Background.It has been shown that simultaneous blockade of CD28- and CD40-mediated costimulatory signals significantly prolongs allograft survival. Although these results led to an expectation of the establishment of specific immunotolerant therapy for organ transplantation, it became evident that these treatments rarely resulted in indefinite allograft survival. To uncover the mechanisms underlying these costimulation blockade-resistant allograft rejections, we studied the process of allogenic skin graft rejection in CD28 and CD40 ligand (L) double-deficient (double-knockout [dKO]) mice.Methods.Skin grafts from BALB/c or BALB.B mice were transplanted to C57BL/6 background dKO mice. The frequency of CD4+and CD8+T cells responding to alloantigens presented by direct or indirect pathways were defined by the use of a cytostaining assay.Results.BALB/c skin grafts were rapidly rejected by dKO mice. This CD28 and CD40L independent allograft rejection was inhibited by the depletion of CD8+T cells. In vitro studies indicated that CD8+T cells from BALB/c skin-grafted dKO mice responded to donor antigen presented only by the direct pathway. Unlike major histocompatibility complex (MHC)-mismatched donors, allogenic skin grafts from MHC-matched donors were accepted by dKO mice.Conclusion.In the absence of CD28 and CD40 costimulatory signals, CD8+T cells recognize MHC antigens by the direct pathway, resulting in the rejection of skin grafts from MHC-mismatched donors. In contrast, MHC-matched and non-MHC-mismatched donor skin grafts indefinitely survive in dKO mice. These results indicated that donor-host MHC matching may still be critical to costimulation blockade therapy for organ transplantation.