Abstract Resistance to radiation therapy (RT) remains a challenging problem for high-risk head and neck squamous cell carcinoma (HNSCC) patients. Anti-PD1 is now approved in the first- and second-line settings, indicating that the majority of these tumors are resistant to treatment. The field awaits the results of multiple trials testing whether combining immunotherapy and RT can help avoid resistance to RT, but many preclinical studies (including those from our lab) indicate that treatment resistance still develops despite such combinations. The preferential recruitment of unique immunosuppressive cell subtypes after RT to the tumor microenvironment (TME) in response to RT plays an important role in contributing to the promotion of tumor growth and progression. The tumor endothelium can act as a selective barrier that regulates the entry, stability, and activation status of immune cells, but the mechanistic underpinnings of this barrier activity remain poorly understood. The EphB4 receptor tyrosine kinase and its ligand ephrinB2 define novel molecular targets. Upon cell to cell contact, both EphB4 and ephrinB2 can signal via their cytoplasmic domains. Though they have been extensively studied in cell migration and angiogenesis in early embryonic development, little has been published on their role in modulating the cancer immune microenvironment. Our data show that ephrinB2 is expressed on the tumor vasculature and is upregulated by RT and that EphB4 is expressed on immunosuppressive regulatory T cells (Tregs). Inhibiting EphB4-ephrinB2 interaction with a peptide (TNYL-RAW) results in the selective, and exclusive, reduction in infiltration of Tregs while increasing activation of CD8+ and CD4+Foxp3- T cell populations. Tregs, we have demonstrated, play a key role in the development of resistance to RT, and their depletion re-establishes responsiveness to therapy in HNSCC murine models. We hypothesize that RT’s upregulation of ephrinB2 on tumor endothelial cells acts preferentially on EphB4 expressing Tregs, and blocking this interaction reduces their intratumoral infiltration and survival, which allows CD8+ and CD4+ effector cells to induce tumor regression. In Aim 1, we will analyze the mechanistic outcomes of Treg EphB4 and endothelial ephrinB2 interaction on Treg transendothelial trafficking, intratumoral homing, and survival using genetically engineered animal models with ephrinB2 deleted on endothelial cells or EphB4 deletion on Tregs after RT. In Aim 2, we will interrogate the cellular and molecular mechanisms triggered by the interaction between endothelial ephrinB2 and EphB4- expressing Tregs in endothelial-Treg co-culture assays after RT. Targeted inhibition of STAT3, AKT, Erk pathways will be done with pharmacological inhibitors based on our preliminary data. We expect that these studies will elucidate the molecular and cellular parameters of EphB4-ephrinB2 inhibitors and will provide the necessary information to develop a more effectiv...