O459* Aims: The objective of this study is to determine the activation requirements for CD8+ T cells in mediating allograft rejection. Methods: Fully MHC-mismatched skin transplantation was performed using DBA/2 (H-2d) and B6.CD4−/− knockout (H-2b, CD4KO) mice as donors and recipients. To evaluate the CD28, CD154 and CD134 costimulation pathways in supporting CD8+ mediated rejection, costimulatory blockade treatments were administered to recipients: CTLA-4Ig and MR1 (0.5 mg, days 0, 1, 3), and anti-CD134L blocking mAb (0.5 mg, days 0, 2, 6, 8). In some experiments, B6.CD28−/−/CD154−/− double knockout (CD28/CD154DKO) mice were used as hosts. To determine the role mTOR in CD8+ mediated rejection, rapamycin (3mg/kg, days 0, 1, 2, 4, 6, 8, 10, 12, 14), was used to block mTOR-dependent growth factor signaling. To quantitatively analyze the role of costimulation and growth factors in promoting CD8+ T cells activation in response to alloantigen stimulation, CFSE-labeled donor C57BL/6 T cells were adoptively transferred into lethally irradiated allogeneic DBA/2 hosts. In vivo proliferation of donor CD8+ T cells in the host mice was analyzed by flow cytometry 3 days later. Results: CD4KO mice in which rejection is mediated solely by CD8+ T cells promptly rejected the skin allograft with a MST of 12 days. Treatment with CTLA-4Ig/MR1, anti-CD134L or anti-CD134L/MR1 failed to prevent skin allograft rejection (MST<24 days). In stark contrast, transient blockade of CD28/CD154/CD134 costimulatory pathways induced long-term skin allograft survival in CD4KO recipients (MST>98 days). Treatment of CD4KO recipients with rapamycin completely prevented skin allograft rejection (MST>120 days). Rapamycin also induced long-term allograft survival in CD28/CD154DKO mice (MST>120), as compared with untreated CD28/CD154DKO or rapamycin treated wild-type recipients (MST<16 days). CFSE-labeled CD8+ T cells proliferated vigorously in untreated hosts, and treatment of host mice with rapamycin markedly inhibited in vivo proliferation of CD8+ T cells. CFSE-labeled CD8+ T cells from CD28/CD154DKO mice proliferated strongly in untreated hosts, while CD8+ T cells expanded poorly in anti-CD134L treated host mice. Conclusions: Our data suggest that in contrast to the common belief that CD8+ T cells are resistant to costimulatory blockade, activation of CD8+ T cells requires the engagement of costimulatory pathways, and concurrent CD28/CD154/CD134 costimulatory blockade is essential to prevent CD8+ mediated rejection. Activation of a subset of CD8+ T cells is clearly dependent on CD134 costimulation. Similarly, CD8+ mediated rejection requires mTOR signaling. Interestingly, when both CD28/CD154 costimulatory signals and growth factor signaling are blocked, skin allograft rejection can be prevented, even in the presence of CD4+ T cells.