Epithelial organoid supports resident memory CD8 T cell differentiation

Highlights•Vaginal epithelial stem cells form organoids mimicking in vivo vaginal epithelium•CD8 T cells co-cultured with organoids differentiate into resident memory CD8 T cells•In vitro CD8 TRMs phenotypically and transcriptionally resemble in vivo epithelial TRMs•VEO-CD8 co-culture model can be interrogated to reveal fundamental TRM biologySummaryResident memory T cells (TRMs) play a vital role in regional immune defense. Although laboratory rodents have been extensively used to study fundamental TRM biology, poor isolation efficiency and low cell survival rates have limited the implementation of TRM-focused high-throughput assays. Here, we engineer a murine vaginal epithelial organoid (VEO)-CD8 T cell co-culture system that supports CD8 TRM differentiation. These in-vitro-generated TRMs are phenotypically and transcriptionally similar to in vivo TRMs. Pharmacological and genetic approaches showed that transforming growth factor β (TGF-β) signaling plays a crucial role in their differentiation. The VEOs in our model are susceptible to viral infections and the CD8 T cells are amenable to genetic manipulation, both of which will allow a detailed interrogation of antiviral CD8 T cell biology. Altogether we have established a robust in vitro TRM differentiation system that is scalable and can be subjected to high-throughput assays that will rapidly add to our understanding of TRMs.Graphical abstract