mechanisms of transplantation immunity

Springer Semin Immunopathol (1992) 14:17-32 Springer Seminars in Immunopathology @ Springer-Verlag 1992

Mechanisms of transplantation immunity

Elizabeth Simpson

Transplantation Biology, Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3UJ, UK

Summary. In summary, this chapter describes the biology and genetics of the major and minor histocompatibility antigens and the nature of in vitro and in vivo immune responses to them and to tissue-specific antigens. It reviews the nature and action of immune response genes. It gives an account of how tolerance to histocompatibility antigens was originally defined and the prospects of intervention aimed at establishing tolerance to these and tissue-specific antigens in adult animals, including man.

The genetics of transplantation antigens

The basic rules

Transplantation of organs or tissues between individuals has to take into account the basic 'laws' of transplantation which reflect the genetics and expression of histocompatibility (H) antigens [40, 41]. These fall into three broad categories, major histocompatibility complex (MHC) antigens (H-2 in mouse, HLA in man) [35], minor histocompatibility antigens [61] (e. g. the male specific, H-Y antigen) and tissue specific or autoantigens. Since major and minor H antigens are co- dominantly expressed, grafts exchanged between individuals of different inbred mouse strains P1 and P2, and their F 1 progeny show the following characteristics: P1 and P2 grafts will be rejected by P2 and Pl recipients, respectively, but both will be accepted by F l recipients; however, PI and P2 recipients will reject F 1 grafts (Fig. la). In the F2 generation, there is independent segregation of unlinked H gene loci so that depending on the number of H gene loci involved there will be between 25 % with one locus (Fig. lb), 6,25 % with two loci (Fig. lc) an a vanishingly small percentage with, say, ten loci or more of F 2 progeny which will have inherited the same combination of histocompatibility genes as either of the parental strains P1 or P2. Those that have will accept grafts from each other and the appropriate parental strain but not from F~ donors: however, F1 recipients will accept F 2 grafts since they express a complete set of each parental H antigens (see Fig. 1).

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a P1- ;~" " P2

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Pl a/a P2 b/b

F 1 a/b X F~ a/b

F 2 aa ab ab bb

e P1 a/a r/r P2 b/b s/s

F~ a/b, r / sxF , a/b, r/s

F 2 a/a r/r " r/s " r/s " S/S

a/b r/r "~ ' ' r / s [ " r / s~ x2

' ' S/S

b/b r/r 2 " r/s " r / s

" s / s

Fig. l a x . The genetics of transplantation antigens, a ( /---- ), ( ~ ) graft accepted, direction of arrow indicates recipient; ( ~ ), ( ~ ) graft rejected, direction of arrow indicater recipient; asterisks indicate an exception for those F 2 progeny inheriting two complete sets of all Pi (or P2) histocompatibility (H) genes: this will be 25% if only one H gene locus is involved (see b), 6.25% in the case of two independently segregating loci and so on. b Genotype of parental (Pl, P2), F, and F 2 mice, where a and b are different alleles at the same locus: underlinings indicate the parental (homozygous) genotype, e Genotype of parental, (P~, P2), F t and F 2 mice, where a and b are different alleles at one locus and r and s are different alleles at a second independently segregating locus: underlinings indicate the parental (homozygous) genotypes

Major histocompatibility complex antigens

F r o m e x p e r i m e n t s p e r f o r m e d w i t h i nb red m o u s e s t ra ins by Snel l and G o r e r [22, 69] , it was c l ea r that t he re was o n e locus (or t igh t ly l inked g r o u p o f loc i as we n o w k n o w ) e n c o d i n g v e r y s t rong h i s t o c o m p a t i b i l i t y an t igens w h i c h not on ly

e l i c i t ed r ap id g ra f t r e j e c t i o n r e s p o n s e s but a lso an t ibod ies . T h e locus was n a m e d the m a j o r h i s t o c o m p a t i b i l i t y locus ( la ter c o m p l e x , h e n c e M H C ) and as it was the s e c o n d o n e d i s c o v e r e d that e l i c i t ed a l l oan t ibod ie s in m o u s e it b e c a m e k n o w n as

Mechanisms of transplantation immunity 19

H-2 [22]. Subsequently, a homologous MHC has been found in every mammalian species examined and MHC antigens are characteristically very polymorphic [35]. In man, Ceppellini et al. [14] established that skin grafts exchanged between siblings matched at serologically defined HLA antigens survived statistically longer than those between HLA disparate siblings. This functionally defined the strong or major nature of this H antigen system in man. It was, however, the antibody responses to them that allowed MHC antigens, first class I and subsequently class II, to be defined immunochemically as cell surface transmembrane glycoproteins and enabled the genes encoding them to be cloned, sequenced and chromosomally mapped [75]. Antibodies also allowed the isolation of sufficient HLA-A2 material for the crystalographic studies which have illuminated our understanding of how MHC molecules can perform their physiological function as restriction elements, or antigen (peptide)-presenting molecules to T lymphocytes [10, 11].

Minor histocompatibility antigens

The existence of H antigens other than those of the M HC was clear from the earliest studies with mice, when it was found that so very few F 2 progeny would accept skin grafts from each other or the parental strains [40, 41]. Judging from the number of such Fz mice, an estimate of 14 or 15 loci was made, but subsequent work by Bailey has increased this number to dozens, of which many have been isolated genetically in congenic mouse strains, each differing from one parental strain at a single minor H locus, backcrossed from the other parental strain [3]. In mice at least it is possible using congenic strains to study individual minor H antigens and the immune responses they elicit, both in vivo and in vitro. One of the difficulties has been that no antibodies are made to minor H antigens. However, in addition to the graft rejection responses elicited by them, it is now possible to isolate and propagate in vitro T cell clones specific for minor H antigens and restricted by self MHC molecules from both mouse and man [24, 64, 67]. Such T cell clones can be used to type cells from individuals for the presence of minor H antigens, much the same as antibodies, but with the constraints of needing to match for MHC-restriction molecules [45, 49, 65-67].

In man, minor H antigens as the targets of graft-rejection (host versus graft, HvG) responses in recipients of MHC-matched tissues or organs, thus necessi- tating continuous use of immunosuppression in such patients. They are also the targets of the severe graft versus host disease (GvH) which occurs in recipients of MHC-matched bone marrow. It is from patients undergoing HvG and GvH responses that minor H antigen-specific T cell clones have been isolated [23, 24, 66]. The strength of these GvH and HvG responses is not trivial because between any human donor/recipient pair there are many different minor H antigen mismatches and the immune response to each of them is specific, separate and potentially additive.

Whereas the molecular identity of the genes and proteins of the MHC antigens as well as their physiological role as guidance or restriction molecules for T cell responses are well established, comparable data on minor H antigens is sparse. In mice, the chromosomal localisation of a number of minor H genes is established: they are distributed throughout the genome and not clustered like MHC genes

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[3]. In humans only the male-specific antigen gene HYA is mapped [66] but as in vivo GvH and HvG responses and in vitro T cell responses specific for minor H antigens between MHC-matched donor/recipient pairs are comparable in man and mouse [23, 67], it is likely that minor H genes in man are likewise dispersed throughout the genome. The characteristic MHC restriction of T cell responses to minor H antigens implies that they are probably peptides derived from endogenous proteins having small allelic sequence differences not affecting their physiological function. This has yet to be proven, but it is a useful paradigm for the tissue-specific autoantigens that are the targets of T cell-mediated autoimmune disease.

Tissue-specific antigens

Different tissues of the body display different arrays of self molecules as a reflec- tion of their various differentiation pathways. For example, work on human leucocyte differentiation antigens recognised by mouse monoclonal antibodies has identified dozens of diverse cell surface expressed molecules which mark different subsets of leucocytes [50]: similar approaches to non-lymphoid tissues have also identified molecules which are cell type specific or have a limited tissue distribution [70]. The immune system is generally tolerant to all self molecules even when they are expressed in a very tissue-specific way. Induction of tolerance at the level of T cells is very important because T cells are the conductors of the immunological orchestra; effector functions including antibody production do not occur in the absence of T cell help. T cell tolerance to self molecules occurs by clonal deletion of those T cells whose receptors would otherwise be self reactive at high affinity or by clonal anergy. Clonal deletion occurs principally in the thymus [30], whilst in the periphery ways of inducing anergy have been demonstrated in a number of experimental situations [42, 52, 77] and could provide a physiological fail-safe mechanism to prevent autoimmune reactions (see the contribution by John Kappler in this volume).

Autoimmune disease follows the breakdown of clonal deletion and/or induction of clonal anergy and may be induced by the abnormal expression of tissue-specific antigens. The molecular identity of these triggering antigens is unclear except in a few situations: in experimental allergic encephalomyelitis (EAE) induced experimentally in rats and mice by myelin basic protein (MBP), the encephalitogen is a well-defined peptide of MBP, which binds to certain alleles of MHC class II molecules and is presented by them to T cells bearing receptors using a limited number of V region genes. By analogy, tissue-specific antigens are likely to be peptides of proteins endogenously expressed and processed to be presented by MHC class I or class II molecules at the cell surface. They would, thus, be similar to minor H antigens but their recognition by T cells would be due not to allelic differences between individuals but to the breakdown or absence of tolerance within one individual. There are examples of both class I and class II MHC molecules being expressed in a tissue-specific way [47]. This is consistent with the notion that class I and class II molecules carry endogenous peptides in their antigen-presenting grooves and that the identity of the peptide(s) varies between different tissues, depending on the translation products of their transcribed genes.


T cells specific for allogeneic MHC molecules are also now considered to be recognising endogenous peptides presented in the antigen-binding grooves of MHC cell surface molecules [6, 43]. This hypothesis accounts for the very high frequency of alloreactive T ceils and predicts that T cell responses to allogeneic MHC molecules will be clonally very heterogeneous, the response being the sum of clones specific for different peptides in the antigen-binding grooves of similar MHC molecules (see [48] for the original formulation of this idea).

In vivo and in vitro responses to trans ~lantation antigens

Historical perspective

Gorer and Snell [22] first showed that mice exposed to tissues bearing foreign H-2 antigens (MHC disparate) made H-2-specific antibodies. These antibodies provided powerful tools for the analysis of MHC genetics [34]. Medawar and his colleagues [8, 51] demonstrated that in vivo graft rejection was mediated by a cellular rather than a humoral (antibody) response, since specific responsiveness to an allograft could be transferred with lymphocyte suspensions but not with serum. They also showed that secondary (second set) graft rejection of a graft genetically identical with the first was very much more rapid than the primary (first set), thus establishing graft rejection as a reaction characterised by specific immunological memory [20]. This work focussed attention on effector lymphocytes, thus paving the way for the separation of T cells from B cells and the analysis of T cell subpopulations, initially with Thy-l-, Ly-l-, and Ly-2-specific allo- antibodies in mouse [13] and subsequently with CD4, CD8 and the plethora of other monoclonal antibodies marking potential differentiation antigens of leucocytes of man, mouse, rat and a number of other species [50].

CD4 + and CD8 + T cell responses to MHC antigens in vitro

The use of antibodies to the CD4 and CD8 T cell differentiation molecules has allowed detailed analyses of in vivo and in vitro functions of the two principal subclasses of T lymphocytes: CD4 § T cells which respond to MHC class II molecules and have a predominantly T helper function (although under certain conditions they can be cytotoxic [72]) and CD8 § T cells which respond to MHC class I molecules and are predominantly cytotoxic in function (although those which produce lymphokines can mediate helper effects).

The original analysis of the bi-partite reaction to MHC alloantigens came from the findings that, whilst primary in vitro one-way mixed lymphocyte responses (MLR) between cells from pairs of individuals disparate across the whole MHC led to the generation of cytotoxic T cells against the MHC antigens we now know as class I, no cytotoxic response was seen if mixed lymphocyte cultures were set up between pairs differing genetically at class I or class II loci only. However, if responder lymphocytes were added to cultures containing two types of irradiated stimulator cells, one genotypically different just at a class I locus, the other just at a class II locus, then cytotoxic T cells specific for the class I antigens were generated [56]. This result implied that two types of stimulatory signal were needed

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for an optimal cytotoxic response and the analysis was subsequently taken further when two different T cell types were shown to respond to the two signals: T helper cells (now designated CD4 +) to class II, and T cytotoxic cells (now CD8 +) to class I [13]. Later it was shown that the activity of T helper cells could be replaced by a lymphokine, IL-2 in the cultures, and this knowledge has facilitated the isolation and continuous growth in vitro of T cell clones specific not only for MHC alloantigens but for self MHC-restricted T cell clones specific for minor H, viral and other peptide antigens [17].

In vitro MLR can readily be obtained from using cells from unimmunised individuals (a primary MLR) when responder and stimulator lymphocytes are taken from MHC disparate individuals. This implies that a relatively high proportion of responder T cells can be activated by allogeneic MHC molecules. In contrast, it is not possible to obtain primary in vitro responses to MHC-matched, minor H-disparate cells [21], or to proteins or viral antigens outside the group known as "superantigens" (Staphylococcal and Streptococcal enterotoxins, which stimulate in an apparently non MHC-restricted manner but have affinity for T cells bearing receptors of particular Va types [80]). Limiting dilution analysis of primary MLR between cells from MHC-mismatched individuals has confirmed the high precursor frequency of T cells with MHC alloreactivity: the fine specificity of these T cells is likely to involve MHC-restricted reactivity to endogenous peptides, as has already been discussed [6, 43].

CD4 + and CD8 + T cell responses to minor H antigens in vitro

In vitro MLR to minor H antigens can only be obtained using responder T cells from humans or mice previously immunised in vivo by a therapeutic intervention such as grafting or in mice by grafting or spleen cell injection [21, 23, 24, 63, 67]. These secondary MLR cultures generate strong minor H-specific, MHC- restricted cytotoxic T cells, and from them can be cloned minor H-specific class I and II MHC-restricted T cells [24, 67]. In mice both CD4 + (class II restricted) and CD8 § (class I restricted) minor H-specific clones have been isolated and maintained [64, 67]; in man a predominance of class I-restricted minor H-specific T cell clones has been reported [23, 24]. In mice, the cultures from which such clones are derived can use cells from responder/stimulator pairs of strains differing either at a single minor H antigen, since inbred and minor H congenic strains are available, or from pairs matched at the MHC but differing at multiple minor loci. Humans, as an outbred population can only provide responder/stimulator pairs differing at multiple minor loci. From work in mice, however, it is clear that even when there are multiple minor disparities, T cell responses to only few immunodominant antigens are seen, suggesting a hierarchy of responsiveness mediated possibly through antigenic competition [79].

Minor H-specific T cells have been used to chromosomally map certain minor H genes, in both humans and mice [45, 49, 66, 67], and are also being used in prospective studies of human bone marrow-transplant recipients from MHC- matched donors to determine whether the number of identifiable mismatches for minor H antigens correlates with the severity of GvH disease. The initial data


suggest this correlation exists despite incomplete identification of all relevant minor H antigens [23, 24].

In vivo and in vitro correlates of immunity to H antigens

The extent to which in vitro-generated CD4 T helper (proliferative) and CD8 cytotoxic T cells reflect the in vivo graft-rejection response has been a matter of discussion and debate. It is clear that both populations have potential effector functions, CD8 by direct cytotoxicity and CD4 by cytokine production and possibly also direct cytotoxicity [72].

Experiments designed to test whether CD4 or CD8 cells alone can effect allograft rejection in vivo have shown that either can do so but that in situations in which both cell types can respond, both contribute to rejection. These experiments have included (a) grafting between mouse strains differing only at class I or class II MHC loci (and thus activating predominantly CD8 or CD4 cells, respectively) - in each case rejection is seen, for example between C57BL/6 recipients and the class I bml or the class II bml2 mutant strains; (b) grafting recipients whose CD4 or CD8 T cells have been depleted by the administration of cytotoxic antibodies to these molecules [16]; and (c) grafting irradiated recipients which have been repopulated with each subset [44]. The balance of which T cell effector type is most important depends on factors which include the tissue or organ grafted, whether the response is primary or secondary [16, 55] and the genetic difference between donor and recipient [16, 77]. In addition to antigen-specific CD4 and CD8 cells, antigen-non-specific cells such as macrophages are drawn into the rejection response, cytokine production, by specific T cells leading to their recruitment.

Regulatory pathways affecting specific T effector function can modify graft- rejection responses. Allo-antibodies themselves are rarely responsible for graft rejection, with the exception of hyperacute rejection particularly of organ grafts such as kidneys, which can occur in sensitised patients with high levels of cytotoxic antibodies, see [12]; however, they are made during the course of graft rejection where there are MHC class I and/or II differences between donor and recipient. Such antibodies may down-regulate T cell responses by blocking antigenic sites on the graft but can also amplify T cell responses by binding antigen which is then processed and represented [32]. When this is done by B cells sharing any MHC antigens with the donor, they in turn become targets for destruction by T cells [57]. T cells themselves can become the targets for destruction by antigen- specific CD4 and CD8 effector T cells if they acquire the specific antigen by infection (as in HIV infection in man) or by processing [36]. These types of regulatory interactions probably account for the 'suppressive' properties variously attributed to CD8 and CD4 subpopulations, being an inevitable concomitant of their ability to specifically destroy any cell possessing the peptide antigen in an appropriate class I or class II antigen-binding groove [62].

Rejection of different organs and tissues

The ease with which graft rejection of different organs and tissues can be modified varies. This is in part due to the number of cells in the graft which express class

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II, in addition to class I MHC molecules: CD8 reponses directed at class I molecules are amplified by CD4 responses to class II molecules, in vivo as well as in vitro [13, 16, 37, 60, 77]. Grafts which express class I but little class II are less immunogenic. Skin is a notoriously difficult tissue to transplant, partly because it contains a wealth of specialised antigen-presenting cells, the Langerhans cells, which are class I and class II positive, partly because the epidermis is readily induced to express class II molecules in the presence of inflammatory infiltrates in some species [81], and partly because the grafted donor skin suffers a period of relative anoxia before being revascularised by the recipient. Liver transplants are irnmunologically less challenging: parenchymal cells express class I but not class II molecules: in some species liver graft recipients do not require any immunosuppression. Kidney grafts, like liver and heart, are immediately vascularised, but they contain a number of interstitial cells which express class II molecules and are as a result more immunogenic [37]. Segmental grafts of pancreas are prone to chronic graft rejection in patients receiving levels of immunosuppressive drugs adequate to maintain kidney grafts: however, recipients of a kidney and pancreas graft from the same donor fare better, the presence of the kidney graft in some way protecting the pancreas [12].

Current immunosuppressive regimes allow for the transplantation of heart, liver and pancreatic transplants from MHC incompatible donors with mismatching for all HLA antigens, or matched for only some (i. e. partly matched). The 5-year survival times depend on the organ but on average are over 50%. In recipients of kidney grafts from living related donors, usually MHC matched, the results are appreciably better [ 12] than those from partly MHC-matched cadaveric donors, although the continued need to use immunosuppression in MHC-matched recipients is a measure of the strength of multiple minor H barriers. Bone marrow transplantation is more difficult to achieve: HvG and GvH reactions are very strong if donor/recipient pairs are not matched at the MHC, leading to the loss of grafts and death of recipients in a high proportion of such cases [ 12, 77]. Where recipients receive bone marrow from HLA-matched siblings, results are better, although GvH can be severe, particularly if mature T cells inevitably present in the donor bone marrow are not removed. In these cases the donor T cells respond to minor H antigens of the recipient in an HLA-restricted manner, donor and host being identical at the MHC complex. Rejection of the donor graft by the recipient can also occur by MHC-restricted responses to minor H antigens, especially if the recipient has been presensitised, for example by transfusions [23-25]. Under these circumstances the sensitised T cells of the recipient survive the preconditioning by doses of irradiation which would be adequate in non-sensitised recipients.

I m m u n e response genes

Definitions The genetic differences between donor and recipient determine not only histoin- compatible target antigens of the donor but also immune response genes of the recipient. Immune response genes are those whose products affect the ability to


mount an immune response and they can be broadly categorised as MHC Ir genes and non-MHC Ir genes.

MHC Ir genes

The MHC Ir genes are those encoding the MHC class I and II molecules, which serve as restriction elements in presenting peptide antigens to T cells [38, 74]. Certain alleles of class I and II molecules bind particular peptides well, leading to the formation of a highly stimulatory complex and, thus, a strong response, whilst others fail to bind particular peptides sufficiently well to stimulate a response. The former are referred to as high-responder alleles, the latter as low- responder alleles, and such distinctions have been observed in the control of immune responses to viruses, exogenous protein antigens and minor H antigens [38, 61, 63, 74]. MHC molecules also exert a control over immune responses in another way, by positive and negative selection of T cell clones as they mature in the thymus. The details of these events have been described by John Kappler in this volume. These selections result in a peripheral T cell repertoire which is skewed towards the use of self-MHC molecules as restriction elements (by positive selection) but excludes T cell clones which would recognise other self molecules in association with self-MHC molecules (by negative selection).

Non-MHC Ir Genes

The non-MHC Ir genes are very much more heterogeneous and less well understood. They were discovered by finding that amongst inbred mouse strains sharing the same MHC, there was variation between strains in the ability to mount responses to certain antigens. These differences could be genetically mapped, using F2, backcross and recombinant inbred strain analysis, to various chromosomes other than chromosome 17 in which the MHC is located in mice [18, 19]. The identity of the genes or their products is not yet known, but some clearly interact with MHC Ir genes [18]. This would be consistent with at least some of them being endogenous peptides competing for binding in the antigen- binding grooves of class I and/or class II molecules and, thereby, limiting the binding of peptides derived from the antigen against which the response is controlled by non-MHC Ir genes. Such an explanation is likely for the control of cytotoxic T cell responses to the male-specific minor antigen, H-Y, in mice with MHC haplotypes other than H-2 b [18]. The T helper response to the synthetic polymer GT in the H-2 d strains BALB/c and DBA/2 is controlled by a self antigen in the non-responder BALB/c which mimics GT associated with H-2 d class II molecules leading to deletion of GT-specific T cell clones in BALB/c, but not in the responder DBA/2 [76]. Other non-MHC gene products have been shown to influence the T cell repertoire by causing deletion in the thymus of all T cells expressing receptors using particular V~ genes. This is true of the Mls-1 a and Mls-2 a alleles [31, 46, 54].

Another class of non-MHC Ir genes are those encoding immunoglobulin and T cell receptor (TCR) genes. Amongst the latter in particular it is well documented that some mouse strains have variable numbers of TCR V~ genes deleted [26]. This excludes the the participation of the deleted variable region genes in the forma-

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tion of the T cell repertoire and might, thereby, lead to 'holes in the repertoire'. However, it has to be remembered that the T cell repertoire is enormously plastic, with substantial junctional region diversity in addition to diversity generated from combinatorial association of Ve and V~ genes. It is, therefore, unlikely that significant numbers of holes could be created in the repertoire to induce non- responsiveness to a particular antigen, for even with very simple antigens the T cell response is not monoclonal. Extreme examples of mice carrying a very limited T cell repertoire are TCR transgenics. In those expressing receptors derived from a TCR transgene taken from either a class I- or a class II-restricted T cell clone, rearrangement of the endogenous TCR /3 genes is prevented but not that of endogenous TCR ot genes and this allows for a T cell repertoire which includes an ability to respond to antigens other than that against which the transgenic TCR is directed [33]. However, the limitations of the repertoire in such transgenics remains to be fully explored.

Other classes of non-MHC Ir genes include those affecting antigen processing and those affecting the expression or function of accessory molecules involved in the cell-cell interactions between cells of the immune system [71]. Outright defects or deletions of such genes would result in generalised immuno-incom- petence or even developmental lethal effects. Susceptibility to bacterial infections attributable to defective LFA-1 molecules is well documented [71], but other more subtle and selective defects would be expected of less drastic mutations. This is an area requiring further exploration.

The effect o f Ir genes on immune responses to transplantation antigens and autoantigens

The evidence that MHC and non-MHC Ir genes influence immune responses against transplantation antigens comes from both clinical and experimental studies, Among human kidney graft patients, the presence of DR6 (an MHC class II allele) in recipients is associated with more graft-rejection episodes [28]. In mice, the ability to make a cytotoxic T cell respones to H-Y in H-2 d mice is dependent on a non-MHC Ir gene on chromosome 2, mapping close to/3z-microglobulin and the minor H antigen H-3 [19]. It is possible that one class of non-MHC Ir genes controlling responses to minor H antigens are other minor H antigens which, as peptides bound in the grooves of MHC molecules, could operate either by competing for binding sites or by causing clonal deletion and so affecting the T cell repertoire. The influence of MHC and non-MHC Ir genes on immune responses to autoantigens is seen in humans and mice [73]. The association of certain human MHC alleles with type I insulin-dependent diabetes, multiple sclerosis and rheumatoid arthritis indicates the involvement of MHC Ir genes (see contributions by John Todd, John Bell and Laurence Steinman in this volume). In the case of diabetes the existence of particular sequences within the crucial antigen-binding grooves of class II molecules is strongly predisposing to the disease and these sequences are also present in the non-obese diabetic (NOD) inbred mouse strain which provides a very close model of the human disease (see section by J. A. Todd). However, not all humans or even HLA-identical sibs of diabetic patients carrying the predisposing MHC class II alleles develop the


disease, and this and the difference in incidence of concordance between identical twins and HLA-identical sibs implies the influence of non-MHC Ir genes. In NOD mice, in addition to the MHC Ir gene(s), genetic studies have shown that there are at least two more independently segregating recessive Ir genes, not linked to the MHC on chromosome 17 [53]. A similar story is likely in man. In both species, environmental factors are also implicated, including dietary components and infections: both may operate on the immune system via MHC and/or non-MHC Ir genes.

Tolerance to transplantation antigens

Historical perspective

The ability to induce specific tolerance in mice to skin grafts carrying MHC and minor H antigen differences was first reported nearly 40 years ago by Medawar and his colleagues [7, 9]. This was the first indication that the 'transplantation problem' was capable of solution and encouraged the development of clinical kidney graft transplantation. The classic experiment in mice involved injection into newborns of one strain, P1, of lymphoid cells from F 1 hybrid mice of PI and a second, unrelated strain, P2. As young adults the injected mice were grafted with skin from the P2 strain and in a proportion of recipients the grafts were accepted indefinitely. They would not, however, accept genetically unrelated third party grafts, i. e. tolerance was immunologically specific. These mice were shown to be chimeric with respect to the injected P2 lymphoid ceils and tolerance was dependent on the persistence of chimerism. The interpretation placed on the results was that the immune system of the young mice developing in the presence of F1 cells bearing P2 antigens was educated to accept the 'foreign' P2 antigens as self. The need for continued chimerism implied that new waves of maturing lymphoid cells occurred during adult life and needed 'education'. At that time, this mode of tolerance induction was considered to be due to clonal deletion. Clearly in a clinical situation injection of neonates to induce tolerance was not practicable. Also, it was found that only species with a poorly developed immune system at birth, like rats and mice, could be tolerised to alloantigens in this way: lambs and primates (and by implication, man) had well-developed immune systems, including the T cell compartment, well before birth so that tolerisation to cellular antigens in the neonatal period was not possible. Attempts to induce tolerance following chimerism in adult mice by injection of allogeneic lymphoid cells following conditioning, immunosuppressive regimes such as irradiation [68], injection of corticosteroids or antilymphocyte (ALS) [39] serum were not so strikingly successful as the neonatal injections for tolerance induction, although a number of regimes produced prolonged graft survival (for review see [78]). The clinical picture was improved substantially by the empirical use of cytotoxic immunosuppressive drugs and thoughts of the induction of specific tolerance were held in abeyance, along with those on induction of specific enhancement of kidney grafts which looked very promising in rats [4] but again proved difficult to produce in other species, including man.

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Monoclonal antibodies and peptides

In the mid 1960s the development of ALS serum showed that biological reagents could be immunosuppressive in vivo [39]. The lymphocyte antigens against which such antisera were directed were heterogeneous and difficult to characterise and standardisation of different batches of ALS was problematical; they have, however, been used clinically, often as an adjunct to immunosuppressive chemicals in the treatment of rejection crises [12]. With the development of monoclonal antibodies to human lymphocyte antigens in the 1980s [50] came the possiblity of more specifically targeting T lymphocytes and their subpopulations and antibodies to CD3 (pan T: associated with the T cell receptor), CD4 (on helper T cells), CD8 (on cytotoxic T cells), CD1 la (LFA-1) and the IL-2 receptor (on activated lymphocytes) have been under test [12, 77, 78]. There is no doubt that some of these antibodies can block graft-rejection responses but generally they have been used at doses or in combinations that render them immunosuppressive and, thus, render the recipient as susceptible to infection as immunosuppressive chemicals. The first hope that more discriminant use of monoclonal antibodies might lead to antigen-specific tolerance induction came with experiments on mice given rather small doses of antibodies to the mouse CD4 molecule, together with a protein antigen human gamma globulin (HGG) [5]. Under this 'umbrella' mice became tolerant and failed to make immune responses to subsequent challenge doses of HGG. This approach has now been extended to induction of specific non- responsiveness to allogeneic MHC and minor H antigens, using bone marrow grafts to induce chimerism under antibody cover and skin grafts to test the tolerance so induced [15, 16, 55, 77]. For transplantation antigens, a judicious mix of CD8 and CD4 antibodies appears to produce the best results- a measure probably of the involvement of both subpopulations in graft-rejection responses [16]. The novel finding in these series of experiments has been that tolerance induction need not depend on ablation of the T cell subsets involved, but on their inactivation in the periphery in the presence of the tolerogen. Maintenance of tolerance requires the continuous presence of antigen, very much like the findings with classical neonatally induced tolerance [7, 9, 77, 78].

A similar approach has been made in attempts to tolerise potential diabetics to the tissue-specific antigen on pancreatic/3 cells. In man grafts of pancreatic tissue between monozygotic twins, only one of which (the recipient) had developed diabetes, show/3 cell-specific destruction in the graft with lymphocytic infiltration of the islets, unless immuno-suppression is used [59]. In NOD mice the lymphoid infiltration of islets can be held in abeyance for a period following the administration of monoclonal antibodies to CD4 [27, 58]. However, the conditions for long- term-specific non-reactivity to/3 cell antigens have yet to be worked out. It is clear that CD8 cells are also involved in the destruction of/3 cells, since spleen cell transfer of the disease with cells from a diabetic NOD to a young unaffected recipient can be blocked by the administration of CD8 antibodies to the recipient [29]. Currently schedules of potentially tolerising monoclonal antibodies are under investigation in this mouse model for type 1 diabetes in man.

An alternative approach to tolerise to tissue-specific antigens involved in autoimmune disease is identification of the putative peptide antigen which, in


association with MHC molecules, forms the target. Such a peptide could competitively block interaction of effector T cells with their target and perhaps induce clonal anergy in the T ceils [2]. Another way of inducing clonal anergy, or perhaps deletion of T cell effector cells with specificity for tissue antigens, is to inject monoclonal antibodies specific for TCR utilising particular Va or V, genes: in EAE at least, there is evidence that clones reactive with the encephalatogenic peptide 1 - 2 0 of MBP utilise a small number of V~ and V~ genes, making the approach a practicable one for this disease ([1] and the section by Laurence Steinman in this volume). In diabetes, it is possible that T cell clones reactive with the ~ cell specific antigen may use a limited repertoire of TCR a and/or/3 genes and, if this is the case, a similar approach would be possible (see section by David Wraith and Laurence Steinman in this volume).

Acknowledgements. I would like to thank Mrs. Vivien Tikerpae for help in preparing the manuscript and Profs. Leslie Brent and Herman Waldmann for critically reading it.

References

1. Acha-Orbea H, Mitchell DJ, Timmermann L, Wraith DC, Tansch GS, Waldor MK, Zamvil SS, McDevitt HO, Steinmann L (1988) Limited heterogeneity of T cell receptors from lymphocytes mediating autoimmune encephalomyelitis allows specific immune intervention. Cell 54:263

2. Adorini L, Muller S, Cardinaux F, Lehmann PV, Falcioni F, Nagy ZA (1988) In vivo competition between self peptides and foreign antigens in T cell activation. Nature 334:623

3. Bailey DW (1975) Genetics of histocompatibility in mice. I. New loci and congenic lines. Immunogenetics 2:249

4. Batchelor JR (1985) Concepts of immunological enhancement. In: Medawar PB, Lehner T (eds) Major Histocompatibility System. The Gorer Symposium. Blackwell, Oxford, pp 2 8 - 3 6

5. Benjamin R, Waldmann H (1986) Induction of tolerance by monoclonal antibody therapy. Nature 320:449

6. Bernard EJ, Le AT, Yannelli JR, Holterman MJ, Hogan KT, Parham P, Engelhard VH (1987) The ability of cytotoxic T cells to recognise HLA-A2 or HLA-B7 antigens expressed on murine cells correlates with their epitope specificity. J Immunol 139:3614

7. Billingham RE, Brent L, Medawar PB (1953) Actively acquired tolerance of foreign ceils. Nature 172:603

8, Billingham RE, Brent L, Medawar PB (1954) Quantitative studies on tissue transplantation immunity. II. The origin, strength and duration of actively and adoptively acquired immuntiy. Proc R Soc Lond [Biol] 143:58

9. Billingham RE, Brent L, Medawar PB (1956) Quantitative studies on tissue transplantation immunity. HI. Actively acquired tolerance. Philos Trans R Soc Lond [B] 239:357

10. Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, Strominger JL, Wiley DC (1987) Structure of the human class I histocompatibility antigen, HLA-A2. Nature 329:506

11. Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, Strominger JL, Wiley DC (1987) The foreign binding site and T cell recognition regions of class I histocompatibility antigens. Nature 329:512

12. Brent L, Sells RA (eds) (1989) Organ transplantation: current clinical and immunological concepts. Bailliere & Tindall, London

13. Cantor H, Boyse EA (1975) Functional subclasses ofT lymphocytes bearing different Ly antigens. I. The generation of functionally distinct T cell subclasses is a differentiative process independent of antigen. J Exp Med 141:1376

14. C eppellini R, Mattiuz PL, Scudeller G, Visetti M (1969) Experimental allotransplantation in man. I. The role of the HLA system in different genetic combinations. Transplant Proc 1:385

15. Cobbold SP, Waldmann H (1986) Skin allograft rejection by L3T4 and Lyt2 T-cell subsets. Transplantation 41 : 634

30 E. Simpson

16. Cobbold SP, Martin G, Qin S, Waldmann H (1986) Monoclonal antibodies to promote marrow engraftment and tissue graft tolerance. Nature 323:164

17. Fathman CG, Fitch FW (1982) Isolation, characterisation and utilisation of T lymphocyte clones. Academic Press, New York

18. Fierz W, Brenan M, Mullbacher A, Simpson E (1982) Non-H-2 and H-2 linked immune response genes control the cytotoxic T cell response to H-Y. Immunogenetics 15:261

19. Fierz W, Farmer GA, Sheena JH, Simpson E (1982) Genetic analysis of the non-H-2 linked Ir genes controlling the cytotoxic T-cell response to H-Y in H-2 d mice. Immunogenetics 16:593

20. Gibson T, Medawar PB (1943) The fate of skin homografts in man. J Anat 77:299 21. Gordon RD, Simpson E, Samelson LE (1975) In vitro cell-mediated immune responses to the

male specific (H-Y) antigen in mice. J Exp Med 142:1108 22. Gorer PA, Lyman S, Snell GD (1948) Studies on the genetic and antigenic basis of tumour

transplantation: linkage between a histocompatibility gene and 'fused' in mice. Proc R Soc Lond [Biol] 135:499

23. Goulmy E (1985) Class I restricted human cytotoxic T lymphocytes directed against minor transplantation antigens and their possible role in organ transplantation. Prog Allergy 36:44

24. Goulmy E (1988) Minor histocompatibility antigens in man and their role in transplantation. Transplant Rev 2 :29

25. Goulmy E, Termijtelen A, Bradley BA, van Rood JJ (1977) Y-antigen killing by T cells of women is restricted by HLA. Nature 266:544

26. Haqqi TM, Banerjee S, Anderson GD, David CS (1989) Rill S/J (H-2r), an inbred mouse strain with a massive deletion of T cell receptor V/3 genes. J Exp Med 169:1903

27. Hayward AR, Cobbold SP, Waldmann H, Cooke A, Simpson E (1988) Delay in onset of insulitis in NOD mice following a single injection of CD4 nd CD8 antibodies. J Autoimmun 1:91

28. Hendriks GFJ, Schreuder GMTH, D'Amaro J, van Rood JJ (1986) The regulatory role of HLA- DRw6 in renal transplantation. Tissue Antigens 27:121

29. Hutchings PR, Simpson E, O'Reilly LA, Lund T, Waldmann H, Cooke A (1990) Involvement of Ly2 § T cells in 13 cell destruction. J Autoimmun 3: [Suppl] 101-109

30. Kappler JW, Roehm N, Marrack PC (1987) T cell tolerance by clonal elimination in the thymus, Cell 49:273

31. Kappler JW, Staerz U, White J, Marrack PC (1988) Self tolerance eliminates T cells specific for the MHC complex. Nature 332:35

32.Kawamura H, Berzofsky JA (1986) Enhancement of antigenic potency in vitro and immunogenicity in vivo by coupling the antigen to anti-immunoglobulin. J Immunol 136:58

33. Kisielow P, Bluthmann H, Staerz UD, Steinmetz M, von Boehmer H (1988) Tolerance in T-cell receptor transgenic mice involve deletion of non mature CD4§ + thymocytes. Nature 333:742

34. Klein J (1975) Biology of the mouse histocompatibility-2 complex. Springer, New York Berlin Heidelberg

35. Klein J (1986) Natural history of the major histocompatibility complex. John Wiley and Sons, New York

36. Lanzavecchia A, Roosnek E, Gregory T, Berman P, Abrignani S (1988) T cells can present antigens such as HIVgpl20 targeted to their own surface molecules. Nature 334:530

37. I.e~hler RI, Batchelor JR (1982) Restoration of immunogenicity to passenger cell-depleted kidney allografts by the addition of donor strain dendritic cells. J Exp Med 155:31

38. Le Meur M, Gerlinger P, Benoist C, Mathis D (1985) Correcting an immune-response deficiency by creating E~ gene transgenic mice. Nature 316:38

39. Levey RH, Medawar PB (1966) Some experiments on the action of anti-lymphoid antisera. Ann NY Acad Sci 129:164

40, Little CC, Johnson (1922) The inheritance of susceptibility to implants of splenic tissue in mice. I. Japanese Waltzing mice, albinos and their F.1 generation hybrids. Proc Soc Exp Biol Med 19:163

41. Little CC, Tyzzer EE (1916) Further experimental evidence on the inheritance of susceptibility to a transplantable tumour, carcinoma (J. W. A.) of a Japanese Waltzing mouse. J Med Res 33:393

42. Lo D, Burkly LC, Widera GT, Cowing C, Flavell RA, Palmiter R, Bunster RL (1988) Diabetes and tolerance in transgenic mice expressing class II MHC molecules in pancreatic beta cells. Cell 53:159


43. Lombardi G, Sidhu S, Lamb JR, Batchelor JR, Lechler R (1988) Co-recognition of endogenous peptides with HLA-DRI by alloreactive human T cell clones. J Immunol 142:753

44. Loveland BE, Hogarth PM, Ceredig R, McKenzie IFC (1981) Cells mediating graft rejection in the mouse. I. Lyt-1 § cells mediate skin graft rejection. J Exp Med 143:1044

45. Loveland BE, Sponaas A-M, Simpson E (1985) Mapping H-1 with the distal break point of chromosome 7 in Cattanach's insertion. Immunogenetics 22:503

46. MacDonald HR, Schneider R, Lees KR, Howe RC, Acha-Orbea H, Festenstein H, Zinkernagel RM, Hengartner H (1988) T cell receptor Vf3 use predicts reactivity and tolerance to Mls a encoded antigens. Nature 332:40

47. Marrack P, Kappler J (1988) T cells can distinguish between allogeneic major histocompatibility complex products on different cell types. Nature 332:840

48. Matzinger P, Bevan M (1977) Why do so many lymphocytes respond to the major histocompatibility antigens? Cell Immunol 29:1

49. McLaren A, Simpson E, Epplen JT, Studer R, Koopman P, Evans EP, Burgoyne PS (1988) Loca- tion of the genes controlling H-Y antigen expression and testis determination on the mouse Y chromosome. Proc Natl Acad Sci USA 85:6442

50. McMichael AJ (ed) (1986) Leucocyte typing III. White cell differentiation antigens. Proceedings of the Third International Workshop and Conference on Human Leucocyte Differentiation Antigens, Oxford. University Press, Oxford

51. Mitchison NA (1954) Passive transfer of transplantation immunity. Proc R Soc Lond [Biol] 142:72 52. Morahan G, Brennan FE, Bhathal PS, Allison J, Cox KO, Miller JFAP (1989) Expression in

transgenic mice of class I histocompatibility antigens controlled by the metallothionein promoter. Proc Nati Acad Sci USA 86:3782

53. Prochazka M, Leiter EH, Serreze DV, Coleman DL (1987) Three recessive loci required for insulin-dependent diabetes in nonobese diabetic mice. Science 237:286

54. Pullen AM, Marrack P, Kappler JW (1988) The T cell repertoire is heavily influenced by tolerance to polymorphic self antigens. Nature 335:796

55. Qin S, Cobbold SP, Thighe H, Benjamin R, Waldmann H (1987) CD4 monoclonal antibody pairs for immunosuppression and tolerance induction. Eur J Immunol 17:1159

56. Schendel DJ, Alter BJ, Bach FH (1973) The involvement of LD- and SD-region differences in MLC and CML: a three cell experiment. Transplant Proc 4:1651

57. Shinohara N, Watanabe M, Sachs DH, Hozumi N (1988) Killing of antigen-reactive B ceils by class II-restricted, soluble antigen-specific CD8 § cytolytic T lymphocytes. Nature 336:481

58. Shizuru JA, Taylor-Edwards C, Banks BA, Gregory AK, Fathman CG (1988) Immunotherapy of the non-obese diabetic mouse: treatment with an antibody to T helper lymphocytes. Science 240:659

59. Sibley RK, Sutherland DER, Goetz F, Michael AF (1985) Recurrent diabetes mellitus in the pancreas iso- and allograft: a light and electronmicroscopic and immunohistochemical analysis of four cases. Lab Invest 53:132

60. Simon MM, Edwards AJ, H/immerling U, McKenzie IFC, Eichmann K, Simpson E (1981) Generation of effector cells from T cell subsets: III. Synergy between Lyt 1 and Lyt 123/23 lymphocytes in the generation of H-2 restricted an alloreactive cytotoxic T cells. Eur J Immunol 11:246

61. Simpson E (1985) Recognition of minor transplantation antigens: the role of H-2 and other Ir genes. In: Medawar PB, Lehner T (eds) Major Histocompatibility System. The Gorer Symposium. Blackwell, Oxford, p 37

62. Simpson E. (1988) Suppression of the immune response by cytotoxic T cells. Nature 336:426 63. Simpson E, Gordon RD (1977) Responsiveness to H-Y antigen: Ir gene complementation and

target cell specificity. Immunol Rev 35:59 64. Simpson E, Tomonari K (1989) Mapping minor H genes. Immunology [Suppl] 2:42 65. Simpson E, McLaren A, Chandler P, Tomonari K (1984) Expression of H-Y antigen by female

mice carrying Sxr. Transplantation 37:17 66. Simpson E, Chandler P, Goulmy E, Disteche CM, Ferguson-Smith MA, Page DC (1987) Separa-

tion of the genetic loci for the H-Y antigen and in testis determination on the human Y chromosome. Nature 326:876

67. Simpson E, Tomonari K, Lovering E (1989) Minor transplantation antigens: their role in shaping the T cell repertoire. Immunol Lett 21:39

32 E. Simpson

68. Slavin S, Strober S, Fuks Z, Kaplan HS (1977) Induction of specific tissue transplantation tolerance using fractionated total lymphoid irradiation in adult mice. Long-term survial of allogeneic bone marrow and skin grafts. J Exp Med 146:34

69. Snell GP (1948) Methods for the study of histocompatibility genes. J Genet 4 9 : 8 7 70. Souhami RL, Beverley PCL, Bobrow L (1988) First international workshop on small cell lung

cancer antigens. Lung Cancer 4 : 1 71. Springer TA, Dustin ML, Kishimoto TK, Marlin SD (1987) The lymphocyte function-associated

LFA-1, CD2 and LFA-3 molecules: cell adhesion receptors of the immune system. Annu Rev Immunol 5 :223

72. Tite JP, Janeway CA Jr (1984) Cloned helper T cells can kill B lymphoma cells in the presence of specific antigen: Ia restriction and cognate vs noncognate interactions in cytolysis. Eur J Immunol 14:878

73. Todd JA, Bell JI, McDevitt HO (1988) A molecular basis for genetic susceptibility in insulin- dependent diabetes mellitus. Trends Genet 4 : 1 2 9

74. Townsend ARM, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ (1986) The epitopes of influenza nucleoprotein recognised by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell 44 :959

75. Trowsdale J, Campbell RD (1988) Physical map of the human HLA region. Immunol Today 9:34 76. Vidovic D, Matzinger P (1988) Unresponsiveness to a foreign antigen can be caused by self

tolerance. Nature 336:222 77. Waldmann H (1989) Manipulation of T cell responses with monoclonal antibodies. Annu Rev

Immunol 7 :407 78. Waldmann H, Cobbold SP, Qin S, Benjamin R J, Wise M (1989) Tolerance induction in the adult

using monoclonal antibodies to CD4, CD8 and CD 11 a (LFA-1). Cold Spring Harbor Syrup Quant Biol 2 :885-892

79. Wettstein PJ, Bailey DW (1982) Immunodominance in the immune response to "multiple" histocompatibility antigens. Immunogenetics 16:47

80. White J, Herman A, Pullen AM, Kubo R, Kappler JW, Marrack P (1989) The V~-specific superantigen staphylococcal enterotoxin B: stimulation of mature T cells and clonal deletion in neonatal mice. Cell 5 6 : 2 7

81, Yeoman H, Anderton JG, Stanley MA (1989) MHC class II antigen expression is not induced on murine epidermal keratinocytes by IFN-3, alone or in combination with TNFc~. Immunology 66 :100

Note added in proof

Subsequent to the writing of this chapter in 1989 there have been several advances in our knowledge of the field: 1. The chromosomal localization of several non-MHC-linked susceptibility genes in diabetes-prone NOD mice and humans: Todd JA et al. (1991) Nature 351: 542; Garchon HJ et al. (1991) Nature 353: 260; Cornall RJ et al. (1991) Nature 353: 262; and Julier et al. (1991) Nature 354: 155. 2. The identification of minor histocompatibility 'loci' as encoding at least two products, one processed and presented through the MHC class I, the other through the class II pathway: see Roopenian DC (1992) Immunol Today 13: 7. 3. The identification of endogenous superantigens in mice as products of mouse mammary tumour proviral integrations. These include Mls antigens and a group of Mls-like antigens, each of which cause T-cell receptor V~-specific deletions of the T-cell repertoire: see Nature 349 (1991), papers by Marrack et al., Frankel et al,, Woodland et al., and Dyson et al. on pp 524, 526, 529 and 531, respectively.

mechanisms of transplantation immunity

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