Single-cell expression profiling of visceral adipose tissue and splenic CD4+ T cells from Treg-bmal1WT and Treg-bmal1KO mice at ZT0 and ZT12.

we reported single-cell gene expression of CD4+ T cells from the visceral adipose tissue of from male Foxp3-Cre.YFP bmal1WT or Foxp3-Cre.YFP bmal1flox/flox mice, and from spleen from bmal1WT mice, at ZT0 or ZT12. We found that VAT Tregs could be subdivided into five subtypes: p1 ST2+, p2 ST2+, Tbet+, IL18r+ and resting clusters. We found diurnal variation within the ST2+ subgroups, where the more activated p1 ST2+ Tregs were more represented at ZT0 in WT VAT Tregs. Such variations were not observed in splenic Tregs. In contrast, bmal1KO VAT Tregs were were enriched in the ST2+ Tregs, and had a constitutively high proportion of the p1 ST2+ subtype at ZT0 and ZT12. Animals were housed in reverse-light cycle rooms. 34,000 VAT and 32,000 splenic CD4+ T cells were single-sorted. Cells from each genotype, time-point and tissue were multiplexed using TotalSeq hashing antibodies separately and then pooled. Cell encapsulation, RNA isolation, and hash library construction and sequencing were performed using the 10X Genomics platform.