Project Summary The central hypothesis of my F99-phase proposal is that the lack of T-cell infiltration into human tumors may be caused by the presence of tumor-derived T-cell excluders (TCEs) in the TME, which are overexpressed in tumor tissues and impair the function of T cells including their ability to migrate into tumor tissues. This provides a potential explanation why both spontaneous and therapeutic T cell-mediated anti-tumor immunity fail frequently in patients with immunologically “cold” tumors. By employing a secretome-wide in vitro T-cell transwell migration high-throughput screening (HTS) platform, we tested over a thousand soluble human proteins on the migration of activated T cells towards chemokine signal. SLIT2 was identified as a candidate target and validated to directly inhibit T-cell chemotaxis towards CXCL11, a common chemokine found in human tumor tissues. The N-terminal fragment of SLIT2 was first confirmed to mediate T-cell chemotaxis inhibition. Detailed functional domain mapping demonstrated that the first two leucine-rich repeat (LRR) domains, where the canonical receptor ROBO1 binds, are dispensable to SLIT2’s function in regulating T-cell chemotaxis. Meanwhile, ROBO1 expression could not be detected on T-cell surface with FACS staining, and soluble ROBO1 extracellular fragment fusion protein failed to neutralize SLIT2’s inhibitory effect on T cells, collectively suggesting a novel T- cell specific SLIT2 signaling axis independent of ROBO1. In a syngeneic mouse pancreatic cancer model, dramatically elevated T-cell infiltrated was observed in SLIT2 knock-out Pan02 tumors. SLIT2-KO tumors were rejected by the immune-competent C57BL/6 mice, while the SLIT2 wild-type tumors grew. Such difference diminished when the SLIT2-WT/KO tumors were inoculated into immune-compromised NSG mice. Anti-SLIT2 monoclonal antibody 11C8 neutralized the inhibitory effect of SLIT2 on T-cell chemotaxis in vitro, and its single- agent treatment led to Pan02 tumor regression in vivo. The functional SLIT2 receptor expressed on T cells, the broader impact of tumoral SLIT2 to the immune contexture within the TME, and the potential synergistic effect of anti-SLIT2/anti-PD-1 combo are subject to further investigation during the F99 phase training. In the K00 phase of this proposal, I would like to expand my study to understand dysfunction of T cells during tumor progression. These studies might include the discovery of additional TME-specific extracellular factors that drive dysfunctional anti-tumor immunity in T-cell inflamed tumors. I propose to establish in vitro HTS platforms to identify candidate targets that (1) promote central memory T-cell phenotype that favors egression into secondary lymphoid organs, (2) reduce T-cell proliferative capacity and induce apoptotic signatures, (3) suppress T-cell effector functions and drive exhaustion phenotype. Multi-omics studies and inducible expression animal models will be utilized in parallel for targ...