H. Nishimura

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.

Beneficial effects of perioperative low-dose inhaled carbon monoxide on pulmonary allograft survival in MHC-inbred CLAWN miniature swine.

Background. We have recently reported that perioperative low-dose carbon monoxide (CO) inhalation decreases lung ischemia-reperfusion injury in miniature swine. The aims of this study were to establish a large animal model of pulmonary allograft rejection using polymerase chain reaction-typed major histocompatibility complex (MHC)-inbred CLAWN miniature swine and to examine the effects of CO on allograft survival. Methods. Eleven CLAWN miniature swines received fully MHC-mismatched lungs followed by 12 days of tacrolimus (days 0–11; blood level 35–45 ng/mL). Six recipients received tacrolimus alone (control group). Five recipients were additionally treated with inhaled CO (180 min for donors until graft harvest; 390 min for recipients until 2 hr after reperfusion). Results. All recipients treated with tacrolimus alone uniformly rejected their grafts by postoperative day 63 with development of cytotoxic antidonor antibodies. CO treatment was effective in prolonging allograft survival from a mean of 47±7 to 82±13 days (P=0.017), with one CO-treated animal maintaining function until postoperative day 120. Development of antidonor antibodies and donor-specific responsiveness by cell-mediated lympholysis and mixed lymphocyte reaction assays was delayed in animals that received CO therapy. Furthermore, serum concentrations of proinflammatory cytokines (interleukin-1&bgr; and -6) 1 day after transplant were significantly decreased in the CO-treated group. Conclusions. Fully MHC-mismatched lungs in CLAWN miniature swine were consistently rejected within 63 days, suggesting that this is a robust large animal model ideal for investigating mechanisms and treatment of lung rejection. Perioperative low-dose CO inhalation prolonged graft survival and inhibited antidonor antibody production and was associated with decreased proinflammatory mediators in this model.

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.

Hepatocyte growth factor sustains T regulatory cells and prolongs the survival of kidney allografts in major histocompatibility complex-inbred CLAWN-miniature swine.

Background. Although 12 days of high dose of FK506 permits the induction of tolerance of fully major histocompatibility complex (MHC)-mismatched allogeneic kidneys in MGH-miniature swine, we found that the same dose of FK506 is insufficient to induce such tolerance CLAWN-miniature swine. The CLAWN swine model was therefore chosen to study the potential immunoregulatory effects of human-recombinant hepatocyte growth factor (HGF). Methods. Ten CLAWN miniature swine received fully MHC-mismatched kidneys with 12 days (days 0–11) of FK506. Among these 10 recipients, 4 received 7 or 14 days of human-recombinant HGF starting at day 11. Graft function was assessed by daily serum creatinine and biopsies. Immunologic assays, including CD4/CD25 DP and FoxP3+ cells and development of antidonor antibodies, were performed. Results. Without HGF, all six CLAWN recipients developed severe acute rejection (Cre >9 mg/dL) within 3 weeks of transplantation. In contrast, in the four animals that received HGF for 7 to 14 days, stable renal function was observed for more than 50 days, although all grafts were ultimately rejected by postoperative day 80. Percent FoxP3+ cells in the CD4+CD25+ double positive population (T regulatory cells) in peripheral blood monocyte cells decreased in recipients with FK506 induction monotherapy while no reduction was observed in recipients treated with FK506 and HGF. Conclusion. This study demonstrates that in CLAWN swine treated with a dose of FK506 insufficient to induce tolerance across a fully MHC mismatched barrier, a short course of HGF may inhibit acute rejection while maintaining T regulatory cells. To our knowledge, this study provides the first evidence in a large animal transplantation model of HGFs immunoprotective effects.

Protective effect of neutralization of the extracellular high-mobility group box 1 on renal ischemia-reperfusion injury in miniature swine.

Background Strategies that reduce ischemia-reperfusion injury (IRI) have the potential to expand the numbers of available organs for transplantation. Recent reports in rodent models have demonstrated that high-mobility group box 1 (HMGB1) acts as an alarm in initiating the inflammatory response resulting from ischemic injury. The aim of this study was to evaluate the cytoprotective effects of anti-HMGB1 antibodies on renal IRI in preclinical large animals. Methods One hundred twenty minutes of warm and 60 min of cold renal ischemia were induced in 8 CLAWN miniature swine. Three of eight animals received intravenous anti-HMGB1 antibody at 1 mg/kg just before the reperfusion of renal blood flow. Renal function was assessed by serum creatinine and renal biopsy. Serum levels of interleukin (IL)-1&bgr;, IL-6, and HMGB1 were measured. Results The concentration of HMGB1 increased as early as 30 min after reperfusion and before the elevation of IL-1&bgr; and IL-6. Serum creatinine levels were markedly elevated, peaking at a median of 5 days (peak creatinine levels: 11.6±1.6 mg/dL) and recovering by day 14. Anti-HMGB1 antibody injection dramatically decreased renal damage as well as serum levels of HMGB1 associated with IRI. Renal function returned to near normal by day 9, and peak creatinine levels were markedly lower (7.4±0.2 mg/dL), and biopsies possessed fewer pathologic changes when compared to the control group. Conclusion In this study, we demonstrated the beneficial effects of perioperative administration of anti-HMGB1 antibody in reducing renal IRI in a clinically relevant, large animal model.

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

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+).

ANTI-HIGH-MOBILITY GROUP BOX-1 ANTIBODIES REDUCE PULMONARY ISCHEMIA-REPERFUSION INJURY IN MINIATURE SWINE: 3191

H. Sahara1, M. Oku1, K. Setoyama1, M. Tasaki1, H. Nishimura1, O. Wunimenghe2, M. Okumi3, A. Shimizu4, K. Yamada5 1Xenotransplantation Surgery Unit, Frontier Science Research Center, Kagoshima University, Kagoshima/JAPAN, 2Frontier Science Research Center, Kagoshima University, Kagoshima/JAPAN, 3Urology, Osaka University Graduate School of Medicine, Suita/ JAPAN, 4, Nippon medical school, Tokyo/JAPAN, 5Transplantation Biology Research Center, Harvard Medical School, Boston/MA/ UNITED STATES OF AMERICA