Project Summary: This proposal is a renewal application of R01 (AI130197). In the previous funding cycle, we sought to identify the mechanism(s) by which the small adaptor protein Crk controls NK cell activation and inhibition, using human NK cells and NK cell-specific conditional knockout mice. We found that Crk phosphorylation plays a key role in function and that Crk haploinsufficiency leads to NK cell deficits and impaired integrin signaling. Importantly, we identified new small molecule inhibitors of pCrk (Patent #: 63/305,354). In the proposed renewal, we use this new tool to further dissect Crk’s ability to integrate activating and inhibitory input at the immune synapse (IS) of both NK and T cells in a variety of relevant settings: in high-resolution live human primary cell imaging assays, in human Crk haploinsufficiency, and in triple negative breast cancer (TNBC). We test our central hypothesis that Crk functions as a master regulatory switch, fine-tuning activating and inhibitory influences via interactions with downstream molecules and the actin cytoskeleton, and propose the new hypothesis that pCrk inhibition can restore exhausted immune responses in cancer via novel signaling pathways. Guided by strong preliminary data, we propose three Specific Aims: 1) How does Crk function as a master regulatory switch to control NK cell function? Here, we use cutting-edge imaging systems, lipid bilayers, live human primary cells, RNA-Seq, and our new CrkL inhibitors to determine where Crk, pCrk, and their upstream and downstream partners localize in the cytotoxic, inhibitory, and exhausted IS, and how pCrk inhibitors influence NK signaling in healthy and exhausted NK cells. 2) How does Crk influence immune function and integrin signaling in pDGS T cells? Our previous program showed that NK cells from patients with pDGS – a common genetic syndrome resulting from haploinsufficiency of CrkL-containing locus 22q11 – exhibit functional NK cell deficiencies, as well as defective integrin activation at the IS. We expand these studies to T cells, using primary pDGS patient cells and integrin conformation-specific antibodies to assess how Crk functions as a two-way regulatory switch for CTL function. We present data that haploinsufficiency of CRKL results in functional T cell deficits, and examine integrin signaling, T cell function, and IS quality with an eye towards clinical application of small molecule LFA-1 agonists to restore pDGS-related functional immune cell deficiencies. 3) Discover the role of pCrkL inhibitors in cancer immunotherapy. Our preliminary data show that pCrk inhibitors significantly inhibit tumor growth, restore cytotoxicity of inhibitor receptor-dominant immune cells, and enhance survival. We hypothesize that pCrk inhibitor-mediated solid tumor suppression results from both direct tumor killing and restoration of exhausted immune cell function. In Aim 3, we use a battery of TNBC mouse models to elucidate Crk’s molecular mechanisms ...