OBJECTIVES/SPECIFIC AIMS: In the first aim, we will evaluate the proportion of highly HIV-susceptible memory CD4+ T cells present in the rectal mucosa, based on the proliferation status and expression of the HIV susceptibility markers, CCR5 and α4β7, between HIV-negative adolescent MSM and adult MSM engaging in RAI. The second aim will assess differences between the two study groups in the ratio of Th17 cells (CD4+ IL17+) to Treg cells (CD4+ FoxP3+ CD25+) in the rectal mucosa as a determinant of mucosal inflammation. Finally, in the third aim, we will utilize ex vivo rectal biopsy explant challenge experiments to examine whether HIV target cell availability and the Th17/Treg ratio influence rectal mucosal HIV susceptibility. METHODS/STUDY POPULATION: Rectal biopsy specimens are being collected from healthy, HIV-negative men that comprise the two study groups: 40 adolescent MSM 18-21 years of age who have engaged in RAI at least once previously in their lifetime and 40 adult MSM ≥35 years of age who have engaged in RAI for the previous 5 consecutive years with a minimum of 12 episodes annually. To identify CD4+ subsets of interest for aims 1 and 2, rectal mucosal mononuclear cells are isolated and phenotyped with CD45, CD3, CD4, CD45RA, CCR7, CD69, CCR5, α4β7, Ki67, FOXP3, and CD25 antibodies. To identify the Th17 cell subtype, the cells are stimulated with PMA/Ionamycin and stained with an antibody specific to IL-17A. Using cross-sectional analyses, we will compare the frequencies of mucosal CD4+ T cells that express certain phenotypic characteristics and evaluate differences in the Th17/Treg ratio between adolescent and adult MSM. For aim 3, rectal biopsy specimens are inoculated with HIV virus and the culture supernatant is assayed for p24 concentration on days 3, 7, 10 14, and 18. Longitudinal analyses will be performed to detect differences in p24 concentration at each time point and assess associations with mucosal target cell availability and with the Th17/Treg ratio. RESULTS/ANTICIPATED RESULTS: We hypothesize that younger age will be associated with enhanced memory CD4+ T cell proliferation and increased expression of HIV susceptibility markers (CCR5 and/or α4β7). In addition, we expect that the rectal mucosa of adolescent MSM will demonstrate a higher Th17/Treg ratio as compared to adult MSM, which could facilitate HIV transmission. It is also anticipated that rectal mucosal immune phenotypes characterized by increased HIV target cell availability and high Th17/Treg ratios will be associated with enhanced mucosal HIV susceptibility in the explant challenge model. DISCUSSION/SIGNIFICANCE OF IMPACT: There is a paucity of information regarding the mechanisms of rectal HIV transmission, and no studies to date investigate the immunologic effects of aging on transmission in the rectal mucosa. The results from this study will provide important information regarding age-related differences in the immune cell composition of the rectal mucosa as a critical step in better understanding immunologic factors that influence rectal HIV transmission.