Two subsets of regulatory CD8⁺ T cells with differential transcriptome revealed by single cell analysis

Although CD8⁺ regulatory T cells (Tregs) were described in the 1970's, they remain poorly defined compared to CD4⁺ Tregs. Their phenotypic heterogeneity and lack of consensus markers have hindered mechanistic studies and slowed clinical development despite their therapeutic potential. In this study, we performed single-cell RNA sequencing coupled with CITE-seq and TCR-seq on peripheral blood CD8⁺ T cells from four healthy donors, including the CD45RC marker to distinguish pro-inflammatory from pro-regulatory subsets. We analyzed ∼7000 freshly isolated, non-stimulated CD8⁺ T cells and identified two distincts CD8⁺ Tregs subsets, defined by HELIOS or TNFR2 expression, with unique transcriptional and surface marker profiles. Functional assays revealed potent suppressive capacity of the TNFR2⁺CD29^lowCD45RC^low/- subset. These findings were independently validated using a publicly available single-cell dataset from four additional individuals. This work provides the most comprehensive profiling of human CD8⁺ Tregs to date and supports their translation to clinical application.