Interrogating mucosal resident memory CD8 T cell biology with 3D organoids

ABSTRACT
Resident Memory CD8 T cells (TRM) in the female reproductive tract (FRT) have a proven protective role
against viral infections. As such positioning of CD8 TRM of high quantity and quality that are durably
maintained is a key goal to achieve protective antiviral immunity in the FRT. Detailed understanding of
molecular cues that guide FRT TRM differentiation is essential to attain this objective. Cells in the local
environment i.e., reproductive mucosa is thought to be a big source of signals that shape CD8 TRM
differentiation. Rodent models have emerged as key to understanding these molecular signals and local
interactions. However, the complex nature of the in vivo vaginal microenvironment along with technical issues
associated with inefficient FRT TRM isolation process have limited execution of high throughput studies
focused on identifying these cellular communications. We have established an in vitro three-dimensional
vaginal epithelial organoid system (VEO) that accurately captures the features of in vivo multilayered stratified
vaginal epithelium. By culturing these VEOs with CD8 T cells, we were able to induce CD8 TRM differentiation
and the resulting TRM phenotypically and transcriptionally resembled antiviral TRM generated in mouse. We
aim to leverage this VEO-CD8 coculture model to rapidly uncover fate-specifying regulators of FRT TRM and
investigate fundamental interactions between the vaginal epithelium and CD8 T cells that govern TRM
differentiation. In aim-1, we will execute a targeted RNAi screening approach to rapidly define transcription
factors (TFs) that instruct TRM formation in the VEO system. We have previously found that the TF Runx3
supports TRM differentiation in diverse non-lymphoid tissues, and here, we will evaluate an unappreciated role
for Runx3 in driving FRT TRM differentiation in vitro. Lastly, transcriptional profiling of VEO-induced TRM found
an undescribed role for retinoic acid (RA) in promoting TRM formation in the vaginal epithelium. In aim-2, we
will utilize the VEO-CD8 coculture system as well as in vivo infection models to test if CD8 T cell intrinsic or
extrinsic RA signaling regulates TRM formation. The proposed study will establish a robust reductionist
alternative to the in vivo mouse models currently in use and will provide novel mechanistic insights into
epithelial-CD8 T cell interaction in the vaginal mucosa.

Grant Number: 5R21AI183017-02
NIH Institute/Center: NIH
Principal Investigator: Lalit Beura