Single-cell Transcriptomic Analysis Reveals Disparate Effector Differentiation Pathways in Human Treg Compartment [Treg_HD]. Single-cell Transcriptomic Analysis Reveals Disparate Effector Differentiation Pathways in Human Treg Compartment [Treg_HD]

Human FOXP3+ regulatory T (Treg) cells are central to immune tolerance. However, their heterogeneity and differentiation remain incompletely understood. Here we use single-cell RNA and T cell receptor sequencing to resolve Treg cells from healthy individuals and patients with or without acute graft-versus-host disease (aGVHD) who undergo stem cell transplantation. These analyses, combined with functional assays, separate Treg cells into naïve/activated/effector stages, and resolve the HLA-DRhi, LIMS1hi, highly suppressive FOXP3hi, and highly proliferative MKI67hi effector subsets. Trajectory analysis assembles Treg subsets into two differentiation paths (I/II) with distinctive phenotypic/functional programs, ending with the FOXP3hi and MKI67hi subsets, respectively. Transcription factors FOXP3 and SUB1 contribute to some Path I and Path II phenotypes, respectively. These FOXP3hi and MKI67hi subsets and two differentiation pathways are conserved in transplanted patients, despite having functional/migratory impairments under aGVHD. The findings expand the understanding of Treg cell heterogeneity and differentiation and provide a single-cell atlas for the dissection of Treg complexity in health and disease. Overall design: Here, we performed droplet-based scRNA-seq of human FOXP3+ regulatory T (Treg) cells to construct a molecular landscape and dissecting the heterogeneity of Treg cells from healthy donors and transplanted patients with or without acute graft-versus-host disease (aGVHD).

人FOXP3阳性调节性T细胞（FOXP3+ regulatory T cells, Treg）在免疫耐受调控中发挥核心作用。然而，其异质性与分化机制尚未完全阐明。本研究采用单细胞RNA测序与T细胞受体测序技术，对健康个体以及接受干细胞移植且伴/不伴急性移植物抗宿主病（acute graft-versus-host disease, aGVHD）的患者体内的Treg细胞进行解析。结合功能实验，本研究将Treg细胞划分为初始/活化/效应三个阶段，并解析出HLA-DR高表达、LIMS1高表达、具有强抑制功能的FOXP3高表达亚群，以及高增殖活性的MKI67高表达效应亚群。拟时序分析将Treg亚群整合为两条具有独特表型与功能特征的分化通路（通路I与通路II），其终末亚群分别为FOXP3高表达亚群与MKI67高表达亚群。转录因子FOXP3与SUB1分别参与调控通路I与通路II的部分表型特征。尽管伴aGVHD的移植患者体内Treg细胞存在功能与迁移缺陷，但上述FOXP3高表达、MKI67高表达亚群以及两条分化通路在该群体中仍保守存在。本研究结果深化了对Treg细胞异质性与分化机制的认知，并为解析健康与疾病状态下Treg细胞的复杂性提供了单细胞图谱资源。实验设计概述：本研究对人FOXP3阳性调节性T细胞（Treg）开展基于液滴的单细胞RNA测序，以构建分子图谱，并解析健康供者以及接受干细胞移植且伴/不伴急性移植物抗宿主病（aGVHD）的患者体内Treg细胞的异质性。