Multimodal profiling reveals site-specific adaptation and tissue residency hallmarks of ?d T cells across organs in mice

?d T cells perform heterogeneous functions in homeostasis and disease across tissues. However, it is unclear whether these roles correspond to distinct ?d subsets or to a homogeneous population of cells exerting context-dependent functions. Here, by cross-organ multimodal single-cell profiling, we reveal that various mouse tissues harbor unique site-adapted ?d subsets. Epidermal and intestinal intraepithelial ?d T cells are transcriptionally homogeneous and exhibit epigenetic hallmarks of functional diversity. Through parabiosis experiments, we uncovered cellular states associated with cytotoxicity, innate-like rapid IFN-? production and tissue repair functions displaying tissue residency hallmarks. Notably, our observations nuance the link between IL-17-producing ?d T cells and tissue residency. Moreover, transcriptional programs associated with tissue-resident ?d T cells are analogous to those of CD8+ tissue-resident memory T cells. Altogether, this study provides the first multimodal landscape of tissue-adapted ?d T cells, revealing heterogeneity, lineage relationships and their tissue residency program. Overall design: To identify and compare various subsets of ?d T cells present across different tissues, we took a single-cell multimodal approach and sorted gd T cells from lymph nodes, spleen, liver, lung, skin, small and large intestine using flow cytometry. Sorted cells were subjected to single-cell sequencing using two different approaches. In the first approach, ?d T cells from different organs were barcoded with distinct oligonucleotide-conjugated antibodies as well as 26 other antibodies. The transcriptomes and epitopes were simultaneously profiled using single-cell RNA sequencing (scRNA-seq) and TotalSeq, respectively. In the second approach, simultaneous single-nucleus RNA sequencing (snRNA-seq) and assay for transposase-accessible chromatin using sequencing (scATAC-seq) were performed on pooled ?d T cells from different organs without sample barcoding. Furthermore, simultaneous profiling of RNA and gamma delta T cell receptor (TCR) profiling was also performed at a single-cell resolution.