B. Gillon

First experience with the use of a recombinant CD3 immunotoxin as induction therapy in pig-to-primate xenotransplantation: the effect of T-cell depletion on outcome.

Background. We have previously reported life-supporting kidney xenograft-survival greater than 80 days using a steroid-free antithymocyte globulin (ATG)-based induction regimen (ATG regimen) in a GalT-KO pig-to-baboon thymokidney (TK) model. We evaluated two induction regimens, a newly developed anti-monkey CD3 recombinant immunotoxin (anti-CD3 rIT) and an anti-human CD2 antibody (LoCD2), by assessing T-cell depletion (TCD) and graft survival. Methods. Four baboons received anti-CD3 rIT; the time course of TCD was studied in two animals and the other two received GalT-KO TK transplants. Two additional baboons underwent GalT-KO TK transplantation after treatment with LoCD2. All other treatments were identical to previous TCD studies with ATG. TCD was assessed by flow-cytometry; renal function was evaluated by serum creatinine and histology. Results. Baboons that received the anti-CD3 rIT died from pneumonia or cardiac failure on days 15 and 23. Both animals in the rIT group died with functioning grafts. Thymokidney grafts from baboons treated with the LoCD2 regimen were rejected by day 14. TCD levels in baboons receiving the anti-CD3 rIT regimen were 150 to 250 cells/&mgr;L for at least 14 days, whereas baboons receiving the LoCD2 recovered to more than 300 cells/&mgr;L by day 7. Conclusions. The newly developed anti-CD3 rIT could be a useful TCD agent in baboons. However, optimal dosage, treatment duration, and bioactivity must be studied to avoid side effects. A LoCD2-based regimen was not effective for preventing xenogeneic rejection. Optimal TCD levels less than 250/&mgr;L during the induction period seem to be important for success of xeno-thymokidney transplantation.

Abrogation of Renal Allograft Tolerance in MGH Miniature Swine: The Role of Intra-Graft and Peripheral Factors in Long-Term Tolerance

We have previously demonstrated that long‐term tolerance (LTT) of an MHC class‐I mismatched renal allograft can be achieved with a short course of cyclosporine. In order to examine regulatory mechanisms underlying tolerance in this model, we assessed the contributions of factors within the graft and in the peripheral blood for their relative roles in the maintenance of stable tolerance. Twelve LTT recipients of MHC class‐I mismatched primary kidneys were subjected to a treatment consisting of donor‐specific transfusion followed by leukapheresis, in order to remove peripheral leukocytes, including putative regulatory T cells (Tregs). Following treatment, 2 controls were followed clinically and 10 animals had the primary graft removed and received a second, donor‐MHC‐matched kidney. Neither control animal showed evidence of rejection, while 8 of 10 retransplanted animals developed either rejection crisis or full rejection of the second transplant. In vitro assays confirmed that the removed leukocytes were suppressive and that CD4+Foxp3+ Treg reconstitution in blood and kidney grafts correlated with return to normal renal function in animals experiencing transient rejection crises. These data indicate that components of accepted kidney grafts as well as peripheral regulatory components both contribute to the tolerogenic environment required for tolerance of MHC class‐I mismatched allotransplants.

VASCULARIZED COMPOSITE ALLOGRAFT TRANSPLANT SURVIVAL IN MINIATURE SWINE: IS MHC TOLERANCE SUFFICIENT FOR ACCEPTANCE OF EPIDERMIS?

Background We have previously reported that Massachusetts General Hospital miniature swine, which had accepted class I–mismatched kidneys long-term after 12 days of high-dose cyclosporine A, uniformly accepted donor-major histocompatibility complex (MHC)–matched kidneys without immunosuppression but rejected donor MHC-matched split-thickness skin grafts by day 25, without changes in renal graft function or antidonor in vitro responses. We have now tested whether this “split tolerance” would also be observed for the primarily vascularized skin of vascularized composite allografts (VCAs). Methods Group 1 animals (n=3) received donor MHC-matched VCAs less than 70 days after primary kidney transplant (KTx). Group 2 animals (n=3) received a second donor-matched kidney transplant followed by a donor-matched VCA more than 200 days after primary KTx. Results Animals in Group 1 lost the epidermis on days 28, 30, and 40, with all other components of the VCAs remaining viable. Histology showed cellular infiltration localized to dermal-epidermal junction. One of three recipients of VCAs in Group 2, accepted all components of the VCA, including epidermis (>200 days). The other two recipients lost only the epidermis on days 45 and 85, with survival of the remainder of the VCA long-term. Conclusions All tissues of a VCA are accepted long-term on animals tolerant of class I–mismatched kidneys, with the exception of epidermis, the survival of which is markedly prolonged compared with split-thickness skin grafts but not indefinite. Exposure of tolerant animals to second donor-matched kidneys before VCA increases the longevity of the VCA epidermis, suggesting an increase in the immunomodulatory mechanisms associated with tolerance of the kidney.

Adoptive Transfer of Renal Allograft Tolerance in a Large Animal Model

Our recent studies in an inbred swine model demonstrated that both peripheral and intra‐graft regulatory cells were required for the adoptive transfer of tolerance to a second, naïve donor‐matched kidney. Here, we have asked whether both peripheral and intra‐graft regulatory elements are required for adoptive transfer of tolerance when only a long‐term tolerant (LTT) kidney is transplanted. Nine highly‐inbred swine underwent a tolerance‐inducing regimen to prepare LTT kidney grafts which were then transplanted to histocompatible recipients, with or without the peripheral cell populations required for adoptive transfer of tolerance to a naïve kidney. In contrast to our previous studies, tolerance of the LTT kidney transplants alone was achieved without transfer of additional peripheral cells and without strategies to increase the number/potency of regulatory T cells in the donor. This tolerance was systemic, since most subsequent, donor‐matched challenge kidney grafts were accepted. These results confirm the presence of a potent tolerance‐inducing and/or tolerance‐maintaining cell population within LTT renal allografts. They suggest further that additional peripheral tolerance mechanisms, required for adoptive transfer of tolerance to a naïve donor‐matched kidney, depend on peripheral cells that, if not transferred with the LTT kidney, require time to develop in the adoptive host.

The rejuvenating effects of leuprolide acetate on the aged baboon's thymus

BACKGROUND We have previously demonstrated that the juvenile thymus plays an essential role in tolerance induced by both renal transplantation and a short course of calcineurin inhibitors. Aged thymi have a decreased ability to induce tolerance. Luteinizing hormone-releasing hormone (LHRH) is known to pharmacologically rejuvenate the thymus in rodents. In order to develop a clinically applicable regimen of transplantation tolerance in adults, we sought to determine if thymic rejuvenation would occur with LHRH agonism in non-human primates. METHODS AND RESULTS Thymic rejuvenation was evaluated by magnetic resonance imaging (MRI), histology, as well as in-vitro cellular and molecular tests. Four aged male hamadryas baboons underwent subcutaneous injection of a 3-month depot of Lupron (11.25mg; LI) and were followed for 3 months. Thymi increased volumetrically by MRI. After LI, thymic cellularity markedly increased within the cortical and medullary thymus. Additionally, a significant increase in the CD4(+)/CD45RA(hi+) population in the peripheral blood occurred for 50 days after LI, and flow cytometry of thymic tissue revealed a large increase in the percentage of CD4(+)/CD8(+) cells. TREC assay corroborated enhancement in thymic function. CONCLUSION These data indicate that LI is associated with thymic rejuvenation in baboons, and further confirm that extrinsic factors play an important role in thymic rejuvenation in a non-human primate model.

Tolerogenicity of donor major histocompatibility complex-matched skin grafts in previously tolerant Massachusetts general hospital miniature swine.

Background Long-term tolerance of class I disparate renal allografts in miniature swine can be induced by a short course of cyclosporine and persists for 3 to 4 months after grafts are removed. Donor class I peptide immunization 6 weeks after graftectomy of tolerated kidneys leads to sensitization, but donor skin grafts do not. Here, we tested the hypothesis that skin grafts prevent rejection after simultaneous peptide administration and skin grafting. Methods Miniature swine underwent bilateral nephrectomy and class I–mismatched renal transplantation with a 12-day course of cyclosporine A to induce long-term tolerance. Tolerated allografts were then replaced with recipient-matched kidneys, and animals were challenged with simultaneous donor-type skin grafts and peptide. Six weeks later, second donor-matched kidneys were transplanted without immunosuppression, and immune responses were characterized. Results Animals treated only with peptide (n=2) rejected subsequent renal transplants in 3 to 5 days with strong in vitro antidonor responses. Of five recipients of skin-plus-peptide regimen, two accepted kidneys long term, one demonstrated a modestly prolonged survival (11 days), and two rejected rapidly (5–7 days). The two long-term acceptors maintained donor-specific hyporesponsiveness in vitro. Conclusions Sensitization by class I peptide in previously tolerant swine could be prevented by simultaneous class I skin grafts. These data suggest that skin grafts may actually augment rather than abrogate downregulation in some cases. A mechanistic hypothesis for this surprising result is that recognition of class I antigens through the direct rather than the indirect pathway of antigen presentation promotes tolerance by expanding regulatory T cells.

Development of antidonor antibody directed toward non-major histocompatibility complex antigens in tolerant animals.

Background The clinical significance of antibodies directed against antigens other than major histocompatibility complex (MHC) antigens is poorly understood, and there are few large animal models in which such antibodies can be examined. We studied, both retrospectively and prospectively, the development of antibodies to non-MHC antigens in tolerant miniature swine. Methods Our database was assessed for cases of antidonor antibody formation in tolerant animals over the last 20 years. Flow cytometry, absorption assays, and familial analyses for inheritance pattern of the gene(s) potentially responsible for the antibody reactivities were carried out, and an animal determined to be negative for this reactivity was immunized by a skin graft and subcutaneous injections of peripheral blood monocyte cells from an antigen-positive donor. Results Sixteen of 469 tolerant animals tested were found to have developed antidonor antibodies. These antibodies were found to be specific for the same, presumably single, non-MHC antigen. Familial analyses indicated that the gene encoding this antigen was expressed in an autosomal-dominant manner in approximately 95% of the herd. In a prospective study, antidonor antibodies with the same specificity as those observed retrospectively were successfully induced in an antigen-negative animal after immunization with peripheral blood monocyte cells. Conclusion To our knowledge, this is the first report of the development of antibodies to a highly prevalent, non-MHC antigen present on peripheral blood mononuclear cells and developing in tolerant animals without signs of graft dysfunction. Considering the concern often raised by the appearance of antidonor antibodies in transplant recipients, these data could have important implications for clinical transplantation.

HIGH LEVELS OF CYTOTOXIC ANTI-NON GAL PREFORMED NATURAL ANTIBODY DID NOT INDUCE HYPERACUTE REJECTION BUT DID CAUSE EARLY GRAFT LOSS IN A GALT-KO PIG-TO-BABOON THYMOKIDNEY MODEL: 2413

H. Nishimura1, A. Shimizu2, A. Griesemer3, J.R. Scalea1, S. Moran4, J. Weiner5, B. Gillon1, J. Fishman3, S.C. Robson3, M. Sykes3, D.H. Sachs5, K. Yamada5 1Surgery, Harvard University, Charlestown/UNITED STATES OF AMERICA, 2, Nippon medical school, Tokyo/JAPAN, 3Transplantation Biology Research Center, Harvard Medical School/MGH, Boston/ MA/UNITED STATES OF AMERICA, 4Transplant Biology Research Center, Massachusetts General Hospital, Boston/UNITED STATES OF AMERICA, 5Transplantation Biology Research Center, Harvard Medical School, Boston/MA/UNITED STATES OF AMERICA

Prevention of Early Development of Proteinuria by Rituximab in a Pig to Baboon Thymokidney Model Likely Associated with Modulating Podocyte Function in An SMPDL-3b-Dependent Manner: 1259

Background: Signal regulatory protein (SIRP)α is a critical immunoinhibitory receptor expressed on macrophages, and its interaction with CD47, a ligand for SIRPα, prevents autologous phagocytosis. We have previously proven that the interspecies incompatibility of CD47 causes in vitro phagocytosis of porcine xenogeneic cells by human macrophages. In this study, we used an in vivo mouse model to investigate whether genetic manipulation of rat insulinoma cells for mouse CD47 expression could inhibit macrophage-mediated xenograft rejection. Methods: To determine whether rat CD47 can interact with mouse SIRPa, we compared SIRPa tyrosine phosphorylation in mouse macrophages after interaction of the macrophages with mouse or rat red blood cells (RBCs). To determine whether expression of mouse CD47 on rat insulinoma (INS-1E) cells could confer protection against phagocytosis by mouse macrophages, we generated mouse CD47expressing INS-1E (mCD47-INS-1E) cells. The phagocytotic activity of mouse macrophages towards INS-1E cells was evaluated by in vivo assays. Carboxyfluorescein succinimidyl ester (CFSE)-labeled mCD47-INS-1E cells and control vector-transfected INS-1E (cont-INS1E) cells were injected into the peritoneal cavity of Rag2gc -/mice that had streptozotocin-induced diabetes and lacked T, B, and natural killer (NK) cells. The blood glucose levels were intermittently monitored using a blood glucose test meter. To further investigate whether inhibition of CD47-SIRPα signaling would abrogate the effect of genetic manipulation of CD47 expression, we injected anti-mouse SIRPa monoclonal antibody (mAb) into the peritoneal cavity of Rag2gc -/mice that had streptozotocininduced diabetes. Results: Western blotting showed that incubation of mouse macrophages with mouse RBCs and mCD47-INS-1E cells caused significant SIRPa tyrosine phosphorylation. However, SIRPa tyrosine phosphorylation was not induced in mouse macrophages incubated with rat RBCs and cont-INS-1E cells, similar to that observed in the control macrophages incubated with medium alone. Mouse CD47 expression on INS-1E cells radically reduced the susceptibility of these cells to phagocytosis by mouse macrophages. Diabetic Rag2gc -/mice became normoglycemic immediately after receiving mCD47-INS-1E cells. In contrast, the mice that received cont-INS-1E cells failed to achieve normoglycemia. However, mice that had received mCD47-INS-1E cells and were injected with anti-mouse SIRPa mAb failed to achieve normoglycemia, similar to that observed in mice that had received cont-CD47-INS-1E cells. Conclusions: These results show that interspecies incompatibility of CD47 significantly contributes to in vivo rejection of xenogeneic cells by macrophages. Thus, genetic induction of recipient CD47 on donor cells for generating SIPRα-inhibitory signals on recipient macrophages is necessary to prevent macrophage-mediated xenograft rejection. 1259

BREAKING MHC-CLASS I TOLERANCE WITH DST FOLLOWED BY LEUKAPHERESIS: 1429

Introduction: Understanding the cellular mechanisms underlying the development and loss of tolerance may lead to clinical protocols of transplant tolerance, obviating the need for immunosuppression. In this study, we attempted to break tolerance in an established renal tolerance model in MHC inbred MGH miniature swine. Methods: Tolerance to class I mismatched kidney allografts was induced by a 12-day course of high dose Cyclosporine in 6 MGH miniature swine. Long-Term Tolerance (LTT) was defined as stable renal function for 100 days following primary transplant and in-vitro evidence of donor specific unresponsiveness by both CML and MLR. LTT animals underwent DST (10ml/kg donor type whole blood) followed by leukapheresis (8 days after DST) of 1.6-3.15x109cells/kg. One day after leukapheresis, 4 animals underwent nephrectomy and retransplant of a donor-type kidney without immunosuppression. Two animals, serving as controls, did not receive a second graft. Renal function was evaluated by serum creatinine and open biopsies. Characterization of cell populations, including CD4+/FoxP3+, cells was assessed by FACS at multiple time points before and after DST, leukapheresis, and Ktxp. Presence of antibody was evaluated using flow cytometry. Results: All 6 recipients became tolerant. Leukapheresis decreased lymphocyte count peripherally by > 50% in all cases. 2 controls (no retransplant) showed no increase in creatinine over baseline (1.0-1.3mg/dL) > 30days after DST and leukapheresis. Of 4 animals retransplanted, 3 had a rejection crisis between POD4 and POD8 (Peak creatinine 3.7mg/dL-11.0mg/dL). 1 of these 3 animals proceeded to develop chronic renal failure and reject completely by 3 months. The remaining animal did not have a rejection crisis following DST+leukapheresis. This animal had only 1.6x109cells/kg removed with leukapheresis. Decrement in the absolute number of CD4+/FoxP3+ was observed following DST and leukapheresis. Absolute CD8+ count increased following DST. All cell populations decreased with leukapheresis. Immunohistochemistry of the explanted grafts from tolerant animals showed bright staining of FoxP3+ cells, whereas biopsies of the retransplanted grafts did not. Animals did not develop antibody (IgM or IgG) before or after DST, leukapheresis, or transplantation. Conclusions: DST + Leukapheresis does not break tolerance in a LTT animal unless the primary graft is removed and replaced with a donor-matched kidney. Further, if leukapheresis was not extensive (i.e. <1.6x109cells/kg) rejection crisis did not occur. These data suggest that both graft infiltrating/protecting cells in tolerant kidneys and peripheral T-regs may be involved in the maintenance of tolerance. Lack of antibody development following retransplant suggests that rejection crises were due to a T-cell mediated response. As animals developed increased CD8+ cells, but decreased T-regs following DST, the loss of tolerance may be associated with a change in the balance between alloreactive T-cells and T-regs (CD4+/ FoxP3+).