Approximately 50% of mice treated with sMR1 alone accepted heart allografts for over 100 days

Approximately 50% of mice treated with sMR1 alone accepted heart allografts for over 100 days. with anti-CD154 therapy in promoting long-term allograft survival. The addition of calcineurin inhibitors did not abolish this synergistic effect. Intense CD154-CD40 blockade by a multiple-dose routine of anti-CD154 resulted in long-term graft survival and profound alloreactive T-cell unresponsiveness and overcame the opposite effects of calcineurin inhibitors. CTLA4Ig induced long-term graft survival, and the CX3CL1 effect was not affected by the concomitant use of any immunosuppressive drugs. Conclusions The common view that calcineurin inhibitors abrogate the effects of T-cell costimulatory blockade should be revisited. Sufficient costimulatory blockade and synergy induced by CD154 blockade and rapamycin promote allograft tolerance and prevent chronic rejection. Blocking T-cell costimulatory activation pathways is an effective strategy in preventing allograft rejection, promoting long-term survival, and inducing tolerance in some experimental transplant models. 1C8 The mechanisms of action of T-cell costimulatory blockade in vivo include induction of T-cell anergy, apoptosis, regulatory cells, and immune deviation. 9,10 Recent studies also exhibited the efficacy of CD28-B7 and CD154-CD40 blockade in prolonging primate renal and islet allograft survival as relevant preclinical models for future translation to humans. 11C15 Furthermore, costimulatory blockade has been extensively studied as a encouraging therapeutic strategy not only in transplantation but also in autoimmunity, allergy, and infections. 9,16 Indeed, the efficacy of CTLA4Ig therapy has already been confirmed clinically in the autoimmune disease psoriasis vulgaris. 17C19 Phase I-II studies are underway with CTLA4Ig, humanized anti-B7, and anti-CD154 monoclonal antibodies (mAbs) in transplantation and autoimmunity. One of the major difficulties to developing T-cell costimulatory blockade strategies for the medical center, especially in the transplant setting, is usually understanding the interactions between brokers that block T-cell costimulation and standard immunosuppressive drugs currently in clinical use. 20 This is an extremely important and clinically relevant issue since immunosuppressive drugs may abrogate, synergize with, or not affect the functions of such brokers. Previous reports showed that cyclosporine but not rapamycin abrogated the effect of combined blockade of CD28-B7 (by CTLA4Ig) and CD154-CD40 (by anti-CD154 mAb) costimulatory pathways in rodent transplantation models. 6,21,22 Smiley et al. also reported the distinct effects of some immunosuppressive drugs on anti-CD154 mAb therapy and showed that cyclosporine and steroids but not rapamycin abrogated the effect of anti-CD154 mAb plus concomitant administration of donor cells in promoting long-term allograft survival in a mouse heart transplant model. 23 The effect of the immunosuppressive drugs on CD154 mAb therapy alone was not investigated in that study. Kirk et al. recently reported that the additional use of steroids or tacrolimus to humanized anti-CD154 mAb might have a detrimental effect on graft survival in a primate renal transplant model. 13 Addition of cyclosporine or rapamycin to CTLA4Ig was reported to enhance allograft survival in a class I MHC-mismatched skin transplant model. 24 In this study, we investigated systematically the interactions between T-cell costimulatory blockade (CTLA4Ig to block CD28-B7 or MR1 to block CD154-CD40) and the immunosuppressive brokers cyclosporine, tacrolimus, rapamycin, steroids, and IL-2R mAb in vivo. We used Tolfenpyrad a model of vascularized cardiac transplantation in a fully allogeneic mouse strain combination, C57BL/6 into BALB/c. Our data spotlight the complex interactions between B7 or CD154 blockade on the one hand and immunosuppressive drugs on the other in acute and chronic rejection, and provide clinically relevant novel data to translate to large animals and humans. METHODS Transplantation Model C57BL/6 (H-2b) and BALB/c (H-2d) mice aged 6 to 8 8 weeks were purchased from Taconic Farms (Germantown, NY). BALB/c mice were used as recipients and C57BL/6 mice as donors. The cardiac allografts were placed in an intraabdominal location, as previously described. 25 Graft function was assessed by palpation of the heartbeat. Rejection was determined by total cessation of palpable Tolfenpyrad beat and was confirmed by direct visualization after laparotomy. 26 Fusion Proteins, mAbs, and Immunosuppressive Drugs Anti-CD154 mAb (MR1, a kind gift of Dr. R. Noelle) and anti-IL-2R mAb (PC61, a kind gift of Dr. L. Turka) were manufactured from their respective hybridomas by Bioexpress Cell Culture Services (West Lebanon, NH). Murine CTLA4Ig was a nice gift of Dr. R. Peach (Bristol Myers Squibb, Princeton, NJ). Cyclosporine (Novartis), methylprednisolone (Upjohn), and tacrolimus (Fujisawa) were obtained from the Brigham and Womens Hospital pharmacy. Rapamycin was generously provided by Wyeth-Ayerst (Princeton, NJ). Cyclosporine, methylprednisolone, and tacrolimus were prepared as Tolfenpyrad a.