Tissue-resident memory CD8 T Cell Diversity is Spatiotemporally Imprinted

Tissue-resident memory CD8 T cells (TRM) provide protection from infection at barrier sites. In the small intestine, TRM cells are found in at least two distinct subpopulations: one with higher expression of effector molecules and another with greater memory potential. However, the origins of this diversity remain unknown. We proposed that distinct tissue niches drive TRM phenotypic heterogeneity. To test this, we leveraged spatial transcriptomics of human samples, a murine model of acute systemic viral infection, and a newly established strategy for pooled optically-encoded gene perturbations to profile the location, interaction, and transcriptome of pathogen-specific TRM 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 intestinal architecture’s regionalized signaling supports two distinct TRM cell states: differentiated TRM and progenitor-like TRM cells, located in the upper versus lower villus, respectively. This diversity is mediated by distinct ligand-receptor activities, cytokine gradients, and specialized cellular contacts. Blocking TGFb or Cxcl9/10-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 has revealed that T cell location and functional state are fundamentally intertwined. Spatial transcriptomics (Xenium) of mouse small intestine at day 6, 8, 30 and 90 after infection with LCMV and adoptive transfer of P14 CD8 T cells. Spatial transcriptomics (Xenium) of uninfected mouse small intestine. Spatial transcriptomics (Xenium) of mouse small intestine at day 7 after infection with LCMV and adoptive transfer of perturbed P14 CD8 T cells. Spatial transcriptomics (MERSCOPE) of mouse small intestine at day 8 after infection with LCMV and adoptive transfer of Tgfbr2 KO or WT P14 T cells. Spatial transcriptomics (Xenium) of human ileum biopsies.