IL-21-mediated expansion of Vγ9Vδ2 T cells is limited by the Tim-3 pathway

V gamma 9V delta 2 T cells are the main gamma delta T subset in the peripheral blood and lymphoid organs. Previous studies have shown that V gamma 9V delta 2 T cells could expand in the presence of phosphoantigens and IL-2 and exert antitumor functions. However, their potency was limited because sustained proliferation could not be achieved, possibly due to exhaustion caused by prolonged antigenic stimulation. In this study, we examined the proliferative response of V gamma 9V delta 2 T cells to IL-21, a cytokine previously shown to promote NK cell and CD8 T cell cytotoxicity. We found that IL-21 could significantly improve the proliferation of phosphoantigen-stimulated V gamma 9V delta 2 T cells in a dose-dependent manner. However, in acute myeloid leukemia (AML) patients, the efficacy of IL-21 was significantly reduced. V gamma 9V delta 2 T cells from AML patients exhibited lower expression of IL-21R, and required higher levels of IL-21 for expansion. IL-21-treated V gamma 9V delta 2 T cells from AML patients presented lower increase in STAT1 phosphorylation than V gamma 9V delta 2 T cells from healthy volunteers. Interestingly, AML V gamma 9V delta 2 T cells presented significantly higher Tim-3 expression than healthy V gamma 9V delta 2 T cells. IL-21 treatment further induced Tim-3 upregulation. Blocking Tim-3 increased the proliferation and the STAT phosphorylation in V gamma 9V delta 2 T cells in response to IL-21. Together, these results demonstrated that IL-21 could significantly expand the V gamma 9V delta 2 T cells, but its efficacy was limited since it also increased the expression of checkpoint molecule Tim-3.