PROJECT SUMMARY Effective cell-based immunity depends on two major systems: (i) the ability to generate a diverse TCR repertoire capable of recognizing antigens from previously unencountered pathogens, and (ii) the ability to discriminate between self- and non-self-antigens and prevent autoimmunity. Inappropriate balance between these two systems causes a variety of disease states including ineffective tumor immune surveillance and poor pathogen clearance due to gaps in the TCR repertoire, or the onset of autoimmunity and off target immunopathology during infection. Autoimmunity can be curtailed by deleting autoreactive TCRs or diverting them into the Treg lineage in the thymus. However, given the high degree of cross-reactivity of TCRs this process cannot be truly efficient at removing all self-reactive TCRs as this would remove much of the potential diversity of the conventional TCR repertoire, thereby impairing immunity to pathogens. Thus, a key biological question is how thymic selection functions to balance protection against autoimmunity while providing effective immunity against pathogens. We hypothesize that a unique population of thymic recirculating Treg cells (RT-Treg) act as a rheostat to decrease the stringency of selection once peripheral Treg cell tolerance is established, thereby allowing for a more diverse and pathogen-reactive conventional immune system. This will be examined in two specific aims: Aim 1: Define RT-Treg heterogeneity and functional consequences of RT-Treg accumulation. Aim 2: Identify the mechanisms by which RT-Treg cells affect immune repertoires and mTEC numbers. The proposed experiments will elucidate the role that RT-Treg play in governing the stringency of central tolerance, both promoting effector cell diversity and ensuring maintenance of self-tolerance by Treg cells.