Identification of human exTreg cells as CD16+CD56+ cytotoxic CD4+ T cells

In atherosclerosis, some regulatory T (T-reg) cells become exT(reg) cells. We crossed inducible T-reg and exT(reg) cell lineage-tracker mice (FoxP3(eGFP-Cre-ERT2)ROSA26(CAG-fl-stop-fl-tdTomato)) to atherosclerosis-prone Apoe(-/-) mice, sorted T-reg cells and exT(reg) cells and determined their transcriptomes by bulk RNA sequencing (RNA-seq). Genes that were differentially expressed between mouse T-reg cells and exT(reg) cells and filtered for their presence in a human single-cell RNA-sequencing (scRNA-seq) panel identified exT(reg) cell signature genes as CST7, NKG7, GZMA, PRF1, TBX21 and CCL4. Projecting these genes onto the human scRNA-seq with CITE-seq data identified human exT(reg) cells as CD3(+)CD4(+)CD16(+)CD56(+), which was validated by flow cytometry. Bulk RNA-seq of sorted human exT(reg) cells identified them as inflammatory and cytotoxic CD4(+)T cells that were significantly distinct from both natural killer and T-reg cells. DNA sequencing for T cell receptor-beta showed clonal expansion of T-reg cell CDR3 sequences in exT(reg) cells. Cytotoxicity was functionally demonstrated in cell killing and CD107a degranulation assays, which identifies human exT(reg) cells as cytotoxic CD4(+)T cells. In inflammation, some regulatory T (T-reg) cells lose FoxP3 expression and become exT(reg) cells. Ley and colleagues mapped mouse T-reg and exT(reg) cell transcriptomes to a human peripheral blood mononuclear cell single-cell RNA-sequencing dataset with surface markers (CITE-seq) and identify human exT(reg) cells as cytotoxic CD4(+) T cells.