Single cell and spatial transcriptomics analysis of kidney double negative T lymphocytes in normal and ischemic mouse kidneys [spatial transcriptomics]

T cells are important in the pathogenesis of acute kidney injury (AKI), and TCR+CD4-CD8- (double negative-DN) are T cells that have a regulatory role. However, little is understood about these cells in comparison to traditional CD4+ and CD8+ cells. To elucidate the molecular and spatial dynamics of DN T cells during AKI, we performed single-cell RNA sequencing (scRNA-seq) on sorted murine DN, CD4+, and CD8+ cells, combined with spatial transcriptomic profiling of normal and post-AKI mouse kidneys. scRNA-seq revealed distinct transcriptional profiles for DN, CD4+, and CD8+ T cells of mouse kidneys, with enrichment of Kcnq5, Klrb1c, Fcer1g, and Klre1 expression in DN T cells compared to CD4+ and CD8+ T cells in normal kidney tissue. We validated the expression of these genes in mouse and human kidney DN, CD4+ and CD8+ T cells using RT-PCR and in the NIH kidney precision medicine project (KPMP). Spatial transcriptomics in normal and ischemic mouse kidney tissue showed a localized cluster of T cells in the outer medulla expressing DN T cell genes including Fcer1g. These results provide a template for future studies in DN T as well as CD4+ and CD8+ cells in the normal and diseased kidney. Overall design: Normal kidneys and kidneys treated with ischemic reperfusion injury were analyzed using spatial transcriptomics.