FROM THE FOREHEAD OF ZEUS: THE ONTOGENY OF THE IMMUNE RESPONSE

ARTHUR M. SILVERSTEIN Baltimore, Maryland

SUMMARY side-chain theory of formation! postulated Many of the most important developments that result in that all possible specificities pre-exist as surface a fully functioning vertebrate take receptors, presumably requiring no special develop­ place in the developing fetus. From a variety of gene mental intercession. Then, when the specificity segments there is assembled in B cells a congeries of repertoire seemed too large to support an intrinsic antibody combining sites, one to a cell, which form the basis, extrinsic factors were invoked in the form of greater part of the large repertoire of immunological antigenic instruction of the specific antibody binding specificities that characterise the system. This capability site,2,3 once again demanding the services of no is further expanded later by somatic mutations. Just as special ontogenetic mechanisms. immunoglobulin isotypes are produced sequentially Two developments in postwar science caused a (IgM, IgD, IgG, IgE, IgA) as they are read along the radical change in immunological thinking about the chromosome, so does the fetus and neonate manifest origins of the specificity repertoire, and each shone a immunological competence sequentially to different spotlight on developmental processes in the mam­ by employing variable region germline genes malian fetus. The first was the discovery of the as they appear along the chromosome. The generation phenomenon of immunological tolerance, which of receptor diversity is accomplished by a similar seemed to imply that something occurs in the mechanism of gene segment translocations. Each stage B developing fetus to ensure that anti-self in the lineage of T and cells is associated with the cannot attack tissues and organs with destructive appearance of unique combinations of surface mole­ autoimmune diseases. The second development was cular markers, which in T cells characterise also the the finding that all protein formation involves genetic specialised functions of different subsets. If the immune control of the amino acid sequence, causing mole­ system does not spring forth quite fully formed from the evolved vertebrate genome, as Athena did from the cular immunobiologists to focus attention on the forehead of Zeus, ontogenetic mechanisms have made nature and number of the fetal germline genes it very nearly complete. involved in antibody formation. The principal conceptual architect of the new During the past 35 years or so, the Leitmotif of was , whose clonal immunological speculation has centred around the selection theory of antibody formation.4,5 (stimulated origin of the astonishingly large repertoire of by Niels Jerne's earlier natural selection theory6) immunological specificities. This led naturally to an posed most of the critical ontogenetic questions - interest in those ontogenetic events which might and purported to answer many of them. Whereas influence the maturation of the immune system Burnet paid little attention to the nature of antibody during fetal (and, often, neonatal) development. specificity or to the size of its repertoire, David But it has not always been thus; for the first 75 Talmage addressed the former question7 and Joshua years (1880-1955), ontogenetic influences were not Lederberg the latter8 in detail. Lederberg suggested called into question. At the outset, Paul Ehrlich's that the chief generator of immunological diversity is a high rate of spontaneous and random mutation in Correspondence to: Professor Arthur M. Silverstein, Institute 'a gene for globulin synthesis'. This notion of a of the History of Medicine, Johns Hopkins University, 1900 East Monument Street, Baltimore, MD 21204, USA. somatic generation of diversity would serve thence-

Eye (1995) 9, 147-151 © 1995 Royal College of Ophthalmologists 148 A. M. SILVERSTEIN forth as the focus of a strenuous and heuristically entire lineage development. The closest valuable debate between paucigene theories invol­ analogue of the avian bursa in mammals appears to ving much somatic mutation and/or recombination') reside in the . and multigene theories which held that everything The several stages of B cell development have pre-exists in the germline. I 0, I I As we shall see, the been identified by the appearance either of distinc­ final outcome represents something of a compromise tive morphological changes (e.g. cell size or nucleus/ between the two extremes. cytoplasm relationships) or more usually by the appearance of distinctive markers of differentia­ 13 HAEMOCYTE/ LINEAGES tion. We have already mentioned those involving cytoplasmic or surface Igs; of equal importance has Haemopoietic Stem Cells been the development of reagents to identify surface It is now well understood that there develops early markers that come and go at various stages of during fetal life a multi potential stem cell car:able of differentiation. Originally called lymphocyte (Ly) giving rise to multiple lineages of blood cells. 2 These antigens from mouse studies, the advent of mono­ stem cells have been located (with some variation in clonal techniques has permitted the identification of different vertebrate species) first in the yolk sac and a variety of surface antigenic markers in the human. then in the fetal liver and spleen, thence to be Theses are now organised with a 'cluster of dispersed throughout the body. The first fork in the differentiation' (CD) nomenclature. Thus, CD34 is pathway of haemocyte differentiation is represented present during the transition from lymphoid stem cell by the appearance from these multipotential stem through the large pre-B cell, and CD20 from the cells of myeloid stem cells and lymphoid stem cells. large pre-B cell through to the mature B cell, etc.14,15 The myeloid stem cell and its descendants begin T cells. The evidence is strong that most T cells are their activity first in the fetal liver, but quickly move born in the ;16,17 First, thymectomy very early to the developing bone marrow. There they produce in life in many species prevents the appearance of T erythrocytes, /, , cells in peripheral lymphoid tissues; the same is true /mast cells and megakaryocytes, while in congenital athymia (e.g. nu/nu mice and human sufficient myeloid stem cells persist to reproduce DiGeorge's syndrome patients). Secondly, studies colonies of each of these tyes under suitable show that labelled within the thymus conditions. actually leave it to migrate peripherally, so that T The origin of natural killer (NK) cells is thus far cells may now be considered to be thymus-derived unclear; whether they develop directly from the rather than merely thymus-dependent. multipotential stem cell or via the lymphoid stem cell The picture of T cell ontogeny has been best pathway has not been determined. worked out in the mouse. After entering the thymus, stem cells soon express Thy-l surface markers and and Lymphoid Organs undergo extensive proliferation. At day 14 of B Cells. These immunocytes, ultimately the produ­ gestation, most thymocytes are Thy-1+CD4-CDS' cers of circulating antibody, have an exceedingly blast cells. Two to three days later, the cells change complicated ancestry. From the lymphoid stem cell into small lymphocytes expressing either CD4 or comes a pro-B cell, in which immunoglobulin heavy CDS or both molecules, and at the same time they chain V-D-J rearrangement is initiated (see below). express T cell receptors on their membranes. By day These then differentiate to form pre-B cells, which 1S-19 of the 20 day gestation period, the mouse T contain intracytoplasmic J.L chains. Next comes the cells appear fully functional, and popUlation of immature B cell, characterised by the presence of peripheral lymphoid organs is well under way. IgM membrane receptors (sIg), and then mature B cells with both IgM and IgD receptors. Finally, antigenic stimulus activates the mature B cell to form THE IMMUNOLOGICAL SPECIFICITY plasma cells which export large quantities of anti­ REPERTO IRE body to the circulation. B Cells The earliest differentiation of B cell lineages The precise size of the immunological specificity occurs in the fetal liver, but then quickly shifts to repertoire has been the subject of much speculation, the bone marrow, spleen and regional lymph nodes, ranging from the order of 105 completely different where distinctive areas devoted to B cell dynamics antibody combining sites all the way to 1016 such are formed, associated primarily with germinal sites. III As mentioned above, ontogenists fought with centres. IgA-forming B cells develop in and home molecular geneticists over the number of genes primarily to the gut-associated lymphoid tissues to present in the germline that encode for antibody form the secretory immunoglobulin system. Avian variable regions. The geneticists suggested that the species are exceptional in that they possess a hindgut genome could not afford to devote up to 10% of its bursa of Fabricius whose early excision aborts the DNA to the immune response, and therefore they ONTOGENY OF 149 postulated initially that only a few genes, acted upon the theory is that a single cell will by random somatic mutations and/or recombinations, produce only a single H- and L-chain V region give rise to antibody diversity. For their part, the combination, i.e. only a homoligating H L antibody. 2 2 ontogenists pointed to the lack of random develop­ But each B cell has two chromosomes whose Ig ment of the repertoire in fetal and neonatal region may rearrange. How then can both chromo­ maturation; there appears to be a well-ordered and somes be prevented from contributing different V species- or strain-specificsequence of appearance of regions to the final antibody molecule? Apparently a antibodies specific for one or other , both in regulatory mechanism exists at the pro-B and pre-B the perinatal mouse19 and in the larger fetal lamb?O cell stages, wherein the formation of a f.L H chain on Molecular biological approaches disclosed a novel one chromosome signals the termination not only of solution: the number of genes lay between the two H chain rearrangements on both chromosomes, but extremes earlier proposed; somatic mutation and also the initiation of light chain rearrangements, with recombination both contribute to the generation of kappa generally preceding lambda. Since a compar­ diversity; and the programmed sequence of appear­ able regulatory mechanism exists to ensure that only ance of repertoire components represents the initial a single light chain is formed within the cell, allelic 4 utilisation of the most proximal germline variable exclusion characterises all B cell activity? region gene segments as read down the chromo­ some.21 T Cells Genetics of Specificity Diversity. The germ line material encoding light and heavy chains has been The T Cell Receptor (TCR). Unlike B cells, the T cell best worked out in the mouse. The kappa light chain does not recognise the surface areas of native sequence includes some 100 V genes and 4 active J proteins. Rather, the receptor is involved in a (joining) regions, or 400 possible combinations. For trimolecular interaction between the TCR and H chains, we assume conservatively 100 VH genes, 4 molecules of the host's major histocompatibility JH genes and 12 D (diversity) regions, or 4800 complex (MHC) , in which a cleft contains an oligopeptide breakdown product of the antigen possible combinations. Assuming random association involved. Thus, whereas the B cell responds to of L and H chains, some 2 X 106 different specificity intact antigens in solution, the T cell responds to combinations are possible. We may multiply this by antigenic peptides presented on the cell surface an unknown number of recombinations and alter­ membranes of antigen presenting cells (APCs) or native reading frame variants, so that it is apparent virus-infected cells. that a fairly broad universe of immunological Evolution of the TCR and its functions has much specificities is already possible utilising only the in common with that of Ig receptors. The most available germline material. common TCR is composed of a heterodimer Then, to all of these gene combinations, somatic consisting of an a and a J3 chain, members of the Ig point mutations of VL and VH genes are known to supergene family. The TCR is associated with a set of occur, especially at the hypervariable regions that transmembrane proteins collectively designated as contribute to the antibody combining site. These the CD3 complex, which play a critical role in signal mutations serve not only to expand the specificity transduction. Like Ig light and heavy chains, repertoire, but ,play an overriding role in the repertoire diversiti5 in the TCR a and J3 chains is maturation of the average affinity of binding to generated by multiple combinational possibilities of antigen of a given antibody response. As clonal V and J exons in the former and of V, D and J exons selection theory predicts, those B cells with higher in the latter. Unlike Ig genes, somatic mutation affinity receptors for antigen will be more efficiently appears not to play a significant role in the selected for activation by that antigen. generation of TCR diversity. Allelic exclusion is a !sotype Switching The presence in serum of various characteristic of the ontogeny of the TCR, as it is of immunoglobulin isotypes (e.g. IgM, IgG, IgA) the Ig receptor of B cells. suggested that different genes control the expression TCR Function. As mentioned above, the maturation of the different heavy chain constant regions. With of T cells in the fetal thymus is accompanied by the the finding that switching occurs in a single appearance of surface markers that help to define a clone of B cells of the same specificity,22 it became variety of functions. Some cells differentiate to carry apparent that to a certain extent spontaneously, and the CD4 marker; these respond to antigenic peptides especially during the active immune response, the presented in association with Class II MHC mole­ heavy chain constant region genes may be utilised cules. CD4+ cells play an important role as 'helper T sequentially. These exist on chromosome 12 in the cells', mediating the response of B cells to proteins mouse in the order 5'-f.L, 8, "13,"II, -y2b, -y2a,E, and a- and other T-dependent antigens?6,27 CD4+ cells also 3,.23 appear to function as suppressor T cells in the down­ Allelic Exclusion. One of the central predictions of regulation of certain immune responses (see below). 150 A. M. SILVERSTEIN

In addition, these cells are the main participants in mental induction of tolerance in the mouse fetus by mediating the inflammatory reactions associated with Medawar and co-workers?2 This mechanism for the delayed-type . Finally, these cells screening of anti-self-reactive T cells prior to their participate in allograft rejection where class II exit from the thymus is not completely foolproo( MHC alloantigens are involved, apparently by a since auto-reactive T cells may be found in the combination of cytotoxic activity and periphery. There, they must be subject to a variety of release. regulatory controls (suppressor T cells, TCR mod­ Those cells which differentiate to carry the CD8 ulation, idiotypic network regulation, etc.) to prevent molecular marker recognise antigenic peptides only them from inciting disease. in the context of class I MHC molecules. Their At the same time that negative selection of TCRs principal function is the cytolytic destruction of cells is occurring in the fetal thymus, a process of positive which present foreign Reptides (e.g. viral or alloanti­ selection also takes place?3 This involves those gens) on their surface. 8 Much of the action of both receptors which show an affinity for self-MHC CD4 and CD8 lymphocytes depends on the release molecules which are found in abundance in the of groups of lymphokines which subserve a large dense network of antigen presenting cells; however, . variety of activating and down-regulating functions. cells with too high an affinity for self MHC would be The description above of the various functions of dangerous to the host, and these are deselected. TCRs is meant to apply only to those composed of Q Since only 1-2% of all thymocytes are exported, it is and 13 chains. The functions of the rarer 'Y/8 TCRs are probable that cells that are neither positively nor substantially unknown. negatively selected are 'neglected', and die within days. IMMUNOWGICAL TOL ERANCE B Cell Tolerance From the above" description of the several mechan­ isms which have evolved for the generation of The existence of clonal deletion as an explanation of diversity of both T and B cell receptors, it is evident B cell tolerance has been debated for several that nothing prevents the spontaneous generation of decades. The initial studies appeared to demonstrate specificitiescapable of reacting with the self antigens that experimental antigen-induced tolerance resulted of the host. Thus, in order to obviate the otherwise only in tolerance of specific helper T cells, thus inevitable development of autoimmune diseases, a rendering the B cells unable to mount an antibody complicated set of regulatory mechanisms had to response.34 Since circulating anti-self B cells specific evolve in. parallel. Some of these involve central for many self antigens can be found in the circulation ontogenetic events, while others depend upon a of normal individuals, it was assumed that no broad range of peripheral regulatory functions to mechanism exists in ontogeny for the central suppress or inhibit the autoaggressive tendencies of induction of B cell clonal deletion. However, the immune system. We shall review briefly here only subsequent findings employing transgenic mice35 those mechanisms that occur during the fetal and suggest that clonal deletion of anti-self B cells may neonatal development of the immune system; the occur amongst those cells displaying the highest­ more general features of immunological tolerance affinity specific receptors. As might have been will be reviewed extensively elsewhere in this issue. expected, early treatment of the developing immune system with anti-Il. antibodies results in destruction of IgM-bearing cells as they form and thus the abortion Intrathymic Selection of T cells of all B cell maturation, including all Ig isotypes The concept of clonal deletion (abortion) in utero of located further down the chromosome?6 anti-self clonal precursors was first advanced by It has become increasingly clear, however, that Burnet to explain the relative rarity of autoimmune down-regulation of B cells is primarily concerned disease. While its applicability to antibody-forming B with events in the periphery, including such phenom­ cells is open to question, it has been shown to be an ena as receptor blockade, B cell anergy, anti-idiotypic important mechanism of T cell tolerance. From the suppression, antibody feedback and terminal differ­ first appearance of a TCR specific for a self peptide entiation. in the thymus, both double positive (CD4+8+) and single positive (CD4+8- and CD4-8+) cells are negatively selected following interaction with their Key words: B cell development, Fetal development, ­ esis, Molecular genetics, Ontogeny, T cell development. specificpeptides on MHC molecules?9 These may be either self peptides, or those present as the result of REFERENCES intrafetal administration, thus explaining the tolerant chimaerism in non-identical twin cattle observed by 1. Ehrlich P. Die Wertbemessung des Diphtherieheil serums. Klin lahrb 1897;60:299-326. 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