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

Tcf1, the DNA-binding partner of ß-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, Tgfß receptor, TH17, and Wnt/ß-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. Overall design: 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.

Tcf1是β-连环蛋白（β-catenin）的DNA结合伴侣，对调节性T细胞（Tregs）的发育与功能至关重要。然而，目前对于Tcf1如何调控Treg功能特化的机制尚不完全明晰。本研究在Tregs中特异性敲除Tcf1，以阐明其在健康小鼠及结肠癌小鼠中对Treg特化的调控作用。通过RNA测序（RNA-seq）与单细胞RNA测序（scRNA-seq）分析发现，Tcf1缺陷的Tregs可维持其核心转录特征与细胞多样性，但能够增强T细胞受体、转化生长因子β受体、辅助性T细胞17（Th17）以及Wnt/β-连环蛋白信号通路的活性。具体而言，低表达Kruppel样因子2（Klf2）的中枢记忆型Tregs，以及高表达巨噬细胞迁移抑制因子（Mif）的效应性Tregs，将获得Th17细胞特征，并分化为强表达c-Maf转录因子的效应性Treg亚群。Tcf1缺陷的Tregs对T细胞增殖与细胞毒性的抑制能力增强，但在调控CD4+ T细胞极化及炎症反应方面存在功能缺陷。在息肉易感型小鼠模型中，Tcf1缺陷的Tregs会促进炎症反应与肿瘤生长。综上，Tcf1可通过调控Treg功能，进而影响Th17相关炎症以及结直肠癌的病程与转归。

总体实验设计：从Foxp3-Cre重组酶工具小鼠（Foxp3Cre）与Foxp3-Cre介导的Tcf7条件性敲除小鼠（Foxp3CreTcf7fl/fl）的肠系膜淋巴结（MLN）中，分离经磁激活细胞分选（MACS）预纯化的CD4+ T细胞（小鼠CD4+ T细胞分离试剂盒，美天旎Miltenyi），再通过荧光激活细胞分选（FACS）分选出CD25+YFP+ Tregs。按照制造商说明书，使用PicoPure RNA提取试剂盒（Arcturus）提取总RNA。文库构建与测序工作由芝加哥大学基因组学核心实验室完成。Foxp3Cre与Foxp3CreTcf7fl/fl小鼠的样本均设置3次生物学重复并进行测序。将完整活细胞用1×PBS+0.04%牛血清白蛋白（BSA）洗涤两次后，立即送至单细胞分选核心实验室。对于单细胞RNA测序，首先使用Vi-Cell XR细胞活力分析仪（贝克曼库尔特Beckman-Coulter）以及光学显微镜下的血细胞计数板，对细胞进行计数并检测细胞活力。将冻存于-80℃的带条形码的凝胶珠解冻，按照Chromium单细胞3'端v2文库制备试剂盒（10x Genomics）的制造商说明书制备反转录预混液。本研究对4种基因型的5.5月龄小鼠（每组n=2个生物学重复）的肠系膜淋巴结Tregs进行测序，分别为Foxp3CreTcf7fl/fl小鼠及其对照Foxp3Cre小鼠、息肉易感型APC468小鼠及其对照野生型C57BL/6J（B6）小鼠。每个样本均带有唯一的索引标签。