Establishment of a vaginal organoid system to study local resident memory T cell differentiation

Abstract

Abstract Over a million sexually transmitted diseases (STDs) are acquired every day worldwide, many of which preferentially target the female reproductive tract (FRT). Resident memory T cells (TRM) in the vaginal mucosa have proven protective roles against these STDs. Accordingly, a crucial objective of STD vaccinations is to establish a higher quantity and quality of TRM in the FRT. The vaginal epithelium has been implicated in regulation of local TRM differentiation and function, but the biological nature of this interaction has not been investigated. Here, we established a murine three dimensional (3D) vaginal organoid-CD8 coculture system to examine epithelial-T cell interactions at the molecular level. Differentiation of single epithelial stem cells led to the formation of 3D epithelial organoids. These organoids can be maintained long-term through serial passage and structurally resembled vaginal epithelium with a multilayer specialized epithelium. After culturing activated CD8 T cells with the organoids, we assessed the transcriptional and phenotypic composition of the T cells. We found that cocultured CD8 T cells were integrated into the extracellular matrix and acquired phenotypic and functional traits of in vivo TRM. This TRM-biased differentiation could be suppressed by a small molecule inhibitor of the cytokine TGF-β. Overall, the vaginal organoid coculture model recapitulates the in vivo environment far better than traditional 2D cell culture, while retaining high-throughput abilities. Importantly, it will enable highly reductionist experiments to examine the complex epithelial cell-T cell crosstalk to devise targeted approaches for improving CD8 T cell mediated immunosurveillance in the FRT. Supported by grants from NIH (2P20GM109035 & P20GM121298) and by the Searle Scholars award.