IL-12/18/21 pre-activation enhances the anti-tumor efficacy of expanded γδT cells and overcomes resistance to anti-PD-L1 treatment

γδT cells are promising candidates for cellular immunotherapy due to their immune regulation through cytokine production and MHC-independent direct cytotoxicity against a broad spectrum of tumors. However, current γδT cell-based cancer immunotherapy has limited efficacies and novel strategies are needed to improve its clinical outcomes. Here, we report that cytokine pre-treatment with IL-12/18, IL-12/15/18, IL-12/18/21 and IL-12/15/18/21 could effectively enhance the activation and cytotoxicity of in vitro expanded murine and human γδT cells. However, only adoptive transfer of IL-12/18/21 pre-activated γδT cells significantly inhibited tumor growth in a murine melanoma model and a hepatocellular carcinoma model. Both IL-12/18/21 pre-activated antibody-expanded and zoledronate-expanded human γδT cells effectively controlled tumor growth in a humanized mouse model. IL-12/18/21 pre-activation promoted γδT cell proliferation and cytokine production in vivo and enhanced IFN-γ and TNF-α production, as well as granzyme B expression by endogenous CD8 + T cells in a cell-cell contact dependent manner. Furthermore, the adoptive transfer of IL-12/18/21 pre-activated γδT cells could overcome the resistance to anti-PD-L1 therapy, and the combination therapy had a synergistic effect on the therapeutic outcomes. Moreover, the enhanced anti-tumor function of adoptively transferred IL-12/18/21 pre-activated γδT cells was largely diminished in the absent of endogenous CD8 + T cells when administered alone or in combination with anti-PD-L1, suggesting a CD8 + T cell-dependent mechanism. Taken together, IL-12/18/21 pre-activation could promote γδT cell anti-tumor function and overcome the resistance to checkpoint blockade therapy, indicating an effective combinational cancer immunotherapeutic strategy. Synopsis IL-12/18/21 pre-activation enhances the anti-tumor efficacy of adoptively transferred γδT cells by promoting its proliferation, activation, and cytotoxicity, as well as activating endogenous CD8 + T cells. Adoptive transfer of IL-12/18/21 pre-activated γδT cells could overcome the resistance to anti-PD-L1 therapy, indicating an effective combinational cancer immunotherapeutic strategy.