Workshop K: Tolerance and Regulatory T Cells

Workshop K Tolerance and Regulatory T Cells

Department of Nephrology and Clinical Immunology, Universitätsklinikum, Aachen, Germany

K. 1 Isolation and characterization of dendritic cells capable of crosspresentation of self antigens D. BENKE, A. LANG, E.E. HAMILTON-WILLIAMS, M. WIRTZ, T. KRÜGER, and C. KURTS Transgenic RIP-mOVA mice express the model self antigen, OVA, in the kidney and pancreas. CD11c-positive dendritic cells (DCs) can present OVA-derived peptides in the draining lymph nodes with MHC class I molecules. Such cross-presentation can induce activation and proliferation of OVA-specific CD8 T cells (OT-I cells), but ultimately leads to their deletion; a process referred to as cross-tolerance. To further investigate such cross-tolerising DCs, we established techniques to purify them from the kidneys and from their lymph nodes. A combination of collagenase digest, density centrifugation and magnetic bead separation for CD11c+ cells yielded approximately 5¥104 DCs per kidney and 15¥103 DCs per renal lymph node with a purity of 70–80%. In a co-culture system, these cells induced the proliferation of OT-I cells, demonstrating that the isolated DCs must have taken up OVA in vivo. All these DCs were MHC IIhi, CD11b+, CD40+, CD80+ and CD86+. Some DCs were CD205+ and F4/80+. Immunohistology showed that kidney DCs were localized in the tubulo-interstitial, but not in the glomerular compartment of the kidney. Their phagocytic capacity was higher, whereas their ability to stimulate T cells in an allo-response was lower than that of splenic DCs. Stimulation with Toll-like-receptor ligands such as LPS increased the stimulatory potential of renal DCs. Consequently, these mediators could break cross-tolerance in vivo. These results identify a DC in non-lymphoid tissues that can cross-tolerize autoreactive CD8 T cells in the absence of tissue inflammation or infection. Furthermore, they suggest that «third signals» such as ligands for Toll-like receptors can determine the outcome of immune responses induced by cross-presentation.

Tumor Immunology Program, German Cancer Research Center, Heidelberg, Germany

K. 2 «Promiscuous» expression of tissue antigens by thymic epithelial cells: A judicious or random selection? J. DERBINSKI and B. KYEWSKI The prevailing notion that tolerance to «peripheral» self-antigens is solely covered by post-thymic mechanisms has been questioned by the recent finding that «tissue-specific» antigens are expressed by thymic stromal cells, a phenomenon termed «promiscuous gene expression». Interestingly, this «promiscuous» expression is a property of thymic epithelial cells rather than bone marrow-derived antigen presenting cells, implying cell-type specific regulation. In particular thymic medullary epithelial cells have been identified as a cell type expressing a wide range of

146 · 33rd Annual Meeting of the German Society of Immunology tissue antigens at the mRNA level when compared to other antigen presenting cells or epithelial cells of other tissues. Antigen expression in this cell type only is sufficient to confer specific T cell tolerance attesting to its functional role. We studied gene expression in thymic stromal cells at the population level by RT-PCR and gene array analysis. Genes expressed in this promiscuous manner at the mRNA level are highly diverse and encompass many known or suspected autoantigens of major autoimmune diseases, fetal antigens and tumor-associated antigens. They have no obvious structural or functional commonalities and also include abundant self-antigens which circulate at tolerogenic levels. Thus, the selection does not appear to be judicious in the sense that it serves tolerance induction only to «weakly tolerogenic» self antigens. We suspect epigenetic rather than genetic gene regulation mechanisms to dictate this selection.

Department of General and Thoracic Surgery, Christian-Albrechts-University, Kiel, Germany

K. 3 The thymus is not needed to induce donor-specific tolerance with mixed hematopoietic chimerism B.G. EXNER, A. MELZER, K. BRÖTZMANN, G. ZEHLE, and F. FÄNDRICH Mixed allogeneic chimerism achieved by partial ablative conditioning can induce donor specific tolerance. Some groups postulate a critical role of the thymus in this model. The following study was designed to investigate, if the thymus is needed to induce donor specific tolerance with mixed hematopoetic chimerism. A surgical thymectomy was performed in C57BL/6 (H2b) recipient mice of the experimental group, while C57BL/6 recipient mice of the control group remained unmanipulated. Recipients were conditioned with 500 cGy irradiation, transplanted with 15¥106 bone marrow cells from C3H (H2k) donors and injected with 200 mg/kg Cyclophosphamide intraperitoneally on day +2. Chimerism was accessed by flow cytometric analysis of peripheral blood. Skin transplants were performed 4 weeks after bone marrow transplantation (BMT). Animals received donor specific (C3H; H2k) and third party (BALB/c; H2d) skin transplants. The level of donor chimerism 16 weeks after BMT was 31.0±5.0% in thymectomized animals and 40.3±10.2% in controls. The leukocyte count was 4400±504/ml in thymectomized mice and 6800±716/ml in controls as compared to 8100±505/ml in naïve C57BL/6 mice. T cells in the thymectomized animals were detectable only in low levels by CD3, CD4, CD8, ab-TCR and gd-TCR, while the cells positive for these markers showed a normal distribution pattern in control mice. Thymectomized mice showed higher levels of DX5, GR1 and MAC1 positive cells than controls. All but one animal in the control group accepted donor specific skin for more than 120 days, while third party skin was rejected in 20±1.2 days. Thymectomized mice accepted donor specific skin transplants in the same way (all but one were accepted more than 120 days), but the rejection of the third party skin was slower than in controls (26.8±3.7 days). We conclude that the thymus is not needed to induce donor specific tolerance with mixed hematopoietic chimerism.

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Department of Surgery, Universität of Würzburg, Würzburg, Germany, 2Department of Pathology, The Children’s Hospital, Philadelphia PA, and 3Laboratory of Immunogenetics and Transplantation, Brigham and Women’s Hospital, Harvard Medical School, Boston MA, USA

K. 4 Regulatory functions of allopeptide-specific Th2 cells and induction of tolerance in experimental kidney transplantation M. GASSER1, W.W. HANCOCK2, A. CHANDRAKER3, D. MEYER1, A. TRUMPFHELLER1, A. THIEDE1, M.H. SAYEGH3, and A.M. WAAGA-GASSER1 We have recently published the characteristics and functions of Th1/Th2 clones generated from allografts of rejecting (Th1-type) and tolerant (Th2-type) animals. T cell clones were established by stimulating graft infiltrating cells of acutely rejected (WFÆLEW) and tolerant (CTLA-4Ig treated WFÆLEW) kidneys with donor-derived immunodominant class II MHC allopeptide (RT1.Duß20–44) presented by recipient antigen-presenting cells (indirect allorecognition). Here, we analyzed the in vivo functional/regulatory capacities of these T cell clones in an established WFÆLEW kidney transplant model. Recipients were treated with low dose cyclosporine (CsA, 1mg/kg day 0–3 and 5mg/kg, day 4–7, s.c.). Unmodified LEW recipients acutely rejected WF renal allografts (6.3±0.8 days, n=8). Short-term CsA therapy resulted in prolongation of graft survival (121.7±34.6 days, n=8) but all grafts were ultimately rejected with evidence of progressive changes of chronic allograft nephropathy not apparent in the syngeneic controls which remained virtually normal. Adoptive transfer of Th2 cell clones (30¥106, i.p., day 0) prevented allograft rejection (>250 days, n=8) and induced tolerance proven by acceptance of donor specific skin (>60 days, n=6) in long-term survivors. Renal allografts from these animals remained fully functioning; urine protein excretion remained virtually at baseline. Long-term survivors developed only minor morphological changes of chronic vasculopathy but no evidence of glomerular sclerosis or fibrosis. Enrichment of regulatory T cells producing Th2 type cytokines was observed immunohistologically in the target organ. In contrast, animals injected with Th1 cell clones chronically rejected their allografts in an accelerated manner (65.0±20.4 days vs CsA controls 121.7±34.6 days, p<0.0001, n=8/group). These results are the first description of the functions of Th1/Th2 alloreactive T cell clones generated via the indirect pathway of allorecognition in an allogeneic transplant setting and provide a causal link between Th1 cell clones and development of chronic rejection and Th2 cell clones and regulation of alloimmune responses in vivo.

Department of Surgery, University of Regensburg, Regensburg, Germany

K. 5 Soluble donor MHC class I antigen produced via adenoviral gene transfer markedly inhibits allo-CTL and prolongs heart transplant survival C. GRAEB, M.N. SCHERER, M. JUSTL, S. TANGE, J. ANDRASSY, E. FRANK, K.W. JAUCH, and E.K. GEISSLER Introduction: We have previously shown with ex vivo liposome-gene transfer that soluble donor MHC class I (sdMHC I) molecules prolong heart transplant (Tx) survival in a donor-specific

148 · 33rd Annual Meeting of the German Society of Immunology manner when combined with low-dose cyclosporine. The need for concurrent immunosuppression is likely related to the short duration and low-level of sdMHC I expression with this gene transfer method. Here we sought to improve the immunosuppressive effect of sdMHC I by increasing expression via adenoviral gene transfer. Methods: We constructed (AdEasy system) an E1-E3-deleted adenovirus containing genes for both green fluorescent protein and a secreted form of the rat MHC class I molecule, RT1.Aa (AdRQ). An empty adenovirus was used for controls. Lewis (RT1.Al) rats were injected with 2¥109 fluorescence forming units of AdRQ and serum was tested for RT1.Aa by ELISA. Some animals were challenged with a fully allogeneic heterotopic ACI (RT1a) heart Tx 14 days after adenovirus injection, and either allograft survival was determined, or spleens were removed 4 days postTx for testing in a CTL limiting dilution assay. Results: Serum samples taken from AdRQ-injected rats (n=3) showed high expression of RT1.Aa after 1 day (936±181 ng/ml), which increased slightly by day 7 (1334±543 ng/ml), but decreased over the next 7 days to <316 ng/ml. Empty adenovirus injected controls showed no RT1.Aa signal. Regarding the in vivo effect of RT1.Aa on allo-CTL, the anti-ACI CTL precursor frequency decreased as much as 16-fold after AdRQ treatment. ACI heart Tx survival was prolonged to 10.2±1.5 days (n=5) with AdRQ, versus controls (6.0±0.7, n=5). Conclusions: Adenoviral gene transfer produces in vivo serum RT1.Aa levels that are approximately 100-fold higher, compared to our previously reported ex vivo gene transfer method. Prolongation of heart Tx survival without concurrent immunosuppression in a high-responder combination, and the marked reduction of allo-CTL, suggests a therapeutically useful effect of sdMHC I.

Third Medical Department, Justus-Liebig-University, Giessen, Germany

K. 6 Long-term acceptance of allogeneic rat islets after a single postoperative ALS injection without donor-tissue APC depletion H. JAHR, B. HUSSMANN, and R.G. BRETZEL The passenger leukocyte concept proposed that tissue immunogenicity will strongly be reduced by depletion of donor tissue from antigen-presenting cells (APC). The observation that longterm (10–14 days) cultured (and therefore APC-depleted) isolated pancreatic islets were accepted spontaneously in allogeneic mice, and in rats after a single ALS injection, was generally regarded as a proof of this concept. The aim of our study was to investigate whether a similar acceptance of allogeneic rat islets is also possible without APC depletion. Therefore, experiments were carried out with purified but only short-term cultured islets from Lewis rats transplanted into the portal vein of MHC-incompatible Wistar-Furth rats, an immunological high responder strain. Islets were isolated from donor pancreata by collagenase digestion followed by gradient centrifugation, and cultured in TCM-199/5%FCS; first overnight at 37°C, and then one day at 22°C. Cultured islets were practically endotoxin-free and without necrotic cells. Immunohistological studies showed the persisting intraislet presence of macrophages (ED1and ED2 markers) and of MHC class II positive cells. After this short-term culture, about 800 islets each (from one donor each per recipient) were injected into the portal vein of streptozotocin-diabetic (plasma glucose >350 mg/dl) WF-rats. One day after islet transplantation, rats got a single intraperitoneal 0.5 ml shot of rabbit anti-rat lymphocyte serum. Plasma glucose levels in the islet recipients (n=7) were

33rd Annual Meeting of the German Society of Immunology · 149 normalized (<130 mg/dl) within one or two days after islet transplantation, and remained normoglycemic throughout the observation period. After 100 days of graft acceptance, the rats were injected intraperitoneally with 5¥106 donor-type spleen cells. Within one week, plasma glucose levels raised again to diabetic values (>300 mg/dl). Thus, prolonged graft acceptance did not lead to immunological tolerance. Taken together, it was shown that donor tissue APC-depletion is not a precondition for permanent islet graft acceptance in allogeneic rats treated with a single postoperative ALS-injection.

1

Abteilung Immunologie, Max-Planck-Institut für Infektionsbiologie and 2Deutsches Rheuma-Forschungszentrum, Berlin, Germany

K. 7 Regulatory CD4+CD25+ T cells restrict memory CD8+ T cell responses M. KURSAR1, K. BONHAGEN2, J. FENSTERLE1, A. KÖHLER1, R. HURWITZ1, T. KAMRADT2, S.H.E. KAUFMANN1, and H.-W. MITTRÜCKER1 CD4+ T cell help is important for the generation of CD8+ T cell responses. We used depleting anti-CD4 mAb to analyze the role of CD4+ T cells for memory CD8+ T cell responses after secondary infection of mice with the intracellular bacterium Listeria monocytogenes, or after CD4+ T cell help is important for the generation of CD8+ T cell responses. We used depleting antiCD4 mAb to analyze the role of CD4+ T cells for memory CD8+ T cell responses after secondary infection of mice with the intracellular bacterium Listeria monocytogenes, or after boost immunization by specific peptide or DNA vaccination. Surprisingly, anti-CD4 mAb treatment during secondary CD8+ T cell responses markedly enlarged the size of antigen-specific CD8+ T cell population. After boost immunization with peptide or DNA, this effect was particularly profound, and antigen-specific CD8+ T cell populations were enlarged at least 30-fold. In terms of cytokine production and cytotoxicity, the enlarged CD8+ T cell population consisted of functional effector T cells. In depletion and transfer experiments, the suppressive function could be allocated to the CD4+CD25+ T cell population. Our results demonstrate that CD4+ T cells control the CD8+ T cell response in two directions. They initially promote the generation of a CD8+ T cell responses and later they restrain the strength of the CD8+ T cell memory response. Down modulation of CD8+ T cell responses during infection could prevent harmful consequences after eradication of the pathogen.

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Department of Immunology and Allergology, Institute of Environmental Health Research, Düsseldorf, Germany and 2PharmAAware B.V., University of Utrecht, Utrecht, Holland

K. 8 Prevention of drug-induced antinuclear autoantibody formation by the adoptive transfer of previously exposed CD4+CD25+ regulatory T cells L.E. LAYLAND1, M. WULFERINK2, S. DIERKES1, and E. GLEICHMANN1 Drug-induced autoimmunity is often observed after the administration of certain drugs such as procainamide, mercury and gold. Although these xenobiotic compounds are different in structure and, perhaps, mechanistic action, the adverse immune reactions to these drugs share the development of antinuclear autoantibodies and the intricate involvement of CD4+ T cells. Our research focuses upon the involvement of T cells in procainamide-induced autoimmunity. We have found that after long-term oral treatment, CD4+ T cells derived from treated A/J mice but not age-related controls, could respond ex vivo to neoantigens from procainamide-pulsed macrophages or the reactive metabolite of procainamide, N-hydroxylamine-PA. Here, we report that adoptive transfer of the CD4+CD25– T cell subpopulation from procainamide-treated donors into syngenic recipients, that were or were not already treated with procainamide for one week, initiated the development of autoantibodies. In contrast, the transfer of regulatory CD4+CD25+ T cells were able to prevent the autoantibody development in treated or untreated recipients. Therefore, to observe whether this suppressive phenomena could occur with other xenobiotics, similar experiments using HgCl2 and gold sodium thiomalate (GST) have been investigated. After several weeks of HgCl2 treatment, autoantibodies developed in the donor C57BL/6 mice but not the controls. Following adoptive transfer into recipients also under HgCl2 treatment, all recipient groups developed autoantibodies, but those injected with the regulatory CD4+CD25+ T cells from HgCl2-treated donors were considerably reduced. Recipients not injected with HgCl2 also developed autoantibodies, but only when injected with the CD4+CD25– T cells from HgCl2-treated donors. Experiments using GST are continuing. In conclusion, CD4+CD25+ T cells, previously exposed to a certain xenobiotic, are able to hinder the development of autoantibodies in recipients undergoing treatment with the same xenobiotic. To our knowledge, this is the first time in which CD4+CD25+ T cells have been reported to play a role in drug-induced autoimmune diseases.

1

Experimental Rheumatology, Department of Rheumatology and Clinical Immunology and Institute of Pathology, Charité, Humboldt-University, and 2Deutsches RheumaForschungszentrum, Berlin, Germany 3

K. 9 Expression of the integrin aEb7 identifies unique subsets of CD25+ as well as CD25– regulatory T cells J.C.U. LEHMANN1, J. HÜHN1, M. DE LA ROSA2, F. MASZYNA2, U. KRETSCHMER1, V. KRENN3, M.C. BRUNNER-WEINZIERL2, A. SCHEFFOLD2, and A. HAMANN1 Regulatory CD25+CD4+ T cells are considered as important players in T cell homeostasis and self-tolerance. We here report that the integrin aEb7 which recognizes epithelial cadherin identi-

33rd Annual Meeting of the German Society of Immunology · 151 fies the most potent subpopulation of regulatory CD25+ T cells. Strikingly, also CD25-negative aE+CD4+ T cells displayed regulatory activity. Both aE+ subsets, CD25+ and CD25–, express CTLA-4, suppress T cell proliferation in vitro and protect mice from colitis in the SCID model in vivo. Whereas aE+CD25+ T cells produce almost no cytokines, aE+CD25– T cells represent a unique subset in which high IL-2, IFN-g and Th2-cytokine production is linked with suppressive function. Thus, the integrin aEb7 can be regarded as a novel marker for subsets of highly potent, functionally distinct regulatory T cells specialized for crosstalk with epithelial environments.

Institut für Virologie und Immunbiologie, Universität Würzburg, Würzburg, Germany

K. 10 Rat CD25+CD4+ regulatory T cells: Characterization, efficient expansion, and modulation of their response by CD25– cells C.H. LIN and T. HÜNIG In mice and humans, CD25+CD4+ regulatory T cells are constitute about 10% of peripheral CD4+ T cells and play a crucial role in controlling autoimmune and inflammatory diseases. Using a new anti-rat CTLA-4 mAb, we demonstrate here that, consistent with the characteristics of T regulatory cells described in mice and humans, rat CD25+CD4+ cells express CTLA-4, produce high levels of IL-10 and fail to secrete IL-2. Upon co-stimulation, rat CD25+CD4+ cells show reduced proliferation compared to CD25– cells, and the reduced proliferation could be overcome by adding high concentrations of exogenous IL-2. In contrast, CD25+CD4+ cells proliferated as well as CD25– cells upon stimulation with «superagonistic» CD28 specific mAb which can activate T cells without TCR ligation. Furthermore, superagonistic anti-CD28 also efficiently expanded CD25+CD4+ cells in vivo. Activated CD25+CD4+ remained anergic upon co-stimulation and were much more potent in suppressing CD25– indicator cells than «naïve» or anti-TCR plus anti-CD28 pre-activated CD25+CD4+ cells. This suggests that superagonistic anti-CD28 is suitable for the expansion of CD25+CD4+ regulatory T cells for therapeutic purposes. Finally, we found that while CD25– were deactivated by co-culture with CD25+ cells, CD25+ cells became more activated. Thus, co-culture of CD25+ with CD25– cells leads to reciprocal modulation of activation of both cell populations.

Klinik für Viszeral- und Transplantationschirurgie, Medizinische Hochschule, Hannover, Germany

K. 11 Thy1-positive donor derived cells in livers of stable MHC class I dimorphic irradiation chimeras pretreated with Retrorsine F.C. POPP, M.H. DAHLKE, H. ASELMANN, K. WONIGEIT, P. PISO, and H.J. SCHLITT It was recently reported that adult bone marrow contains hepatic progenitors capable of differentiation into mature hepatocytes. It was also shown that bone marrow bound hematopoetic

152 · 33rd Annual Meeting of the German Society of Immunology progenitors can transdifferentiate into hepatocytes. In the present study Retrorsine pretreatment and exposure to carbontetrachloride were combined in a new rat bone marrow transplantation model to achieve donor derived hepatocytes. LEW.1A (RT1aaa) female rat bone marrow recipients were treated with 2 doses of Retrorsine two weeks apart followed by 7 Gy of total body irradiation on day 0. Bone marrow of LEW.1WR2 male rats (RT1uaa) was transplanted by i.v. injection on day 1. Four weeks later rats were exposed to carbontetrachloride dosed 0,6 ml/kg BW followed by a booster injection of bone marrow 3 days later. Liver cryosections were doublestained with donor specific antibody (antiRT1Au mAb) and Thy1 (CD90) mAb. Two weeks after exposure to CCl4 recipient livers showed a multilineage inflammatory infiltrate consisting of donor and recipient hematopoetic cells mainly of granulocytic lineage. Four weeks after injection of CCl4 numbers of hematopoetic cells within the parenchyma were almost back to pretreatment levels. At this timepoint ovally shaped Thy1 positive cells of donor origin occurred in clusters associated with bile ducts. Bone marrow derived cells of donor origin are present in the Retrorsine/CCl4 model four weeks after hepatic injury. As these cells occur in clusters and coexpress Thy1 (CD90) it is likely that they resemble donor derived oval cells. If these oval cells are result of transdifferentiation or seeding of the recipient liver with donor hepatic progenitors remains open.

Klinik für Viszeral- und Transplantationschirurgie, Medizinische Hochschule, Hannover, Germany

K. 12 Isolated conditioning by anti-CD45 MAb for tolerance induction by hematopoietic chimerism T. RÖSELER, K. TIMROTT, M.H. DAHLKE, J.Y. LIU, M. NEIPP, K. WONIGEIT, H.J. SCHLITT, and M.D. JÄGER We could recently show that a single injection of an anti-CD45 MAb allows long-term acceptance for fully allogeneic heart grafts. Since CD45 might represent a selective target for BMTconditioning, we tested the chimerism inducing potential of this MAb in order to achieve robust tolerance. The model consisted of various MHC congenic rat strains on LEW background. Recipient strains possessed the RT7a allele of CD45, donor strains the RT7b one. Conditioning consisted of one or two injections of anti-RT7.1 MAb detecting RT7a. 1¥108 BMC of different MHC mismatches were injected; control groups received MAb only. Animals were tested for BM engraftment monthly by FACS. Aplasia incidence as major side effect of the anti-CD45 MAb conditioning was monitored. Tolerance was confirmed by skin transplantation on day +50. With increasing MHC barriers a strict dependance of necessary MAb dosage could be shown for allogeneic BM engraftment. Whereas a single MAb injection was sufficient in the setting of no or an isolated MHC class I mismatch, only two times injections enabled for chimerism in combined MHC mismatched settings. Reciprocally to decreasing engraftment rates the risk of aplasia was present as far as the immunological barrier involved MHC class II mismatches. Long-term chimeras of fully MHC mismatched group accepted donor-specific skin grafts (MST >200 days), while promptly rejecting third party skin (MST 9 days). Control groups receiving syngeneic BM or no BM underlined the intact immunological reconstitution after anti-CD45 MAb conditioning by acutely rejecting allogeneic skin grafts. In conclusion, selective targeting of the leukocyte

33rd Annual Meeting of the German Society of Immunology · 153 system by this anti-CD45 MAb enabled permanent engraftment of MHC-mismatched BM in a strictly dosage dependent manner. Donor-specific tolerance was based on mixed hematopoietic chimerism. The risk of aplasia by this isolated MAb conditioning seemed dependent from MAb dosage in combination with increasing MHC mismatch barriers, namely MHC class II.

Department of Surgery, University of Regensburg, Regensburg, Germany

K. 13 Soluble MHC class I gene therapy can prevent hyperacute rat heart allograft rejection in fully sensitized recipients M.N. SCHERER, C. GRAEB, A. KRÖMER, M. JUSTL, E. FRANK, K.W. JAUCH, and E.K. GEISSLER Introduction: Presensitized patients face the low probability of receiving a compatible organ transplant (Tx), since preformed antibodies and primed T cells rapidly destroy an allograft. Here our aim was to test whether soluble donor MHC class I gene therapy is a possible strategy to prevent hyperacute rejection (HR) of rat heart allografts in fully sensitized recipients. Methods: Using the AdEasy system, we made an E1 – E3-deleted, replication-deficient, adenovirus containing the genes for both green fluorescent protein and for a soluble form of the rat MHC class I molecule, RT1.Aa (Ad-RQ). Lewis (RT1l) rats, presensitized with 3 consecutive (12 day interval) ACI (RT1a) skin allografts, received an optimal 2¥109 fluorescence-forming units dose (i.v.) of Ad-RQ 14, 17 or 19 days post-3rd skin Tx. ACI heterotopic heart Tx was performed 21 days after the 3rd skin Tx. Serum RT1.Aa levels were measured by ELISA in non-sensitized Lewis rats injected with Ad-RQ. Results: Recipients not receiving Ad-RQ consistently (10/11) rejected the ACI heart allograft within 6 h after Tx. In contrast, 4/5, 4/4 and 4/5 recipients receiving Ad-RQ on day -2, -4 and -7 relative to heart Tx, respectively, did not hyperacutely reject the allograft; those hearts beat for 3–5 days before rejection. Infection with an empty adenovirus did not prevent HR. Regarding RT1.Aa expression, serum samples from Ad-RQ-injected non-sensitized rats showed that high expression of RT1.Aa (731–1919 ng/ml, n=3) could be maintained for at least 7 days, before decreasing. Conclusions: Adenoviral gene transfer produces high in vivo RT1.Aa expression levels that effectively prevent HR of heart allografts in sensitized recipients. Interestingly, the levels of soluble donor RT1.Aa with Ad-RQ infection are similar to those observed following liver transplantation, which is resistant to HR. Further development of this strategy could improve the success of organ Tx in highly sensitized patients.

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Swiss Institute of Allergy and Asthma Research, Davos, Switzerland, 2Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain, and 3Department of Immunology, The Hospital for Sick Children, Toronto, Canada

b suppression mediator of CD25+ K. 14 CD105 expression acts as a TGF-b regulatory T cells C.B. SCHMIDT-WEBER1, S. KUNZMANN1, P. SCHMID1, B. RÜCKERT1, C. BERNABÉU2, M. LETARTE3, and K. BLASER1 TGF-b mediated suppression of antigen-specific T cell responses by regulatory T cells is an important step in the control of mucosal tolerance such as allergic rhinitis or asthma. The TGF-b co-receptor endoglin (CD105) binds TGF-b and modulates TGF-b susceptibility. The present study reveals that CD105 is expressed by memory T cells and affects TGF-b mediated suppression of T cells. The regulation of CD105 was investigated following in vitro stimulation of freshly isolated CD4+ T cells. CD105 expression coincided with that of the T cell activation markers CD25+. It also increased following TCR engagement. Transient overexpression of CD105 in freshly isolated CD4+ T cells inhibited the induction of a TGF-b sensitive reporter gene construct. Interestingly, T cells overexpressing CD105 presented TGF-b to recipient T cells, which in turn showed increased activity of a TGF-b sensitive reporter gene, that was previously transfected into these cells. These results demonstrate that CD105 protects T cells from TGF-b mediated signal transduction, but keeps its biological activity alive, by presenting it to CD105– T cells. This mechanism is important for T cell responses in TGF-b rich environments and for the contact-dependent activity of CD25+ regulatory T cells.

Institute of Immunology, Westfälische Wilhelms-Universität, Münster, Germany

K. 15 Idiotype-specific CD4+CD25+ Ts cells inhibit the production of antinuclear autoantibodies in a response to a bacterial polysaccharide antigen C. SPECHT, K. BRÜNING, M. ROLFING, and E. KÖLSCH CD25+ T suppressor cells are regulating the immune response against a(1Æ3)dextran (Dex) in BALB/c mice. These T cells on the one hand restrict the Dex-specific IgG antibody repertoire towards the dominance of the J558 Idiotype and on the other hand reduce Dex-specific IgG. Antibodies with non-J558 structures in VH region only occur when the T cell control fails; e.g. in nude mice. This does not result in the appearance of antibodies with enhanced affinity to dextran, but can result in crossreactivity of Dex-specific antibodies with nuclear proteins. We now report a CD25+ Ts cell mediated restriction of VH usage, preventing the appearance of Dex-specific IgG antibodies, crossreacting as antinuclear autoantibodies (ANA). Adoptive transfer of both, an idiotype-specific CD25+ T cell clone (178–4 Ts) and freshly isolated CD4+CD25+ T cells restrict the Dex-specific IgG repertoire and prevent the occurrence of autoantibodies. This emphasizes the advantage of T cell mediated antibody invariance in responses to antigens with highly conserved epitopes like bacterial polysaccharides.

33rd Annual Meeting of the German Society of Immunology · 155 Klinik für Viszeral- und Transplantationschirurgie, Medizinische Hochschule, Hannover, Germany

K. 16 Tolerization of the CD4+ T cell subset is essential for long-term acceptance of organ grafts in stable hematopoietic chimeras K. TIMROTT, T. RÖSELER, T.Y. TSUI, J.Y. LIU, K. WONIGEIT, H.J. SCHLITT, and M.D. JÄGER Mixed allogeneic chimerism achieved by bone marrow transplantation (BMT) is associated with tolerance for solid organ grafts syngeneic with the BM inoculum. In this study, the significance of intrathymic tolerization of CD4+ or CD8+ T cell subsets was evaluated for heart graft acceptance in chimeric systems. Primarily, mixed BM chimeras on LEWIS background were generated using sublethal irradiation dosages. Following MHC settings were used: 1. completely MHC incompatible, 2. MHC class II incompatible, 3. MHC class I incompatible. On day 50 stable chimeras received heart grafts, which were completely MHC mismatched to the recipient in each group, but shared different degrees of MHC antigens with BM donors. Long-term heart graft acceptance was observed in group 2 sharing the MHC class II antigen between BM and heart graft donors comparable to group 1 with the complete MHC matched setting between BM and heart graft (n=10, MST >150 days). In contrast, heart grafts sharing only MHC class I antigens between BM and heart graft donors (group 3) were acutely rejected independently of chimerism degree (n=8, MST=12 days). Histological analysis of long-term accepted heart grafts did not reveal significant differences for obliterative arteriopathy as chronic rejection sign nor definitive lymphocyte infiltrations as hint for local immunomodulation. Acutely rejected grafts showed massive lymphocyte mixed infiltrations consisting of CD4+ and CD8+ T cells with increased CD25 expression. Therefore, the key point for tolerance by stable mixed chimerism is the tolerization of the alloreactive CD4+ T cell subset. How far immunoregulation within the peripherally tolerized CD8+ T cell subset support the organ graft acceptance in this model could not be clarified so far.

Department of Pathology, State Medical University, Tbilisi, Georgia

K. 17 The novel mechanism of peripheral tolerance D. USHARAULI The circulating pool of lymphocytes contains self as well as non-self reactive T cells. T cells recirculate via HEV (high endothelial venules) to the LN. The priming of naïve T cells occurs in the peripheral lymphoid organs, e.g. lymph nodes (LN), where DC migrate to after activation and maturation. The mature DCs also present nonself as well as self epitopes to naïve T cells. To avoid autoimmunity organism has to keep the mature DCs afar from self-specific T cells, I had proposed that the different anatomical distribution of the immature (tolerogenic) and mature (immunogenic) DC in the peripheral lymphoid tissues may contribute to this mechanism. The main principle of the novel hypothesis is that the layer of mature DC is localized behind the layer of immature DC in the peripheral lymphoid organs. I had proposed that T cells to reach the mature DC should pass through the layer of immature DCs, which constitutively phagocytose and

156 · 33rd Annual Meeting of the German Society of Immunology transport apoptotic cells to the regional LN and present only «self» epitopes. Thus, self-specific T cells will be trapped by the immature DC layer and be deleted or become anergic. The layer of immature DC will be crossed only by those T cells that are not self-specific. Thus, that distribution of immature and mature DC in peripheral lymphoid organs could control peripheral self-tolerance.

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Institute of Biomedical Aging Research, Austrian Academy of Sciences, 2Division of Hematology and Oncology, Department of Internal Medicine, Leopold-Franzens University, Innsbruck, and 3Department of Surgery, Landeskrankenhaus, Hall in Tyrol, Austria

K. 18 Increase of regulatory T cells in the peripheral blood of cancer patients A.M. WOLF1, D. WOLF2, M. STEURER2, G. GASTL2, A. MARKL3, E. GUNSILIUS2, and B. GRUBECK-LOEBENSTEIN1 Recently, the existence of a CD4+CD25+CD45RA– regulatory T cell (Treg) population has been described in rodents and humans. In healthy humans this population accounts for 5–10% of peripheral CD4+ T cells. The observation, that regulatory T cell-depleted mice developed a broad range of autoimmune pathologies revealed, that this T cell subset has an important checkpointfunction for the control of T cell-mediated autoimmunity. Moreover, experimental tumor models in mice revealed that Tregs are potent inhibitors of an effective anti-tumor immune response. We therefore designed the current study to determine, whether cancer patients exhibit an expanded Treg-pool. Our report demonstrates for the first time that patients suffering from epithelial malignancies show an increase of CD4+CD25+ T cells in the peripheral blood sharing characteristic properties with Tregs, i.e. they are CD45RA+, CTLA-4+ and TGF-b+. Proliferation assays revealed that in contrast to CD4+CD25+ T cells, CD4+CD25++ T cells were anergic towards T cell receptor stimulation by anti-CD3 as well as by allogeneic DCs. In addition, CD4+CD25+ T cells also suppressed the proliferation of cocultured CD4+CD25+ T cells. When cultured together with CD56+ NK cells, CD4+CD25+ T cells isolated from cancer patients effectively inhibited NK cellmediated lysis in a flow-cytometric cytotoxicity assay. Thus, we provide evidence of an increased pool of CD4+CD25+ regulatory T cells in the peripheral blood of cancer patients with potent immunosuppressive features which might counterbalance the generation of an efficient endogenous anti-tumor response. Therefore, depletion of Tregs may be a promising therapeutical strategy to enhance tumor immunity (i.e. induced by peptide pulsed DCs).