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

In atherosclerosis, some regulatory T (Treg) cells become exTregcells. We crossed inducible Tregand exTregcell lineage-tracker mice (FoxP3eGFP−Cre-ERT2ROSA26CAG-fl-stop-fl-tdTomato) to atherosclerosis-prone Apoe−/−mice, sorted Tregcells and exTregcells and determined their transcriptomes by bulk RNA sequencing (RNA-seq). Genes that were differentially expressed between mouse Tregcells and exTregcells and filtered for their presence in a human single-cell RNA-sequencing (scRNA-seq) panel identified exTregcell signature genes as CST7, NKG7, GZMA, PRF1, TBX21and CCL4. Projecting these genes onto the human scRNA-seq with CITE-seq data identified human exTregcells as CD3+CD4+CD16+CD56+, which was validated by flow cytometry. Bulk RNA-seq of sorted human exTregcells identified them as inflammatory and cytotoxic CD4+T cells that were significantly distinct from both natural killer and Tregcells. DNA sequencing for T cell receptor-β showed clonal expansion of Tregcell CDR3 sequences in exTregcells. Cytotoxicity was functionally demonstrated in cell killing and CD107a degranulation assays, which identifies human exTregcells as cytotoxic CD4+T cells.