Single cell RNA-Sequencing of adipose innate T cells for investigation of circadian rhythm.

The circadian rhythm of the immune system helps to protect against pathogens; however, the role of circadian rhythms in immune homeostasis is less well understood. Innate T cells are tissue-resident lymphocytes with key roles in tissue homeostasis4–7. Here we use single-cell RNA sequencing, a molecular-clock reporter and genetic manipulations to show that innate IL-17-producing T cells—including ?d T cells, invariant natural killer T cells and mucosal-associated invariant T cells—are enriched for molecular-clock genes, compared with their IFN?-producing counterparts. We reveal that IL-17-producing ?d T (?d17 T) cells, in particular, rely on the molecular clock to maintain adipose tissue homeostasis, and exhibit a robust circadian rhythm for ROR?t and IL-17A across adipose depots, which peaks at night. In mice, loss of the molecular clock in the CD45 compartment (Bmal1?Vav1) affects the production of IL-17 by adipose ?d17 T cells, but not that of cytokines by aß T or by IFN?-producing ?d T (?dIFN? T) cells. Circadian IL-17 is essential for de novo lipogenesis in adipose tissue, and mice with an adipocyte-specific deficiency in IL-17 receptor C (IL-17RC) have defects in de novo lipogenesis. Whole-body metabolic analysis in vivo shows that Il17a/f-/- mice (which lack the expression of IL-17A and IL-17F) have defects in their circadian rhythm for de novo lipogenesis, which results in disruptions to their whole-body metabolic rhythm and core-body temperature rhythm. This study identifies a crucial role for IL-17 in whole-body metabolic homeostasis, and shows that de novo lipogenesis is a major target of IL-17. Overall design: We performed 10x single cell RNA-Sequencing of ?d T cells and iNKT cells from murine epididymal white adipose tissue. Both innate T cell populations were sorted using FACS and then combined and sequenced as sample. A total of five visceral adipose tissue deposits from five independent biological replicates were pooled for sequencing.