Identification of human exT_reg 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-β 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.