurons by the CS. The sensory neurons are, in turn, linked to efferent neurons which regulate the behavior. The technique of classical conditioning has until recently been applied only to responses known to be under direct neural control. Thus, the ability to demonstrate classical conditioning of immunological responses has been interpreted to suggest that these responses may be regulated by the nervous system. There is a long history of anecdotal evidence of conditioned immune responses, such as the ability to elicit allergic responses in patients through the presentation of pictures or artificial representations of allergens. One of the first experimental demonstrations of conditioned effects on the immune system was the production of immunosuppression in rats by conditioning by Ader and Cohen in 1975. Saccharin-flavored drinking water (the CS) was paired with injection of the immunosuppressive drug cyclophosphamide (the US). Conditioned animals given saccharin solution at the time of immunization with sheep red blood cells (SRBC) showed a decreased response to SRBC in comparison with non-conditioned animals exposed to the CS or conditioned animals not exposed to the CS. Conditioned immunosuppression using similar CS/US paradigms has been demonstrated subsequently in both rats and mice for a variety of immune parameters, including both T-dependent and T-independent responses to antigen challenges, T-cell proliferation responses to mitogens, graft-vs-host responses, and natural killer cell (NK) activity. However, not all attempts at conditioned immunosuppression have been successful, as some studies have failed to demonstrate significant effects on antibody responses, particularly to T-independent antigens, and delayed-type hypersensitivity. Furthermore, although statistically significant, the magnitude of
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immunosuppression achieved through conditioning was relatively small in comparison to the normal effects of the immunosuppressive drugs. Another problem with the paradigms of conditioned immunosuppression is that they are connected to taste aversion, making it difficult to determine whether the CS-induced immunosuppression is the result of a non-specific stress response or a process involving specific learning and memory of immune function. The ability to demonstrate immunosuppression following exposure to the CS without significant taste aversion in some studies has been taken as evidence against a non-specific stress response mediating the conditioning effects. However, a specific neurological mechanism for conditioned immunosuppression has not yet been demonstrated. Attempts have also been made to condition stimulation or enhancement of immune function, which might further support the idea of neuronal regulation of immune function. To date, there has been no convincing evidence for the ability to condition antigen-specific responses. Perhaps the best example of immunoenhancement has been the CS-induced increase in cytotoxic T cells following conditioning by repeated allogeneic skin grafting; in this paradigm, the grafting procedure represented the CS and the presentation of histoincompatible donor skin the US.
STRESS AND IMMUNE FUNCTION The idea that stress could affect the immune system grew out of attempts to explain the observation that stress was associated with illness. Selye and others demonstrated in animals that stressors induced activation of the hypothalamic-pituitaryadrenal axis and led to pathological changes in various organ systems. In the 1970s, epidemiological studies established a correlation between stressful life events and an increased risk of subsequent illness or death. The term "stress" generally refers to noxious or arousing stimuli, which can be physical or emotional in nature. One major area of research has been to study the effects of stress on in vitro immune parameters in rodents, particularly lymphocyte proliferation responses to mitogens and NK cell activity. These assays can be performed on lymphocytes obtained from peripheral blood samples or spleen, making them relatively easy to perform. Both NK cells and mitogen-induced lymphocyte proliferation represent more primitive components of the immune system; neither involves antigen-specific recognition. The clinical significance of these parameters is not fully understood, although both responses are significantly decreased in severely ill or immunocompromised subjects. Rodents have been subjected to a variety of stressors, including inescapable electric shock, noise, isolation, or crowding. The results have been mixed; both decreased and increased immune responses were observed in early studies. More recent studies have attempted to assess the effects of duration or intensity of the stressor in rodents. Although increased intensity of the stressor was associated with increased suppression of mitogen responses by peripheral blood lymphocytes
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(PBLs), a longer duration of the stressor resulted in return to normal levels of mitogen responses or even enhanced responses in other studies. Lymphocytes derived from spleen appeared to be less susceptible to stress-induced suppression of mitogen responses than PBLs. NK cells obtained from rat spleen, however, showed decreased abilities to lyse target cells following acute or chronic stress. In vivo tests performed in rodents have assessed effects of stressors on tumor growth and response to bacterial/viral challenges. A number of studies have suggested that tumor growth and susceptibility to infection are increased with stress, although opposite effects (i.e., decreased rates of infection or tumor growth in stressed animals) have also been reported. The variability of the results has been interpreted as suggesting that acute stress is immunosuppressive, whereas chronic stress may result in immunoenhancement. The differing natures of the stressors and the challenging agents applied makes it difficult to make generalizations about the epidemiology of stress effects on host responses. Furthermore, most of these studies have not included direct measures of immune function, which makes it difficult to know whether changes in the immune system are primarily responsible for the observed effects. To address the effects of stress on the immune system more specifically, in vivo tests of immune function have been performed on both rodents and humans exposed to stressors. Once again, the results have been mixed. The preponderance of studies has documented immune suppression, including decreased antibody production following antigenic challenge, decreased delayed-type hypersensitivity (DTH), and reduced graft-versus-host responses. A few studies have shown immunoenhancement following stress, however. An alternative view regarding stress and immune function has been proposed recently by Dhabhar and colleagues, who hypothesize that stress may actually have immunoenhancing effects. Their studies have demonstrated decreases in peripheral blood lymphycytes, NK cells and monocytes following acute stress with redistribution of these cells to other compartments such as skin. Given that immune responses do not tend to occur in blood, the stress-induced migration of leukocytes to target organs may enhance the ability of the organism to respond to antigenic challenge. As predicted, in their studies, rats subjected to restraint or shaking stress also showed increased DTH responses. The seemingly contradictory nature of the results should not be particularly surprising. Immune responses in vivo involve sequential steps of antigen recognition, processing, and presentation, as well as the involvement of different interacting lymphocyte and accessory cell populations. Stress effects could interact with the immune system in any number of ways, including altering patterns of lymphocyte release from lymphoid organs and recirculation, impacting on the function of specific cell types through neuroendocrine mechanisms, or modifying the integrity of host barriers. Clearly, stress has demonstrable effects on the immune system
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which cannot be categorized reHably until the mechanisms for these effects are better understood. Stress Effects on Humans Because of the potential clinical importance of stress effects on disease susceptibility and progression, a number of paradigms of stress and immune function are being studied in humans. Most have been confined to measuring mitogen responses and/or NK activity in peripheral blood lymphocytes, or serum antibody titers. Some of the earliest clinical studies performed were assessments of lymphocyte proliferation to mitogens in recently bereaved spouses. There is epidemiological evidence that bereaved spouses have a greater incidence of morbidity and mortality immediately after the death of their spouses than age-matched nonbereaved subjects. In the earliest bereavement study, Bartrop and colleagues found that mitogen responses in widowers were decreased significantly in the weeks following the deaths of their wives in comparison to nonbereaved controls. Prospective studies of both men and women who were about to lose their spouses to cancer showed that mitogen responses immediately following the death of the spouses were decreased in comparison to responses measured in the same subject prior to the spouse's death. Although these studies have received methodological criticisms (i.e., the period immediately prior to the death of the spouse is not particularly stress-free), they nevertheless suggest that changes in some compartments of the immune system predictably occur following bereavement. Since bereavement is associated with increases in depressive symptomatology, it is reasonable to hypothesize that bereavement affects the immune system by causing depression, which can result in a number of neuroendocrine changes that could impact on immune function. A number of investigators have assessed immune parameters in patients with major depression.^ The results for depression have been less consistent than for bereavement, however. Studies of peripheral blood lymphocyte stimulation by mitogens have shown decreased responses in some studies and no changes in others. Similarly, NK activity in depression has been reported as either decreased or unchanged in comparison to normal controls. Counts of white blood cells, lymphocytes, neutrophils, and NK cells have also not been consistently altered in peripheral blood samples obtained from depressed patients. Since depressed patients may not be necessarily homogenous in either their symptomatology or pathophysiology, some studies have attempted to determine whether particular patient characteristics are more likely to be associated with immune system changes. There is some evidence that increased age and severity of depression may be associated with decreased mitogen responsivity. Several studies have assessed the role of HPA axis activation on immune function in depression, since corticosteroids are known to affect immune function. However, no consistent changes in lymphocyte counts or mitogen responses were found in
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either depressed patients with increased Cortisol levels or those who failed to show Cortisol suppression following dexamethasone administration. The effects of various situational stressors on immune function have also been studied. Kiecolt-Glaser and colleagues found decreased mitogen responses by peripheral blood lymphocytes, decreased cellular immune responses to herpesvirus, and reduced NK activity in medical students during their final examination period in comparison with levels obtained earlier. Increases in several herpesvirus antibodies were also found, and were interpreted as indicating decreased cellular control of latent infection, although the results could also be interpreted as a non-specific enhancement of antibody production. Loneliness and social isolation have also been correlated with decreased NK activity and mitogen responses in studies of students, psychiatric patients, and divorced subjects. Decreased immune function has also been shown in primates following maternal separation and/or isolation. Exercise has also been postulated to have similar effects to stress on the immune system. Studies on behavioral states and immune function have not all revealed consistent or significant effects on immune parameters. These studies have been criticized for the failure to correlate any of the observed immune system changes with clinical effects. For example, it is not known whether the small decrements in mitogen responses or NK activity observed in some studies can lead to increased susceptibility to infection or malignancy. Furthermore, the epidemiological relationships between bereavement, depression, or acute stress and diseases specifically associated with immune system dysfunction tend to be weak at best. Finally, behavioral states such as depression or bereavement are probably not equivalent amongst afflicted individuals, which may also account for the failure to document robust or consistent effects on the immune system related to these conditions. Yet another approach to studying immune system-nervous system interactions is to assess the relationships between specific behavioral states and immune function. The effects of sleep and sleep deprivation on immune responses have been widely studied. Sleep is a universal behavior in animals, and although the ultimate function of sleep has not been identified, it is known that sleep is necessary for survival; rats deprived of sleep die within 3 to 4 weeks. Furthermore, sleep loss is a common result of stress and is associated with most psychiatric disorders, particularly depression. A number of studies have looked at the effects of sleep deprivation on immune function in animals and humans. Short-term sleep deprivation in humans has been reported to cause decreases in mitogen responses andNK acfivity in several studies, but in a recent study, subjects deprived of sleep for 60 hours showed an increase in NK activity compared to pre-deprivation levels. Rats subjected to several hours of sleep deprivation showed decreases in secondary but not primary antibody responses to a viral challenge. Rats deprived of sleep to the point of near-death have yielded several interesting—^and seemingly contradictory—results. First, they
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showed no decline in mitogen responses, or in vitro or in vivo primary antibody responses to antigens. However, in another study, 5 of 6 sleep-deprived rats showed bacteremia shortly before death. Taken together, these results suggest that prolonged sleep deprivation in rats may lead to a breakdown in innate defense barriers, yet not necessarily affect all acquired immune responses, such as the ability to produce antibodies in response to antigenic challenge.
CONCLUSION In spite of a great deal of research, the nature of the relationship between the nervous and immune systems is not yet fiilly understood. Obviously, they are both highly complex systems which have the potential for interacting with each other in multiple ways. This complexity creates the potential for seemingly contradictory results, as well as the possibility for providing a scientific basis for reunifying behavioral and physiological approaches to health and disease. There are several important caveats which must be considered in evaluating studies of immune-nervous system interactions, however. First, decreases in immune measures, particularly as indicated by in vitro tests of peripheral blood lymphocytes, may not correlate with how an individual responds to a clinical challenge such as an infection. Conversely, increases in immune measures do not necessarily indicate an improved immune status, since they may represent a compensation for a deficit in another compartment of the immune system, or could even predispose the individual to autoimmune disease. Second, changes in one set of immune responses may not necessarily represent the immune system as a whole, since the immune system can simultaneously upregulate some responses while downregulating others. Third, although much of the popular appeal of psychoneuroimmunology has been its potential application for cancer patients, there is little evidence that the immune system plays a significant role in combatting many forms of cancer, particularly established tumors. An accurate understanding of the current limits of our knowledge of psychoneuroimmunology is particularly important for the practice of clinical medicine. It is possible for patients to believe that their illnesses—especially cancer—^have been caused by their inability to "handle stress" or a poor psychological state, thus causing them to blame themselves for their illnesses. Although some studies have suggested that various behavioral and psychosocial interventions such as psychotherapy, relaxation training, or guided imagery techniques (i.e., imagining one's immune system successfully fighting off a tumor) might have small but significant positive effects for some patients, in the majority of cases, cancer survival is most closely associated with tumor pathology rather than psychological state. There is no consistent evidence to date that particular psychological states or psychosocial interventions are predictably and consistently associated with specific changes in the immune system. Thus, although psychological and behavioral treatments may be helpful for improving a cancer patient's sense of well-being and thereby improve
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quality of life, it is important to keep in mind that a failure of these interventions to cure or even significantly retard disease progression does not mean that the individual has "failed" to adhere to the treatments correctly. Although the nervous system does not appear to be the primary regulator of the immune system (and vice versa), it does not mean that immune-nervous system interactions are unimportant or irrelevant to clinical medicine. On the contrary, perturbations of one system can have significant impact on the other. The potential for applications to various areas of clinical medicine—such as infectious diseases, autoimmune diseases, transplantation, and AIDS, for example—^has hardly begun to be explored. Much further work is needed to define the mechanisms of the interactions between the two systems, which in turn should lead to more effective treatment strategies.
NOTE 1. Major depression is defined by the presence of depressed mood or loss of interest or pleasure for a period of two weeks or more, accompanied by at least five of the following symptoms: significant change in appetite or weight, insomnia or hypersomnia, psychomotor agitation or retardation, fatigue, sense of worthlessness or guilt, decreased concentration, and/or suicidal ideation.
RECOMMENDED READINGS Ader, R. (1992). On the clinical relevance of psychoneuroimmunology. Clinical. Immunol. Immunopathol. 64, 6-8. Ader, R., Felten, D.L., & Cohen, N. (eds). (1991). Psychoneuroimmunology (2nd edn.) Academic Press, Inc., New York. Cotman, C.W., Brinton, R.E., Galaburda, A., McEwen, B., & Schneider, D.M. (eds). (1987). The Neuro-Immune-Endocrine Connection. Raven Press, New York. Dhabhar, F.S., & McEwen, B.S. (1996). Stress-induced enhancement of antigen-specific cell-mediated immunity. J. Immunol. 156, 2608-2615. Hoffman-Goetz, L., & Pedersen, B.K. (1994). Exercise and the immune system: A model of the stress response? Immunol. Today 15, 382-387. Husband, A.J. (ed). (1993). Psychoimmunology: CNS-Immune Interactions. CRC Press, Boca Raton. Kiecolt-Glaser, J.K., & Glaser, R. (1995). Psychoneuroimmunology and health consequences: Data and shared mechanisms. Psychosom. Med. 57, 269-274. Kreuger, J.M., Toth, L.A., Floyd, R., Fang, J., Kapas, L., Brendow, S., & Obal, F. (1994). Sleep, Microbes and Cytokines. Neuroimmunomodulation. 1, 100-109. Lloyd, R. (ed). (1994). Explorations in Psychoneuroimmunology. Grune and Stratton, Orlando. Maier, S.F., Watkins, L.R., & Fleshner, M. (1994). Psychoneuroimmunology. Am. Psychol. 49, 1004-1017.
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INDEX
Acquired Immunodeficiency Syndrome (AIDS), 17 Activation induced cell death (AICD), 270 Adenosine deaminase (ADA), 17 Affinity maturation, 35, 54, 284 mimicking, 294 AITD, 243-252 (see also "Autoimmune diseases....") Allelic exclusion, 94 Allografts, 76 rejection, use of antibodies in, 285 Alloresponsiveness, 120 Allotype, 75 Amiodarone, 251 Anaphylactoid reactions, 232 Anaphylatoxins, 200-201 Anaphylaxis, 186, 223, 229-236 causes, classification of, 231-235 anaphylactoid reactions, 232 angioedema, 233 "aspirin triad," 232 blood transfusions, 231-232 catamenial, 233 clinical findings, 233 complement-mediated, 231-232 differential diagnosis, 234-235 direct mast cell activators, 232233
exercise-induced, 233 haptens, 231 hereditary angioedema, 234-235 histamine, 233, 234, 235 idiopathic, 233 IgE mediated, 231 non-immunologically mediated mast cell reactions, 232-233 nonsteroidal antiinflammatory drugs (NSAID), 232-233 pathogenesis, 233-234 radiocontrast media, iodinated, 232 systemic mastocytosis, 234-235 tryptase, 235 urticaria, 233 introduction, 230-231 components, 230 definition, 230 mast cell degranulation, 230 summary, 235 treatment, 235 epinephrine, subcutaneous, 235 Anergy, 70-71, 74-75, 78, 80, 242, 253 Antagonist peptides, 77 Antibodies, designer, 279-300 bispecific, 297 chimeric and humanized, 287-288 conclusion, 298 315
316
effector functions, antibodytargeted, 295-298 antibody-effector molecule conjugates, 296-297 cancer vaccines, implications for, 297 GM-CSF, fusion of, 297 immunotoxins, 295-296 radioisotopes, 296 ricin, 296 toxicity, 296 Escherichia Coli, expression of fragments in, 288-289 advantages, 288 Fv fragments, 289 single-chain Fv fragments, 289 technical overview, 288-289 transfectoma, 288 in gene therapy, 298 immunoglobulin fragment combinatorial libraries, 289-295 affinity maturation, mimicking, 294 antibody secretion vs. cell surface expression, mimicking, 291 antigen-driven selection, mimicking, 291 bypassing immunization, 291294 chain shuffling, 294 filamentous bacteriophage, 290 fragment expression library, 290-291 murine expression systems, 294295 phage library, 294 Polymerase Chain Reaction (PCR), 294 SCID mice and transgenic mice, using, 295 intracellular, 298 HIV, 298
INDEX
introduction, 280-281 monoclonal antibodies in therapeutics, 284-287 in allograft rejection, 285 Campath-1 antibody, 287 in cancer, 285 concerns and limitations, 286287 costimulation, 285 diagnostic imaging, 286 HAMA response, 286, 287 hybridoma technology, 286 immunotoxin, 285 quantity, 286 radioisotopes, 285 transplanatation, 285 uses, other, 285-286 structure, general, and biosynthesis, 281-284 affinity maturation, 284 biosynthesis, 284 complementarity determining regions (CDR), 281 constant domains, 281, 282-284 effector functions, 282-284 fragments, 282 hypervariable regions, 281, 283 immunoglobulin domains, 281 introduction, 281-282 variable domain, 281, 283 Antiidiotypy, 117 Antigen-antibody complex, 105-118 antibody structure, 106-111 Complementarity Determining Regions of variable domains (CDR), 107-108, 117 constant domains, 107-108 Fv fragments, 109-110 IgG, 106 immunoglobulin domain, 107, 108 quaternary arrangement, 106
Index
VL-VH pairing, 110-111, 118 variable domains, 107-108 antibody-antigen-complexes, 114117 antiidiotypy, 117 conformational changes, 116 Fv fragments, 116 hemoglobin, 117 somatic hypermutation, 116 antigen structure, 111-114 hemagglutinin, 111-112 influenza viruses, 111,113 monoclonal antibodies, 112 neuraminidase, 111-112 introduction, 105-106 summary, 117-118 Antigens, 84 Antigen-presenting cells, 65, 67, 87, 199-200 (see also "Immunological tolerance") Apoptosis, 88, 98, 240, 263-278 {see afco "Cell death ") Arachidonic acid, 222-223 Arthus reaction, 201 "Aspirin triad," 232 Autocrine proliferation, 53 Autoimmune diseases, 17-18, 237261 {see also "Thymus...") autoimmunity, 238 disease characteristics, 238 "horror autotoxicus," 238 immunological unresponsiveness, 238 self-tolerance, 238 thyroglobulin (Tg), 238 Witebsky's postulates, 238 disease, 242-243 diabetes, Type I, 242 effector mechanisms, 243 multiple sclerosis, 242 introduction, 238
317
negative selection and selftolerance, 240-242 affinity theory, 241 clonal anergy, 242, 253 clonal balance, 242, 253 clonal deletion, 241-242, 253 thymus, 242 positive selection, mechanisms of, 239-240 CD4 and CD8, 239 tyrosine kinase, 239-240 self and non-self, 238-239 negative selection, 239, 240-242 positive selection, 239-240 T cell receptor, 239 summary, 253-254 thyroglobulin, 252-253 thyroxine, as prohormone for, 252 thyroid disease, autoimmune (AITD), 243-252 amiodarone, 251 animal models, 245 autoantigens, 244 cytokines, role of, 247 drugs, 251 endocrine abnormality, 249-250 environmental factors, 250-251 Epstein-Barr virus infection, 250 genetic control of susceptibility, 249 genetic predisposition, 248-249 Graves' disease, 243 Hashimoto's thyroiditis (HT), 243 histopathological changes, 243244 HLA genes, 248 hormones in regulation of, 250 immunological response, 246248 incidence, 243 infectious agents, 250
318
iodine, 250-251 long-acting thyroid stimulator (LATS), 243 multifactorial etiology, 245-252 prolactin, 250 stress, 251 suppressor cell defect, 247-248 thyroglobulin (Tg), 244 thyroid peroxidase (TPO), 244 thyroid-stimulating hormone receptor (TSHR), 244 toxins, 251-252 Azurophil granules, 218 B cell in immunity, 5-6, 21-44, 65 antigen, responses to, 31-33 early stages, 32 T-dependent and T-independent responses, 32-33 in bone marrow, 23-30 anergy, 28 autoreactivity, 28 CDIO, 26, 27 CD19, 26 CD23, 29 CD40, 29 clonal deletion, 28 HLA-DR, 27 Ig-transgenic, 28 IgD, 29 IgE, 29 IgM, 29 IL7, 26 immature cells, 25 MHC class II antigens, 27 pre-B cells, 25, 26, 27-28 pro-B cells, 25, 26 scid mutation, 26-27, 40 stem cell factor (SCF), 25 stem cells, 24 tolerance, 28 virgin cells, 25, 26
INDEX
cell migration, 30-31 bursa of Fabricius, 31 dendritic cells, 30 follicles, 30 follicular dendritic cell (FDC), 30,32 IgM'igD'', 30 interdigitating dendritic cells (IDCs), 30, 32 marginal sinus, 30 marginal zone, 30 periarteriolar lymphocytic sheath (PALS),30 deficiencies, 39-42 bacterial meningitis, 42 btK 41 common variable immunodeficiency (CVID), 42 IgA deficiency, 41 IgM and IgC deficiencies, 42 immunoglobulin isotype deficiencies, selective, 41-42 X-LA, 39-41 X-LA with hyper-IgM, 39, 4142 {see also "X-LA with hyper-IgM") introduction, 22-23 antibody, 22, 23 antigen, 22, 23 in bone marrow, 22, 23-30 germinal center, 22, 23 immunoglobulin gene recombination, 32 isotype switching, 22 memory B cells, 22 plasma cell, 22, 26 repertoire, 22 somatic mutation, 22, 23 schematic, 23, 37 secondary response, 37-38 summary, 42 and T cells, interaction between, 5356 {see also "Cell-to-cell....")
Index
T-cell dependent responses, 33-37 affinity maturation, 35 B7,33 centroblasts, 35 centrocytes, 35 complementarity determining regions (CDRs), 36, 37 germinal center, 34, 35 {see also "Germinal....") IgM, 33, 34 IL4, 34 interdigitating dendritic cells (IDCs), 33 isotype switching, 34 Langerhans cells, 33 memory B cell, 36-37 PALS, 33 somatic mutation, 35, 37 Tingible bodies, 34-35 tolerogenic, 33 T cell independent responses, 38 lipopolysaccharide (LPS), 38 type 1 and type 2 antigens, 38 xW, 38,41 types, other, 39 B-CLLs, 39 CD5, 39 Ly-IB cell, 39 B cell signaling and T cell signaling at molecular level, 135-147 BCR and TCR receptors, 136138 CD3, 136 CD4 and CD8, 137 major histocompatibility complex (MHC), 137 structure, 137 inositol phospholipid hydrolysis and calcium mobilization, 138-139 phospholipase C(PLC), 138-139 protein kinase C (PKC), 138
319
introduction, 136 B cell antigen receptor complex (BCR), 136-138 T cell antigen receptor complex (TCR), 136 lymphocytes, other PTKs in, 142 nucleus, toward, 145-147 Grb2, 146 c-Raf, 146 mitogen activated protein (MAP) kinases, 146 Ras protein, 146 She, 146 Sos, 146 TCR-induced tyrosine phosphorylation cascade, 145-146 Vav, 146 Src family of nonreceptor PTKs, 141-142 Blk, 142 CD45 PTPase, 144 Csk PTK, 144 Fyn, 141, 142 Lck, 141 Lyn, 142 phosphatidylinositol 3-kinase (PI3-K), 143 regulation of, 142-144 SH2 and SH3, 143 summary, 147 pathways, three, of intracellular signal transduction, 147 SYK family of nonreceptor PTKs, 142 Zap, 142 tyrosine phosphorylation, 139-141 phosphotyrosine phosphatases (PTPases), 140 protein tyrosine kinases (PTKs), 139-140 Src family kinases, 140-142 {see afao "...Src family....")
320
Syk family kinases, 140-141 TCR/CD3 complex, 139 Bacterial killing by complement, IS3-IU (see also "Complement...") Bacterial meningitis, 42 Basophils, 204, 206-209, 218 Bat (B Associated Transcript) series of genes, 123 BCR, 136-138 (see also "B cell signaling....") bel-2, 266 Bereavement, effect of, 307-308 Bispecific antibodies, 297 (see also "Antibodies...") "Bjorkman's groove," 125, 126 Blood transfusion, anaphylaxis and, 231-232 Bombay blood group, 215 Bone marrow, 86 (see also "Diversity ") transplantation, 129 graft versus host disease, 129 Booster vaccination, 90 btk, 41 Burnet, Sir MacFarlane, 86 Bursa, 4-5 of Fabricius, 31, 150 Bystander killing, 274 Bystander lysis, 179 c-myc, 266 Cancer, cell death and, 275-276 antibodies, use of, 285 Cardiac transplantation, 129 Catamenial anaphylaxis, 233 CD5, 39 CD45 PTPase, 144 (see also "B cell signaling....") CDRs, 36, 84 Cell adhesion superfamilies, 46-47 immunoglobulin, 46 "barrel," 46
INDEX
integrins, 47 Leukocyte Function Antigen-1 (LFA-1), 47 selectins, 47 carbohydrate, binding, 47 transferase enzyme, 47 Cell-to-cell interactions in immune system, 45-60 antigen presentation to lymphocytes, 49-53 alpha helices, 51 autocrine proliferation, 53 to B cells, 50 CD3, 52 CD28, 52 clonal selection, 49 costimulation, 52 on dendritic cells, 51 endogenous peptides, 51-52 endoplasmic reticulum, 52 epitopes, 50 follicular dendritic cells (FDC), 50 ICAM family, 52 IL-2 and IL-2 receptors, 53 immunological memory, 50 introduction, 49 Langerhans cells, 51 LFA-1, 52 MHC molecules, 50-53 peptides, 51-52 to T cells, 50-53 T cell receptor complex, 52 leukocytes and endothelium, 58-59 E-selectin, 58, 59 high endotheUal venules (HEV), 58 L-selectin, 58, 59 lymphoid organs, 58 memory cells vs. naive cells, 58 model, three-step, 58-59 molecular, basis of, 46-47 adhesion, 46
Index
cell adhesion superfamilies, 4647 (see also "Cell adhesion ") cell surface molecules, 46-47 cytokines, 48-49 (see also "Cytokines") extracellular matrix, 46 as receptors for soluble factors, 46 T cell, 46 T cell antigen receptor, 46 summary, 59 in secondary lymphoid tissue, 59 T cells, effector mechanisms of, 53-58 affinity maturation, 54 CD40L, 54-55 class switching, 54 cytokines, 54-55 cytotoxic cells, 56-58 interferon-gamma (IFN-a), 5455 Leishmania infections, 56 and macrophages, 56 natural killer cells, 57 phagocytosis, 56 plasma cells, 54 T and B cells, interaction between, 53-56 T independent antigens, 54 Thl and Th2 cells, 56 tetanus toxoid, 54 transforming growth factor-beta (TGF-/?), 54-55 Cell death and immune system, 263278 biochemistry of apoptosis, 266-268 bel-2, 266, 270 c-myc, 266 DNA cleavage, 266 oxidative stress as mediator, 268
321
p53, 266 phagocytosis, 268 characteristics of apoptosis and necrosis, 265 clinical immunology, apoptosis in, 274-276 autoimmune disease, 276 cancer, 275-276 Epstein-Barr virus, 275 HIV, 275 host-graft interactions, 276 infection, 275 inflammation, 275 definitions, 264 apoptosis, 264 inflammation, 264 morphology of apoptosis and/ or necrosis, 264, 265, 267 necrosis, 264 programmed cell death, 264 of immune cells, 268-272 activation induced cell death (AICD), 270, 272 B cell selection, 270-271 cell types, other, 271 during development and maturation, 268-271 of mature cells, 271-272 negative selection, 270 T cell selection in thymus, 269270 introduction, 264 apoptosis, 264 summary, 276-277 target cell death, 272-274 bystander killing, 274 cell-mediated cytotoxicity, 273 Cell-mediated immunity, 155-157 Chain shuffling, 294 Charcot-Leyden crystal, 206 Chemoattractants, 195-227 (see also "Phagocytes....")
322
Chemokines, 155, 159-160 C-C branch, 159 C-X-C branch, 159 chemotaxis, 159 monokines TNF-a and IL-1, 160 stem cell growth inhibitors, 159 in vivo, 160 Chemokinesis, 209 Chemotaxis, 87, 159, 184, 199, 209 Chimerism, 75-76, 80 Chronic Granulomatous Disease (CGD), 221 Class switching, 54 Classical conditioning, 305-306 Clonal abortion, 66, 78 Clonal anergy, 67, 70-71, 74-75, 78, 80 Clonal deletion, 66, 70-71, 72-74, 241-242 intra-thymic, 68-69, 270 Clonal exhaustion, 76-77 Clonal ignorance, 66 Clonal selection, 49 Clonal Selection Theory, 100 Clusterin, 182 Collaborative Transplant Study, 129 Collagenase, 212-213 Colony-stimulating factors (CSFs), 150 (see also "Cytokines") Common variable immunodeficiency (CVID), 42 Complement system, activation and control of, 169-193 activation, 173-180 alternative pathway, 177-178 amplification on activator surfaces, 178 bystander lysis, 179 CI, 173-175 C2, 176-177 C3, 176, 177 C4, 175 C5, 177, 178-180
INDEX
C5b67, 179 C6, 179 C7, 179 C8, 179 C9, 179 clasical pathway, 173-177 factor B and factor D, 178 IgG, 174 IgM, 174 mannose binding protein (MBP), 175 MBP-associated serine protease (MASP), 175 membrane attack complexes (MACs), 179-180, 181, 185 membrane attack pathway, 179180 "tickover" phenomenon, 178 control, 180-183 in alternative pathway, 182 CI-inhibitor, 180 C4b-binding protein, 180-181 CD59, 183 in classical pathway, 180-182 clusterin, 182 complement receptor 1 (CRl), 181, 182 decay accelerating factor (DAF), 181 Factor 1, 180, 182 Factor H, 180-181,182 homologous restriction factor (HRF), 183 in membrane attack pathway, 182-183 membrane cofactor protein (MCP), 181, 182 properdin (P), 182 regulation of, 181 Regulators of Complement Activation (RCA), 182 S protein, 182
Index
serine protease inhibitor (Serpin) family, 180 vitronectin, 182 deficiencies, 185-189 (see also "....pathology") introduction and basic concepts, 170-173 innate immune defense, central role in, 173 pathways, three, 170, 173 purpose, 173 pathology, 185-189 in autoimmune diseases, 187 C3, 186 control of activation in vivo, 188-189 CRl, 189 CVF, 188 deficiencies, 185-187 Echovirus, 188 Epstein-Barr virus, 188 evasion of by microorganisms, 187-188 in hemodialysis, 188 hereditary angioedema (HAE), 186 HIV, 187 hyperacute rejection, 188 iatrogenic activation, 188 measles, 188 meningococcal meningitis, 186 Neisseria inkciions, 186 opsonization, 183-189 overactivation, 189 paroxysmal nocturnal hemoglobinuria (PNH), 1816-187 properdin deficiency, 186 systemic lupus erythematosus (SLE), 186 in vivo, control of activation in, 188-189 xenotransplantation, 188
323
physiological roles, 183-185 anaphylatoxin inactivator, 184 anaphylaxis, 184 bacterial killing, 183-184 C5a,184 cell activation, 184-185 chemotaxis, 184 coating with complement, 184 CR3, 183 erythrocytes, 184 immune complex solubilization. 184 and immune response, 185 Neisseria, exception of, 183 opsonization, 183-189 summary, 189 Complementarity determining regions (CDRs), 36, 84, 107108,117,281 Conditioned immune responses, 305306 Congenic partners, 65-66 Cortex of thymus, 2, 3 Crossmatch, 130 CSFs, 150 (see also "Cytokines") Csk PTK, 144 (see also "B cell signaling..") CVID (common variable immunodeficiency), 42 Cyclic neutropenia, 203 Cytokines, 15, 48-49, 149-167 autocrine, 48 in autoimmune thyroid disease (AITD), 247 clinical applications, 162-164 G-CSF, 163 IL-2, 162-163 families and their cellular sources, 150-160 chemokines, 159-160 (see also "Chemokines") colony-stimulating factors (CSFs), 150
324
interleukins, 150 lymphokines, 150, 152-157 (see also "Lymphokines") lymphotoxin, 150 monokines, 157-158 (see also "Monokines") transforming growth factors, 150 tumor necrosis factor a, 150 IL-lra, 48 IL-6, 48 in immune system, 49 interleukin-1, 48, 150, 152 (see also "Interleukins") introduction, 149-150 bursa of Fabricius, 150 lymphocytes, 149-150 T cells and B cells, 149-150 macrophages, 48 mRNA, 48 paracrine, 48 proinflammatory, 158 receptors and signaling pathways, 161-162 Duffy antigen, 162 IL-2, 161 tumor necrosis factor (TNF), 48, 150, 157, 158, 160 Cytotoxic cells, 56-58, 87 Darier's sign, 235 Dendritic cells, 3, 51, 68, 87 Depression, 308 Designer antibodies, 279-300 (see also "Antibodies....") Desmosomes, 3 Diabetes, type I, 242 Diapedesis, 199, 212-213 diGeorge syndrome, 17 (see also "Thymus...") Directed mutagenesis, 100-101 (see also "Diversity ") Disease, human, phagocytes and, 198
INDEX
Diversity in immune system, generation of, 83-104 clonal selection, 85-90 affinity maturation, 88-89 antibody molecules, 86 antigen-presenting cells, 87 bone marrow, 86 chemotaxis, 87 Clonal Selection Theory, 86 cytotoxic T cells, 87 follicular dendritic cell (FDC), 88 germinal centers, 88, 90, 101 helper T cells, 87, 90 human immunodeficiency viruses (HIV), 90 immunoglobulin (Ig), 86 lymphocytes, 85-86 medullary cords, 88 MHC antigens, 87 opsonization, 87 phagocytosis, 87 somatic hypermutation, 88 T cell receptor (TcR), 86 target cell lysis, 87 directed mutagenesis, 100-101 Clonal Selection Theory, 100 isotypes of immunoglobulins, functional and structural, 90-100 allelic exclusion, 94 diversity genes, 93-95 germline repertoire, 92-93 Human Genome Project (HUGO), 92-93 IgA, 91-92 IgD, 91-92 IgE, 91 IgG, 91-92 IgM, 91 isotype switch, 91-92 joining gene, 93-95 junctional diversity, 95-96
Index
Mannik Phenomenon, 95 N regions, 95-96 palindromes, 97 rearrangements, 91, 93-95 receptor editing, 97 secondary rearrangements, 97 somatic hypermutation of rearranged V(D)J genes, 98-100 somatic mutations, 97, 98-100 somatic repertoire, generation of, 93-100 systemic lupus erythematosus, rearrangement from, 95-96 terminal deoxynucleotidyl transferase, 95 V-DJ rearrangement, 96-97, 99 variable gene, 93-95 Wu-Kabat structures, 98-99 introduction, 84-85 antigen receptors, diagrams of, 84 antigens, 84 B cell and T cell receptors, 85 complementarity determining regions (CDR), 84 constant region, 84 epitopes, 85 framework regions (FR), 84 heavy (H) chains, 84 light (L) chains, 84 self antigens, 84 variable region, 84 summary, 101 Duffy antigen, 162, 212 (see also "Cytokines") E-selectin, 58, 213 Echovirus, 188 Ehrlich, Paul, 62-63, 212-213, 217, 238, 298, 299 Elastase, 212-213 Endocrine abnormality in AITD, 249-250
325
Endogenous peptides, 52 Endothelium and leukocytes, interaction between, 58-59 (see afao "Cell-to-cell....") Environmental factors in autoimmune thyroid disease (AITD), 250-251 Eosinophils, 204, 205-206, 218, 219 cationic protein (ECP), 219 -derived neurotoxin (EDN), 219 peroxidase (EPO), 219 Epidermal growth factor (EGF) receptor, 146-147 Epitopes, 50, 62 Epstein-Barr virus, 188, 250, 275 Erythrocytes, 184 Exercise-induced anaphylaxis, 233 Extracellular matrix, 46 Fenton reaction, 221 Follicles, 30 Follicular dendritic cell (FDC), 30, 50,88 Framework regions (FR), 84 Fv fragments, 109-110 single-chain, 289 Fyn family PTK, 141 GALT, 34 Gelsolin, 218 Genetic predisposition to AITD, 248-249 Germinal center, 22, 34, 35-37 centroblasts, 35-36 centrocytes, 35-36 functioning, model for, 35-36 complementarity determining regions (CDRs), 36 as site of cell death, 35 "Germlike genes," 10 Glucocorticoids, 304 Graft-versus-host disease (GVHD), 76, 129
326
Granule contents, 217-19 (see also "Phagocytes...") Graves' disease (GD), 243 Gut associated lymphoid tissues (GALT), 34 HAE, 186 HAMA response, 286 Haptens, 231 Haptotaxis, 209 Haplotype, 120 Hashimoto's thyroiditis (HT), 243,253 Hassall's corpuscles, 3 Heat shock protein 70, 123 Helminths, defense against, 206, 218 Helper T cells (Th), 87, 90 Hereditary angioedema (HAE), 186, 234-235 Hewson, William, 197 High endothelial venules (HEV), 58 Histiocytes, 207 HIV, 90, 275, 298 and complement, 187 HLA genes and AITD, 248 HLA-ABCgtms, 120-122 HLA system in transplantation, 128130 (see also "Major Histocompatibility ") Homologous restriction factor (HRF), 183 Hormones, 304-305 in regulation of autoimmune process, 250 "Horror autotoxicus," 62-63, 238 (see also "Immunological tolerance") HRF, 183 HTLV-1, 17 Human Genome Project (HUGO), 92-93 Human immunodeficiency viruses (HIV), 90, 275, 298 (see also "HIV")
INDEX
H-Y, 68-69 (see also "Immunological tolerance") Hybridoma technology, 286 Hyperacute rejection, 188 Hypermutation, 35 Hypothalamic-pituitary-adrenalcortical axis, 302, 306 ICAM family, 52, 214 Idiopathic anaphylaxis, 233 Idiotype antigen, 77 IgA deficiency, 41-42 IgE, mediated anaphylaxis and, 231 IL-1, 303 IL-2, 161-164 IL-6, 48, 303 Immune deviation, 77-78 (see also "Immunological tolerance") Immune system, cell-to-cell interactions in,45-60 (see also "Cell-to-cell ") Immune system, cell death and, 263278 (5^^flfao "Cell death...") Immune system-nervous system interactions, 302-305 (see also "Psychoneuroimmunology") Immunity: B cell in, 21-44 (see also "B cell....") definition, 199 phagocytes, 195-227 (see also "Phagocytes....") Immunoglobulin (Ig), 86 (see also "Diversity....") Immunoglobulin superfamily, 46-47 (see also "Cell adhesion superfamily") Immunological memory, 50 (see also "Cell-to-cell....") Immunological tolerance, 61-82 artificially induced, 75-79 antagonist peptides, 77 anti-idiotypic responses, generation of, 77
Index
chimerism, 75-76 clonal abortion, 78 clonal exhaustion, 76-77 graft-versus-host disease, 76 idiotype, 77 immune deviation, 77-78 oral tolerance, 76 persistence of antigen essential, 78 pre-B cells, 78 soluble antigens, 76 T cell accessory molecules, antibodies to, 76 T cell independent antigens, 77 targeting antigen to naive B cells, 76 thymectomy, 78 in tissue culture, 78-79 veto and suppression, 77 in vivo, 75-78 autoimmunity and breakdown of immunological self tolerance, 79-80 immunoregulatory T cells, 79-80 molecular mimicry, 79 Thl and Th2 cells, 80 historical background, 62-66 allografts, 63 antigen-presenting cells (APC), 65,67 clonal selection theory, 64 co-stimulator signal, 65 congenic partners, 65-66 "horror autotoxicus," 62 immune repertoire, 64 LCMV, 63 T and B cells, 65 thymus, role of, 65 transgenic technology, introduction of, 65-66 introduction, 62 co-stimulatory molecules, 62, 67 epitopes, 62
327
"self," 62-66 self-reactivity, 62 self-reacting lymphocytes, possible fate of, 66-67 affinity, 67 B7 molecule, 67 CD28, 67 clonal abortion, 66 clonal anergy, 67, 70-71 clonal deletion, 66, 70-71 clonal ignorance, 66 lymphokines, 67 suppression, 67, 71 self-tolerance in B cells, 72-75 absence of T cell help, 72 allotype, 75 anti-idiotypic B cells, 73 clonal anergy, 74-75 clonal deletion, 72-74 hypermutation, 72-73 IgC class as T-cell dependent, 72 IgM antibodies, 72 interleukins, 72 self-tolerance in T cells, 68-71 binding, 68 CD4^ and CD^^ cells, death of, 68 at cortico-meduUary junction, 68 cytotoxic, 68 dendritic cells, 68 "forbidden clones" of Burnet, 69 H-Y, 68-69 helper, 68 intra-thymic clonal deletion, 6869 macrophages, 68, 71 major histocompatibility complex (MHC), 68 positive selection, role of, 68 post-thymic tolerance, 70-71 programmed cell death, 68
INDEX
328
TCR gene rearrangement, 68 TCR genes, 68 TGF-/?, 71 Thl and Th2 cells, 71,80 "veto" cells, 68 summary, 82 therapeutic applications, potential, 80 non-depleting CD4 antibodies, 80 Immunoregulation, 223 Immunoregulatory T cells, 79-80 Immunosuppression, 305-306 Immunotoxins, 295-296 Infection: apoptosis and, 275 phagocytes and, 197-199 Infectious agents, role of in autoimmune disease, 250 Inflammation, 196, 275 definition, 264 {see also "Cell death....") phagocytes and, 200-201 Influenza viruses. 111, 113 Insulin, receptor for, 142 Integrins, 47, 214 {see also "Cell adhesion superfamily") Intercellular adhesion molecules, 214 Interdigitating dendritic cells (IDCs), 30,33,51 Interferons, 154-155 a, 158 )8 1, 158 -7(IFN-7), 11,54-55 receptors, 146-147 Interleukins: definition, 152 -1 (IL-1), 48, 72, 160 -la, 157-158, 160 -1)8, 157-158, 160 -2, 11, 15,72,303 -3, 156 -4, 154
-5, 156 -6,157,158 -12, 156-157 receptors, 146-147 Iodine and autoimmune thyroid disease, 250-251 Jerne, Niels, 86 Koch's Postulates, 196 Kostmann's syndrome, 205 Kupffer cells, 207 L-selectin, 58, 213 Lactoferrin, 219 LAD-1 and LAD-2, 214-215 Langerhans cells, 33, 51, 207 LATS, 243 LBP, 215 Lck family PTKs, 141 Leishmania infections, 56, 250 Leukocyte adhesion deficiency 1 and 2 (LAD-1 and LAD-2), 214215 Leukocyte Function Antigen-1 (LFA-1), 47, 52 Leukocyte integrins, 214 Leukocytes, 197 and endothelium, interaction between, 58-59 Leukotriene B4, 223 LFA-1, 47, 52,214 Lipopolysaccharide-binding protein (LBP), 215 Lithium therapy and autoimmune thyroid disease, 251 Liver transplantation, 129, 130 Ly-IB cell, 39 Lymph nodes, 51 Lymphoblastic leukemia, 17 Lymphocytes, 2, 61-82, 149-150 {see also "Cytokines" and "Immunological tolerance")
Index
Lymphokines, 150, 152-157 (see also "Cytokines") in B cell responses, 153-155 CD4 and CD8 T cells, 152-153 CD40, 156 in cell-mediated immunity, 155157 GM-CSF production, 155-156 IL-2, 152 IL-3, 156 IL-4, 154 IL-5, 156 IL-12, 156-157 interferon-7, 154, 155 interleukins, 152 interleukin-3, 156 in T cell responses, 152-153 source, 152 Lymphoma, 17 Lymphotoxin (TNF ^8), 150 MACS, 179-180, 181, 185 Mac-1, 214 Macrophages, 3, 68, 71, 87, 207-209 and T cells, interaction between, 56 Major basic protein (MBP), 219 Major Histocompatibility Complex (MHC), 119-134 disease associations, HLA and, 130-131 antigen presentation, restriction of, 130-131 of autoimmune nature, 130 class III association, 131 molecular mimicry, 130 Systemic Lupus Erythematosus (SLE), 131 function of Class I and Class II molecules, 128 endoplasmic reticulum (ER), synthesized in, 128 LMP2andLMP7, 128 Tap 1 and Tap 2, 128
329 genetic structure of, 120-123 C2 and C4, 123 class I region, 120-122 class II region, 122-123 class III region, 123 DO/DN sub-region, 123 DP sub-region, 122-123 DQ sub-region, 122 DR sub-region, 122 haplotype, 120 heat shock protein 70, 123 HLA-ABC gQuts, 120-122 microlymphocytotoxicity assay, 122, 127 Nomenclature Committee reports, 123 one dimensional isoelectric focusing (lEF), 122 primed lymphocyte test (PLT), 123 properdin factor (Bf), 123 tumor necrosis factor A and B, 123 21-OHA and 21-OHB, 123 HLA and disease associations, 130-131 {see also "...disease ") HLA system in transplantation, \2^-U0 (see also ".. ..transplanation...") introduction, 120 alloresponsiveness, 120 polymorphism, 120 molecules, 50-53 nomenclature and polymorphism, 126-127 allele sequences, 126-127 class I, 126-127 class II, 127 protein structure of molecules, 124-126 "Bjorkman's groove," 125, 126 class I molecules, 124-125
330
class II molecules, 125-126 peptide binding groove, 125 summary, 131 techniques for detecting polymorphism, 127-128 molecular techniques, 127-128 Polymerase Chain Reaction— Sequence Specific Oligonucleotide technique (PCRSSO), 128, 129 serology, 127 transplantation, HLA system in, 128-130 bone marrow, 129 cardiac, 129 Collaborative Transplant Study, 129 crossmatch, 130 graft versus host disease (GVHD), 129 influences, two, 128 liver, 129, 130 matching, 129 renal transplants, 129 sensitization, 129-130 solid organs, 129 Mannik Phenomenon, 95 (see also "Diversity....") Mannose binding protein (MBP), 175,215 Mast cell degranulation, 230 MBP, 219 MBP-associated serine protease (MASP), 175 MCP, 181 Measles, 188 Medulla of thymus, 2, 3 Membrane attack complexes (MACs), 179-180, 181, 185 Membrane cofactor protein (MCP), 181 Memory B cell, 22, 36-37 (see also "Bcell...")
INDEX
Meningitis, bacterial, 42 Metachromasia, 206 Metchnikoff, Elie, 197 MHC molecules, 50-53 Microlymphocytotoxicity assay, 122, 127 Mitogen activated protein (MAP) kinases, 146 "Molecular mimicry," 79, 87, 130, 212, 242 Monocytes, 207 Monokines, 157-158 interferons a and )8-l, 158 interleukins-la and -l/J, 157-158 interleukin-6, 157, 158 proinflammatory cytokines, 158 TNF-a, 157, 158 Multiple sclerosis, 242 Mycobacteria, 56 Mycoplasma, 250 Myelomas, 280 Myeloperoxidase, 218, 221 Natural killer (NK) cells, 57, 303 Necrosis, 263-278 (see also "Cell death....") Necrotaxis, 207 Nervous system-immune system interactions, 302-305 (see also "Psychoneuroimmunology") Neuropeptides, 304-305 Neurotransmitters, 304-305 Neutropenia, 205 Neutrophil-Specific Granule Deficiency (SGD), 219 Neutrophils, 204-205, 217 Nomenclature Committee reports, 123 Nonsteroidal antiinflammatory drugs (NSAID), anaphylaxis and, 232-233 Norepinephrine, 303
Index
NSAID, 232-233 "Nurse cells," 2 One dimensional isoelectric focusing (lEF), 122 Opelz, Professor G., 129 Opsonization, 87, 155, 183-189, 199 phagocytosis, 215-217 P-selectin, 213 p 2 r ^ 146 p53, 266 p 7 y ' , 146 FAF, 223 PALS, 30 Paroxysmal nocturnal hemoglobinuria (PNH), 186-187 Pasteur, Louis, 196 Penicillins, 231 Peptide binding groove, 125, 126 Periarteriolar lymphocytic sheath (PALS), 30 Phagocytes in immunity and inflammation, 195-227 development, 201-209 basophils, 204, 206-209 bone marrow, differentiation from stem cells in, 201-203 Charcot-Leyden crystal, 206 colony forming unit, 203 colony-stimulating factors (CSF), 203 cyclic neutropenia, 203 eosinophils, 204, 205-206 glucocorticoids, 205 GMCSF and GCSF, 203 helminths, defense against, 206 hematopoietic inductive microenvironment, 203 histamine, 206 histiocytes, 207 IL-5, 205-206 Kostmann's syndrome, 205
331
Kupffer cells, 207 Langerhans cells, 207 lipopolysaccharide, 205 macrophages, 207-209 marginated pool, 205 mature, 203-209 metachromasia, 206 microglial cells, 207 monocytes, 207 multinucleated giant cells, 207 necrotaxis, 207 neutropenia, 205 neutrophils, 204-205 progenitor cell compartment, 201 promonocyte, 207 pulmonary alveolar macrophages, 207 stem cell compartment, 201-203 functions of, specialized, 209-223 antimicrobial oxidants, production of, 219-222 arachidonic acid, 222-223 azurophil granules, 218 C3b and C3bi, 215 cell migration, 209-215 chemoattractants, 210-212 chemokinesis, 209 chemotaxis, 209 Chronic Granulomatous Disease (CGD), 221 coUagenase, 212-213 complement receptors, 215, 216 CR3, 216 DAG, 212 degranulation, 217-219 Duffy red cell antigen, 212 elastase, 212-213 Fenton reaction, 221 flavocytochrome, 221-222 G proteins, 211-212 gelatinase, 218 gelsolin, 218
332
INDEX
glutathione, 222 haptotaxis, 209 hexose monophosphate shunt, 221 IL-1, 222 IL-6, 222 IL-8,211 immunoregulation, 223 integrins, 214 intercellular adhesion molecules (ICAM-1 and ICAM-2), 214 lactoferrin, 219, 221 lamellipodium, 212 leukocyte adhesion deficiency 1 and 2 (LAD-1 and LAD-2), 214-215 leukotriene B4, 223 lipopolysaccharide-binding protein (LBP), 215 major basic protein (MBP), 219 mannose binding protein (MBP), 215 molecular mimicry, 212 myeloperoxidase, 218, 221 NADPH oxidase, 220-222 Neutrophil-Specific Granule Deficiency (SGD), 219 nitric oxide synthase, 222 opsonins, 215 oxidative killing mechanisms of, 220 phagocytosis / opsonization, 215217 platelet-activating factor (PAF), 223 pro-inflammatory mediators, production of, 222-223 prostaglandins, 222 protein kinase C, 212 pseudopodium, 212 respiratory burst, 219-220 selectins, 213
sialyl Lewis "^ blood group antigen, 213 specific granules, 218-219 superoxide dismutase, 221 synexins, 218 tertiary granule, 218 thromboxanes, 222 tumor necrosis factor, 213 uropod, 212 introduction, 196-201 anaphylatoxins, 200-201 antigen presentation, 199-200 Arthus reaction, 201 chemoattractants, 199, 210-212 chemotaxis, 199 complement system, 200 delayed type hypersensitivity reactions, 201 diapedesis, 199, 212-213 germ theory of disease, 196 granuloma, 201 historical background, 196-197 homeostasis, general role in, 196-201 and human disease, 198 immediate type hypersensitivity reactions, 201 and immunity, 199-200 and infection, 197-199 and inflammation, 200-201 leukocytes, 197 NADPH oxidase, 199 opsonization, 199 phagosome, 199 post-streptococcal glomerulonephritis, 200 smallpox, 196-197 vaccination, 196-197 white corpuscles, 197 summary, 223-224 Phagocytosis, 56, 87 opsonization, 215-217 Phagosome, 199
333
Index
Phosphatidylinositol 3-kinase (PI3K), 143 Phospholipase C (PLC), 138-139 Phosphotyrosine phosphatases (PTPases), 140 Plasma cell, 22 Platelet-activating factor (PAF), 223 Pneumococcus, polysaccharide, 38 PNH, 186-187 Polymerase Chain Reaction (PCR), 294 —Sequence Specific Oligonucleotide technique (PCR-SS), 128, 129 Post-streptococcal glomerulonephritis, 200 Primed lymphocyte test (PLT), 123 Programmed cell death, 98, 240, 263-278 (see also "Cell death....") Prolactin, 250, 304 Promonocyte, 207 Properdin, 182-186 deficiency, 186 factor (Bf), 123 Protein kinase C, 138, 212 Protein tyrosine kinases (PTKs), 139-140 Psychoneuroimmunology, 301-311 conclusion, 309-311 conditioned immune responses, 305-306 classical conditioning, 305-306 immunosuppression, 305 immune system-nervous system interactions, evidence for, 302-305 cytokines, 303 glucocorticoids, 304 hormones, 304-305 interleukin-2, 303 lymphocytes, 303
natural killer (NK) cell activity, 303 neuropeptides, 304-305 neurotransmitters, 304-305 norepinephrine, 303 during sleep, 304 vasoactive intestinal peptide (VIP), 303 introduction, 301-302 biopsychosocial model of illness, 302 hypothalamic-pituitary-adrenal cortical axis, 302, 306 psychosomatic disorders, 302 stress and immune function, 306309 bereavement, effect of, 307-308 definition, 306 depression, 308 humans, effects of on, 307-309 infection, increased susceptibility to, 307 sleep and sleep deprivation, 309 studies' results mixed, 307 Receptor editing, 97 Renal transplants, 129 S protein, 182 Salt wasting, 123 SCID mice,using, 295 Scid mutations, 40 Selectins, 47, 213 (see also "Cell adhesion superfamily") Self-antigens, 84 Self-tolerance, 238 Serine protease inhibitor (Serpin) family, 180 Serology, 127 SGD, 219 Sialyl Lewis "" blood group antigen, 213 SLE, 131
334
Sleep and sleep deprivation, 304,309 Smallpox, 196-197 Soluble antigens, 76 Somatic hypermutation, 88, 284 of rearranged V(D)J genes, 98-100 Somatic mutation, 22, 35, 37, 97, 98100 Southern blotting, 66 {see also "Cellto-cell...") Spleen, 13-14 periarteriolar lymphocyte sheath (PALS), 13-14 red and white pulps, 13 Stem cells, 24, 201-203 Stress and autoimmune thyroid disease, 251 Stress and immune function, 306-309 {see also "Psychoneuroimmunology") Stromal cells, 2 Suppression, 67, 71 Suppressor T cells, 77 defect in as basis for AITD, 247248 Syk family PTKs, 142 Synexins, 218 Systemic Lupus Erythematosus (SLE), 131, 186,276 Systemic mastocytosis, 234-235 T cell independent antigens, 77 T cell receptor (TcR), 86 and autoimmunity, 239 T cell signaling at molecular level, 135-147 (^eea&o"B cell signaling....") T cells, 6-7, 65 antigen presentation to, 50-53 and B cells, interaction between, 53-56 receptor complex, 52 Talmage, David, 86 Target cell lysis, 87
INDEX
TCR, 9-10 Tetanus toxoid, 54 Thymectomy, 4-5, 78 {see also "Thymus....") neo-natal, 242 Thymus, discovery of role of, 65 intra-thymic clonal deletion, 68-69 Thymus in immunity, 1-20 antigen recognition and major histocompatibility complex (MHC), 7-10 antigen-presenting cell (APC), 8 CD3 complex, 9-10 endocytosis, 8 endogenous pathway, 8 endoplasmic reticulum, 8 exogenous pathway, 8 "germlike genes," 10 MNC restriction, 8 restriction elements, 8 superantigens, 7 TCR, 9-10, 12, 18 in disease states, 17-18 Acquired Immunodeficiency Syndrome (AIDS), 17-18 adenosine deaminase (ADA), 17 autoimmune diseases, 18 diGeorge syndrome, 17 HTLV-1, 17 immune deficiency states, 17 lymphoblastic leukemia, 17 lymphoma, 17 thymomas, 17 historical background, 4-7 B cells, 5-6 in birds, 5-6 bursa, 4 cell-mediated immune responses, 5 humoral immune responses, 5 immunoglobulin, 6 obscure prior to 1960, 4 T cells, 6-7
335
Index
intrathymic events, 15-17 cytokines, 15 double-negative (DN) cells, 15 double-positive cells, 15 MHC polymorphism, 15 negative selection, 16-17 positive selection, 16-17 programmed cell death, 16 introduction, 2-4 cortex, 2,3 dendritic cells, 3, 51 desmosomes, 3 as epitheUal organ, 3-4 HassalFs corpuscles, 3 layers, three, 2 location, 2 lymphocytes, 2 macrophages, 3 medulla, 2,3 "nurse cells," 2 as primary or central lymphoid organ, 4 self-reactive lymphocytes, 3 stem cells, 4 stromal cells, 2 structure, 3 peripheral T cell subsets, 10-12 CD4+ and CD8+ cells, 10-12, 18 interferon-7, 11 interleukin-2, 11, 15 MEL 14, 11 "naive" or "virgin" cells, 11 summary, 18 T cell migration, 12-15 B cell dependent area, 13 high endothelial venules (HEV), 13 integrin, 14 L-selectin, 13 memory-type T cells, 14-15 recirculation of naive T cells, 13-14
spleen, 13-14 {see also "Spleen") T cell dependent area, 13 tissue-selective homing of activated and memory T cells, 14-15 Thyroglobulin (Tg), 244 Thyroid disease, autoimmune, 243252 (see also "Autoimmune diseases....") Thyroid peroxidase (TPO), 244 Thyroid-stimulating hormone receptor (TSHR), 244 Thyroiditis, 242 Thyroxine, thyroglobulin as prohormone for, 252 Tingible bodies, 34-35 Tolerance, 61-82 (see also "Immunological tolerance") Toxins and autoimmune thyroid disease, 251-252 TPO, 244 Transfectoma, 288 Transforming growth factor (TGF), 150 -)8 (TGF-jS), 54-55 Transgenic mice, using, 295 Transgenic technology, introduction of, 65-66 Transplant rejection, cytotoxic cells and,57 Tumor cells, lysis of, cytotoxic cells and,57 Tumor necrosis factor (TNF), 48, 125, 213, 272, 274, 303 A and B, 123, 150, 157, 158, 160 Tyrosine kinase, 239-240 Tyrosine phosphorylation, 139-141 Urticaria, 233 Vaccination, 196-197, 199 Vasoactive intestinal peptide (VIP), 303
INDEX
336
"Veto" cells, 68-69 Viral infection, apoptosis and, 275 Virus infection of thyroid cells, AITD and, 250 Vitronectin, 182 Wheal, 233 White corpuscles, 197 Witebsky"s postulates, 238 Wu-Kabat structures, 98-99 X-LA (X-linked agammaglobulinemia), 39-41
X-LA with hyper-IgM, 41-42 common variable immunodeficiency (CVID), 42 meningitis, bacterial, 42 gp39, 41 IgA deficiency, 41-42 IgM and IgC deficiencies, 42 immunoglobulin isotype deficiencies, 41-42 symptoms, 41 Xenotransplantation, 188 Zap, 142
