JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 8, NUMBER 2
MARCH 1992
BASIC SCIENCE REVIEW
THE NEW IMMUNOLOGY Katherine A. Siminovitch Among the biomedical sciences, immunology stands out as a discipline in which knowledge emanating from fundamental research has rapidly been transferred to the clinical paradigm, with consequent improvement in human health. Virtually all medical subspecialties have benefitted from diagnostic reagents and technologies provided by basic immunology. In terms of numbers of lives saved, immunologic-based therapeutic strategies, most notably vaccination, rank among the most effective measures ever achieved by medical intervention. Yet, despite immunology's profound impact on medicine and the longstanding recognition of many of the general principles and cellular components involved in immunity, until relatively recently, the operational workings of the immune system eluded precise definition. The abstract nature of the immune system rendered the field intangible or, at the very least, confusing, to the nonimmunologic medical community. However, in recent years, this situation has changed radically, as cell cloning, hybridoma, and recombinant DNA technologies have provided the means to delineate the precise immunologic cellular structures and interactions. The purpose of this review is to highlight a few of the most significant advances in immunology during the past decade, and to show how they have made possible the translation of abstract concepts of classical immunology into tangible, structural information. Striking gains in the understanding of antigen recognition, one of the most fundamental aspects of immunity, are described as an illustrative case.
THE IMMUNE SYSTEM—GENERAL PROPERTIES.
In
higher organisms, immune protection against foreign cells, foreign substances, or antigens, is provided by two systems of defense (Fig. 1). The first of these, innate immunity, protects by virtue of nonspecific and constitutively-expressed mechanisms such as phagocytosis by neutrophils and monocytes, or chemical degradation by gastric acid or lysozyme. However, many infectious microbial agents can overcome these natural defenses, and their elimination requires invoking the more powerful mechanisms of the adaptive immune system. Adaptive immunity involves mechanisms that are not constitutively expressed, but instead are induced in response to a given antigen exposure (immunization). They are specifically targeted toward elimination of that particular antigen, a process referred to as a specific "immune response". Effective induction of adaptive immune responses requires coordinate interactions between many cell types, as well as soluble mediators, but the critical elements underlying such responses are B (bone-marrow-derived) and T (thymusderived) lymphocytes. By convention, immunity engendered by B cells is referred to as humoral (antibodymediated) immunity, and that provided by T cells is
termed cell-mediated immunity. The B and T cell arms of the immune response share a number of distinct properties that are integral to the immune system's capacity for protection and that embody the way in which most of us define immunity. These properties are: • specificity for distinguishing one antigen from another by virtue of antigen-specific recognition receptors; • diversity, so that the individual's repertoire of lymphocytes includes cells with antigen receptors collectively capable of recognizing a huge spectrum (at least 109) of distinct antigens; • amplification, so that antigen exposure induces proliferation of an initially small number of responsive lymphocytes to generate an expanded population of cells capable of recognizing/eliminating the antigen; • effector mechanisms for the elimination or neutralization of antigens; • memory, so that the immune response to a particular antigen improves upon reexposure; and • tolerance for self, so that the immune system dis- 121
Department of Medicine, University of Toronto, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto Reprint requests-. Dr. Siminovitch, Mt. Sinai Hospital, Rm.656A, 600 University Ave, Toronto, Ontario, Canada M5G 1X5 Accepted for publication August 30,1991 Copyright © 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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ABSTRACT
JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 8, NUMBER 2
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tinguishes and does not react against self-components, but only against those that are foreign or non-self. THE IMMUNE SYSTEM—ISSUES OF THE '80S. While the key features of B and T lymphocyte-mediatedadaptive immune responses were already recognized by 1980, very little was known about the operational mechanisms whereby these functional behaviors were achieved. More specifically, some of the critical issues facing immunologists in 1980 can be framed as follows:
1. Diversity: How is a pool (or repertoire) of B and T lymphocytes generated that has sufficient diversity to specifically recognize and distinguish among the enormous variety of antigens to which an individual may be exposed? 2. T-cell antigen-recognition: How do T lymphocytes which lack cell-surface immunoglobulin "see" and interact with antigen? 3. Tolerance: How do B and T lymphocytes develop and maintain the capacity to distinguish self from non-self? How does tolerance break down and autoimmunity develop? 4. Coordination of the immune response: What is the nature of the soluble factors mediating communication/co-operation between cells of the immune system? 5. Immunosuppression: How can the specificity intrinsic to B and T lymphocytes be exploited, to selectively target and eliminate pathogenic cells? THE ANTIGEN-RECOGNITION RECEPTORS—SOLUTIONS
122
OF THE '80s. Most of the questions listed above relate to the most essential facet of immunity, that is, the capacity of the immune system to recognize foreign or non-self substances and to distinguish these from self.
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Figure 1. The battle against a constant onslaught of foreign pathogens is waged by two tiers of defense mechanisms: non-specific (or innate) immunity and specific (adaptive) immunity. Innate strategies for defence are non-specific and always present, while adaptive mechanisms are specific and expressed only in response to the offending pathogen. The principal components of the adaptive immune system are B and T lymphocytes.
It is therefore not surprising that, among all the advances in immunology since 1980, identification of the genes and genetic mechanisms involved in the expression of B- and T-cell antigen-recognition receptors stands out as having had the most significant ramifications in terms of understanding immune function. More specifically, the characterization of these genes has provided the solution to the first two issues described above and the framework for resolving the others. In this context, the ensuing discussion is, for the most part, focused on the B- and T-cell antigen receptors, and it is directed toward illustrating how the information about their structure has not only enhanced our understanding of lymphocyte biology, but has also paved the way for development of immunotherapeutic strategies targeted at selective elimination of pathogenic lymphocyte subpopulations.
The B Cell and its Antigen Receptor B-CELL DEVELOPMENT AND FUNCTION.
B-lineage
lymphocytes originate from pluripotential hemopoietic stem cells and, in adult humans, complete all but the most terminal stage of their differentiation in the bone marrow and in the absence of antigen. The key event in B-cell maturation is the synthesis of antibody (immunoglobulin) molecules, which are expressed on the cell surface of all mature B lymphocytes prior to their migration into the bloodstream and peripheral lymphoid organs. It is this antibody anchored in the B-cell membrane that constitutes the B-cell recognition and binding receptor for antigen, and thus represents the cardinal molecule in expression of B-cell-mediated immunity. The membrane antibodies expressed on mature B cells originating from different marrow precursors are not structurally identical and, in fact, pro-
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THE IMMUNE SYSTEM
MARCH 1992
IMMUNOLOGY/SIMINOVITCH
In humoral immunity, then, the antigen receptors or antibodies have both a membrane and soluble form and serve dual functions, mediating both the recognition, as well as elimination, of antigen. B-CELL FUNCTION AND THE CONCEPT OF CLONALITY.
To comprehend why antibody diversity presented such
a problematic issue for immunologists, some understanding of the term "clonality" is necessary. In cell biology, clonal or monoclonal refers to a population of cells that arise from a single precursor cell and are genetically identical. In specific reference to B lymphocytes, a clonal population is comprised of cells that express essentially identical antibody molecules on their surface. The classic example of a B-lineage clonal population is found in multiple myeloma, a condition in which a single plasma cell undergoes malignant transformation. As shown in Figure 3, the unchecked proliferation of the transformed parent plasmacyte yields large numbers of identical progeny cells, all secreting identical or monoclonal antibodies. Along similar lines, the activation of a single mature B cell by antigen results in the clonal expansion of this cell and in the production of B cells and plasma cells that produce identical (monoclonal) antibodies to those expressed by the parent cell. However, in general, the configuration of most antigens is such that the antigen is recognized by and binds to several structurally distinct antigen receptors, and thereby activates multiple, genetically-unrelated B cells. Thus, during a normal immune response, while each activated parent B cell undergoes clonal expansion, the response as a whole is polyclonal, and associated with the appearance of structurally distinct, or polyclonal, antibodies. As described below, the activation and clonal expansion of a B cell by antigen requires the antigen to
THE ADAPTIVE
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vide a cell-specific marker that distinguishes each individual B cell from others. It is by virtue of their structural differences that antibodies manifest specificity, that is, the differential capacity to recognize and interact with given antigens. The humoral or B-cell-mediated arm of the immune response is initiated when antigen entering the system "finds" and binds to the appropriate antigenreceptors on one or more B cells in the periphery. As indicated in Figure 2, this interaction signals the B cell to become "activated" so that the cell proliferates, producing an expanded population of identical cells that either undergo terminal differentiation to yield plasma cells or become memory cells. Plasma cells do not express antibodies on their cell surfaces, but instead secrete these proteins into the circulation. Secreted, soluble antibodies bind to their target antigen and thereby render it susceptible to digestion and elimination by phagocytic cells or by complementmediated lysis. Alternatively, some of the antigenactivated peripheral B cells remain in the circulation as long-lived memory cells, engendering a faster and better quality antibody response to a subsequent exposure to the same antigen.
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Figure 2. The adaptive immune response involves antigen recognition by lymphocytes, lymphocyte activation, and antigen elimination. Left panel: The B-cell (humoral) response begins when antigen binds to the antigen receptor (antibody) on B cells and induces B-cell activation. Activated B cells proliferate and differentiate into antibody-secreting plasma cells. Secreted antibodies bind antigen and facilitate its elimination by phagocytosis and complement lysis. Right panel: The T-cell (cellular) response begins only after antigen is taken up by an antigen-presenting cell, broken down into peptide fragments, complexed to a major histocompatibility complex (MHC) protein, and the complex reexpressed on the surface of the presenting cell. Contact of antigen-MHC complexes with the T-cell antigen receptor induces T-cell activation, proliferation, and expression of T-cell effector functions: help or cytotoxicity. Helper T cells secrete factors (cytokines) that induce further B-cell activation and differentiation. Cytotoxic T cells induce lysis of antigen-bearing cells.
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MARCH 1992
124
find or "select" among the spectrum of available B-cell types) of heavy chains (mu, delta, gamma, alpha, and antigen receptors those having the appropriate bind- epsilon) and two types of light chains (kappa, lambda) ing sites, a phenomenon which is referred to as clonal are recognized. Within a single antibody, the two heavy selection and which represents a key facet of the adaptive and light chains are of the same isotype and type, immune system's modus operandi. Implicit in the con- respectively; antibodies are described accordingly, so cept of clonal selection is the understanding that the that an antibody composed of mu heavy chains and spectrum or repertoire of available antigen receptors kappa light chains is referred to as an IgMk antibody. is preestablished in the absence of antigen so that The structural distinction of the antibody polymature B cells leave the bone marrow already pro- peptides into V and C regions directly reflects the grammed to react with particular antigens. In other duality of antibody function, that is, the mediation of words, antigens do not invoke the expression of their both antigen recognition and its elimination. The first antigen-receptor counterparts but, instead, the indi- of these two functions is, not surprisingly, provided by vidual must generate a repertoire of B cells bearing the heavy- and light-chain V region amino acids. These antigen receptors with sufficient structural diversity to residues form the antibody's combining site for antirecognize all potential antigen exposures. As de- gen; their intrinsic sequence variation is consistent scribed below, the cloning of the antibody genes has with the expression of structurally diverse antibodies provided the answer to how such an enormous degree that can react specifically with a wide spectrum of of diversity can be achieved. antigens. In fact, it is amino acid residues mapping ANTIBODY STRUCTURE AND FUNCTION. The basic within the most highly variable segments (hypervaristructure of antibodies, at least at the protein level, able) of the V regions, that appear most critical to was elucidated many years ago and is described here antigen binding. While the V regions of antibodies are briefly in relation to antibody function. As shown in responsible for antibody specificity, it is sequences Figure 4, antibodies (or immunoglobulins) are Y-shaped within the heavy-chain constant regions that mediate proteins composed of two heavy and light chain poly- the elimination or neutralization of antigen by complepeptide pairs joined together by disulfide bonds. By ment lysis or phagocytosis. There are isotype-related the mid-1960's, data from studies of antibody structure differences in antibody effector functions, but in all had revealed that the amino-acid residues comprising antibodies, effector activity is mediated by the heavyone end (amino-terminal) of both the heavy and light chain constant region portion, as is consistent with a chains varied considerably from one antibody to an- lack of requirement for structural diversity. other, while the residues within the remaining and Clarification of the relationship between antibody carboxy-terminal regions of these chains were very structure and function represented a major step tosimilar among different immunoglobulins (see Fig. 4). ward understanding the genesis of antibody diversity, On this basis, these regions are now referred to as the but did not entirely resolve the issue. As noted above, variable (V) and constant (C) regions, respectively. the requirement for B cells to spontaneously manifest Each heavy and light chain therefore contains a V and a a repertoire of antigen-receptors with specificities for C region. Some structural variations also occur among the vast array of potential antigens, was difficult to the C regions of different antibodies and, depending reconcile with the genetic wisdom of the pre-recombion which C regions are used, five major classes (iso- nant DNA era, which dictated that every distinct poly-
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Figure 3. Malignant transformation of a single plasma cell in multiple myeloma results in the generation of very large numbers of geneticallyidentical cells, producing identical or monoclonal antibodies. A monoclonalantibody population can be detected as a spike on a serum-electrophoretic tracing. In contrast, a normal immune response involves activation and proliferation of different B cells, producing distinct or polyclonal antibodies that migrate differentially during electrophoresis.
IMMUNOLOGY/SIMINOVITCH
peptide chain was the product of one gene. If this were true, then the generation of an antibody repertoire with sufficient diversity to specifically interact with millions of distinct antigens would require most of our useable genetic material. ANTIBODY GENES AND THE GENERATION OF DIVERSITY.
The resolution of the diversity dilemma was rendered possible by the isolation of the genes encoding antibodies and the realization that contrary to the one gene/polypeptide doctrine, each individual heavy and light chain of an antibody is derived by virtue of the rearrangement and assembly of multiple gene segments. The principal facets of antibody gene assembly are illustrated in Figure 5. As shown, the genetic elements involved in the formation of an antibody polypeptide chain are not continuous, but are widely separated from one another in the genome. Among these elements, for example, at the kappa-chain gene locus,
Figure 5. The gene segments encoding antibody molecules are widely separated in the germline. During B-cell differentiation, these segments are rearranged so that a V-D-J and a V-) element are juxtaposed and encode the heavy- and light-chain V regions, respectively. After transcription, splicing, and translation, the heavy and light chains are assembled to construct an antibody molecule.
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Figure 4. A single antibody molecule is composed of two heavy and two light chains. In the lower left, the amino acid sequences encoding a small part of the variable and a small part of the constant regions of four different antibodies, are compared. Differences in shading indicate the use of a different amino acid than that used in the first antibody.
there are many (about 100) that differ slightly from one another in sequence, but that share the capacity to encode the V-region portion of a kappa protein. However, there is but a single gene segment corresponding to the C region. In between the V and C segments are J (joining) segments that also encode a small portion of the kappa-chain V region. This, then, represents the "germ-line" configuration of the kappachain genes, that is, the organization of the kappa gene elements in the DNA of all the cells in our body. However, during the course of B-cell differentiation, the DNA at the kappa gene locus undergoes a rearrangement, so that a single V and J segment are juxtaposed, thereby creating a V-) gene that will ultimately encode the V region of the kappa chain produced by this cell. Transcription and splicing of the rearranged DNA segment then yields a V-J-C messenger RNA which will be translated into kappa polypeptide. The very same ge-
125
netic mechanism is used to generate heavy chains (see Fig. 5) and lambda light chains, although the germ-line organization and chromosomal location of the gene segments encoding each of these polypeptides are distinct. The availability of multiple distinct V, I and, in the case of heavy chains, D (Diversity), segments, that can be reshuffled at random in many different ways, provides a mechanism for creation of millions of distinct antibody V regions from relatively little genetic material. In addition to rearrangement per se, imprecise rejoining of V-J orV-D-J segments, differential combining of particular heavy chains with particular light chains and, most significant, point mutations in the V region nucleotide sequence, contribute to the diversification of the antibody repertoire. Somatic mutation differs from the other diversification mechanisms, in that it occurs, for the most part, in conjunction with B-cell proliferation after antigen-induced activation. It appears to "fine-tune" the V regions so that antibodyantigen binding is strengthened. V-region mutations are most commonly found in antibodies formed after reexposure to antigen, and they provide a structural explanation for the improved quality of secondary over primary immune responses. In any case, as is easily discernable, the various genetic mechanisms used for diversifying the antibody repertoire together provide the means to generate a potentially enormous array of distinct antigen-combining sites, and their identification has resolved at least one of the major dilemmas in immunology.
The T Cell and its Antigen Receptor T-CELL DEVELOPMENT AND FUNCTION.
126
The second
principal player in the expression of adaptive immunity is the T lymphocyte—a cell type that, like the B-lineage, originates from multipotential hematopoietic stem cells in the marrow. However, T cells become immunocompetent and recognizable as being of the T lineage, only after marrow-derived T precursors have undergone differentiation in the thymus. T lymphocytes operate by virtue of many of the same principles as B cells. Mature T cells, for example, do not interact with foreign antigen while in the thymus but instead, like B cells, emigrate into the bloodstream and enter the lymphatic system and secondary lymphoid organs where antigen encounter may occur. As illustrated in Figure 2, T cells are also subject to clonal selection by antigen and, in response to antigen, undergo activation, clonal expansion, and expression of effector functions directed at antigen elimination. However, while there are parallels in the manner in which B and T cells engender immunity, the precise mechanisms whereby these two lymphocyte subclasses accomplish antigen recognition and elimination are markedly different. For
MARCH 1992
example, while T and B cells share the ability to specifically recognize a diverse spectrum of antigens, T cells, unlike B cells, cannot "see" native antigen alone, but require that the antigen be processed, that is, broken down into smaller peptide fragments and complexed to a self-protein, specifically a protein encoded by the major histocompatibility complex (MHC). Moreover, T cells do not synthesize or secrete antibodies and interact with antigen-MHC complexes only when they come in direct contact with the T-cell surface. Thus, in contrast to B cells, T lymphocytes exercise their effector functions directly (hence the term "cell-mediated immunity"), and they do not rely on any intermediary proteins, such as antibodies. The major effector functions carried out by T cells include the killing of antigen-infected cells (cytotoxicity), and the induction of B-cell activation, differentiation, and antibody secretion (helper). Therefore, T cells play both an effector and a regulatory role in the immune response. These facets of T-cell behavior were already recognized by 1980. In addition, it was understood that T cells were the critical players in the genesis of tolerance to self, the most fundamental aspect of immune function, and they were also the critical culprits underlying a spectrum of clinical problems (notably autoimmune disease and allograft rejection). However, while it was generally agreed that some T-cell counterpart to the B-cell antigen receptor must exist, its nature was entirely unknown. And since the identification of the molecule(s) whereby T cells recognize antigen was clearly a prerequisite to the understanding of so many critical issues in immunology, the search for the T-cell antigen receptor represented one of the most prominent objectives fueling immunology research within this last decade. T-CELL ANTIGEN RECOGNITION AND THE MAJOR HISTOAS noted above, T cells recognize foreign antigen only when it is combined with another molecule, specifically with one of the proteins encoded by genes of the major histocompatibility complex (MHC). The MHC glycoproteins, also known as human leucocyte antigens (HLA), are highly polymorphic (variable) among the population, but are generally subdivided into two types, class. I and class II. The class I MHC proteins are encoded by three gene loci, HLA-A, B, and C, are found on all nucleated cells, and are structurally distinct from the class II MHC proteins. The latter are encoded at the HLA-DR, DQ, and DP loci and are present only on immunocompetent cells, such as B cells, macrophages, and activated T cells. Because the MHC proteins are so variable, they constitute a marker that distinguishes one individual from the other, in other words a marker of "self". Although initially identified in the context of tissue transplantation and rejection, MHC molecules are now recognized as key to normal immune function. More specifically, MHC proteins facilitate "presentaCOMPATIBILITY COMPLEX.
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JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 8, NUMBER 2
MARCH 1992
Figure 7. To obtain monoclonal antibodies reactive with a particular human protein, mice are immunized with the protein of interest (triangles in lower right), and their B cells are subsequently isolated. PEG is used to fuse these B cells with myeloma cells that secrete no antibody and that die when grown in HAT medium. The cells are plated in microwell plates and cultured in HAT-containing medium, so that the unfused B cells and myeloma cells die, and only B-cell-myeloma hybrids survive. Supernatants from each well are then screened for the presence of antibodies that bind the protein of interest. Hybrids from the microwells positive for antibody production are replated at a density of one hybrid/well (cloning), to obtain a clonal-cell population secreting monoclonal antibodies.
fusing B cells producing an antibody of a given specificity, with myeloma cells that have been selected for their inability to produce antibodies and to grow in a selective medium called HAT (Hypoxanthine/Aminopterin/Thymidine). After fusion, cells are cultured in HAT medium. While normal B cells can grow in HAT, they cannot survive in culture. Conversely, the myeloma cells can survive in normal, but not in HATcontaining, culture medium. Myeloma-B cell hybrids, however, can survive and continue to produce antibody. Of course the myeloma cells will have fused with several B cells, each with its own specificity. However, cultures containing the desired hybrids are identified by screening for the antibody directed to the specific antigen, and they are then plated to isolate a single hybrid that can be grown and expanded to yield a monoclonal cell population secreting monoclonal antibodies. Such antibodies have provided a powerful source of reagents, both for probing basic cell function and for the diagnosis/investigation of a spectrum of clinical problems. TARGETING LYMPHOCYTE ANTIGEN RECEPTORS.
I 28
His-
torically, the immunointerventive strategies used to eliminate pathologic {e.g., malignant, autoreactive) lymphocyte populations, have invoked non-specific manipulations that affect the normal, as well as abnormal, cells and are thus associated with considerable toxicity. However, the realization that lymphocyte antigen receptors constitute cell-specific markers, combined with the availability of monoclonal antibodies, has provided the means to address this problem. A
good example is provided by B-cell neoplasms, such as B-cell lymphoma, in which the membrane antibody on the tumor cells distinguishes malignant from normal lymphocytes. Antibodies, and most particularly the V-region portions, are immunogenic and thus, as illustrated in Figure 8, immunization of an animal with the lymphoma membrane antibody, and subsequent hybridoma fusion, can be used to generate monoclonal antibodies that bind specifically to the lymphoma membrane antibody. This monoclonal reagent may be used alone or coupled to a cell toxin, to specifically target and eliminate the lymphoma cells while leaving their normal cell counterparts intact. The potential therapeutic value of selective immunointervention extends to a broad spectrum of diseases and, in particular, to a number of autoimmune diseases in which the clinical abnormalities can be ascribed largely to the actions of pathogenic, autoreactive T cells. Extensive efforts are currently underway to determine whether such latter populations are clonal or oligoclonal and whether they might express only one or a few antigen receptors that could serve as targets for monoclonal antibody therapy. Based on preliminary findings in animal models of autoimmunity, it seems likely that at least some autoimmune diseases will be amenable to this therapeutic approach.
SUMMARY During this past decade, we have witnessed a virtual explosion in our understanding of the immune
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JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 8, NUMBER 2
IMMUNOLOGY/SIMINOVITCH
system. Although this review has specifically focused on the genetic characterization of the lymphocyte antigen receptors, recent progress in immunology has resulted in a plethora of other remarkable breakthroughs, including the identification of cytokines and their role in intercellular communication, of molecules involved in lymphocyte homing and adhesion, and of the crystal structure of MHC molecules. These and the many other recent advances have served to demystify immunology and to further meld the fundamental sciences with clinical care. Such developments provide the framework for the continued resolution of the mechanisms
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Figure 8. Membrane antibodies constitute a cell-specific marker that distinguishes a B-lymphoma cell from all other B cells. Mice can be immunized with the lymphoma-associated antibody (right panel), to create a monoclonal antibody that reacts with the "antigenic" portion (usually the variable regions) of the membrane antibody on the lymphoma-cell surface. The monoclonal antibody can be used alone or couple to a cell toxin, to selectively destroy the lymphoma cells.
that mediate and regulate the immune response and for addressing the therapeutic challenges presented by the spectrum of immunologically-mediated human diseases. K. Siminovitch is a Career Scientist of the Ministry of Health and a recipient of a Canadian Life and Health Insurance Association Medical Scholarship, as well as grants from the Medical Research Council, Arthritis Society, and the National Cancer Institute of Canada. The review of the manuscript by Drs. M. Denise Daley and Lou Siminovitch, and the help in manuscript preparation provided by Bev Bessey and IMS. Creative Communications are appreciated.
129
MARCH 1992
BASIC SCIENCE REVIEW
THE NEW IMMUNOLOGY Katherine A. Siminovitch Among the biomedical sciences, immunology stands out as a discipline in which knowledge emanating from fundamental research has rapidly been transferred to the clinical paradigm, with consequent improvement in human health. Virtually all medical subspecialties have benefitted from diagnostic reagents and technologies provided by basic immunology. In terms of numbers of lives saved, immunologic-based therapeutic strategies, most notably vaccination, rank among the most effective measures ever achieved by medical intervention. Yet, despite immunology's profound impact on medicine and the longstanding recognition of many of the general principles and cellular components involved in immunity, until relatively recently, the operational workings of the immune system eluded precise definition. The abstract nature of the immune system rendered the field intangible or, at the very least, confusing, to the nonimmunologic medical community. However, in recent years, this situation has changed radically, as cell cloning, hybridoma, and recombinant DNA technologies have provided the means to delineate the precise immunologic cellular structures and interactions. The purpose of this review is to highlight a few of the most significant advances in immunology during the past decade, and to show how they have made possible the translation of abstract concepts of classical immunology into tangible, structural information. Striking gains in the understanding of antigen recognition, one of the most fundamental aspects of immunity, are described as an illustrative case.
THE IMMUNE SYSTEM—GENERAL PROPERTIES.
In
higher organisms, immune protection against foreign cells, foreign substances, or antigens, is provided by two systems of defense (Fig. 1). The first of these, innate immunity, protects by virtue of nonspecific and constitutively-expressed mechanisms such as phagocytosis by neutrophils and monocytes, or chemical degradation by gastric acid or lysozyme. However, many infectious microbial agents can overcome these natural defenses, and their elimination requires invoking the more powerful mechanisms of the adaptive immune system. Adaptive immunity involves mechanisms that are not constitutively expressed, but instead are induced in response to a given antigen exposure (immunization). They are specifically targeted toward elimination of that particular antigen, a process referred to as a specific "immune response". Effective induction of adaptive immune responses requires coordinate interactions between many cell types, as well as soluble mediators, but the critical elements underlying such responses are B (bone-marrow-derived) and T (thymusderived) lymphocytes. By convention, immunity engendered by B cells is referred to as humoral (antibodymediated) immunity, and that provided by T cells is
termed cell-mediated immunity. The B and T cell arms of the immune response share a number of distinct properties that are integral to the immune system's capacity for protection and that embody the way in which most of us define immunity. These properties are: • specificity for distinguishing one antigen from another by virtue of antigen-specific recognition receptors; • diversity, so that the individual's repertoire of lymphocytes includes cells with antigen receptors collectively capable of recognizing a huge spectrum (at least 109) of distinct antigens; • amplification, so that antigen exposure induces proliferation of an initially small number of responsive lymphocytes to generate an expanded population of cells capable of recognizing/eliminating the antigen; • effector mechanisms for the elimination or neutralization of antigens; • memory, so that the immune response to a particular antigen improves upon reexposure; and • tolerance for self, so that the immune system dis- 121
Department of Medicine, University of Toronto, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto Reprint requests-. Dr. Siminovitch, Mt. Sinai Hospital, Rm.656A, 600 University Ave, Toronto, Ontario, Canada M5G 1X5 Accepted for publication August 30,1991 Copyright © 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
Downloaded by: National University of Singapore. Copyrighted material.
ABSTRACT
JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 8, NUMBER 2
V
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tinguishes and does not react against self-components, but only against those that are foreign or non-self. THE IMMUNE SYSTEM—ISSUES OF THE '80S. While the key features of B and T lymphocyte-mediatedadaptive immune responses were already recognized by 1980, very little was known about the operational mechanisms whereby these functional behaviors were achieved. More specifically, some of the critical issues facing immunologists in 1980 can be framed as follows:
1. Diversity: How is a pool (or repertoire) of B and T lymphocytes generated that has sufficient diversity to specifically recognize and distinguish among the enormous variety of antigens to which an individual may be exposed? 2. T-cell antigen-recognition: How do T lymphocytes which lack cell-surface immunoglobulin "see" and interact with antigen? 3. Tolerance: How do B and T lymphocytes develop and maintain the capacity to distinguish self from non-self? How does tolerance break down and autoimmunity develop? 4. Coordination of the immune response: What is the nature of the soluble factors mediating communication/co-operation between cells of the immune system? 5. Immunosuppression: How can the specificity intrinsic to B and T lymphocytes be exploited, to selectively target and eliminate pathogenic cells? THE ANTIGEN-RECOGNITION RECEPTORS—SOLUTIONS
122
OF THE '80s. Most of the questions listed above relate to the most essential facet of immunity, that is, the capacity of the immune system to recognize foreign or non-self substances and to distinguish these from self.
\
Figure 1. The battle against a constant onslaught of foreign pathogens is waged by two tiers of defense mechanisms: non-specific (or innate) immunity and specific (adaptive) immunity. Innate strategies for defence are non-specific and always present, while adaptive mechanisms are specific and expressed only in response to the offending pathogen. The principal components of the adaptive immune system are B and T lymphocytes.
It is therefore not surprising that, among all the advances in immunology since 1980, identification of the genes and genetic mechanisms involved in the expression of B- and T-cell antigen-recognition receptors stands out as having had the most significant ramifications in terms of understanding immune function. More specifically, the characterization of these genes has provided the solution to the first two issues described above and the framework for resolving the others. In this context, the ensuing discussion is, for the most part, focused on the B- and T-cell antigen receptors, and it is directed toward illustrating how the information about their structure has not only enhanced our understanding of lymphocyte biology, but has also paved the way for development of immunotherapeutic strategies targeted at selective elimination of pathogenic lymphocyte subpopulations.
The B Cell and its Antigen Receptor B-CELL DEVELOPMENT AND FUNCTION.
B-lineage
lymphocytes originate from pluripotential hemopoietic stem cells and, in adult humans, complete all but the most terminal stage of their differentiation in the bone marrow and in the absence of antigen. The key event in B-cell maturation is the synthesis of antibody (immunoglobulin) molecules, which are expressed on the cell surface of all mature B lymphocytes prior to their migration into the bloodstream and peripheral lymphoid organs. It is this antibody anchored in the B-cell membrane that constitutes the B-cell recognition and binding receptor for antigen, and thus represents the cardinal molecule in expression of B-cell-mediated immunity. The membrane antibodies expressed on mature B cells originating from different marrow precursors are not structurally identical and, in fact, pro-
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THE IMMUNE SYSTEM
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IMMUNOLOGY/SIMINOVITCH
In humoral immunity, then, the antigen receptors or antibodies have both a membrane and soluble form and serve dual functions, mediating both the recognition, as well as elimination, of antigen. B-CELL FUNCTION AND THE CONCEPT OF CLONALITY.
To comprehend why antibody diversity presented such
a problematic issue for immunologists, some understanding of the term "clonality" is necessary. In cell biology, clonal or monoclonal refers to a population of cells that arise from a single precursor cell and are genetically identical. In specific reference to B lymphocytes, a clonal population is comprised of cells that express essentially identical antibody molecules on their surface. The classic example of a B-lineage clonal population is found in multiple myeloma, a condition in which a single plasma cell undergoes malignant transformation. As shown in Figure 3, the unchecked proliferation of the transformed parent plasmacyte yields large numbers of identical progeny cells, all secreting identical or monoclonal antibodies. Along similar lines, the activation of a single mature B cell by antigen results in the clonal expansion of this cell and in the production of B cells and plasma cells that produce identical (monoclonal) antibodies to those expressed by the parent cell. However, in general, the configuration of most antigens is such that the antigen is recognized by and binds to several structurally distinct antigen receptors, and thereby activates multiple, genetically-unrelated B cells. Thus, during a normal immune response, while each activated parent B cell undergoes clonal expansion, the response as a whole is polyclonal, and associated with the appearance of structurally distinct, or polyclonal, antibodies. As described below, the activation and clonal expansion of a B cell by antigen requires the antigen to
THE ADAPTIVE
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vide a cell-specific marker that distinguishes each individual B cell from others. It is by virtue of their structural differences that antibodies manifest specificity, that is, the differential capacity to recognize and interact with given antigens. The humoral or B-cell-mediated arm of the immune response is initiated when antigen entering the system "finds" and binds to the appropriate antigenreceptors on one or more B cells in the periphery. As indicated in Figure 2, this interaction signals the B cell to become "activated" so that the cell proliferates, producing an expanded population of identical cells that either undergo terminal differentiation to yield plasma cells or become memory cells. Plasma cells do not express antibodies on their cell surfaces, but instead secrete these proteins into the circulation. Secreted, soluble antibodies bind to their target antigen and thereby render it susceptible to digestion and elimination by phagocytic cells or by complementmediated lysis. Alternatively, some of the antigenactivated peripheral B cells remain in the circulation as long-lived memory cells, engendering a faster and better quality antibody response to a subsequent exposure to the same antigen.
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Figure 2. The adaptive immune response involves antigen recognition by lymphocytes, lymphocyte activation, and antigen elimination. Left panel: The B-cell (humoral) response begins when antigen binds to the antigen receptor (antibody) on B cells and induces B-cell activation. Activated B cells proliferate and differentiate into antibody-secreting plasma cells. Secreted antibodies bind antigen and facilitate its elimination by phagocytosis and complement lysis. Right panel: The T-cell (cellular) response begins only after antigen is taken up by an antigen-presenting cell, broken down into peptide fragments, complexed to a major histocompatibility complex (MHC) protein, and the complex reexpressed on the surface of the presenting cell. Contact of antigen-MHC complexes with the T-cell antigen receptor induces T-cell activation, proliferation, and expression of T-cell effector functions: help or cytotoxicity. Helper T cells secrete factors (cytokines) that induce further B-cell activation and differentiation. Cytotoxic T cells induce lysis of antigen-bearing cells.
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find or "select" among the spectrum of available B-cell types) of heavy chains (mu, delta, gamma, alpha, and antigen receptors those having the appropriate bind- epsilon) and two types of light chains (kappa, lambda) ing sites, a phenomenon which is referred to as clonal are recognized. Within a single antibody, the two heavy selection and which represents a key facet of the adaptive and light chains are of the same isotype and type, immune system's modus operandi. Implicit in the con- respectively; antibodies are described accordingly, so cept of clonal selection is the understanding that the that an antibody composed of mu heavy chains and spectrum or repertoire of available antigen receptors kappa light chains is referred to as an IgMk antibody. is preestablished in the absence of antigen so that The structural distinction of the antibody polymature B cells leave the bone marrow already pro- peptides into V and C regions directly reflects the grammed to react with particular antigens. In other duality of antibody function, that is, the mediation of words, antigens do not invoke the expression of their both antigen recognition and its elimination. The first antigen-receptor counterparts but, instead, the indi- of these two functions is, not surprisingly, provided by vidual must generate a repertoire of B cells bearing the heavy- and light-chain V region amino acids. These antigen receptors with sufficient structural diversity to residues form the antibody's combining site for antirecognize all potential antigen exposures. As de- gen; their intrinsic sequence variation is consistent scribed below, the cloning of the antibody genes has with the expression of structurally diverse antibodies provided the answer to how such an enormous degree that can react specifically with a wide spectrum of of diversity can be achieved. antigens. In fact, it is amino acid residues mapping ANTIBODY STRUCTURE AND FUNCTION. The basic within the most highly variable segments (hypervaristructure of antibodies, at least at the protein level, able) of the V regions, that appear most critical to was elucidated many years ago and is described here antigen binding. While the V regions of antibodies are briefly in relation to antibody function. As shown in responsible for antibody specificity, it is sequences Figure 4, antibodies (or immunoglobulins) are Y-shaped within the heavy-chain constant regions that mediate proteins composed of two heavy and light chain poly- the elimination or neutralization of antigen by complepeptide pairs joined together by disulfide bonds. By ment lysis or phagocytosis. There are isotype-related the mid-1960's, data from studies of antibody structure differences in antibody effector functions, but in all had revealed that the amino-acid residues comprising antibodies, effector activity is mediated by the heavyone end (amino-terminal) of both the heavy and light chain constant region portion, as is consistent with a chains varied considerably from one antibody to an- lack of requirement for structural diversity. other, while the residues within the remaining and Clarification of the relationship between antibody carboxy-terminal regions of these chains were very structure and function represented a major step tosimilar among different immunoglobulins (see Fig. 4). ward understanding the genesis of antibody diversity, On this basis, these regions are now referred to as the but did not entirely resolve the issue. As noted above, variable (V) and constant (C) regions, respectively. the requirement for B cells to spontaneously manifest Each heavy and light chain therefore contains a V and a a repertoire of antigen-receptors with specificities for C region. Some structural variations also occur among the vast array of potential antigens, was difficult to the C regions of different antibodies and, depending reconcile with the genetic wisdom of the pre-recombion which C regions are used, five major classes (iso- nant DNA era, which dictated that every distinct poly-
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Figure 3. Malignant transformation of a single plasma cell in multiple myeloma results in the generation of very large numbers of geneticallyidentical cells, producing identical or monoclonal antibodies. A monoclonalantibody population can be detected as a spike on a serum-electrophoretic tracing. In contrast, a normal immune response involves activation and proliferation of different B cells, producing distinct or polyclonal antibodies that migrate differentially during electrophoresis.
IMMUNOLOGY/SIMINOVITCH
peptide chain was the product of one gene. If this were true, then the generation of an antibody repertoire with sufficient diversity to specifically interact with millions of distinct antigens would require most of our useable genetic material. ANTIBODY GENES AND THE GENERATION OF DIVERSITY.
The resolution of the diversity dilemma was rendered possible by the isolation of the genes encoding antibodies and the realization that contrary to the one gene/polypeptide doctrine, each individual heavy and light chain of an antibody is derived by virtue of the rearrangement and assembly of multiple gene segments. The principal facets of antibody gene assembly are illustrated in Figure 5. As shown, the genetic elements involved in the formation of an antibody polypeptide chain are not continuous, but are widely separated from one another in the genome. Among these elements, for example, at the kappa-chain gene locus,
Figure 5. The gene segments encoding antibody molecules are widely separated in the germline. During B-cell differentiation, these segments are rearranged so that a V-D-J and a V-) element are juxtaposed and encode the heavy- and light-chain V regions, respectively. After transcription, splicing, and translation, the heavy and light chains are assembled to construct an antibody molecule.
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Figure 4. A single antibody molecule is composed of two heavy and two light chains. In the lower left, the amino acid sequences encoding a small part of the variable and a small part of the constant regions of four different antibodies, are compared. Differences in shading indicate the use of a different amino acid than that used in the first antibody.
there are many (about 100) that differ slightly from one another in sequence, but that share the capacity to encode the V-region portion of a kappa protein. However, there is but a single gene segment corresponding to the C region. In between the V and C segments are J (joining) segments that also encode a small portion of the kappa-chain V region. This, then, represents the "germ-line" configuration of the kappachain genes, that is, the organization of the kappa gene elements in the DNA of all the cells in our body. However, during the course of B-cell differentiation, the DNA at the kappa gene locus undergoes a rearrangement, so that a single V and J segment are juxtaposed, thereby creating a V-) gene that will ultimately encode the V region of the kappa chain produced by this cell. Transcription and splicing of the rearranged DNA segment then yields a V-J-C messenger RNA which will be translated into kappa polypeptide. The very same ge-
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netic mechanism is used to generate heavy chains (see Fig. 5) and lambda light chains, although the germ-line organization and chromosomal location of the gene segments encoding each of these polypeptides are distinct. The availability of multiple distinct V, I and, in the case of heavy chains, D (Diversity), segments, that can be reshuffled at random in many different ways, provides a mechanism for creation of millions of distinct antibody V regions from relatively little genetic material. In addition to rearrangement per se, imprecise rejoining of V-J orV-D-J segments, differential combining of particular heavy chains with particular light chains and, most significant, point mutations in the V region nucleotide sequence, contribute to the diversification of the antibody repertoire. Somatic mutation differs from the other diversification mechanisms, in that it occurs, for the most part, in conjunction with B-cell proliferation after antigen-induced activation. It appears to "fine-tune" the V regions so that antibodyantigen binding is strengthened. V-region mutations are most commonly found in antibodies formed after reexposure to antigen, and they provide a structural explanation for the improved quality of secondary over primary immune responses. In any case, as is easily discernable, the various genetic mechanisms used for diversifying the antibody repertoire together provide the means to generate a potentially enormous array of distinct antigen-combining sites, and their identification has resolved at least one of the major dilemmas in immunology.
The T Cell and its Antigen Receptor T-CELL DEVELOPMENT AND FUNCTION.
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The second
principal player in the expression of adaptive immunity is the T lymphocyte—a cell type that, like the B-lineage, originates from multipotential hematopoietic stem cells in the marrow. However, T cells become immunocompetent and recognizable as being of the T lineage, only after marrow-derived T precursors have undergone differentiation in the thymus. T lymphocytes operate by virtue of many of the same principles as B cells. Mature T cells, for example, do not interact with foreign antigen while in the thymus but instead, like B cells, emigrate into the bloodstream and enter the lymphatic system and secondary lymphoid organs where antigen encounter may occur. As illustrated in Figure 2, T cells are also subject to clonal selection by antigen and, in response to antigen, undergo activation, clonal expansion, and expression of effector functions directed at antigen elimination. However, while there are parallels in the manner in which B and T cells engender immunity, the precise mechanisms whereby these two lymphocyte subclasses accomplish antigen recognition and elimination are markedly different. For
MARCH 1992
example, while T and B cells share the ability to specifically recognize a diverse spectrum of antigens, T cells, unlike B cells, cannot "see" native antigen alone, but require that the antigen be processed, that is, broken down into smaller peptide fragments and complexed to a self-protein, specifically a protein encoded by the major histocompatibility complex (MHC). Moreover, T cells do not synthesize or secrete antibodies and interact with antigen-MHC complexes only when they come in direct contact with the T-cell surface. Thus, in contrast to B cells, T lymphocytes exercise their effector functions directly (hence the term "cell-mediated immunity"), and they do not rely on any intermediary proteins, such as antibodies. The major effector functions carried out by T cells include the killing of antigen-infected cells (cytotoxicity), and the induction of B-cell activation, differentiation, and antibody secretion (helper). Therefore, T cells play both an effector and a regulatory role in the immune response. These facets of T-cell behavior were already recognized by 1980. In addition, it was understood that T cells were the critical players in the genesis of tolerance to self, the most fundamental aspect of immune function, and they were also the critical culprits underlying a spectrum of clinical problems (notably autoimmune disease and allograft rejection). However, while it was generally agreed that some T-cell counterpart to the B-cell antigen receptor must exist, its nature was entirely unknown. And since the identification of the molecule(s) whereby T cells recognize antigen was clearly a prerequisite to the understanding of so many critical issues in immunology, the search for the T-cell antigen receptor represented one of the most prominent objectives fueling immunology research within this last decade. T-CELL ANTIGEN RECOGNITION AND THE MAJOR HISTOAS noted above, T cells recognize foreign antigen only when it is combined with another molecule, specifically with one of the proteins encoded by genes of the major histocompatibility complex (MHC). The MHC glycoproteins, also known as human leucocyte antigens (HLA), are highly polymorphic (variable) among the population, but are generally subdivided into two types, class. I and class II. The class I MHC proteins are encoded by three gene loci, HLA-A, B, and C, are found on all nucleated cells, and are structurally distinct from the class II MHC proteins. The latter are encoded at the HLA-DR, DQ, and DP loci and are present only on immunocompetent cells, such as B cells, macrophages, and activated T cells. Because the MHC proteins are so variable, they constitute a marker that distinguishes one individual from the other, in other words a marker of "self". Although initially identified in the context of tissue transplantation and rejection, MHC molecules are now recognized as key to normal immune function. More specifically, MHC proteins facilitate "presentaCOMPATIBILITY COMPLEX.
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Figure 7. To obtain monoclonal antibodies reactive with a particular human protein, mice are immunized with the protein of interest (triangles in lower right), and their B cells are subsequently isolated. PEG is used to fuse these B cells with myeloma cells that secrete no antibody and that die when grown in HAT medium. The cells are plated in microwell plates and cultured in HAT-containing medium, so that the unfused B cells and myeloma cells die, and only B-cell-myeloma hybrids survive. Supernatants from each well are then screened for the presence of antibodies that bind the protein of interest. Hybrids from the microwells positive for antibody production are replated at a density of one hybrid/well (cloning), to obtain a clonal-cell population secreting monoclonal antibodies.
fusing B cells producing an antibody of a given specificity, with myeloma cells that have been selected for their inability to produce antibodies and to grow in a selective medium called HAT (Hypoxanthine/Aminopterin/Thymidine). After fusion, cells are cultured in HAT medium. While normal B cells can grow in HAT, they cannot survive in culture. Conversely, the myeloma cells can survive in normal, but not in HATcontaining, culture medium. Myeloma-B cell hybrids, however, can survive and continue to produce antibody. Of course the myeloma cells will have fused with several B cells, each with its own specificity. However, cultures containing the desired hybrids are identified by screening for the antibody directed to the specific antigen, and they are then plated to isolate a single hybrid that can be grown and expanded to yield a monoclonal cell population secreting monoclonal antibodies. Such antibodies have provided a powerful source of reagents, both for probing basic cell function and for the diagnosis/investigation of a spectrum of clinical problems. TARGETING LYMPHOCYTE ANTIGEN RECEPTORS.
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His-
torically, the immunointerventive strategies used to eliminate pathologic {e.g., malignant, autoreactive) lymphocyte populations, have invoked non-specific manipulations that affect the normal, as well as abnormal, cells and are thus associated with considerable toxicity. However, the realization that lymphocyte antigen receptors constitute cell-specific markers, combined with the availability of monoclonal antibodies, has provided the means to address this problem. A
good example is provided by B-cell neoplasms, such as B-cell lymphoma, in which the membrane antibody on the tumor cells distinguishes malignant from normal lymphocytes. Antibodies, and most particularly the V-region portions, are immunogenic and thus, as illustrated in Figure 8, immunization of an animal with the lymphoma membrane antibody, and subsequent hybridoma fusion, can be used to generate monoclonal antibodies that bind specifically to the lymphoma membrane antibody. This monoclonal reagent may be used alone or coupled to a cell toxin, to specifically target and eliminate the lymphoma cells while leaving their normal cell counterparts intact. The potential therapeutic value of selective immunointervention extends to a broad spectrum of diseases and, in particular, to a number of autoimmune diseases in which the clinical abnormalities can be ascribed largely to the actions of pathogenic, autoreactive T cells. Extensive efforts are currently underway to determine whether such latter populations are clonal or oligoclonal and whether they might express only one or a few antigen receptors that could serve as targets for monoclonal antibody therapy. Based on preliminary findings in animal models of autoimmunity, it seems likely that at least some autoimmune diseases will be amenable to this therapeutic approach.
SUMMARY During this past decade, we have witnessed a virtual explosion in our understanding of the immune
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system. Although this review has specifically focused on the genetic characterization of the lymphocyte antigen receptors, recent progress in immunology has resulted in a plethora of other remarkable breakthroughs, including the identification of cytokines and their role in intercellular communication, of molecules involved in lymphocyte homing and adhesion, and of the crystal structure of MHC molecules. These and the many other recent advances have served to demystify immunology and to further meld the fundamental sciences with clinical care. Such developments provide the framework for the continued resolution of the mechanisms
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Figure 8. Membrane antibodies constitute a cell-specific marker that distinguishes a B-lymphoma cell from all other B cells. Mice can be immunized with the lymphoma-associated antibody (right panel), to create a monoclonal antibody that reacts with the "antigenic" portion (usually the variable regions) of the membrane antibody on the lymphoma-cell surface. The monoclonal antibody can be used alone or couple to a cell toxin, to selectively destroy the lymphoma cells.
that mediate and regulate the immune response and for addressing the therapeutic challenges presented by the spectrum of immunologically-mediated human diseases. K. Siminovitch is a Career Scientist of the Ministry of Health and a recipient of a Canadian Life and Health Insurance Association Medical Scholarship, as well as grants from the Medical Research Council, Arthritis Society, and the National Cancer Institute of Canada. The review of the manuscript by Drs. M. Denise Daley and Lou Siminovitch, and the help in manuscript preparation provided by Bev Bessey and IMS. Creative Communications are appreciated.
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