Regulatory NK1.1⁻CD4⁺NKG2D⁺ subset induced by NKG2DL⁺ cells promotes tumor evasion in mice

Regulatory T cells play critical roles in self-tolerance and tumor evasion. CD4⁺NKG2D⁺ cells with regulatory activity are present in patients with NKG2DL⁺ tumors and juvenile systemic lupus erythematosus. We previously showed that TGF-β-producing CD4⁺NKG2D⁺ T cells are present in pCD86-Rae-1ε transgenic mice. Here, we performed both ex vivo and in vivo studies on pCD86-Rae-1ε transgenic mice and an MC38 tumor-bearing mouse model and show that NK1.1⁻CD4⁺NKG2D⁺ T cells have regulatory activity in pCD86-Rae-1ε transgenic mice. Furthermore, this T-cell subset was induced in mice transplanted with NKG2DL⁺ tumor cells and produced TGF-β and FasL, and secreted low amounts of IFN-γ. This T-cell subset downregulated the function of effector T cells and dendritic cells, which were abolished by anti-TGF-β antibody. In vivo, adoptive transfer of NK1.1⁻CD4⁺NKG2D⁺ T cells promoted TGF-β-dependent tumor growth in mice. We further found that ex vivo induction of NK1.1⁻CD4⁺NKG2D⁺ T cells was dependent on both anti-CD3 and NKG2DL stimulation. Furthermore, regulatory NK1.1⁻CD4⁺NKG2D⁺ T cells did not express Foxp3 or CD25 and expressed intermediate levels of T-bet. Western-blotting showed that STAT3 signaling was activated in NK1.1⁻CD4⁺NKG2D⁺ T cells of MC38 tumor-bearing and pCD86-Rae-1ε transgenic mice. In conclusion, we describe a regulatory NK1.1⁻CD4⁺NKG2D⁺ T-cell population, different from other regulatory T cells and abnormally elevated in pCD86-Rae-1ε transgenic and MC38 tumor-bearing mice.