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.