The tissue-resident innate-like T cell subsets (i.e., NKT, MAIT, and innate-like T) constitute major lymphocyte populations in barrier tissues like the lung, gut, and skin, where they provide a critical first line of defense against bacteria, fungi, and viruses and where they are active players in anti-tumor immunity. Innate- like T cell subsets all share the ability to rapidly producing large amounts of cytokines and chemokines, and thereby possess the ability to shape both the quantity and quality of both the innate and adaptive immune responses. Although the NKT and mucosal-associated invariant T (MAIT) innate-like T cell subsets are well-studied, far less is known of the innate-like T cell populations, and their identity remains ill-defined. We recently completed a comprehensive investigation into the role of the SLAM/SAP signaling pathway in T cell development and function. This study revealed that the SLAM/SAP signaling pathway is a critical determinant in the thymic development of both IL-17- and IFN--producing T cells. Since one of the hallmarks of nearly all innate-like T cell subsets is a dependence on the SLAM/SAP signaling pathway for their development, these results raise the possibility that SAP regulates the development of, as yet undefined innate-like T cell subsets. Therefore, the objective of this proposal is to unambiguously define the SAP-dependent T cell subsets. Because a characteristic feature of all innate-like T cells identified so far is the expression of a signature invariant of semi-invariant TCR, we propose to: identify the SAP-dependent and SAP-independent TCR clonotypes using single-cell next-generation sequencing. We will sort single T cells from C57BL/6J and B6.SAP-/- mice, and determine the paired TCR clonotypes by sequencing barcoded amplicon libraries generated using V- and V-specific primers. We will examine thymic T cells at different stages of development (embryonic day 17, birth, day 6 neonate, and adult) to capture distinct waves of T cell development, and we will examine lung and spleen to identify the SAP-dependent subsets in the periphery. Upon completion of these studies, we will have unambiguously identified the SAP-dependent TCR clonotypes, which will be critical in to our understanding how SLAM/SAP signaling regulates the developmental programming of T cells. In addition, we will have provided an invaluable high-resolution dataset of the paired TCR clonotypes in both the thymus and periphery to investigators seeking to understand better the role of T cells in the immune response.