Two major obstacles to successful prevention of transplant rejection using costimulation blockade-based therapy are 1) the presence of donor-reactive memory T cells prior to transplantation that precipitate acute rejection, and 2) PTLD resulting from compromised EBV-specific immunity in the setting of CD28 blockade. Thus, the identification of strategies to overcome these obstacles is of critical importance in order to optimize the use of belatacept in the clinic. Our preliminary studies revealed that CTLA-4 Ig-resistant CD8+ breakthrough responses are inhibited by selective CD28 blockade using novel CD28 domain antibody (dAb) technology. Using powerful transgenic murine systems in which we can specifically identify, track, and characterize both CD4+ and CD8+ donor-reactive T cells following transplantation, we have identified a coinhibitory molecule (2B4, SLAMf4) that is specifically expressed following selective CD28 blockade. Interrogating the role of 2B4 in CD28 dAb-mediated prolongation of graft survival is innovative in that 2B4 has been shown to play an important role in autoimmunity and in chronic viral infections in both mice and humans, yet has never before been studied in transplantation. Interestingly, 2B4 is expressed at increased levels on secondary CD8+ effectors relative to primary effectors, and a recent study demonstrated that the expression of 2B4 plays a functional role in inhibiting CD8+ secondary effectors during recall responses. These intriguing data raise the possibility that this inhibitory pathway could be harnessed to inhibit donor-specific memory T cell recall responses during transplantation. Finally, we propose to directly compare the effects of selective CD28 blockade vs. CTLA-4 Ig on protective immune responses to a murine homolog of EBV (MHV). A recent study revealed that while CD80/86-/- animals exhibited a profound inability to control MHV replication and reactivation from latency, CD28-/- animals exhibited undetectable viral loads equivalent to wild-type mice. These important data suggest that selective CD28 blockade may better preserve EBV-specific protective immune responses following transplantation. Thus, many questions remain unanswered with regard to the impact of selective CD28 blockade during transplantation. How does selective CD28 blockade in the presence of CTLA-4 coinhibitory signals impact the expression of other costimulatory and coinhibitory molecules to alter T cell programming? How does CD28 blockade differentially impact secondary recall responses, given the fact that memory T cells may be CTLA-4hi and therefore more reliant on this pathway for control of the response? What is the impact of selective CD28 blockade on protective immunity to EBV? Answers to these questions will provide fundamental insights into the biology of costimuatory and coinhibitory receptors in transplantation, and will provide a mechanistic foundation for the further development of novel selective CD28 blockers for use in clinical transplantation.