Wiskott-Aldrich syndrome (WAS) is an X-linked immune deficiency arising from mutations in the WAS protein (WASp) gene, required for the proper functioning of T helper cell (Th), important for shaping the adaptive immune response. The clinical severity of disease can range from mild to severe, even in patients with the identical WASp mutation. The biological basis for this range of adaptive immune dysfunction is not known. The Th:DC interaction initiates multiple signaling pathways specific for Th1 differentiation. The classical Th1 signaling pathway requires the formation of a mature Th1-signalosome (IFN3R+TCR2) at the Th immune synapse (ThIS) and downstream activation of STAT1 and T-bet, leading to transcription of IFN-3. In parallel, ITK-dependent phosphorylation of T-bet is required for deactivation of GATA-3, the Th2 transcription factor. Finally, the Notch signaling pathway has effects on both Th1 and Th2 differentiation. How are these seemingly disparate signaling pathways organized into a functional Th1 developmental paradigm? In this proposal we will test the hypothesis that WASp is critical for both TCR-dependent and Notch-dependent pathways of Th1 activation, and propose that the adaptor function of WASp organizes Th1 developmental responses by fostering interactions between Notch, ITK and T-bet. We posit that the severity of WAS clinical phenotype mirrors the degree of Th dysfunction imparted by WASp mutations that disrupt these interactions. In Aim 1, we will test the hypothesis that WASp is critical for the proper functioning of the TCR-dependent pathway of Th1 differentiation. Aim 2 will test the hypothesis that WASp is critical for Notch-signaling in Th1 differentiation. Finally, Aim 3 will test the hypothesis that the severity of WAS clinical phenotype is mirrored in the degree of Th dysfunction. These studies should provide new insights into the function of WASp in the development of the adaptive immune response in health and disease, which may be of value in developing new therapeutic targets. Lay-term summary: Wiskott-Aldrich syndrome (WAS) is a genetically transmitted systemic immune deficiency resulting from a defect in a protein called WASP and manifesting clinically in severe recurrent infections, easy bruising and bleeding, autoimmunity, and cancers. The studies in this proposal will examine the function of WASP at the molecular level to determine the causes of the above clinical complications of WAS. These studies should lay the foundation for designing novel therapies in the future, for this life-threatening, debilitating disease of the childhood.