Costimulation blockade of both inducible costimulator and CD40 ligand induces dominant tolerance to islet allografts and prevents spontaneous autoimmune diabetes in the NOD mouse.

Costimulation blockade is a promising strategy for preventing allograft rejection and inducing tolerance. Using a fully allogeneic mouse model, we tested the effectiveness of the combined blockade of the CD40 ligand and the inducible costimulator (ICOS) on islet allograft survival and in the prevention of autoimmune diabetes in the NOD mouse. Recipients treated with blocking monoclonal antibodies (mAbs) to ICOS and the CD40 ligand had significant prolongation of graft survival, with 26 of 28 functioning for >200 days. Long-term engrafted mice maintained antidonor proliferative and cytotoxic responses, but donor-specific immunization did not induce graft rejection, and challenge with second, same donor but not third-party grafts resulted in long-term acceptance. The immunohistology of tolerant grafts demonstrated the presence of CD4(+)CD25(+) T-cells expressing Foxp3, and islet/kidney composite grafts from tolerant mice, but not from mice lacking lymphocytes, were accepted indefinitely when transplanted into naïve B6 mice, suggesting that recipient T-cells were necessary to generate dominant tolerance. Combined anti-ICOS and anti-CD40 ligand mAb therapy also prevented diabetes in NOD mice, with only 11% of treated recipients developing diabetes compared with 75% of controls. These data demonstrate that the blockade of CD40 ligand and ICOS signaling induces islet allograft tolerance involving a dominant mechanism associated with intragraft regulatory cells and prevents autoimmune diabetes in NOD mice.

Robust tolerance to fully allogeneic islet transplants achieved by chimerism with minimal conditioning.

BACKGROUND: Whether mixed chimeras induced by nonmyeloablative conditioning are tolerant to challenge with donor allogeneic islet grafts is unknown. Here we investigate whether our nonmyeloablative, costimulation blockade-free and sirolimus (SRL)-based protocol could facilitate mixed chimerism via bone marrow transplantation (BMT) and induce islet allograft tolerance. METHODS: After low dose (1-3 Gy) total body irradiation (TBI, day -1), with or without prior lymphocyte depletion, C57BL/6 mice were transfused with 40 x 10(6) BALB/c bone marrow cells (day 0) and received SRL (3 mg/kg/day) for 4 weeks. Chimerism was monitored by flow cytometry and the recipients were rendered diabetic chemically and challenged with donor islets. RESULTS: Mixed chimerism was achieved in mice treated with TBI 3 Gy/SRL but it declined over time in 60% (9/15) of them. Long-term stable chimerism was established in 100% of recipients over 50 weeks with either antilymphocyte serum (ALS, 9/9), anti-CD4 (4/4), or anti-CD4 plus anti-CD8 (5/5) prior to BMT. TBI conditioning could be reduced to 1 Gy, with 90% (9/10) maintaining chimerism in the long-term. When TBI was substituted with cyclophosphamide (CTX) or busulfan (BUS), all mice remained chimeric in the long-term. The chimeras showed no proliferative response to donor antigen and accepted both first and second donor-specific islet grafts indefinitely while rejecting third-party grafts. CONCLUSIONS: This data provides the first evidence that stable fully allogeneic chimeras induced with BMT after nonmyeloablative conditioning with SRL and lymphocyte-depleting antibodies exhibit robust donor-specific tolerance to islet grafts.

Multiple combination therapies involving blockade of ICOS/B7RP-1 costimulation facilitate long-term islet allograft survival.

In recent years a series of novel costimulatory molecules have been identified, including inducible costimulator (ICOS). In a fully major histocompatibility complex (MHC)-mismatched mouse model of islet transplantation, we demonstrate that while monotherapy with CTLA4-Ig, CD40 ligand monoclonal antibody (CD40L mAb) or rapamycin each improves islet allograft survival, graft rejection eventually develops. Immunohistologic analysis of rejected grafts revealed increased ICOS expression, suggesting a role for this costimulatory molecule as an alternate pathway for T-cell activation. The combination of a blocking anti-ICOS mAb with each of the above therapies resulted in significantly improved islet allograft survival, confirming the importance of ICOS signaling in islet allograft rejection. Mechanistic studies conducted in mice treated with anti-ICOS mAb and rapamycin demonstrated a lack of donor-specific immunological tolerance and an absence of regulatory T-cell activity. However, a dramatic effect was seen on acute anti-donor responses whereby anti-ICOS mAb and rapamycin significantly reduced the initial expansion and function of alloreactive T cells. These data demonstrate that blockade of the ICOS/B7RP-1 pathway has potential therapeutic benefit given its role in enhancing islet allograft survival and regulating acute alloresponses in vivo.