Tolerance induction in mature CD4+ T cells

34

Tolerance induction

production is completely inhibited by soluble antigen administration. In parallel to prevention of Thl cell tolerance, coadministration of IL-12 blocks the Th2 response induced by soluble protein. IL-12 administered in adjuvant with antigen, or i.p. two days after the immunization is not able to break tolerance in Thl cells whereas it can still inhibit, but does not eliminate, the Th2 response induced by soluble antigen. In contrast to 11-12, coadministration of IL-2 does not affect the switch to Th2 induced by soluble antigen. Thus IL-12, but not 11-2, converts in vivo a tolerogenic signal for Thl cells into an immunogenic one, while blocking a positive signal for Th2 cell development. This suggests the possibility to administer soluble autoantigen associated to IL-12 antagonists to restore more effectively tolerance in autoimmune diseases.

0.5.01.3

Tolerance induction In mature CD4+ T cells

A. Lanoue, A. Sarukhan, H. von Boehmer. lnsfitut iVec/reclNsERM373, Paris, France

Introduction:Induction of tolerance in mature T cells has for years been attempted in many different ways also because of the potential applications in organ-specific autoimmune disease. Here we are repotting studies in TCR transgenic mice that express on CD4+ but also on some CD6+ cells a receptor specific for peptide 111-119 from influenza hemagglutinine (HA) presented by Ed MHC molecules. Our aim was initially to induce tolerance in these single transgenic mice in order to be able to reproduce afterwards the same effect in double transgenic mice (TCR x INSHA) that express the same transgenic TCR but also (HA) under control of the rat insulin promoter and which all develop insulitis and diabetes in 30% of cases. Materials and Methods:Mice expressing HA under control of the lg K promoter were crossed to the TCR transgenic mice to obtain conditions where the antigen is expressed on hemopoietic cells and in order to see if cells bearing the transgenic TCR could be tolerised when the antigen was present throughout their development. In addition, to see if mature T cells could be tolerised when exposed continously to high doses of antigen, lymph nodes cells from RAG-/TCR+/+ were injected i.v. into x-irradiated HA+ recipients. Futther, the peptide and the protein were given orally, intraperitonealy or intravenously to the TCR transgenic mice. The effect of such treatments was analysed by FACS using the clonolypic antibody (6.5) as well as by analysing in vitro proliferation and cytokine production. Reeults:In double transgenic mice where HA was expressed as a self antigen by hemapoiteic cells, we found some mature T cells in periphery that had escaped thymic deletion and that showed signs of activation but which were anergic. Mature CD4+6.5+ cells that wem transfered into antigencontaining recipients went through an initial phase of expansion after which most cells were deleted and those remaining became unresponsive, as previously described for CD&t cells. Inducing tolerance in CD4+6.5+ cells in situ in single transgenic mice proved a diiicult task: classical protocols using single doses of soluble or deaggregrated antigen as well as feeding antigen all failed to induce antigen-specific unresponsiveness. It was only after decreasing cell numbers by CD4 antibody treatment and by repeatedly reintroducing antigen thereafter that unresponsiveness of 6.5+ cells was achieved and maintained. In no case could we observe the appearance of antigen-specific T cells with a Th2 cytokine profile among the remaining cells and therefore conclude that deletion and anergy represent the major mechanisms of tolerance in our studies. We are now stating to study the double transgenic mice (TCR x INSHA). Preliminaty results show that antigen-specific T cells in these mice are more resistant to tolerance induction pres;mabiy because they are partially activated by antigen. Concluelon: Tolerisation is achieved and maintained when shifting the balance between the frequency of reactive T cells and the dose of antigen in favor of the latter. 0.5.01.4

Anti-CD4 monoclonal antibody-induced allograft tolerance despite persistence of donor-reactive T cells

M. Lehmann, E. Graser, K. Risch, W.W. Hancock, A. Miiller, J.W. Kupiec-Weglinski, J. Brock, H.-D. Volk. Inst. Med. Biochem., Univ Roskxk;Inst. Med. Immunol., Charit& Berlin, Germany. Sandoz Ctr. Immunobiol., Dept. Pathol., Boston, USA, Surg. Res. Lab., Dept Surg., Briaham and Women’s Hoso.. . Boston. USA

Introduction:Although CD4-targeted therapy abrogates acute rejection, and may induce permanent graft acceptance in rodents, little is known about the mechanisms of long-term graft survival in these models. Recently, we have shown that treatment with a nondepleting and-CD4 mAb (RIB-6/2) induces longterm survival of renal, heart and skin allografts in strong MHC VII incompatible rat strains. Results: Here, we demonstrate that the development of MHC-specific and tissue-nonspecific tolerance rather than graft adaption is responsible for longterm anti-CD4 mAb-induced transplant survival. Donor-specific but not thirdpatly heart and pancreatic islet grafts were permanently accepted without ad-

Moruhy, 23 June 1997 - Oral presentations

junctive therapy in long-term kidney allografl recipients, and infusion of naive or alloimmune splenocytes failed to break the tolerant state. Interestingly, alloreactive T cells were not depleted in these long-term survivors, as ax viva donor-specific MLC reactivitv was laraelv unaffected. Cvtokine exoression studies at mkNA (quantitative e-PCR) aid brotein (immunohistologyj level demonstrated a strong inhibition of CD25,lL-2, IL-lo, TNFar,and IFNY in anti-CD4 mAb treated rats (x75% of untreated controls) at day 2-5 post-transplant which further decreased during 300 day follow-up. By contrast, IL-4 expression was only moderately (3050% of controls) inhibited. In fact, intragraft IFN g/IL-4 mRNA ratio dropped from 0.4 at day 5 to 0.07 at day 100 (2.3 at day 5 in controls). The RT-PCR analyses of long-term renal ailografts before and after donor-specific antigen challenge (heart grafting at day 100) revealed no changes in CD3 mRNA level, but upregulation of CD25 11-2, IFNy, IL-4 and IL-IO mRNA in the earfy phase, suggesting the presence of alloreactive T cells in toferant rats. At later time points, the expression of IFNy declined rapidly, whereas IL-4 persisted, resultinginareversalofIFNyllL-4 ratio(day 1000.07, day105: 1.I, day200:0.04). Conclusion: Our data demonstrate the stability of anti-CD4 mAb induced tolerance despite persistence of alloreactive T cells, suggesting the role of active tolerance-maintaining mechanisms. The ThlIlI2 shift may be involved in this regulatory process, as anti-CD4 mAb prevents acute graftdeteriorating rejection by effectively blocking Thl responses, and well-functioning grafts may tolelize themselves by inducing regulatory cells.

0.5.01.5

Feptide-antlgen treatment of naive and memory CTL: Antlaen-sPecific tolerization versus immunopitholdgy

P. Aichele, K. Brduscha-Riem, S. Oehen, R.M. Zinkemagel, H. Hengartner, H. Pircher. InsMufe of Experimental Immunology Department of Pathology, University of Ziirich, Schmekbergstr 12, CH-8091 ZOtich, Switzedand, ’ Institute of Med. Micmbiology and Hygiene, University of Freiburg, Hermann-Herder-Sk 11, D-79104 F&burg, Germany

Introduction:Peptide-specific downmgulation of T cell responses may represent a powerful tool to intervene in autoimmune diseases or graft rejections. To achieve T cell tolerance as a therapeutic tool, it is essential to identify the antigens recognized by the disease mediating T cells and to define protocols for antigen application, that specifically tolerize naive as well as antigen-experfenced, memory CTL. Mat&al and Methods: Naive and lymphocvtic choriomeningitis virus (LCMV)-primed C57BU6 mice were treated i.b. thr& times with 50019 LCMV GP33 peptide in IFA, to establish antigen-specific tolerance. CTL activities were analyzed in 51 Cr release assay aier in vitro restimulation of splenocytes. Histological analysis of spleen sections were done, using different moncclonal antibodies specific for the various lymphocytes populations. Resuftaz LCMV GP33 peptide-treatment of naive mice toletized GP33 peptide-reactive CTL In vivo, whereas the same peptide treatment in the presence of GP33 peptide-specific memory CTL in LCMV-primed mice not only abrogated the GP33-specific CTL activity, but had an unspecific immunosuppressive effect on unrelated third-party antigen-specific CTL responses. lmmunohistological analysis of spleen sections from LCMV-primed mice after peptide-treatment revealed a severe destruction of the T cell rich region of the petiarteriolar lymphoid sheath (PALS). Further experiments showed, that GP33 peptids treatment of LCMV-primed mice, activated the specifkz memory CTL within 12-24 h and simultaneously loaded spleen cells in vivo with peptide. These peptide-loaded spleen cells serve as target cells in vivo for the GP33 peptide-activated memory CTL, which may explain the observed immunopathology and immunosuppression after GP33 peptide administration in LCMV-primed mice. Conclusions: Treatment of memory CTL with the corresponding peptide antigen caused severe immunopathdogical damages leading lo a non-specific immune suppression rather than to antigen-specific T cell tolerance. The findings raise a red flag for the use of peptide-immunotherapy in the treatment of ongoing autoimmune diseases or graft rejections. Therefore, other strategies have to be developed to overcome these problems and to render peptids-specific tolerization of memory CTL feasible. 10.5.01.6

1 Anergic T cells actively modulate the response of autoreactive T cells

Leonie S. Taams ‘, A.J.M.L. van Rensen 2, J.P.A. Wagenaar ‘, A. Besseling ‘, D.J.A. Crommelin 2, W. van Eden ‘, M.H.M. Wauben ‘. ’ Dept. of Immunology; institute of Infectious Diseases and lmmunolog~ Fat. of Veterinary Medicine, RO. BoxSO.188, 3508 TD Utmcht, 2Dept. of Pharmaceutics, Fat. of Pharmacy UtmchtUniversity, The Netherlands Introduction: Induction of energy in autoreactive T cells could be a novel strategy for the treatment of T cell mediated autoimmune diseases. We have obtained evidence that anergic T cells are not just passive bystanders during an ongoing immune response, but are able to actively modulate the response of immunocumpetent T cells.