T cell sensitivity to antigens is intrinsically regulated during lymphocyte development and maturation to ensure proper development of immunity and tolerance, but how this is accomplished remains elusive. MicroRNAs (miRNAs), an abundant class of ~22-nucleotide (nt) small noncoding RNAs, have emerged as important players in animal development, the pathogenesis of cancers, and immune responses. Among them, miR-181a is preferentially expressed during lymphocyte development and plays important roles in T and B cell development. Increasing miR-181a expression in mature T cells augments the sensitivity to peptide antigens, whereas inhibiting miR-181a expression in immature T cells reduces sensitivity and impairs both positive and negative selection. Interestingly, quantitative regulation of T cell sensitivity by miR-181a enables mature T cells to recognize antagonists--the inhibitory peptide antigens--as agonists, suggesting that quantitative regulation of antigen sensitivity could result in a shift in the activation threshold in T cells. Supporting the idea that miR-181a may function as an intrinsic antigen-sensitivity `rheostat' during T cell development is that higher miR-181a expression seems to correlate with greater T cell sensitivity in various T cell populations. The proposed research plan will examine the function of the mir-181 family genes in regulating T cell sensitivity to antigens during lymphocyte development, selection, and function, and will further test the effects of tuning miRNA expression on the development of tolerance. Specifically, we will use loss-of-function approaches, including pharmacological (antagomir knock-down) and genetic (targeted deletions in mice) approaches, to reduce or abrogate the expression of the mir-181 family genes in DP cells, in order to determine the roles these miRNA genes play in controlling TCR signaling strength in DP thymocytes and in influencing positive and negative selection (Specific Aim 1). We will also characterize the roles of the mir-181 family genes in the development of the peripheral T cell receptor repertoire as well as naive and memory T cells (Specific Aim 2). Finally, we will investigate the functions of the mir-181 family genes in early T cell development in the thymus by controlling the pre-TCR and Notch signaling pathways (Specific Aim 3).