PROJECT SUMMARY/ABSTRACT There are approximately 1.3 million transgender adults in the US, and about 467,000 of these individuals (~36%) are transgender men. Transgender men are individuals who were assigned female at birth but identify as male. Trans men may transition physiologically from female to male by receiving masculinizing hormone therapy and/or hysterectomy. Those in the trans male community participate in diverse sexual behaviors and lifestyles resulting in unique risks to STIs, especially HIV-1. Currently, there is a significant knowledge gap of the impact of HIV-1 on trans men, including limited knowledge regarding the effects of testosterone therapy on HIV-1 susceptibility and acquisition. Over 70% of trans men receive testosterone to promote masculine characteristics and reduce secondary female sex characteristics. Trans men treated with testosterone report symptoms of vaginal dryness and loss of elasticity, which increase mucosal tissue breaks, which contribute to increased risk of HIV-1 transmission in trans men. Trans men treated with testosterone for at least one year have significantly reduced levels of Lactobacillus comprising the vaginal microbiome, which correlates with bacterial vaginosis, and thus increased risk of HIV transmission. Like other androgens, testosterone is a steroid hormone that interacts with many different cell types, broadly affecting both innate and adaptive immunity through its effect on toll-like receptors, immune-response cells, and pro- and anti-inflammatory cytokines. Testosterone has broad-ranging effects on adaptive and innate immune functions and acts in a dynamic and often antagonistic manner with other androgens, particularly dehydroepiandrosterone (DHEA), to modulate the development and function of immune response cells. The central HYPOTHESIS of this research proposal is that testosterone alters cellular and immunologic responses in the cervical mucosa that affect susceptibility to HIV-1 infection. To interrogate this hypothesis, we propose to characterize certain cellular and innate immunologic properties of cervical mucosal tissue obtained from transgender men receiving gender-affirming masculinizing therapy, and undergoing medically indicated hysterectomies, and to correlate these findings to tissue susceptibility to HIV-1 infection ex vivo. We anticipate identifying specific alterations in the cervical mucosa that correlate with testosterone therapy and altered susceptibility to HIV-1 infection. If successful, our findings will provide new underpinnings for future hypothesis-driven research focused on HIV-1 prevention strategies for transgender men. The research proposed in this R21 grant application is guided by the following SPECIFIC AIMS: 1. Determine the effects of testosterone on the susceptibility of cervical explant tissue to HIV-1 infection and populations of T lymphocytes; and 2. Determine the effects of testosterone treatment on cytokine and chemokine expression in cervical tiss...