The quest continues: Human CD4 ⁺ CD16 ⁺ CD56 ⁺ ‘exTreg’ resemble NKT cells instead

Regulatory T cells are essential for immune tolerance, but their loss of function under inflammatory conditions in murine models signify a risk factor for Treg-based therapies. Recently CD4 ⁺ CD56 ⁺ CD16 ⁺ T cells were suggested to resemble such ex-Treg in human PBMC. Here, we re-evaluate the identity of the CD4 ⁺ CD56 ⁺ CD16 ⁺ population at a phenotypic and transcriptomic level using multiparametric flow cytometry on human PBMC and CITE-seq analysis to demonstrate that the CD4 ⁺ CD56 ⁺ CD16 ⁺ cells mostly constitute NKT cells instead. Further, we evaluated the stability of human Treg under lineage-challenging conditions and observe robust lineage stability in vitro. Finally, we also explore the potential of Tr17 induction using TGF-β and IL-6, a possible therapeutic strategy for Treg ex vivo expansion-based therapies. Together, we conclude that human exTreg remain to be described and instead human Treg present as remarkably stable, further promoting Treg-based adoptive transfer therapies.