Single-cell transcriptomic analysis reveals disparate effector differentiation pathways in human T reg compartment

Human FOXP3 + regulatory T (T reg ) 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 T reg 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 T reg cells into naïve, activated, and effector stages, and resolve the HLA-DR hi , LIMS1 hi , highly suppressive FOXP3 hi , and highly proliferative MKI67 hi effector subsets. Trajectory analysis assembles T reg subsets into two differentiation paths (I/II) with distinctive phenotypic and functional programs, ending with the FOXP3 hi and MKI67 hi subsets, respectively. Transcription factors FOXP3 and SUB1 contribute to some Path I and Path II phenotypes, respectively. These FOXP3 hi and MKI67 hi subsets and two differentiation pathways are conserved in transplanted patients, despite having functional and migratory impairments under aGVHD. These findings expand the understanding of T reg cell heterogeneity and differentiation and provide a single-cell atlas for the dissection of T reg complexity in health and disease.