Dissecting Human Tissue Resident Memory T cell Heterogeneity on the Single Cell Level

Abstract

The majority of T cells throughout the human body persist as non-circulating tissue-resident memory T cells (TRM) in lymphoid and mucosal sites, and are vital for orchestrating protective immune responses. However, given the difficulty of sampling healthy human tissues, a full understanding of TRM identity and function across tissue sites remains lacking. Here, we utilize a unique human tissue resource where tissues are obtained from organ donors to dissect CD4+ and CD8+ TRM heterogeneity using high-dimensional flow cytometry and single cell RNA-sequencing (scRNA-seq). We identify remarkable heterogeneity in expression of key surface markers that define TRM, namely CD103, CD101, CD49a, PD-1 and CXCR6, particularly in the lung and the intestines. These phenotypic groups of TRM denote different functional subsets after activation: CD103+ and CD101+ TRM preferentially produce IL-17A, CD49a+ and CXCR6+ TRM show enhanced IFNγ production, and PD-1+ TRM produce granzyme-B. To investigate the transcriptional programming underlying TRM, we profile ~20,000 resting and activated T cells from the lungs and intestines of human organ donors with scRNA-seq and identify TRM based on our previously defined tissue signature. In both tissues, we detect multiple clusters of TRM enriched for expression of individual or a combination of TRM marker genes. Importantly, these clusters show variable expression of genes coding for cytokines (IFNG, IL23A, IL26), transcription factors (TBX21, GATA3, BATF), and cytotoxic molecules (GNLY, GZMs), suggesting differentially regulated TRM subsets. Our results therefore highlight the heterogeneity of TRM across human tissue sites and define the transcriptional programming underlying TRM function.