PROJECT SUMMARY Despite excellent short-term outcomes after transplantation, the long-term survival of transplanted organs has stagnated for several decades. Recent clinical studies have demonstrated that acute cellular rejection, which is mediated by alloreactive T cells, is a risk factor for later graft failure. In order to effectively prevent acute rejection, deeper understanding of how T cells response to allogeneic antigen are needed. The CD28 pathway blocker belatacept (a CTLA-4 Ig derivative) offers significant long-term benefits versus calcineurin inhibitors for kidney transplant patients, but has limited efficacy in restraining CD8+ T cells and acute cellular rejection. Despite a great body of work investigating the programming of effector CD8+ T cells (TEFF) that respond to infections, relatively little is known about CD8+ TEFF that respond to allogeneic antigen in the context of an allograft. We found that after grafting, a subset of CD8+ TEFF expressing the activated CD43 isoform (defined by the 1B11 epitope) are formed during acute effector timepoints from day 7-21 post-graft. CD43 is a cellular receptor that influences T cell receptor signaling cascades and cellular trafficking, but its role in transplantation is undefined. Relative to other TEFF subsets, CD43+ TEFF appeared highly activated, displayed vigorous effector functions, and drove accelerated graft rejection. In human renal transplant patients, CD43+ CD8+ T cells infiltrate the kidney allografts of patients treated with belatacept, and CD43+ CD8+ T cells are functionally resistant to CTLA-4 Ig in vitro. Together, these data form the premise for our hypothesis that CD43+ TEFF are a potent effector population that can be targeted to limit acute rejection following transplantation. In this proposal, we will use an innovative MHC Class I tetramer to characterize the role of CD43 on graft-specific CD8+ T cells. Specific Aims: First, we will define the effector mechanisms that distinguish CD43+ TEFF function as a population from CD43- TEFF. Using adoptive transfer approaches, we will investigate the differentiation pathways that lead from naïve CD8+ T cells to CD43+ TEFF, the mechanisms by which CD43+ TEFF traffic from the draining lymph nodes into graft tissue, and the role of the Inducible T Cell Costimulator (ICOS) receptor signaling in the fate of CD43+ TEFF. Second, we will investigate the role of CD43 receptor signaling on CD8+ T cells. Using a CD43 knockout mouse model, we will interrogate the requirement of CD43 signaling for graft rejection, how CD43 controls T cell at the clonal level, and the role of CD43 in determining the quality of secondary memory responses. Third, we will explore the potential of CD43 as a therapeutic target to limit alloimmunity in humans and mice. We will evaluate whether targeted deletion of CD43+ TEFF in vivo with an antibody-drug conjugate can elicit tolerance in combination with CD28 blockade. We will explore whether human CD43+ CD8+ T cel...