Transcriptional profiling of mouse ST2+ and ST2- Tregs across tissues

Foxp3+ regulatory T cells (Tregs) are critical mediators of peripheral tolerance and immune homeostasis. Tregs that express the IL-33 receptor ST2 are enriched in peripheral nonlymphoid tissues and can exert a variety of tissue-specific functions from metabolic regulation within adipose tissue to skeletal muscle repair. However, the relationship between ST2+ and ST2- Tregs within and across different tissues remains unclear. To compare murine ST2- and ST2+ Tregs within and across tissues, we performed RNA sequencing (RNAseq) of ST2-CD44hi and ST2+CD44hi Tregs from blood, spleen, lungs, visceral adipose tissue (VAT), colon, and skin. RNAseq was also performed on ST2- CD44lo CD62L+ Tregs from the spleen and lungs. We found that the tissue microenvironment was the major factor shaping the transcriptome of Tregs across tissues. Across the tissues studied, Treg transcriptomes displayed an ordered hierarchy that may represent graded levels of activation or differentiation across tissues. We also identified a core signature that distinguished ST2+ Tregs from ST2- Tregs across tissues and a large number of differentially expressed genes between ST2- and ST2+ Tregs within individual tissues that could support the tissue-specific adaptation and function of ST2+ Tregs. In summary, our work highlights the unique, tissue-specific phenotype of ST2+ Tregs and reveals a core ST2+ Treg transcriptional signature shared across tissues. Single cell suspensions were prepared from the blood, spleen, lungs, abdominal visceral adipose tissue (VAT), colonic lamina propria, and back skin of five Foxp3YFP-cre mice. ST2+CD44hi Tregs and ST2-CD44hi Tregs were flow sorted from all tissues; ST2-CD44loCD62L+ Tregs were sorted from spleen and lungs. RNAseq was performed on libraries prepared from amplified cDNA prepared from the sorted populations. Mouse IDs are provided so that samples from a single mouse can be identified.