ABSTRACT Head and neck cancer (HNSCC) afflicts mostly men. Female patients who develop HNSCC tend to be postmenopausal, with menopausal hormone therapy lowering their risk of developing the disease. We show that premenopausal female HNSCC patients have an improved response to therapy including immunotherapy (IO). Using clinical trial data, we show no difference in response to IO from testosterone. Similarly, our preclinical data show no IO response effect from gonadal castration. But 0.1 mg estradiol (E2) improves response in castrated male mice to that of female mice. Our overall objective is to understand how the premenopausal female hormone E2 contributes to progression and treatment resistance. Our data show that E2 had no effect on cancer cell viability, suggesting the tumor microenvironment (TME) as the site of action. E2 is known to play a major role in autoimmunity and inflammation, where it modulates dendritic cell (DC) and regulatory T cells (Treg) differentiation and function, and lymphatic vessel maturation. These immune cell populations regulate response to immunotherapy in HNSCC, but how they are affected by sex hormones remains largely uncharacterized. Our preclinical data also show that compared to male mice, females have increased DCs, less suppressive Tregs, and respond better to immunotherapeutic strategies with enhanced Teff function. Such response is removed with oophorectomy (OVX) but rescued with E2 of 0.1mg. OVX also changes the TME towards an immunosuppressive one with enhanced immunosuppressive Treg activity, similar to that of male mice. Finally, within the TME, OVX reduces the formation of high endothelial venules (HEVs) in the TME and draining lymph nodes (dLN), which enhance Teff cell priming and subsequent trafficking into the TME. We hypothesize that the sexually dimorphic response is driven by E2 acting via its receptors on Tregs and DCs to enhance antigen presentation and increase Teff activation, thus selectively determining response to immunotherapy. We also hypothesize that E2 will increase homing and trafficking of antigen specific T cells by enhancing HEV formation and maturation. In Aim1, we will interrogate the regulatory mechanisms of E2's effects on the DC biology by using genetically engineered mouse models (GEMMS), in vitro assays, and proteomic analysis to examine receptor subtypes and molecular mediators of DC differentiation, maturation, and antigen presentation. Aim 2 will dissect mechanisms of how E2's effect on Tregs can drive response and selection of sex-specific immunotherapies. We will test this with GEMMS, mouse chimeras, and pharmacological manipulation in in vivo and in vitro co-culture experiments, to identify signaling and downstream effectors. Focusing on T cell trafficking in Aim 3, we will examine E2's effect on HEV formation, and T cell egress from the dLN to the TME and systemic circulation using a combination of flow and immunofluorescence techniques in GEMMs and in vitro systems. ...