The role of thymic epithelium in the acquisition of tolerance

immunology Today, kb/ 11, No. I0 1990

ro 3 Voss, F W., Dombrink-Kurtzman, M.A. and Ballard, D.W. (1989) MoL Immunol. 26, 971-977 4 Watts, T.H. and McConnell, H.M. (1987)Annu. Rev. Imrnunol. 5, 461-475 5 Lancet, D. and Pecht, I. (1976) Proc. NatlAcad. Sci. US,~ 73, 3548-3553 6 PechL I. (1982) The Antigens (Vol. VI), pp. 1-68, Academ,c Press 7 Voss, E.W., Dombrink-Kurtzman, M.A. and Miklasz, S. (1983) ImmunoL Investig. 17, 25-30 8 Stevens,F.J., Chang, C-H. and Schiffer, M. (1985) Proc. Natt Acad. Sci. USA 85, 6895-6900 9 Bedzyk,W.D., Weidner, K.M., Denzin, L.K. et aL J. BioL Chem. (in press)

e~: ~rD~

10 Henon, J N., He, X-M, Mason, M.L etal. (1989)Proteins 5, 271-280 11 Schwartz, R.S.(1935)Annu. R~,' immunol. 3, 237-261 12 Livingstone,A.M and Fathman, C.G. (1987)Annu Rev. Immunol. 5, 477-50 ~ 13 Adorini, L. and N,,gy, Z.A (1990) ImmunoL Tod~y 1!, 21-24 14 Bjorkman, P.J., Saper, M.A., Samraou,, B. etal. (1987) Nature 329, 506-512 15 Garrett, T.P.J., Sapc M.A., Bjorkman, P.J. etaf. (1989) Nature 342, 692-696 16 Germain, R N. (1990) Nature 344, 19-22 17 Sawutz, D.G., Koury, R. and Homcy, C.J. (1987) Biochemistry 26, 5275-5282

The role of thymic epithelium in the acquisition of tolerance

There am two separate mechan/srnsof induction of T-cell tolerance m the thymus. First, MHC moleculesexpressedon bone-marrow-oerivedcells can causeclona!deletion of autoreactivecells.Second,as discussedhereby ElisabethHoussaint andMartinFlajnik,thymicepithelialcellscangeneratea form of tolerancethatdoesnot eliminateself-reactiveclones. Thisnonde!etionalmechanism,whichis alsoa featureof the otherMHC dass-ll-bearingepithelia,maycontributeto theestablishmentof tolerance-maintainingregulator/networks. What role do~s the thymic epithelium play in the acquisition of self tolerance? A few years ago we provided evidence, with experiments involving allogeneic thymic grafts during e~r!y embn/onic life, that the epithelial component of the thymus could induce 'sp!it tolerance 'L2. The chimeras accepted skin grafts bearing the thymus haplotype. The mechanism of tolerance, however, was clearly not clonal deletion s!nce donor-rea~ive cells remained and their activity was detected in vitro. Thymic transplantation studies in quail-chick xenogeneic combinations3-s and in mice 6-9 recently addressed this question in detail, and have largeiy confirmed a nondeletional mechanism for thymic epithelium-directed tolerance induction. Evidencefor a nondeletional mechanismof thymic self tolerance Clonal deletion of self-reactive cells is a major mechanism of self tolerance ;°-~8. Bone-marrow-derived cells (dendritic cells and/or macrophages) found in the medulla and at the corticomedullaFy junction appear to be Fnost efficient at effecting deletion of self-reactive T ceils (reviewed in Ref. 16). The thymic stroma itself can also make self-reactive cells tolerant. Stromal tolerization, however, apparently operates through a different mechanism. Thymic epithelium transplants, in MHC-;~ismatched or xenogeneic combinations, in frogs, birds or mammals established that the thymus grafts were permanently accepted despite their continued expression of foreign 'U 2 i l INSERM,Facultede Medecine, I rue GastonVeil,44035-Nantes Cedex,France;2Universityof Miami, Schoolof Medicine,Bepa,'tmentof Microbiologyand Immunology, MiamL FL33101, USA. (~Z)1990. ElsevierSciencePub ishers Ltd. UK, 0167--4919/901502O0

Elisabeth HoussaintZ and Martin Flajnikz MHC class I and !i antigens 1-S.19-22. Recipients of such transplants were either embryos (frogs and birds) or young adults (mice). Frog (Xenopus) allogeneic chimeras were made microsurgically with 24-hour-old embryos, such that the anterior region (head) containing the thymic anlagen was grafted onto the posterior region (body) containing the anlagen of all hemopoietic cells1. The resulting adult MHC-mismatched chimeras did not reject the heads and later accepted skin grafts of the thymus's MHC type (but rejected third party graf[sX Lymphoojtes from such chimeras, however, proliferated in vitro to MHC antigens of the thymus donor's haplotype. This result was also observed by others who performed thymic transplantation experiments between MHC-mismatched tadpoles23.24. Such 'split tolerance' was also obtained with allogeneic chicl-en chimeras 2. Thymic rudiment, taken before it was colonized by hemopoietic precursors, was grafted into the lateral body wall of a three-day-old MHC-mismatched recipient (Fig. 1). This experimental system, like the studies with frogs, offers the advantage that the thymic rudiment is not yet colonized by the hemopoietic precursors that give rise to the lymphoid and antigen-presenting cell (APC) populations2s.This eliminates the problems of using potentially harmful methods to destroy the hemopoietic cells of the tt~ymus. The second advantage of these models is that the allogeneic thymic graft is implanted from an early stage of the embryonic life. Despite the expression of aliogeneic MHC antigens, the thymic grafts were permanently accepted and ,-olonized by host-derived hemopoietic precursors that differentiated into iymphocytes and APCs. Adult chickens that had received an MHC-incompatible thymic graft during embryonic life tolerated a skin graft of the donor thymus MHC type. Control chickens of the same age that had not received a thymic graft rapidly rejected an allogeneic skin graft. In contrast to the in vivo state of tolerance, lymphocytes 357

Immunology Today, VoL I 1, No. 10 1990

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Donor:.6.5daY B19 thymlc rudiment

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GVH alloreactivity against B19 Fig. 1. Thym~cchimeras~ere ob~ned by grafbng a B19 thymlc rudiment into a BI4 chick emt~ Thegrafted thym~ developedIn the body wall of the reopientand was colonizedby ho.st-~"~d hemopo~bcprecu~ors.At threemonthspost-hatching, t~e B14 chimericchicken te,:~ted a 8 ~9~in graft. Howe~er,thymecytesin the grafted thymusand~Ls werealloreactive aga~-t BI~. :n a G ~ assay.

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within zhe thymic graft, the host thymus or in the peripheral blood were reactive to the donor thymus MHC antigens in a graft-versus-host (GVH) assay performed by injecting the cells into the chorioallantoic vein of 13-day chick embryos~6. They were also reactive to third party antigens but not to host MHC antigens. In these experimental bird chimeras there are two different typc~ of T cell: one set is generated in the grafted thymus and the other is educated in the host thymus. Although no suppressor cells have yet been identified, the in vivo state of tolerance suggests that the latter are somehow controlled by the former. ExpenmenT_s involving xenogeneic transplantation of quai! tnymic rudiment into chick embryonic recipients also led to the conclusion tl,~t thymic epithelium induces transplantation tolerance 3-5. Culture of mouse fetal thymus in deoxyguanosine (dGuo) results in the destruction of all hemopoietic cells but spares the epithelial cells 19. When these remaining stromal cells were transplanted into MHC-mismatched normal or nude mice, the thymic graft was tolerated but failed ~,3 induce ~_lloto!erance, as host helper and killer T cells remained capable of responding to the MHC antigens of zhe thymus donor -~,~.9.2o.If thymuses are cultured without d(=uo, resulting m no destruction of hemopoietic 358

cells, 'full' tolerance to the thymic MHC is observed Contradictory evidence was obtained from transplantation experiments involving mouse fetal thymuses that were precultured at low temperature; this procedure selectively eliminates b~ne-marrow-de.:ved elemenzs-'1. Such thymuses, when grafted into nude mice, induced tolerance, as judged by mixed lymphocyte reaction (MLR) with both thymic and splenic T cells. However, they did not induce tolerance, as judged by the same MLR test, when transplanted into intact histoincompatible mice. Ir, a very recent experiment akin to the studies in frogs and birds, mouse embryonic thymic epithelium was transplanted, before coionization of hemopoietic precursors, into newborn MHC-mismatched nude mice8. These mice later accepted skin grafts from the thymus donors, but were not (in the main) tolerant in in vitro tests to the MHC of the thymus. Transgenic mice constructed to express I-E class ii molecules on bone-marrow-derived cells, but not on thymic epithelium, were apparently made tolerant by deletion of reactive clones. !-E class II molecules present on the thymic epithelial cells were not sufficient to delete all I-E-reactive, V 17a ÷ cells 12 Nevertheless as in previous experiments, the thymus was accepted by animals with T cells reactive against that epithelium. Other groups established radiatior bone marrow chimeras in mice, in which the self MHC antigen te~ed was expressed either on the radioresistant stromal components of the thymus or on bone-marrow-derived, radiation-sensitive, cells. They demonstrated that clonal deletion is achieved after i,lteraction between T-cell receptor (TCR) and MHC expr_~ssed on bcne-marrow-derived cells7 but that tolerance could also be induced by the radioresistant elements. The latter process does not involve deletion but results in clonai anergy7,27. Split tolerance induced by non-thymic MHC expression

A!i of the data, taken together, have established that the thymic epithelium induces tolerance by a poorly understood norJdeletional mechanism. Experiments with frog embryos and tadpoles have also shown that the thymus is not unique in generating the 'split tolerance' phenomenon. The frog eye anlage transplanted during embryonic life also induces in vivo tolerance (the animal accepts skin grafts of the eye's MHC-type) but lymphocytes still exhibit reactivity to stimulator cells bearing the thymus's MHC in MLR28. This is true not only for embryonicaily-transplanted tissue, but also for MHCmismatched skin grafts that render immunocompetent tadpoles tolerant to subsequent skin graft during adult life (reviewed in Ref. 29). Recent studies in mice have shown -chat mature T cells can become unresponsive to self components with a restricted tissue distribution by a nondeletional mechanism 3°-3s. This leads us to propose that any tissue that expresses MHC class II molecules may induce such a split tolerance. In fact, class II molecules are expressecJ, either permanently or occasionally, at many sites throughout the body. Transgenic mice have been constructed that express non-host class II molecules on pancreatic 13cells but not on bone-marrow-derived cells3°-32. The split tolerance obtained in these transgenic mice is similar to tolerance induced by the thymic epithelium. In one study, I-E class II molecules were expressed by islets without obvious autoimmune damage occurring and I-E+ islet cell~ persisted in

Immunology Today, Vol. I 1, No. 10 1990

these mice for months. However, I-E-reactive -[ cells were not deleted from these tolerant mice. Transgenic mice with non-host I-A class II mo!ecules expressed specifically by islet cells were constructed by another group. Again, the mice were not tolerant in MLR to the MHC class II molecules expressed by the pancreatic 13cells33. I-E+ 13cells from transgenic mice were used in an in vitro system as APCs in an attempt to stimulate an I-E-restricted CD4 ÷ T-cell line3~. Paralysis was induced, since reduced reactivity was observed when the T-cell line was later incubated with conventional I-E÷ splenic APC and peptide. The T cells apparently become anergic because the 13 cells failed to deliver costimulatory signals needed for T-cell activation. Cortical thymic epithelial cells also lack full capacity for antigen presentation 36. Expression of the antigen-class II complex on the epithelial cells is not sufficient to stimulate T cells, and leads to inactivation of T-cell clones. Induction of clonal anergy in mature T cells has been reported by other laboratories, most notably by Schwartz and colleagues 35,37. Mature T cells that encounter the antigen-MHC complex on the surface of fixed APCs become anergic. Treatment with Ca2÷ ionophore can also induce anergy, suggesting that Ca2÷ influx in the absence of other signals is tolerogenic. Tolerance can be prevented by interactions with undefined molecules on the surface of the allogeneic spleen cells37, but this is poorly understood at present. The fact that unresponsiveness is induced by the failure to provide T cells with more 'signals' than the antigen-MHC complex essentially confirms the predictions made over 20 years ago by Bretscher and Cohn 33. Tolerance involves different mechanisms We will attempt to summarize events that might occur when T-cell precursors confront self antigens. Interaction between the thymocyte TCR and MHC molecules (or MHC plus peptide) on cortical epithelial cells leads to positive selection and allows further maturation of the precursor cell9.1°,39-42. At this point, there are several alternatives. Interactions with professional APCs at the corticomedullaryjunction and in the medulla can result in the deletion of some of the high-affinity self-reactive clones. Dendritic cells and macrophages should be most efficient at promoting deletion of self-reactive T cells, since they can present endogenous peptides and peptides derived from other self mol~cules of the body that reach the thymus. Thymic APCs appear to be equivalent to all other professional APCs~4,43.Thus we believe that tolerization or activation of T cells depends entirely on their state of r,.~:,Jration. A simple conceivable model is that cos:smulatory signals delivered b7 professional APCs, vhether they are lymphokines or interactions through ue"~ue cell surface molecules, cause the deletion of T cells at one stage of their maturation, and induce activation when the T cells are further developed ~6. As proposed by Schwartz, the defect may be in the ability of the cell to receive costimulatory signals3L It is now clear that the thymic epithelium can induce tolerance by a nondeletional mechanism, either 'clonal anergy' or an unknown form of suppression. How the thymic epithelial cells can be involved both in positive and negative selection is difficult to reconcile. There is stil! no explanation, but we speculate that these two different

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Cortex

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mechanisms take plate in different thymic compartments. Positive selection is ~hought to occur in the cortex and is driven by cortical epithelial cells. Negative selection through a nondeletional mechanism may take place only under the influence of medullary thymic epithelial cells (Fig. 2). In fact, cortical and medullary epithelial cells exhibit different characteristics though they both express class II antigens 22. They may even have distinct embryonic origins since it has been claimed that the ectoderm and endoderm of the branchial pouches contribute to the thymic rudiment ~. Interactions between TCR and self molecules expressed by two different types of thvmic epithelial cell at two different stages of T-cell differentiation might lead to either positive or negative selection. Thus, interactions leading to tolerance by any mechanism may occur only in the medulla or at the corticomedullary junction. All aspects of T-cell tolerance, however, cannot easily be explained by what occurs in the thymus. An age-old problem is how T cells that react with non-thymic tissuespecific antigens become tolerant. As we have emphasized, the thymic epithelium is not the only tissue able to induce nondeietional to!erance~ and m~,, be no more efficient in inducing tolerance than any other tissue that expresses MHC antigens but cannot deliver costimulatory signals. Thus, controversies about whether the thymic epithelium can induce tolerance can be resolved. It is no different in its capacity to induce tolerance than other tissues in the body, but because (1) the thymus has been the best studied model for tolerance induction, and (2) T cells develop in this organ, the thymus has been endowed with 'special' tolerance-inducing capabilities. One model that fits all the data is that the thymus aroaer evolved (!ate in the evolution of ,,ertebrates) as an Organ directing the positive selection of T cells, both for subset selection and so that T cells can be most efficient at recognizing antigen in association with the MHC. Logically, deletional tolerance is carried out by the same types of professional APC that can present antigen in the periphery. Nondeletional tolerance to tissue-specific antigens, including those found in the thymus, occurs after the positive and negative s~'.~.tion events, probably when T cells have become competent to respond to antigen if seen in the proper context Tolerance by the nondeletional mechanism must, presumably, be beneficial to the host. Anergic autoreactive cells may accumulate and stimulate other T cells (perhaps in an anti-idiotypic manner), establishing a network which blocks any potential self-reactive cells escaping clonat deletion 4s. Such a network would explain howT cells from chicken chimeras that have been educated in the host 359

Fros/rm thymus were tolerant of the new, allogeneic MHC on the thymus transplant. Such a 'network' might also explain why serf-reactive cells in many of the chimeras described above do not d,splay devastating autoimmunity ~6. In all the experimental models quoted above, the allogeneic thymic rudiment was ~qtroduced in embryos or in newhorns. Establishment of tolerance to self must require presentation of self antigens during embryonic life or early neonatal life47, It remains to be established whether this favors the generation of regulatory networks. The authors thank L. Du Pasquier, M. Bonneville, and J. Wayne Streilein for stimulating discussions. Our work, menuot~,edin this article, was supported by Wellcome Research Labora,cries and by the Basel Institute for Immunology. MFF is now supported by NSF grant no. 8819366. I Rajnik, M.F., Du Pasquier, L. and Cohen, N. (1985) Eur. J. tmmunol. 15. 540-547

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Immunology Today, Vol. I l, No I0 1990

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Springer-Verlag 38 Bretscher, P. and Cohn, M. (1970) Science 169,

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. a novel class of protein-tyrosine kinase receptor . HIV envelope- cell protein interactions

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. lymphocyte subsets in the blood

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