IL-33 signaling alters regulatory T cell diversity in support of tumor development

Regulatory T cells (Tregs) can impair anti-tumor immune responses and are associated with poor prognosis in multiple cancer types. Tregs in human tumors span diverse transcriptional states distinct from those of peripheral Tregs, but their contribution to tumor development remains unknown. Here, we use single cell RNA-Seq to longitudinally profile dynamic shifts in the distribution of Tregs in a genetically-engineered mouse model of lung adenocarcinoma. In this model, interferon-responsive Tregs are more prevalent early in tumor development, while a specialized effector phenotype characterized by enhanced expression of the interleukin 33 receptor ST2 is predominant in advanced disease. Treg-specific deletion of ST2 alters the evolution of effector Treg diversity, increases infiltration of CD8+ T cells into tumors, and decreases tumor burden. Our study shows that ST2 plays a critical role in Treg-mediated immunosuppression in cancer, highlighting potential paths for therapeutic intervention. Tconv (DAPIneg, i.v.neg, Thy1.2+CD4+Foxp3-GFPneg) and Treg (DAPIneg, i.v.neg, Thy1.2+CD4+Foxp3-GFPpos) cells were single-cell sorted into Buffer TCL (Qiagen) plus 1% β-mercaptoethanol in 96-well plates using a MoFlo Astrios cell sorter. Each plate had 30-100 cell population well and an empty well as controls. Following sorting, plates were spun down for 1” at 2000 RPM and frozen immediately at -80C. Plates were thawed and RNA was purified using 2.2X RNAclean SPRI beads (Beckman Coulter) without final elution (Shalek et al., 2013). SMART-seq2 and Nextera library preparation was performed as previously described (Picelli et al., 2013), with some modifications as described in a previous study (Singer et al., 2017). Plates were pooled into 384 single-cell libraries, and sequenced 50 x 25 paired end reads using a single kit on the NextSeq500 5 instrument.