Tcf1 controls Treg functions that regulate inflammation, CD8 T-cell cytotoxicity, and severity of colon cancer

Tcf1, the DNA-binding partner of β-catenin, is essential for the development and function of T regulatory cells (Tregs). However, how Tcf1 regulates Treg functional specification is less understood. Here, we ablated Tcf1 in Tregs to elucidate its role in Treg specification in healthy mice and mice with colon cancer. RNAseq and single-cell RNAseq revealed that Tcf1 deficient Tregs maintain their core transcriptional signature and diversity, but promote T-cell receptor, Tgfβ receptor, TH17, and Wnt/β-catenin signaling pathways. Central-memory-Tregs with low Klf2 and effector-Tregs with high Mif expression gain TH17 characteristics and mature into effector Treg subpopulations that strongly express cMaf. Tcf1 deficient Tregs exhibit enhanced suppression of T-cell proliferation and cytotoxicity but are compromised in controlling CD4+ T-cell polarization and inflammation. In mice with polyposis, Tcf1 deficient Tregs promote inflammation and tumor growth. Thus, Tcf1 determines Treg functions that regulate TH17 inflammation and the course and outcome of colorectal cancer. CD25+YFP+ Tregs were FACS sorted from MACS-pre-purified CD4+ T cells (mouse CD4+ T Cell Isolation Kit, Miltenyi) isolated from MLN of Foxp3Cre and Foxp3CreTcf7fl/fl mice. Total RNA was isolated using the PicoPure RNA Isolation Kit (Arcturus) following the manufacturer’s instructions. Libraries were generated and sequenced by the University of Chicago Genomics Facility. Samples from Foxp3Cre and Foxp3CreTcf7fl/fl mice are prepared and sequenced in triplicates. Whole live cells were washed twice in 1x PBS + 0.04% BSA and immediately submitted to the Core for Single Cell sorting. For single-cell RNAseq, the cells were first counted and measured for viability using the Vi-Cell XR Cell Viability Analyzer (Beckman-Coulter), as well as a basic hemocytometer with light microscopy. The barcoded Gel Beads were thawed from -80C and the reverse transcription master mix was prepared according to the manufacture’s instruction for Chromium Single Cell 3’ v2 library kit (10x Genomics). Tregs from the mesenteric lymph nodes (MLN) from four different genotypes of mice (n=2 replicates) aged to 5.5 month: Foxp3CreTcf7fl/fl and its control Foxp3Cre mice, the polyposis prone APC468 mice and control WT C57BL/6J (B6) mice are sequenced. Each sample was uniquely indexed.