Speculations on the specificity of suppression

Immunology Today, Vol. 1O, No. 2, 7989

Speculations on the specificity of , suppressm The mechanisms of antigen-speofic T-cell suppression still O

remain inadequately explained. There has been a prolonged and unsuccessful hunt for 'suppressor cell markers'. This has largely deflected attention from a critical question - namely, what molecular structures are specifically recognized by cells mediating antigen-specific T-cell suppression? Here, Richard Batchelor and colleagues present the hypothesis that the structures 'seen'by these cells are/n principle the same as those recognized by other T cells, that is, a major histocompatibility complex (MHC) molecule holding a peptide in its binding cleft. In the particular circumstancesof.specific suppression, the peptide is derived from the variable (idiotypic) regions of the T-cell receptor of the target clone.

J. Richard Batchelor, Giovanna Lombardi and Robert i. Lechler

It is worth emphasizing that not only is the suppression specific in all these experimental systems, but in many cases, studies with purified ceil suspensions have been carried out. These show that T cells are responsible for the suppressive effect. Of course, this does not mean that the T. cells are necessarily the effector cells causing the suppression, only that they are capable of initiating the phenomenon. There are severai different notions cff how suppressor Poking fun at antigen-specific suppressor cells seems to T cells might function. According to one, they might be a popular sport 1. There are good reasons for an belong to a separate lineage or lineages of T cells, increduious attitude. (1) Reproducible data showing that performing different functions from those carried out by T-cell clones mediate specific suppression of an immune the CD4 ÷ or CD8 ÷ subpopulations. In that case, one response in vivo is lacking. (2) The relevance of in-vitro demonstrations of suppression to events in vivo is would expect them to have specific molecular structures that enable them to perform their specialized function doubted by many - or the specificity is Questioned. (3) of suppression; such molecules should distinguish them Earlier claims that 'l-J' was an (MHC)-encoded marker for from other T-cell lineages. Despite a decade of monosuppressor cells have been tacitly withdrawn, overturned clonal antibody technology, there has been no convincby the molecular information on the MHC genes now ing demonstration of such molecules on specific suppresavailable 2. 'i-J' has vanished from the MHC! (4) The sor T cells. profusion of 'suppressor factors' - some claimed to be Lately, interest in the concept of specific suppressor antigen specific, some idiotype specific, none have been adequately characterized in biochemical or genetic terms T-cell lineage(s) was renewed by the discovery that it was possible to distinguish subsets in both the CD4 + and - represents thoroughly unsatisfactory evidence. CD8 ÷ populations. One subset reacted strongly with the Nevertheless, there are too many weii-documented experimental examples of suppression in vivo with ani- monoclonal antibodies 2H4 and HB-11 (CD45R), but very mal models for us to question the existence of the. weakly with 4B4 and UCHL1 (that are specific for the lowest molecular weight form of CD45); the second phenomenon itself. The challenge is to provide 3 satisfacsubset showed an opposite pattern of reactivity. It was tory explanation of the mechanisms in molecular and suggested that CD4 ÷ cells with high expression of 2H4 cellular terms. and HB-11 activated suppre:;sor T cells, whereas the Some of the clearest examples of suppression are subset expressing high levels of 4B4 and UCHL1 'helped' found in transplantation i m m u n o l o g y - namely, the B lymphocytes t~-13. However, recent studies (discussed transfer by lymphoid cells of specific unresponsiveness to in Ref. 14) show that the subsets are not separate allografts. For example, Brent and co-workers 3 showed lineages, but represent stages of immunological differenthat specific unresponsiveness to allogeneic skin grafts tiation from naive to 'memory' cells. It seems that there is could be transferred to naive, syngeneic mice by spleen no positive evidence for a separate lineage of suppressF,r cells from donor mice rendered unresponsive by treatT cells. Although this is not a conclusive objection, it at ment with alloantigen, anti-thymocyte serum and procarbazine. Similarl/, specific unresponsiveness induced by least makes one suspicious of the concept. An alternative hypothesis is that suppressor cells are allogeneic bone marrow and anti-lymphocyte serum treatment leads to the generation of spleen cells able to not a separate lineage, but belong to the wellcharacterized CD4 + or CD8 ÷ populations. There are transfer specific suppression to naive, syngeneic mice~. several require:-~'cnt-~ w~ic~, must be met if this hypothDorsch and Rosers.6 have transferred specific unresponesis is to be sustained. First, an explanation for the siveness with lymphocytes harvested from rats made specificity of suppression is needed; it is critical that any tolerant at birth by the classical method 7. Batchelor and explanation does not conflict with what is already known colleagues have shown that rats with long-surviving kidney allografts, induced by an enhancement protocol 8 concerning the binding of CD4 + or CD8 ÷ T cells to their specific ligands. Second, both in-vitro and in-vivo evior short duration treatment with immunosuppressive dence is needed to demonstrate the existence of T cells drugs 9, also have suppressor ce!!s in their spleens capable with antigen receptors of the specificity postulated to be of transferring specific unresponsiveness; similar results present on suppressor cells. Third, T cells with the have been obtained by Morris's group ~° postulated suppressive specificity must be shown to be Departmentof immunology,RoyalPostgraduateMedicalSchool,Ham- present in rive, at a time when specific suppression is manifest, and to be capable of transferring suppression. mersmithHospital,Du CaneRoad,LondonW12 ONN,UK. C(C~1989, ElsevierScience PublishersLtd, UK 0167 4919/89/$02 O0

37

Immunology Today, Vol. 10, No. 2, 1989

S~ificity of suppression Ultimately, a full clonal analysis of the T cells present in suppressive populations is necessary; but to select and expand the relevant clones, their specificity must be known. A common assumption is that suppressor T cells ~re activated by the alloantigen carried on the incompatible cells to which the host is unresponsive. Another concept, which we discuss in this article, is that they are activated by the idiotypes of the Z-cell receptors (TCRs) of clones causing allograft rejection.

38

Anti-idiotypicT cells The clearest evidence that such anti-idiotypic T cells can be generated in culture comes from Engteman's laboratory 1%16. He and his co-workers have grown human CD8 ÷ T-cell populations and clones that proliferate specifically when stimulated with autologous CD4 ÷ cells harvested from mixed lymphocyte reactions (MLRs). These CD8 ÷ T cells do not themselves respond to alloantigenic stimulators, nor to autologous CD4 ÷ cells proliferating in third-party MLRs. Interestingly, the CD8 ÷ T cells also specifically inhibit their target CD4 ÷ T cells from proliferating in response to alloantigen0 but are not demonstrably cytotoxic. Sucia Foca et al. 17 have a!so cJemonstrated anti-T-cell idiotype responses by autologous human T cells in vitro. In experimenta! systems, T cells with similar specificity have been found in animals which have been rendered specifically unresponsive. For example, rats with long surviving kidney allografts harbour T cells which proliferate in response to autologous T cells specific for the kidney donor alloantigens 18. Recently, in an analysis of the mechanisms of resistance to experimental allergic encephalomyelitis (EAE) in LEW strain rats, Sun eta/. 19 have grown in vitro, a CD4 ÷ T-cell line termed $1 that is specific for the amino acid sequence 68-88 of myelin basic protein (MBP). This line causes EAE when injected into other LEW rats. They also cultured spleen cells from LEW rats which had recovered from Sl-induced EAE and established a line of CD8 ÷ cells that proliferated specifically when co-cultured with $1. The CD8 ÷ cells were not activated by MBP, and inhibited the MBP-driven proliferation of the $1 cell line. The CD8 ÷ cell line was also specifically cytotoxic for the $1 line. There are two further experimental models in rats that indicate that T cells with anti-T-cell :,diotype specificity regulate immune responses in vivo. Kimura and Wilson 2o investigated the resistance to graft-versus-host disease (GVHD) in F1 rats, caused ny first injecting sublethal numbers of parental lymphocytes before challenging with a dose of parental cells sufficient to cause lethal GVHD in control rats that had not received an,, pretreatment. The resistance was specific for GVHD induced by the relevant parental cells, and it could be transferred to syngeneic F~ rats; it was accompanied by the generation of cytotoxic F~ T cells which killed parer, t 1 anti-parent 2 T-cell blasts. In the second model 2~, DA rats tolerized during the neonatal period to PVG strain cells were injected during adult life with normal DA lymphocytes to break tolerance. It was found that 3.8 x 108 cells were necessary to break tolerance; if the tolerant rats were given a preliminary injection of 3 x 107 DA lymphocytes and rested for six weeks, tolerance could not then be broken with 3.8 x 108 cells. These results were consistent with other

evidence that neonatally induced tolerance was maintained by anti-T-cell idiotypic T cells, in this case DA anti-(DA anti-PVG). The failure to break tolerance after pre~reatment with low doses of normal DA lymphocytes as attributed to heightened anti-idiotypic immunity boosted by the small numbers ot precursors of DA anti-PVG alloreactive cells present in the preliminary injections of normal DA cells. This interpretation was supported by experiments in which resistance to the breaking of tolerance could not be induced by small doses of DA lymphocytes if the precursors of DA antiPVG cells were removed earlier by an in-vivo, negative selection procedure 22. Taken together, these experiments provide a substantial body of evidence in support of the concept of a T-cell network, that is driven by 'antigen' consisting of idiotypic variation in TCR of different clones. Furthermore, the anti-idiotypic immunity can regulate the activity of its target population. It is noteworthy too that what was earlier thought to be I-J, has more recently been interpreted as an idiotope on a T-cell receptor 23.

To what molecularstructuredoesthe .~.ppressorT cell bind? The simplest assumption is that, like other CD4 ÷ or CD8 ÷ T cells, suppressor cells 'see' part of the polymorphic, peptide-binding site of an MHC class I or class II molecule together with a peptide held in the cleft. The recent demonstration of the three-dimensional structure of HLA-A2 with electron-dense material in the cleft is consistent with this model 24. Although this model accommodates the presentation of conventional T-dependent antigens, it is not known whether alloreactive T cells see alloantigen in precisely the same way. The very high precursor frequency (in unsensitized subjects) of T cells specific for alloantigen contrasts with very low frequencies for conventional antigen, suggesting that there may be a difference2~; but as the same heterodimeric TCR ,s used by both kinds of T cell, one would expect them to bind to similar ligands. An explanation for this paradox, offered by Matzinger and 8evan 26, suggests that a single MHC molecule can give rise to multiple, different antigenic specificities by forming complexes with mar:~' minor histocompatibility antigens. Recent work from !,/larrack and Kappler27 and our own laboratory 28suppor sthis idea, and also indicatesthat peptides other than those derived from minor histocompatibility antigens bind to MHC molecules, forming compound specificities which can actiJate T cells. A brief summary of our studies indicating that alloreactive T cells co-recognize alIo-MHC plus a peptide fragment follows. A series of human T-cell clones were raised against HLA-DR1 stimulators by two different protocols. The first was entirely conventional, the stimulators being DR1 ÷ blood mononuclear cells; three to six stimulations with the DR1 ÷ cells were carried out before the responder T cells were cloned. In the second protocol, similarly initiated anti-DR1 polyclonal cultures were re-stimulated three times with HLA-DRl-transfected mouse L cells. Cloning of the T cells was then carried out, using the transfectant as a source of stimulating cells. The clones obtained in both protocols were then tested for specificity by conventional proliferation assays in vitro. It was found that some clones proliferated when stimulated by DR1, irrespective of whether the stimulat-

Immunology Today, Vol. I0, No. 2, 1989

ing population was human blood mononuclear cells, Epstein-Barr virus (FBV)-transformed B cells, or the transfected mouse L cell line. Other clones responded to DR1, but only when expressed by a given stimulating cell population - for example human blood mononuclear cells or the murine L-cell transfectant. Numerous controls demonstiated that human cells lacking DR1 or untransfected mouse L cells failed to stimulate any of the clones. Our results could not readily be explained by differences in the level of DR1 expression by the transfected L cells and the DR1 + human cells. Fluorescence activated cell sorter (FACS) analyses showed similar patterns of DR1 expression, and dose response curves in which stimulator to responder ratios were varied showed that the transfected L cells were of comparable stimulating potency to that of human DR1 + cells. These results were consistent with the concept that anti-DR1 reactive T cells co-recognized alIo-MHC plus a peptide, which in some cases was not supplied by the DRl-expressing cell, and thus stimulation failed to occur. In the case of the clones which were stimulated by all types of DRl-expressing cells, there appeared to be two possibilities. First, the co-recognized peptide might be derived from a protein common to human and murine cells. A second possibility was that the mouse L-cell transfectant might ingest, process and present in the context of DR1, a peptide fragment derived from a protein shed by the responding T-cell clone. As the proliferation assays involved 48 hours of culture, there would be an opportunity for this to occur. To test this idea, the transfected mouse L cells were fixed with a range of gluteraldehyde concentrations before using them as stimulators. Fixed cells should be unable to ingest and process extrinsic proteins encountered for the first time during subsequent culture (proliferation assay). They would therefore be expected not to stimulate proliferation when fixed, but to be able to stimulate when not fixed. In contrast, human cells should be internalizing, processing and presenting fragments of autologous human proteins continually, so that fixation would not be predicted to interfere with recognition. The responses of several T-cell clones fulfilled this prediction - the clones were stimulated by unfixed and fixed DR1 ÷ human cells; but they were stimulated only by the unfixed mouse L cell transfected with DR1. An essential control here is that the fixation procedure should not interfere with the presentation function of the DR1 expressed by the transfectant. We found that fixed transfected L cells were capable of presenting an influenza haemagglutinin peptide to the DRl-restricted influenza haemagglutinin peptide-specific human T-cell clone, HA1.7 (Ref. 29). This indicated that the fixation conditions had not nonspecifically impaired the DR1 expression and presenting capability of the L-cell transfectants. We interpret our evidence as being consistent with the concept that alloreactive T cells, like antigen-specific T cells, are activated by a binary complex of a peptide plus MHC molecule. What are the implications of this idea for suppressor T cells? If suppressor T cells belong to the CD4 + and CD8 ÷ lineages as suggested here, it would follow that their TCR would also bind to a binary complex composed of MHC molecule plus peptide. To account for the specificity of suppression, it would only be necessary for the

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Anti-graft T-cell clonal expansion

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T-cell regulatory network with MHC presentation of peptides from TCR Fig.1. Cell 1 - specific for allo-MHC of the graft - undergoes clonal expansion (as shown in the top half of the figure). In the bottom half, the figure illustrates a single cell I expressing both TCR and MHC molecules. Occupying the peptide binding sites of this cell's MHC class I molecules are idiotypic fragments of the TCR of cell 1, and these are co-recognized by o~112that has specific suppressive activity. In addition, fragments of cell 1 TCR are shed extracellularly, then taken up, processed and presented in the context of class II molecules of the accessory cell (cell 3) to cell 4.

peptide to consist of an amino acid sequence derived from the idiotypic, variable regions of the TCR of the alloreactive T-cell clones. Consider now the events during an allograft response. Initially there is an expansion of T-cell clones with high affinity for allogeneic cells (see Fig. 1). The responding T-cell clones carry MHC molecules, whose peptidebinding clefts are likely t~ be occupied by peptides derived from endogenous proteins. Such peptides wilt include those derived from :he idiotypic sequences of the and 13chains of the T-ceil receptor. Thus they represent a potential antigenic stimulJs and target, which could be either MHC class I- or class !-restricted. It may be that the class I-associated TCR fragments become bound intracellularly before the class I molecule reaches the T-cell surface, whereas class h ~ssociated TCR peptides are derived from protein int~ nalized from the cell membrane or shed from the -~Ioreactive T cells, then taken up, processed, and preset.ted by class II positive accessory c e l l s 3 ° . 3 1 . It might be argued tha- natural tolerance to idiotypic variants of TCR would [ e expected. However, in the thymus cell division ceases as soon as TCR expression Occurs 32, and clonal expansion of alloreactive cells does not occur there. Thus the opportunity for tolerance induction would be small. There are several testable predictions that arise from our hypothesis. One, with potential clinical importance, is that the relevant antiidiotypic T-cell population size should correlate inversely

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39

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"~munology Today;Vol. 10, No. 2, 1989

with the strength of allograft rejection. It should be possible to identify and monitor specific, anti-idiotypic T-cell responses in transplant patients (or experimental animals). A second prediction is that suppression of T-cell-mediated autoimmune responses would be expected to have the same basis, and it should be possible to identify corresponding anti-idiotypic T cells specific for the TCR of clones reactive with defined autoantigens. If it is true that suppressor cells share common mechanisms for antigen recognition with 'conventional T cells', and in that sense do not represent a separate lineage, the question arises as to how they exert their functional effects. At least two possibilities exist. Suppression may simply be the result of classical immune effector mechanisms acting on nominal antigen or alloantigen-specific T cells. Lysis or delayed type hypersensitivity-mediated elimination of such cells would be manifest as suppression. Alternatively, presentation h,, T ~---I1~ n f th,',ir n~^ln idintnp~ m~y I~el tn dnwnregulation of the presenting T cell. The observation that specific, MHC-restricted recognition of peptide presented by human T cells leads to tolerance induction implies that T celI-T cell interaction may have special effects. Thus it is possible that the nature of the presenting cell may be an important factor in determining the functional phenotype of the responding T cell. In response to recognition of idiotopes presented by T cells, the lymphokines produced by the responder cell may be inhibitory to the presenting cell. References

1 Moiler, G. (1988)Scand. J. Immunol. 27, 247-250 2 Steinmetz, M., Minard, K., Horwath, S. etal. (1982)Nature 300, 35-42 3 Horsburgh, T., Wood, P.J.and Brent, L. (1981) Transplant. Proc. 13,637-639 4 Monaco, A.P. and Wood, M.L. (1981) Transplant. Proc. 13, 547-555 5 Dorsch S. and Rnser, B. (1982) Transplantation 33,518-524 6 Dorsch, S. and Roser, B. (1982) Transplantation 33, 525-529 7 Billingham, R.E., Brent, L. and Medawar, P.B. (1953)Nature 172, 603-606 8 Batchelor, J.R., Phillips, B. and Grennan, D. (1984) Transplantation 37, 43-46

9 Ch!Ji, Y L. and Batchelor, J.R. (1985) Transplantation40, 150-153 10 Barber, W.H., Hutchinson, I. and Morris, P.J.(1985) Transplant. Proc. 17, 1391-1393 11 Morimcto, C., Letvin, N.L., Distaso, J.A. etal. (1985) J. Immunol. 134, 1508-1515 12 Morimoto, C., Letvin, N.L., Boyd, A.W. etal. (1985) J. Immunol. 134, 3762-3769 13 Mitchison, N.A. and Oliviera, D.B.G. (1986)in Progressin Immunology(Cinader, B. and Miller, R.G., eds), pp. 326-334, Academic Press 14 Sanders, M.E., Makgoba, M.W. and Shaw, S.W. (1988) Irnmunol. Today 9, 195-199 15 Damle, N.K. and Engleman, E.G. (1983)J. Exp. Med. 1.58, 159-173 1G Mohagheghpour, N., Damle, N.K., Takada, S. etal. (1986) J. Exp. Med. 164, 950-955 17 Sociu-Foca, N., Reed, E., Rohowsky-Kochan, C. etal. (1985) Transplant. Proc. 17, 716-719 18 Lancaster, F., Chui, Y.I.. and Batchelor, J.R. (1985)Nature 19 Sun, D., Qin, Y., Chluba, .L etal. (1988)Nature 332, 843-845 2.0 Kimura, H. and Wilson, D.B. (1984) Nature 302,463-464 21 Roser, B., Stephenson, P., Leung, A. etal. (1986)in Progress in Immunology(Cinader, B. and Miller, R.G., eds), pp. 1022-1034, Academic Press 22 Stephenson, S.P. and Roser, B. (1985) Transplant. Proc. 17, 1145-1147 23 Tada, T., Asano, Y., Fuj sawa, I. etal. (1986)in Progres:,in Immunology(Cinader, B. and Miller, R.G., eds), pp. 427--a37, Academic Press 24 Bjorkman, P.J., Saper, M.A., Samraoui, B. etal. (1987) Nature 329, 506-518 :95 Fischer Lindahl, K. and W!',son, D.B. (1977)]. Exp. Med. 145, 500-522 26 Matzinger, P. and Bevan, M.J. (1977) Cellularlmmunol. 29, 1-5 27 Marrack, P. avd Kappler, J. (1988) Nature 332,840-843 :~8 Lombardi, G.i:'Sidhu, S., Lamb, J.R. etal. J. Immunol. (in Flu ~ 1

29 Lamb, J., Eckels, D., Lake, P. etal. (1982)Nature 200, 66-69 30 Germain, R.N. (1986) Nature 322,687 31 Morrison, L.A., Lukacher, A.E., Braciale, V.L. eta/. (1986) J. Exp. Med. 163, 903 32 Parkin, J.G., Owen, J.J.T. and Jenkinson, E.J. (1988) Immunology 64, 97 "J.9

Functional heterogeneityof CD4÷ T ceils in leishmaniasis

In this article F.Y. Liew summarizes the evidence for the functional heterogeneity of CD4+ T cells from studies of immune regulation in the parasitic disease, leishmaniasis. Recent findings suggest that the heterogeneity of CD4+ T cells may extend beyond the current Th7 and Th~ classification and that these T-cell subsets manifest some of their functions via the !ymphokines they secrete, and that the balance of these determines the outcome of the infection. Mature T lymphoc~es express either CD4 or CD8 cell surface glycoprot~ms. The CD4 ÷ T cells are involved in a 40

Departmentof ExperimentalImmunobiology, The WellcomeResearch Laboratories,Lang!eyCourt, Beckenham,KentBR33BS, UK.

F.Y. Liew variety of activities, including antigen-specific or polyclonaI-B-cel! activation, delayed-type hypersensitivity (DTH), killing of appropriate target cells, suppression of DTH and antibody responses and induction of CD8 + killer or suppressor T cells. Early studies showed that T cells mediating help for antibody synthesis in the carrierhapten system are distinct from thos~ involved in the DTH to the c.~rrier protein1, 2. Furthermore, there appears to be a dire-t correlation between DTH reactivity and k~ 1989, Elsevier Science Publishers Ltd, UK 0167 4919/89/50200