Extracellular vesicles from human semen induce unique tolerogenic phenotypes in vaginal dendritic cells and regulatory T lymphocytes

Shahrokh Paktinat¹Michael G Gravett¹Cara Tobey¹Anna Kirby¹Whitney Horner¹Rebecca Shaffer¹Michael Fialkow¹Nam Phuong Nguyen¹German Gornalusse¹Maryam Kalatehjari¹Sean M Hughes¹Florian Hladik^1,2Lucia Vojtech^1*

• ¹University of Washington, Seattle, United States
• ²Fred Hutchinson Cancer Center, Seattle, Washington, United States

The regulation of immune responses to promote tolerance to the fetus is critical for successful pregnancy. An understudied aspect of this process is the initiation of regulation pre-conception via exposure to semen. Our study aimed to understand how semen impacts recipient dendritic cells (DCs) and their subsequent role in shaping CD4 T cell differentiation. Monocyte-derived DCs (MoDCs) were exposed to semen extracellular vesicles (SEV) or vesicle-depleted semen plasma (VDSP). Phenotypic and functional markers were analyzed using flow cytometry. MoDCs exhibited a significant increase of CD141, CD1a, CD38, and ILT4 expression when exposed to SEV or VDSP. A unique feature of semen-treated MoDCs was expression of indoleamine 2,3-dioxygenase (IDO), a potent contributor to the induction of regulatory T cells (Tregs). We also exposed epithelial sheets from vaginal tissue to SEV and VDSP, and measured the number and marker expression of emigrating cells. SEV but not VDSP significantly increased the emigration of intraepithelial DCs. Additionally, SEV significantly enhanced the expression of multiple immunoregulatory markers in the emigrated DCs. Finally, we tested how SEV-or VDSP-exposed DCs altered CD4 T cell differentiation by co-culturing exposed MoDCs or tissue emigrated cells with autologous naïve CD4 T cells. After co-culture, we observed significantly more FOXP3+ Tregs expressing high levels of TIGIT in the groups that were initially exposed to SEV. These findings indicate that exposure to SEV induces a tolerogenic program in DCs that can direct differentiation of a unique memory Treg subset, primed for expansion and presumably destined to support a successful pregnancy.