Project 1 Summary Transplantation tolerance bears the potential to improve the quality of life of transplant recipients. However, we have shown that infections/inflammation can break transplantation tolerance. Understanding the molecular mechanisms induced in alloreactive T cells when tolerance is successful, identifying those that are destabilized during infections, and those missing in T cells from immunosuppressed non-tolerant hosts will help design approaches for more durable tolerance. We have used a mouse model of fully mismatched cardiac transplantation in which a costimulation blockade (CoB)-based treatment results in permanent donor-specific tolerance. In this model, systemic infection with Listeria monocytogenes (Lm), or inflammation, 2 months post- transplantation precipitate graft rejection in 50% of the animals and erode tolerance in the rest. The 1st cycle of this program project demonstrated that transplantation tolerance can exist at different levels of robustness and can be eroded by infection or inflammatory cytokines. In the 2nd (current) cycle of Project 1, we studied the mechanisms that maintain robust tolerance. First, CD4+ T cells with indirect specificity for a donor MHC-I-peptide develop a profound state of T cell dysfunction that combines characteristics of exhaustion and anergy, depending both on graft persistence for ~3 weeks and CD154 blockade. Second, these tolerant T cells maintain better susceptibility to Treg suppression than memory T cells, due to downregulation of the transcription factor Satb1. Third, tolerance durably constrains expansion of high avidity T cell clones in polyclonal populations. Our preliminary data have identified new vulnerabilities that may prevent long-term graft acceptance. We found heterogeneity in the level of dysfunction of alloreactive T cells within a tolerant host and identified 2 causes to this heterogeneity. (i) duration of antigen persistence: high avidity T cells specific for persistently expressed alloantigens (donor MHC-I) have greater loss of function than high avidity T cells specific for more transient alloantigens (donor MHC-II); (ii) TCR strength of signal, a product of T cell affinity/avidity and cognate ligand density, where low avidity T cells are resistant to CoB-mediated dysfunction and preferentially dependent on suppression by Tregs. Because infections and inflammation can inhibit Treg suppressive function and upregulate expression of some donor antigens (MHC class II) on the graft, we reason that infections may enable alloreactive T cells with retained functionality, despite CoB-induced tolerance, to trigger rejection or graft damage. We hypothesize that high affinity/avidity Tconvs specific for chronically expressed alloantigens will develop dysfunction, whereas high avidity T cells recognizing more transiently expressed alloantigens, and low avidity T cells, will retain function and will have to be durably controlled by Tregs. Rejection triggered by infection/...