Tissue-resident memory CD8 T cell diversity is spatiotemporally imprinted

Tissue-resident memory CD8 T (T _RM ) cells provide protection from infection at barrier sites. In the small intestine, T _RM cells are found in at least two distinct subpopulations: one with higher expression of effector molecules and another with greater memory potential ¹ . However, the origins of this diversity remain unknown. Here we proposed that distinct tissue niches drive the phenotypic heterogeneity of T _RM cells. To test this, we leveraged spatial transcriptomics of human samples, a mouse model of acute systemic viral infection and a newly established strategy for pooled optically encoded gene perturbations to profile the locations, interactions and transcriptomes of pathogen-specific T _RM cell differentiation at single-transcript resolution. We developed computational approaches to capture cellular locations along three anatomical axes of the small intestine and to visualize the spatiotemporal distribution of cell types and gene expression. Our study reveals that the regionalized signalling of the intestinal architecture supports two distinct T _RM cell states: differentiated T _RM cells and progenitor-like T _RM cells, located in the upper villus and lower villus, respectively. This diversity is mediated by distinct ligand–receptor activities, cytokine gradients and specialized cellular contacts. Blocking TGFβ or CXCL9 and CXCL10 sensing by antigen-specific CD8 T cells revealed a model consistent with anatomically delineated, early fate specification. Ultimately, our framework for the study of tissue immune networks reveals that T cell location and functional state are fundamentally intertwined.