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. Tumors were microdissected and dissociated into single cell suspensions. CD45+ and CD45- cells were then magnetically separated using MACS CD45 MicroBeads (Miltenyi Biotec) as per manufacturer’s instructions. CD45+ and CD45- single cells were processed through the 10X Genomics Single Cell 3' platform using the Chromium Single Cell 3′ Library & Gel Bead Kit V2 kit (10X Genomics), per manufacturer’s protocol. Briefly, 6,000 cells were loaded onto each channel. Cell lysis and barcoding occur, followed by amplification, fragmentation, adaptor ligation and index library PCR. Libraries were sequenced on an Illumina HiSeqX at a read length of 98 base pairs. De-multiplexing, alignment to the mm10 transcriptome and unique molecular identifier (UMI)-collapsing were performed using the Cellranger toolkit from 10X Genomics version 1.1.0. Please note that the counts data is from both samples together, and the cell name entails both the cell barcode and also which sample this cell came from.