Current models of thymic selection propose that T cells develop via low affinity TCR interactions with self- peptides in the thymus, and that high affinity interactions lead to clonal elimination. This model fails to accommodate emerging data on several non-conventional T cells populations, such as regulatory T cells (Treg), and natural killer T cells (NKT). These T cell subsets are numerical minor, but play a major role in the regulation of immune responses. Evidence suggests that these populations may have receptors with high affinity for self-ligands, though this remains controversial. In this proposal we seek to define the role of TCR signal strength in the thymic selection of Treg and NKT through the development and use of novel tools, including a TCR signal strength reporter mouse, where the level of the fluorescent molecule GFP reflects signaling through the antigen receptor. This tool will be used to study the TCR signals perceived by Treg and NKT cells during development, homeostasis, and infection. Further evidence from our lab suggests that self- stimuli cause NKT cells to continually produce IL-4 in the steady state in some strains of mice. Because of the power of IL-4 to alter the development of other lymphocytes, skew the nature of immune responses to pathogens, and promote allergic responses, it is critical to identify and study the NKT cells that produce IL-4 in the steady state. We will do this by employing an IL-4 reporter and using various genetic and population ablation strategies to define what causes this IL-4 production. PUBLIC HEALTH RELEVANCE: T lymphocytes are crucial to the immune response to pathogens and tumors. Their sophisticated behavior during immune responses is the result of complex selection processes that occur during development in the thymus, where some T cells are instructed to be cytolytic, others to be helpers, others to initiate immune responses, and others to regulate them. This proposal seeks fundamental knowledge about the biochemical and cellular basis of selection of these multiple subsets. Such information is essential not only to understand the developmental process, but to capitalize on it in terms of therapeutic immune reconstitution.