CXCL1, CCL3, CCL5 real-time PCR assays (Applied Biosystems) were run on a 7900HT Real-Time PCR System (ABI). T Cell Intracellular Cytokine Staining To measure IFN- (eBiosciences) and TNF (BD Pharmingen) production by donor reactive cells, solitary cell suspensions of responder splenocytes from transplanted mice were stimulated with BALB/c splenocytes in the presence of 10g/mL Brefeldin A for 4 hours with BALB/c splenocytes and subjected to intracellular cytokine staining (Number 5). with either untreated or DST treated animals. Moreover, only CD154 blockade efficiently inhibited CXCL1 manifestation and neutrophil recruitment into the graft. When combined, anti-CD154 and DST acted synergistically to profoundly diminish the complete quantity of IFN- generating alloreactive CD8+ T cells, and intra-graft manifestation of inflammatory chemokines. These findings demonstrate the previously described ability of anti-CD154 and DST to result in alloreactive T cell deletion involves both delayed kinetics of T cell development and differentiation D149 Dye and inhibited development of KLRG-1low memory space precursor cells. Intro Current immunosuppressive regimens in organ transplantation require life-long administration and result in off-target toxicities such as nephrotoxicity and cardiovascular and metabolic complications . Considering these significant co-morbidities, much work over the years offers focused on the development of novel modes of immunosuppression. The development of costimulation obstructing molecules has been the basis for study by several organizations to specifically target and inhibit the full activation of alloantigen-specific T cells at the time of transplantation. Probably one of the most effective pathways for restorative intervention is the CD154/CD40 pathway, blockade of which results in serious inhibition of graft D149 Dye rejection and in some models the induction of transplantation tolerance C. However, translation of restorative blockade of this pathway has been stymied from the observation of thromboembolic complications in pilot medical trials as a result of the manifestation of CD154 on platelets . However, understanding the modified differentiation programs initiated in alloreactive T cell populations under conditions of CD154 blockade remains an important goal in the ongoing pursuit to D149 Dye harness the restorative potential of this pathway. In order to study the effects of CD40/CD154 pathway blockade on donor-reactive T cell reactions to a transplant, we used an allogeneic pores and skin graft (SG) model in which anti-CD154 monoclonal antibodies (mAb) were administered in combination with donor specific transfusion (DST) as previously explained , , . DST provides a large bolus of antigen offered by relatively inert APCs , stimulating antigen-specific T cell activation by providing signal one. Additional organizations have also shown the potent effects of combined DST and costimulation blockade in the prolongation of islet, cardiac, pores and skin and kidney allograft survival in murine and nonhuman primate models , C. Although it has been generally approved that CD154 costimulation blockade prospects to anergy  or deletion ,  of recently triggered T cells, the mechanism by which DST and anti-CD154 mAb synergize to induce these effects within the alloreactive T cell human population remains incompletely recognized. In order to assess the differential effect of DST and anti-CD154 mAb within the programming of donor-reactive CD8+ T cell development, contraction, and differentiation over time, we performed longitudinal analyses within the donor-reactive CD8+ T cell reactions. We hypothesized the previously observed deletion of graft-reactive CD8+ T cells following anti-CD154/DST treatment was the result of differential programming of these cells following encounter with alloantigen , . Recently, studies of viral-specific CD8+ T cell reactions have revealed programmed differentiation of antigen-specific T cells into either long-lived memory space precursors or short-lived effectors as early as four days post-infection . These differentially programmed cells can be segregated on the basis of their manifestation of KLRG-1 (killer cell lectin-like receptor G-1), in that KLRG-1high cells represent short-lived effectors, while KLRG-1low antigen-specific CD8+ T cells distinguish the long-lived memory space precursors , . As compared to KLRG-1low FRPHE cells, KLRG-1high cells go on to express lower levels of Bcl-2, CD27, and CD62L, and higher levels of GzmB. Functionally, KLRG-1high cells are jeopardized in their ability to produce IL-2, an important T cell autocrine growth element , . Finally, adoptive transfer recipients of KLRG-1high cells have been shown to have poorer recall potential upon secondary rechallenge as compared to those receiving KLRG-1low.