ABSTRACT Renal transplantation remains the only long-term solution to end-stage renal disease (ESRD), which affects approximately 2 million people worldwide. However, long term allograft acceptance requires lifelong immunosuppression, and current standard-of-care immunosuppressive therapies have a multitude of toxicities directly leading to decreased patient and allograft survival. Targeted anti-rejection therapies focusing on T cell co-stimulatory blockade, such as belatacept (CTLA4-Ig) or iscalimab (anti-CD40 mAb) have been developed to prevent T cell mediated rejection. Despite enhanced graft function, rejection episodes under these therapies are more frequent and difficult to treat.Current dogma suggests that these rejections are due to memory CD8+ T cell cross-reactivity from prior infections, which do not require co-stimulation. Here we will examine the cross- reactivity of expanded, graft-infiltrating CD8+ T cells in rejection biopsies from both humans and mice. Our novel preliminary data suggest that expanded CD8+ T cell clonotypes in the graft are alloreactive, and prior viral infection increases the alloresponse. These preliminary findings include: (a) a limited number of unique clonotypes are expanded in the rejecting allograft; (b) the same individual expanded CD8+ T cell clones are observed for months in persistent allograft rejection; (c) expanded clonotypes shift their gene expression to escape immunosuppression; and (d) subcloning of TCRs from expanded clonotypes into Jurkat cells confirmed their allospecificity. Further, we show that using established mouse models of viral infection and transplantation: (e) prior LCMV infection increases the response to alloantigen; and (f) mice without prior LCMV infection reject F1 heart allografts by day 14 post-transplant. Finally, we have samples from patients undergoing rejection after administration of viral-specific T cell therapy to treat infection. Together, our findings and existing literature support a highly novel hypothesis that a significant fraction of pre-existing viral-specific memory T cells home to the allograft shortly after transplant and their cross-reactivity to alloantigens drives rejection events. We propose to test this hypothesis by: (i) identifying alloreactive, viral-reactive, and cross-reactive graft- infiltrating CD8+ T cells in patients undergoing renal allograft rejection (Aim 1) and (ii) determining the impact of prior viral infection on CD8+ T cell infiltration into the allograft of a mouse model of transplantation (Aim 2). Our studies will enhance the knowledge of cross-reactive CD8+ T cells in renal allograft rejection and the characterization of alloreactive T cells on human disease. Given the organ shortage crisis and the limited life expectancies of transplant patients, defining the specificity of alloreactive CD8+ T cells in renal transplant rejection will enable discovery of novel therapeutic targets to directly target alloreactive cells while sparing by...