Immunology Today, vol. 7, Nos. 7 & 8, 1985

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The non-MHCtransplantationantigens: neither weak nor minor The existence of minor histocompatibility (mH) antigens is implied by the very naming of the major histocompatibility antigens. Grafts exchanged between individuals matched at the major histocompatibility complex (MHC) (which encodes the antigens that produce rapid graft rejection) are rejected, albeit at a somewhat slower pace than MHC grafts. There are multiple mH antigen differences between MHC (HLA) matched humans and between MHC (H-2) identical mouse strains. Using mH congenic mouse strains, however, individual mH antigens can be studied, and these provide a basis for interpretation of immune responses to multiple mH antigens in HLA matched human grafts. In this article Bruce Loveland and Elizabeth Simpson discuss four aspects of mH antigens: their immunogenidty; immune response (lr) gene regulation; mH antigens in man; and recent models which might lead to their molecular identity. In man, the transplantation of organs or tissues between HLA identical siblings, matched not only for both entire MHC haplotypes but also for an estimated half of the many non-MHC (mH) transplantation antigens, leads to rejection unless immunosuppression is employed. In the case of bone marrow transplantation, T cells present in the (untreated) donor bone marrow can mount serious graft versus host (GVH) responses against the recipient 1. These reactions are not 'minor' and are life threatening: their strength is almost certainly due to incompatibilities at multiple non-MHC histocompatibility loci. Rejection of grafts, particularly of skin, between H-2 identical mouse strains can be almost as rapid as between strains that differ at the M H C 2'3. Advantageously, in the mouse we can also study the effects of individual mH antigens by using the mH congenic strains which define the histocompatibility antigens H-1 and H-3 to H~I. Immune responses to mH antigens, like those to the MHC antigens, can be elicited in vitro as well as in-vivo. However, responses to mH antigens are apparently exclusively T-cell mediated and can only be regularly obtained in vitro following in-vivo immunization. This contrasts with the strong mixed lymphocyte responses (MLR) obtained with unprimed cells from MHC mismatched individuals. The difference is due to much higher precursor frequencies of T cells (of helper T cells, Th, and of cytotoxic T cells, To) reactive with ailogeneic MHC antigens, in comparison with the cells reactive with individual mH antigens. Another characteristic of mHreactive cells in vitro is that they are invariably MHC restricted, that is, mH antigens are recognized only in the context of self MHC molecules: Th generally with class II and Tc generally with class I. The mH antigens are detected in vivo by acute or chronic transplant rejection, by GVH and delayed type hypersensitivity (DTH) responses: and presumably the same antigens are being recognized by helper and cytotoxic T cells in vitro.

Transplantation Biology Section, Clinical Research Centre, Harrow, MiddlesexHA13UJ, UK ~) 1986, ElsevierScience PublishersB.V, Amsterdam

0167

49191861502.00

Bruce Lovelandand ElizabethSimpson Responses to mH antigens are under the genetic control of both MHC and non-MHC Ir genes. Much of this control operates through 'associative recognition', i.e. via the MHC restriction molecules which are not only highly polymorphic themselves (the MHC Ir gene products) but also are possibly subject to functional modification by other cell surface molecules (non-MHC Ir gene products). Ir gene effects have been studied most extensively in responses to H-Y, the Y chromosome controlled male-specific mH antigen 4, but analysis of responses to other isolated mH antigens also implies interactive MHC/non-MHC Ir gene control s 12. Theoretically derived estimates based on breeding and transplantation studies in mice suggest that a very large number of mH antigens exist, possibly several hundred 13'14. Any two unrelated individuals might be expected to differ at many. However, when occurring in isolation as in congenic strains they can be difficult to detect, and at present there is no good evidence to identify which molecules carry the antigenic determinants.

The immunogenicityof mH antigens

The use of bilineal congenic and other mouse strains The best defined mH antigens are in the mouse and are H-Y expressed by normal males but not females and those defined with the various bilineal congenic strains. There is a large set of congenic strains in which BALB/c mH genes were transferred to the C57BL/6 genetic background and selected by skin graft rejection (the B6.C series) is. Earlier, many independently derived strains were begun by Snell and others using the C57BL/ 10 strain and fixing on it genes which provided transplantation resistance to tumour growth from strains including DBA/2, 129, BALB/c, C3H and LP16'17 (for examples see Table 1). While developing these strains, the most readily detectable histoincompatibility of either skin or tumour cell grafts would be expected to be either the more strongly immunogenic antigens or tightly linked genes coding for several antigens. The bilineal congenic strains of mice have been used to define mH antigens H-1 and H-3 to H - 4 1 ls'16'18 21 This number represents the tip of the incompatibility iceberg. The 8 to 12 generations of backcrossing involved in generating the congenic strains theoretically reduced the genes inherited from the donor to a relatively short length of a particular chromosome and although these strains are generally assumed to define single mH-gene allelisms this is not always correct. The inheritance of a single gene coding for one histocompatibility antigen is deduced by complex breeding and grafting experiments using closely related strains. The analysis depends on differences of more than one H antigen being detected by accelerated secondary graft rejection (a variation of the F1 test) 16. This experimental analysis has provided evidence for the arguments put forward by Bailey 13'22 and Johnson TM for the likely presence of

223

Immunology Today, voL 7, Nos. 7 &~, 1986

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Table I, Examplesof bilinealcongenicstrains

Strain Propername*

Common name

B6.C-/-/-15c B6.C-H-16c

HW13J HW13K HW13 HW13F HW26B HW26C HW21 HW17

4 4 4 4 4 4 4 4

Mup-1 H-15 H-15 H-16 Mup-1 bH-t5 H-16 H-20 H-21 bH-15 H-16 H-21 bH-15 H-16 H-20 H-21 Mup-1 bH-15 H-16 H-20 H-21 H-20 H-18

B10.D2(58N) B10.129(21M) HW80

7 7 7

H-1 Hbb MIv-1 H-1 Hbb c H-1 Hbb c

B6.C-H-3 c

B6.C-H-20 c

B6.C-H-18c BIO.D2-H-1a B10.129-H-Ib B6.C-H-I b

Chromosomalmapping

Locicarryingintroducedalleles

B6.C-H-3c BlO.LP-H-3bH-13 b BIO.LP-H-3 b BlO-paH-3ea t

BIOiP LPa

2 2 2 2

H-3 H-3 H-13 A H-3 B2m Lyre-11 H-3 (H-13 ?)

B6.C-H-7b B10.C-H-7b

HW23 B10.C(47N)

9 9

H-7 Mod-1 Fv-2 H-7 Fv-2

B6.C-H-25c

HW65

1

H-25 Ly-9 Ly-17

*Thepropernameof thebilinealcongenicstraingivestheprimaryparentalstrain(e.g.,C57BL/10(B10),C57BL/6(86)),thedonatingstrain(e.g.,BALB/c(C),129,DBA/2 (D2),LP)andthedefinitivegene(italic):inthesecasesaHgeneencodinga mHantigen.Somestrains,notablyHW13,HW13F,HW26BandHW26C,carrytheallelesof severallinkedmHantigens.(DatafromRef.23,31; andBailey,personalcommunication)

224

several mH differences between each pair of congenic strains whether carried over as linked genes or appearing by mutation 22. For instance, eight strains 'define' the linked mapping of the H-15, H-16, H-18, H-20, and H-21 antigens to mouse chromosome 4 (Table 1) and there is evidence for the existence of at least three more linked mH antigens on this chromosome (D.W. Bailey, personal communication). This implies that there are a large number of mH antigens of extremely weak immunogenicity present but not yet isolated on congenic strains. These have been assumed, reasonably so, to be generally insignificant in the presence of stronger antigens. Providing we are aware that there are likely to be multiple gene differences between any pair of congenic strains, several of which may act as transplantation antigens, we can be prepared for effects due to more than one locus. Polymorphism of the mH antigens appears to be limited to a handful of alleles detected for each of some antigens (e.g. H - l , H-3, H-4, H-7 and H-8) 23 and a low degree of polymorphism among wild strains 24. However, most mH antigens have not been screened extensively for polymorphism. Much of the genetic knowledge gained by the analysis of immune responses in the bilineal congenic strains has assisted our understanding of clinical transplantation responses. The mH antigens exhibit a wide range of immunogenic strengths 16'23'25. For instance, among the B6.C mice, H-1 c and H-25 c produce prompt skin graft rejection after 15 to 30 days whereas H-26 b, H-28 b and H-36 b skin grafts are often not rejected even after 70 days is. In effect, each antigen has a characteristic immunogenicity. The limited examination so far of the responses against definable combinations of mH anti-

gens has been interpreted to show generally additive effects by antigens of equal strength and marginal effects by weak antigens on the stronger 26. This immunodominance is regularly exhibited by the stronger antigens. Although the presence in mice of multiple mH antigen differences occurring between two unrelated but MHCmatched individuals best parallels the clinical situation, in such combinations there are many variables influencing the immunological outcome. These include the host's Ir genes, the number of similar-to-self antigens expressed (in which case responses to them may be excluded from the repertoire or suppressed), the immunogenicity of the donor tissue, any tissue specific antigens and competitive effects (discussed below) which might themselves vary with the tissue and the assay of immune response. By fixing the MHC genes and the majority of background genes, congenic strains allow some control over all these aspects. When assessed by their immunizing potential, different tissues of B6.C mice provided evidence of the wide but perhaps variable tissue expression of the mH antigens, and at this time we cannot assume the ubiquitous distribution of all mH antigens: a few are probably tissue specific 27. Antigens have rarely been found which are immunogenic on some tissues (e.g. the Epa-1 or Skn antigen(s) expressed principally on epidermal cells) but are not readily detectable by other assays of immunological memory such as lymphocyte proliferation, cytotoxicity, or in-vivo HVG lymph node assays28-3°. Another type of inbred mouse strain is invaluable in genetic analysis. The recombinant inbred (RI) strains were derived by inbreeding pairs of F1 mice between two unrelated strains. Thus from the initial random assort-

ImmunologyToday,vol. 7, Nos. 7 & 8, 1986

r ment, genes were fixed in particular combinations and the large related sets of strains so developed (e.g., the CXB, B×D, AKXD and BXH strains) enable the genetic linkage of segregating alleles to be identified 31. Further analysis of mH antigens by responses in-vitro Cytotoxic T-cell responses assayed in vitro after in-vivo priming provide a convenient analysis of mH antigens. For MHC antigens, we know that the target molecules for skin graft rejection and for Tc are the same (namely responses between C57BL16 and the C57BL/6 bm class I mutants16), but except for H-Y, where they also appear to be the same 32, we lack this evidence for other mH antigens. Different aspects of the immune response accessible to analysis in the cytotoxic assay include the in-vivo priming (induction) events, antigen presentation and processing, the in-vitro restimulation stage and antigen expression on the target cell. The induction events in the response to mH antigens can be studied by combining in-vivo and in-vitro steps 33. Competitive effects in the responses to mH antigens Competitive effects between strong and weak H antigens which might be important in clinical transplantation are best illustrated in the extreme case by MHC antigens. H-2 (MHC) differences between mouse strains are generally of sufficient antigenic strength that there is a failure to generate Tc responses to concurrent multiple mH antigen differences. It is apparent that when immunity is induced to multiple mH but not H-2 antigens, only a small number of mH antigens are recognized by effector Tc cells although it has been difficult to identify them individually. For C57BL/6 anti BALB, H-25 c is included amongst the target antigens but in this strain combination there are clearly others not definable by the mH bilineal strains 34, The recently isolated human T-cell lines that recognize mH antigens also provide evidence for a small number of immunodominant mH antigens (see below). By choosing particular B6.C bilineal mouse strains to study pairs of antigens, we have evidence of the consistent immunodominance in Tc assays of H-1 c over H-Y, H-25 c and H-7 a on the C57BL/6 H-2 b background (Loveland, Sponaas and Simpson, unpublished). This may be mainly due to striking differences in the precursor frequencies of antigen-specific To. Additional and contrasting interference effects have been seen both in these and other studies in vitro 25 and in analyses of skin graft rejection: a graft expressing a 'weak' antigen can show a delayed rejection when adjacent to a graft with a stronger antigen, e.g. H--lb beside H-Y (Ref. 35). NonMHC antigens can also provide immunological help at the induction of responses. T¢ specific for the Qa-1 b allele were only detected after immunization of female Qa-la mice with male Qa-lb cells where H-Y was used as the helper determinant 36. Recently, other undefined antigens were found to 'help' anti-H-Y cytotoxic responses in B10.A(2R) and BIO.GD female mice which have been considered non-responders to H-Y (Ref. 37). The factors which influence immunodominance must include the total 'spectrum' of mH antigens present, the MHC haplotype controlling MHC linked Ir gene effects and the non-MHC Ir genes (which may themselves exist as part of the spectrum). Tissue differences in immunogenicity The various tissues and organs used for trans-

vlsl4Ai-

plantation or immunization are possibly differently immunogenic due to several contributing factors. A comparison of the responses to skin, thyroid, heart, liver and lymphoid tissues in C57BL110 recipients leads to the ranking of the H-4, H-7 and H-8 antigens in different orders of strength depending on the grafted tissue 2s. Skin is generally the most immunogenic tissue, with heart or thyroid grafts often surviving much longer or indefinitely 38, Whether this represents quantitative or qualitative characteristics of the antigens on individual cells in the tissues, the graft vascularization, or of the way in which the transplants heal in the host, is quite unclear. It is now widely accepted that the number of MHC class II positive cells in the graft, affecting the induction of immunity, may be an important factor in immunogenicity. Females in general respond more strongly than males (grafts survive longer on males than on females) 39 irrespective of parity, except for responses to H-Y to which multiparous female mice are tolerant 4°. In addition the immunizing route and dosage influences the outcome. Priming with skin grafts and footpad or subcutaneous route injections of lymphoid cells are more successful in eliciting a response than intraperitoneal or intravenous immunizations which can be immunosuppressive (an observation originally made before the 'transfusion effect' on kidney transplantation was noted41). Immune regulation (It gene effects) The male specific H-Y antigen is the most easily studied in mice, principally because of the elegance of every inbred brother/sister being a congenic pair. Immune responses to H-Y are governed both by MHC (H-2) and other autosomal genes (for reviews see Refs 42 and 43). All H-2 b strains of mice respond, defined by skin grafting and in-vitro Tc assays. The rejection of H-Y expressing skin grafts is independent of the cytotoxic T-cell response, since females of the intra H-2 recombinant strains BIO.A(SR) (KbI-AbI-Eb/kD d) and BIO.A(4R) (KkI-AkD b) fail to make Tc responses in vitro, because of the lack of either an appropriate class I allele (BIO.A(SR)) or class II allele (BIO.A(4R)), nevertheless both reject male skin grafts 44. The cell type responsible for graft rejection is either a Th or a separate class of DTH cell, T~TH, restricted by a class II molecule, the precise identity of which is still in question 44a. H-2 k, H-2 d and H-2 s strains are essentially nonresponders to H-Y as measured by primary skin graft rejection, but Tc responses occur in some strains following priming in vivo via the footpad (fp) but not via the intraperitoneal route which suffices for H-2 b strains (Table 2). The existence of responder and non-responder strains of each non-H-2 b haplotype implies the involvement of non-H-2 Ir genes. Since no H-2 haplotype except H-2 b or any genetic 'background' (e.g. BALB, amongst the BALB H-2 congenics or B10 amongst the B10 H-2 congenics) predicts responsiveness, these resuits also imply that responsiveness is a function of the interaction of H-2 and non-H-2 Ir genes. This could operate at the level of antigen presentation, where only certain combinations of non-H-2 Ir gene products and MHC antigens might be permissive for association with H-Y, and/or at the level of the T-cell repertoire selection (e.g. where certain combinations of H-Y with self H-2

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ImmunologyToday,voL 7, Nos. 7 & 8, 1986

-reviews Table 2. CytotoxicT-cellresponsesto H-Y in H-2k, d and s strains following footpad priming H-2 haplotype

Strain

Response*

H-2k

CBA/H BALB.K C58 C3H/He RF CE B10.BR AKR

+ + + +/-

H_2d

C57BL/Ks B10.D2/n B6.C-H-2d BALB/c DBA/2

+ +/+I-

H_2S

BIO.S SJL A.SW

+ +

*Toresponsesbyindividualfemalemicefootpadprimedwithsyngeneicmale cells,asthemajorityof individuals(+), aproportion(+/-), or none(-). (Data from Ref.42) could 'mimic' other self antigens leading to functional deletion of clones which would recognize self H-2 plus H-Y). Such Ir gene effects do operate at the induction of immune responses and not at the level of target cell lysis since responder strain cytotoxic cells can lyse H-2 matched male cells of non-responder strains. By using recombinant inbred mouse strains one of the non-H-2 Ir genes controlling H-Y responses in H-2 d mice has been mapped to chromosome 2 near the 182-microglobulin locus45. Similar regulation almost certainly pertains to the other mH antigens. MHC class I Ir genes have been shown to control Tc responses to H-4 in a manner similar to H-Y 12. Likewise, Tc responses to other mH genes (H-l, H-3, H-7, H-25 etc.) have been shown to be restricted to either or both Kb or Db using the B6.C and BIO bilineal congenic strains (Refs 5-12, 46 and 47; Loveland and Sponaas, unpublished). This implies a class I MHC Ir gene control of these responses.

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Antigen processing Antigen presentation is required at the induction of both primary and memory immune responses. This can be either of 'foreign' antigens acquired and processed by an antigen presenting cell (APC) of the responder, or presentation by donor cells of what one might term 'intrinsic' antigens: molecules of the cell surface membrane which are themselves antigenic and thus would not require processing. Such appears to be the case with responses against mH antigens on stimulator cells sharing MHC molecules. However, when MHC molecules are not shared, the question arises as to whether responses can be efficiently induced against all mH antigens which have been processed by cells of the responder. The published data remain unclear. We know that immune responses are generally obtained to small peptides which often comprise just a portion of the native (soluble) antigenic molecule 48. We cannot yet identify the molecules that behave as mH antigens although by the

methods we can use these appear to be good models for studying MHC restriction, Ir gene effects and the processing of intrinsic molecules. When the recipient and donor tissues have different MHC haplotypes then if mH antigen processing does occur so that native antigenic epitopes of the donor are recognized on the recipient APC, the T clones generated will not recognize the donor mH antigens in the context of the donor MHC molecule(s). Consequently, immunity against such an MHC mismatched, mH different graft would not be directed at the donor MHC/mH complex and if the recipient was tolerant of the donor MHC, such a graft would not be reJected49. This has important implications for the selection of donors in clinical transplantation. An alternative possibility that recipient MHC molecules could be absorbed and expressed by donor cells, allowing intrinsic donor mH antigens to be recognized in the context of host MHC, might lead to a response mounted against such cells of the graft. Several groups have used immunization protocols which resulted in Tc specificities being detected that apparently required antigen to be processed during the induction phase. For example, when H-2 heterozygous F1 responders were immunized with cells homozygous for one parental H-2 haplotype and carrying multiple mH antigens, lysis was obtained not only of the immunizing target but also of the congenic target with the other parental H-2 haplotype 5°. However, when using a single mH antigen, H-Y, such experiments produced no evidence of the cross-priming of Fr responders 5r. Bevan's group further examined the cross-priming of F1 mice with multiple mH antigens by using intravenous immunization followed by stimulation in vitro. They were surprised to find a preference for the induction of cross-primed Tc early in the response 52. In another study of responses to multiple mH antigens but using a different protocol, the cross-priming was not reproduced with Tc but Th were efficiently cross-primed 53. The second protocol involved immunization in the footpad and a brief culture, in the absence of antigen, of cells obtained from the draining lymph nodes. These conflicting results might arise from several causes. They may reflect the complexity of primary versus memory T-cell immunity, the various modes and routes of immunization leading to different dispersal of the immunegen 53, the balances between suppression and responsiveness 54 or of multiple versus single immunizing mH antigens. One hypothesis to account for the crosspriming only of Th could be that it results from the absorption onto donor cells of responder class II antigens. The suppression of immunity can play a powerful role in responses to mH antigens; however, the mechanisms are not yet resolved. The effect is adoptively transferable by T cells, can be long lasting or transient and shows antigen specificity 4 0 ' 52.55 ' ' 56 . One of the unexpected findings with T-cell clones has been the specific suppression in vivo and in vitro against mH antigens mediated by Th clones 57'58. It appears that immune regulation is fine tuned at least partially by the localized concentration of Th cells via yet undefined mechanisms, confirming their central role in regulating immune responses. The analysis of immune responses in strain combinations involving multiple mH antigenic differences implies the generation of specific rather than cross-reactive immunity. However, even when dealing with single mH

Immunology Today, voL 7, Nos. 7 & 8, 1986

antigens, cross-reactions are often seen. The Tc anti-H-Y (D b restricted) cross-reacting with H-2D d are well known, and we regularly obtain similar cross reactions on certain H-2 mismatched targets after immunizing for other mH antigens such as H-4 a, H - 4 b or H-1 c (Loveland, Sponaas and Simpson, unpublished). At the clonal level, Th reactive with H-Y (I-A b restricted) and Tc reactive with H-Y (Db ~restricted) are not infrequently found to also react with cells from some but not all strains of another H-2 haplotype. This is often H-2 k, presumably in association with another mH antigen s9. This implies the common occurrence of crossreactivity of some clones specific for one mH antigen in the context of self MHC, with another mH antigen in the context of a different MHC haplotype s9'6°.

antigen immunogenicity would be a nightmare for the hopeful tissue typer. So far we have barely mentioned tissue specific antigens. Much has been published on the serological and cellular immune reactions that are characterized by a limited tissue distribution and have been detected after transplantation or as autoimmunity and this has been recently reviewed 77. It appears that some of these responses may be to what we have defined as mH antigens but many, especially the serological responses, are against other antigens. For instance, the tissue specific responses only occasionally were correlated with graft rejection crises or tissue damage. Hence, they are qualitatively if not definitively different from the histocompatibility responses discussed here.

Human mH antigens The clinical crises of graft rejection and GVH disease in patients with HLA-matched grafts are evidence for the presence of mH antigens in man. Full HLA-A,B,C and DP, DQ, DR matching is not sufficient to overcome the need for post transplantation immunosuppression, frustrating the aim of establishing well functioning grafts with the minimum of external interference. HLA-matched sibling donors who share about 50% of their mH antigens are a better prospect than unrelated matched combinations in kidney allografting; however, about half of such cases of bone marrow (BM) transplants still result in GVH disease 1,6~. In some cases T-cell immunity to non-HLA antigens has been observed 62 67 Even when the mixed lymphocyte culture (MLC) test is negative between donor and recipient cells (which would indicate HLA antigen matching), and no memory response to mH antigens can be detected, subsequent immune responses are not a small problem and occur frequently. HLA-restricted Tc specific for male (H-Y) cells have been derived from a female recipient of a male BM graft 63, from the PBL of multi-transfused female aplastic anaemia patients68'69, from a female recipient of a male kidney graft 7° and in the PBL of multiparous women 7~. The identification of other non-HLA antigenic specificities is more difficult, requiring a large panel of typed stimulator and target cells. Nevertheless one group has begun this careful task72. Some human mH antigens have been defined by the reactions of T-cell lines and clones established both from the cells of an aplastic anaemia patient after multiple transfusions 7~ and from bone marrow graft recipients undergoing GVH responses61. These cell cultures and clones have equivalent MHC-restricted and specificity reactions to the mouse cell lines studied by others, from which observation we deduce that similar antigens and immune regulation are probably involved. The restriction of Tc responses by HLA A2 occurs at a high frequency although there are examples of restriction to HLA-B7, B27, B40, Bw44 and BW6261'66'73-75. It remains to be seen whether these common HLA haplotypes are 'preferred' by the responding T cells (namely, the parental preference phenomenon in mice76), or of other significance. If they are preferred, prospective typing for a few strongly immunogenic mH antigens may be possible and beneficial, especially when combined with immunosuppression of the recipient and other treatments, e.g. the selective depletion of some T cells from bone marrow grafts. The alternative of a random (normally distributed) HLA haplotype restriction and mH

The molecules carrying mH antigens This section heading is misleading since we know nothing of the molecular nature of the mH antigens. The best evidence that these polymorphic molecules are on the cell surface is that target cells which carry the antigens are killed in cytotoxicity assays, and that both these and proliferative responses are class I or class II MHC restricted. Congenic mouse strains with known, limited polymorphism carry genes encoding definable mH antigens: we deduce that single genes most probably code for single molecular polymorphisms. Moreover, linkage analysis, which in the mouse is most extensive, has not identified correlations of transplantation responses with enzyme markers, congenital disease, viral gene loci (though recent reports suggest a linkage with retrovirus insertion sites78), or other known cell membrane polymorphisms such as the Ly (lymphocyte) markers. Thus the expression of polymorphism appears not to affect the presumed physiological function of the molecules which bear the epitope recognized by responding T cells. We know that the H-3 linked P2microglobulin alleles can be recognized in vitro by Tc but these antigens are not yet proven to be mH antigens 79. Also there is no reason to assume that mH antigens are epitopes of a particular class of molecules: they may be generated by polymorphisms of a functionally very heterogeneous group of molecules. Recent molecular analyses of the immune responses to viral antigens produced surprising data which may be important in finally identifying the mH antigen target molecules. One of the principal antigens of influenza A recognized by Tc is encoded by the viral nucleoprotein gene. The nucleoprotein molecule identified serologically appears only in the nucleus and not on the surface membrane of the target cell8°. Thus the antigens which are recognized by T cells can appear as MHC-restricted cell surface determinants in forms which we do not detect any other way. Whether the nucleoprotein anti gen is recognized in a processed form on the target cell surface, or whether it 'modifies' the expression or conformation of a cell surface molecule (e.g. a MHC class I molecule) is not yet known. Similarly, T-cell responses against Epstein-Barr (EB) virus antigens (LYDMA) may be to particular metabolic components of the virus rather than to intact viral molecules themselves. Not all EB positive cells are Tc targets81. We have also observed a lack of antibody responses to the mH antigens. Protocols to produce monoclonal antibodies have failed to raise reagents, and the sera

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ImmunologyToday,vol. 7, Nos. 7 & 8, 1986

made between H-2 matched strains, purportedly containing anti-mH specificities, could well be directed against other weakly immunogenic but not transplantation antigens. If the mH antigen epitopes are only revealed in association with the MHC molecules then it is relevant to search for MHC-restricted antibodies if such determinants are part of the B-cell repertoire. The Ly series of antigens, in contrast, are expressed predominantly on lymphocytes and induce very good antibody responses82but have not been shown to behave as mH antigens for T-cell mediated responses. A precedent for T-cell recognition in the absence of B-cell recognition is the failure of immunizations between the C57BL/6 wild-type strain and the many H2K bm mutant strains to induce antibodies. Yet the mutant H-2 haplotypes were selected on the basis of skin graft rejection (which was very rapid in some cases) and are characterized by vigorous primary in-vitro MLR and Tc responses between cells of the mutants and the wild strain 16'83. In summary, the mH antigens appear to be ubiquitously expressed, regulated by single genes, and recognized significantly by T lymphocytes and apparently not at all or very poorly by B lymphocytes. One model has recently been proposed which might have the kernel of a solution. It has been suggested that cell membrane receptors, such as the insulin receptor, are in flux as free cell membrane molecules and in exchange with 132-microglobulin in its non-covalent association with class I MHC molecules84. It is not unreasonable that subtle polymorphisms of such receptors, which do not affect their physiological role, are identifiable by T cells during this putative association with class I molecules. The same polymorphisms, though not necessarily the same epitopes, should also be recognized by T cells in association with class II molecules in order to explain class II restricted induction events, proliferation and the subsequent activation of class I restricted T cells. Such polymorphisms, if slightly wrinkling the topography of the complexed mH molecule during its association with the MHC molecules, might well be invisible to B cells. In conclusion, we support the recent hypothesis84 that at least some of the minor histocompatibility antigens are cell membrane molecules which function, for certain periods, in association with either MHC class I or class II molecules. This helps explain the preferential T-cell immunogenicity, the prompt recognition by MHCrestricted T lymphocytes and also suggests a mechanism for the competitive effects seen between individual mH antigens. Whether the mH antigens remain a transplantation barrier depends on the ability to identify them and on our capability to regulate systemic immune responses. However, they cannot be ignored as the targets of acute and more subtle chronic immune reactions. We thank Drs Don Bailey and EIs Goutmy for their critical reading of the manuscript and helpful comments.

References

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