AbstractDespite recent progress in the understanding of γδ T cells’ roles and functions, their interaction with αβ T cells still remains to be elucidated. In this study, we sought to clarify what precisely endows peripheral Vδ2+T cells with immunosuppressive function on autologous αβ T cells. We found that negatively freshly isolated Vδ2+T cells do not exhibit suppressive behavior, even after stimulation with IL-12/IL-18/IL-15 or the sheer contact with butyrophilin-3A1-expressing tumor cell lines (U251 or SK-Mel-28). On the other hand, Vδ2+T cells positively isolated through TCR crosslinking or after prolonged stimulation with isopentenyl pyrophosphate (IPP) mediate strong inhibitory effects on αβ T cell proliferation. Stimulation with IPP in the presence of IL-15 induces the most robust suppressive phenotype of Vδ2+T cells. This indicates that Vδ2+T cells’ suppressive activity is dependent on a TCR signal and that the degree of suppression correlates with its strength. Vδ2+T cell immunosuppression does not correlate with their Foxp3 expression but rather with their PD-L1 protein expression, evidenced by the massive reduction of suppressive activity when using a blocking antibody. In conclusion, pharmacologic stimulation of Vδ2+T cells via the Vδ2 TCR for activation and expansion induces Vδ2+T cells' potent killer activity while simultaneously licensing them to suppress αβ T cell responses. Taken together, the study is a further step to understand—in more detail—the suppressive activity of Vδ2+γδ T cells.