IL12/18/21 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 efficacy, and novel strategies are needed to improve clinical outcomes. Here, we report that cytokine pre-treatment with IL12/18, IL12/15/18, IL12/18/21, and IL12/15/18/21 effectively enhanced the activation and cytotoxicity of in vitro-expanded murine and human γδT cells. However, only adoptive transfer of IL12/18/21 pre-activated γδT cells significantly inhibited tumor growth in a murine melanoma model and a hepatocellular carcinoma model. Both IL12/18/21 pre-activated antibody-expanded and zoledronate-expanded human γδT cells effectively controlled tumor growth in a humanized mouse model. IL12/18/21 pre-activation promoted γδT cell proliferation and cytokine production in vivo and enhanced IFNγ production and activation of endogenous CD8+ T cells in a cell-cell contact- and ICAM-1-dependent manner. Furthermore, the adoptive transfer of IL12/18/21 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 IL12/18/21 pre-activated γδT cells was largely diminished in the absence of endogenous CD8+ T cells when administered alone or in combination with anti-PD-L1, suggesting a CD8+ T cell-dependent mechanism. Taken together, IL12/18/21 pre-activation can promote γδT cell anti-tumor function and overcome the resistance to checkpoint blockade therapy, indicating an effective combinational cancer immunotherapeutic strategy.