512 CD40/CD40L interactions are crucial for the development of CD4+ T cell dependent immune responses such as allograft rejection. For example, simultaneous administration of a CD40L monoclonal antibody (mAb) and the CTLA4-Ig fusion protein synergistically prolongs the survival of skin and cardiac allografts. In this report we establish the efficacy of an anti-CD40 mAb alone and in combination with CTLA4-Ig as an alternative to anti-CD40 ligand as a strategy to inhibit rejection. Immunohistochemical analysis demonstrates that CD40 is strongly induced on both donor and recipient cells in the rejecting allograft. Furthermore, transplants using CD40-/- mice indicate that CD40 on the recipient is functionally more important than on donor tissues. Hearts from CD40-/- donors are rejected promptly, whereas CD40-/- recipients reject wild type (wt) cardiac allografts more slowly than wt B6,129 F1 control recipients. The ability of an anti-CD40 mAb, 7E1, to inhibit allograft rejection was tested in the BALB/c to C3H/HeJ strain combination. Recipient mice were treated with 7E1 (250µg/dose) intraperitoneally (IP) on days 0, 2, 4& 6. 7E1 significantly prolonged cardiac allograft survival relative to untreated controls. However, histological assessment of the 7E1 treated animals demonstrated significant interstitial fibrosis and coronary vasculopathy by postoperative day (POD) 140. As previously reported with a CD40L mAb (MR1), 7E1 synergized with CTLA4-Ig in inhibiting these pathologic changes. C3H/HeJ recipients of BALB/c skin grafts were treated with anti-CD40 mAb, alone and in combination with CTLA4-Ig (250µg/dose IP on days 0, 2, 4& 6). Anti-CD40 mAbs alone prolonged skin graft survival (MST=30d) compared to MR1 treatment (MST-14d). Untreated controls rejected the skin grafts promptly by POD11. As in the cardiac allograft model, 7E1 and CTLA4-Ig synergize to improve skin graft survival (MST=93d). 7E1 was found to prevent the formation of donor specific alloantibodies in the cardiac allograft recipients. Alloantibodies were undetectable on POD 29, 35 & 57, whereas alloantibodies were detected in the control BALB/c cardiac allograft recipients at a dilution of 1:512 at POD 29, 34 & 57. Flow cytometric analysis of peripheral blood of cardiac allograft recipients treated with 7E1 demonstrated partial B cell depletion on POD 8 and 21 which returned to normal by POD55. 7E1 treatment had no effect on T cell and macrophage compartments. These studies suggest that the use of anti-CD40 mAbs is an effective alternative to MR1 in the blockade of the gp39-CD40 pathway alone or in combination with CTLA4Ig.