PROJECT SUMMARY Until recently, the specific HIV prevention needs of transgender populations received insufficient attention, and the biology of HIV transmission, specifically, has been understudied to date. Transgender women who have sex with men (TGWSM) are at elevated risk for HIV acquisition with global HIV prevalence rates approximately 20% and odds of infection 48 times the general population. Engaging in condomless receptive anal intercourse (CRAI) is common among TGWSM, and the physiologic efficiency of HIV transmission across the rectal mucosa facilitates HIV transmission. Historically, TGWSM have been grouped with men who have sex with men (MSM) in HIV prevention studies due to presumed similar risks of rectal HIV exposure despite their unique psychosocial, biologic, and prevention needs. From a biologic perspective, many TGWSM use cross-sex hormone therapy with uncertain rectal mucosal effects. The effects of endogenous and exogenous hormones in the human and animal-model female genital tract has been described with estrogen generally being seen as hindering HIV transmission and progesterone facilitating transmission; however, few studies report effects on the rectal mucosa. In addition, the intestinal mucosa is known to be steroidogenic, and colonic epithelial cells express estrogen receptor β, suggesting that exogenous hormone therapy likely has an effect on the rectal mucosa that could influence HIV transmission. In this application, we will build upon our successful translational mucosal immunology program with a highly successful clinical research and retention infrastructure that was designed to understand factors that may influence rectal HIV transmission and propose to examine the effects of cross-sex hormone therapy on the rectal mucosal resident cellular populations, transcriptome, and microbiome in TGWSM. In addition, we will capitalize on the existing infrastructure of the Emory/CDC HIV/AIDS Clinical Trials Unit and expand our studies to an international site, Bangkok, in order to make global comparisons in our findings. In aim 1, we will compare HIV target cell availability in 1) TGWSM on estrogen therapy, 2) TGWSM on estrogen + progesterone therapy, and 3) cisgender MSM. We will also examine HIV target cell availability in TGWSM before and after initiating cross-sex hormone therapy in a longitudinal cohort. In aim 2, we will compare the transcriptome by RNA-seq between groups in the cross- sectional cohort and before and after initiating cross-sex hormone therapy in the longitudinal cohort in order to identify new targets for biomedical HIV prevention interventions. Finally, in aim 3, we will define the differences in the rectal mucosal microbiota associated with cross-sex hormone therapy in order to inform the design of future HIV prevention intervention clinical trials. The overarching goal of this proposal is to achieve a better understanding of the rectal mucosal effects of cross-sex hormones that will allow for the o...