Epithelial organoid supports resident memory CD8 T cell differentiation

Resident Memory T cells (TRM) play a vital role in regional immune defense in barrier organs. Although laboratory rodents have been extensively used to study fundamental TRM biology, issues like poor isolation efficiency, sampling bias and low survival rates have limited our ability to conduct TRM-focused high-throughput assays. Here we engineered a murine vaginal epithelial organoid (VEO)-CD8 T cell coculture system that supports CD8 TRM differentiation in vitro. The three-dimensional VEOs established from adult stem cells resembled stratified squamous vaginal epithelium and induced gradual differentiation of activated CD8 T cells into epithelial TRM. These in vitro generated TRM were phenotypically and transcriptionally similar to in vivo TRM and their epithelial residence phenotype (CD69+ CD103+) was further enhanced with a second cognate-antigen exposure during coculture. TRM differentiation was not affected even when VEOs and CD8 T cells were separated by a semipermeable barrier, indicating soluble factors' involvement. Pharmacological and genetic approaches showed that TGF-β signaling played a crucial role in their differentiation. We found that the VEOs in our model remain susceptible to viral infections and the CD8 T cells were amenable to genetic manipulation; both of which will allow detailed interrogation of antiviral CD8 T cell biology in a reductionist setting. In summary, we established a robust model which captures bonafide TRM differentiation that is scalable, open to iterative sampling, and can be subjected to high throughput assays that will rapidly add to our fundamental understanding of TRM.