A distinct subpopulation of follicular helper T (Tfh) cells promotes Tfr cell differentiation by producing Sostdc1. A distinct subpopulation of follicular helper T (Tfh) cells promotes Tfr cell differentiation by producing Sostdc1

By using RNA-sequencing assay on sorted Sostdc1-EGFP- Tfh, Sostdc1+ Tfh and Non-Tfh cells and single cell RNA-seq assay on sorted CD4+CD44+T cells from Sostdc1-EGFP reporter mice, support the conclusion that Sostdc1+ Tfh cells are a distinct subpopulation of Tfh cells.After performing the RNA-seq assessment and ATAC-seq on Tfr and Treg cells from WT and Sostdc1 KO mice,demonstrated that Sostdc1 ablation predominantly augmented β-catenin-controlled transcriptome, which in turn resulted in Tfr cell failure. Overall design: We conducted RNA-seq assay on sorted Sostdc1-EGFP- Tfh, Sostdc1+ Tfh, and Non-Tfh cells with two biological replicates per group.CXCR5+PD1+ GITRhi Tfr and CXCR5- PD1- GITRhi Treg cells were sorted from WT and Sostdc1 KO mice and then subjected to RNA-seq and ATAC-seq assessment. CD44+CD4+ T cells were sorted from heterozygous Sostdc1-EGFP reporter mice and then analyzed by single cell RNA-seq (10xgenomics).

本研究通过对来自Sostdc1-EGFP报告基因小鼠的分选后Sostdc1-EGFP阴性滤泡辅助性T细胞（T follicular helper cell, Tfh）、Sostdc1阳性Tfh细胞及非Tfh细胞开展RNA测序（RNA-sequencing, RNA-seq）分析，并对分选获得的CD4+CD44+ T细胞进行单细胞RNA测序，证实Sostdc1+ Tfh细胞是一类独立的Tfh细胞亚群。
本研究同时对野生型（wild type, WT）与Sostdc1基因敲除（knockout, KO）小鼠来源的滤泡调节性T细胞（T follicular regulatory cell, Tfr）和调节性T细胞（regulatory T cell, Treg）进行RNA测序与转座酶可及性测序（assay for transposase-accessible chromatin using sequencing, ATAC-seq），结果显示Sostdc1敲除主要增强了β-连环蛋白（β-catenin）调控的转录组，进而引发Tfr细胞功能缺陷。
实验整体设计如下：1. 对分选得到的Sostdc1-EGFP阴性Tfh、Sostdc1+ Tfh及非Tfh细胞开展RNA测序，每组设置2次生物学重复；2. 从野生型与Sostdc1基因敲除小鼠中分别分选CXCR5+PD1+ GITRhi Tfr细胞与CXCR5-PD1-GITRhi Treg细胞，进行RNA测序与ATAC-seq分析；3. 从杂合子Sostdc1-EGFP报告基因小鼠中分选CD44+CD4+ T细胞，采用10x Genomics平台完成单细胞RNA测序分析。