PROGRAM SUMMARY Transplantation tolerance, a state of long-lasting immune unresponsiveness to donor antigens after cessation of therapy, is an attractive approach for life-long graft acceptance without global immunosuppression. Long-term follow up of tolerant patients has revealed that tolerance can be lost in some individuals after years of graft stability, sometimes after infections, raising 2 possibilities: that tolerance at induction was equally robust but some patients were exposed to more inflammatory events that eroded the state of tolerance, or that tolerance at induction was metastable in some patients and robust in others. The first cycle of our Program Project addressed the first possibility using a mouse model of transplantation tolerance that is robust and resistant to most inflammatory challenges with the exception of Listeria monocytogenes (Lm). Importantly, we tracked alloreactive T cells by analyzing small numbers of congenic TCR-Tg alloreactive T cells seeded before transplantation, or using fluorescent pMHC Class I and Class II multimers to identify endogenous populations of T cells reactive to model donor antigens. By comparing alloreactive T cells before (Project 1) and after (Project 2) Lm infection of tolerant mice, our Program discovered that i) robust transplantation tolerance is maintained by multiple redundant mechanisms of T cell tolerance, including constraining alloreactive T cell numbers, increasing the ratio of regulatory to conventional T cells, inhibiting conventional T cells intrinsically and restraining alloreactive T cell populations to clones with low avidity for alloantigen; ii) robust tolerance is resilient because it spontaneously returned in animals that experienced Lm-dependent graft rejection; iii) tolerance after infection is eroded and dependent on single mechanisms of T cell tolerance such that blockade of PD-L1 or depletion of Tregs was sufficient to precipitate graft rejection in tolerant hosts post-infection but not in uninfected hosts. Globally, our Program has demonstrated that transplantation tolerance is not an all-or-none state, but rather can exist at different levels of robustness. These observations highlight the need to precisely define and monitor the mechanisms underlying graft acceptance in each tolerant recipient and to devise strategies to improve tolerance when it becomes eroded. For this Competitive Renewal, we will address the second possibility, that not all patients achieve robust tolerance at induction. Globally, we hypothesize that the mechanisms restraining alloreactive T cell subsets can distinguish robust tolerance established in naive hosts from eroded tolerance after infection, and from metastable or failed tolerance in sensitized hosts. Project 1 will focus on 2 novel features of alloreactive T cells that we recently discovered as characteristic of robust transplantation tolerance in naïve hosts, namely, cell intrinsic hyporesponsiveness and the constraint of alloreact...