Releasing the restraints of V?9Vd2 T-cells in cancer immunotherapy

Objectives: V?9Vd2 T-cells are a subset of T-cells with a crucial role in immunosurveillance which can be activated and expanded by multiple means to stimulate effector responses. Little is known about the expression of checkpoint molecules on this cell population and whether the ligation of these molecules can regulate their activity. The aim of this study was to assess the expression of both activatory and inhibitory receptors on V?9Vd2 T-cells to assess potential avenues of regulation to target with immunotherapy. Methods: Expression of various activatory and inhibitory receptors was assessed on V?9Vd2 T-cells by flow cytometry following activation and expansion using zoledronic acid (ZA) and Bacillus Calmette-Guérin (BCG). Expression of these markers and production of effector molecules was also examined following co-culture with various tumour cell targets. The effect of immune checkpoint blockade on V?9Vd2 T-cells was also explored. Results: V?9Vd2 T-cells expressed high levels of activatory markers both at baseline and following stimulation. V?9Vd2 T-cells expressed variable levels of inhibitory checkpoint receptors with many being upregulated following stimulation. Expression of these markers is further modulated upon co- culture with tumour cells with changes reflecting activation and effector functions. Despite their high expression of inhibitory receptors when cultured with tumour cells expressing cognate ligands there was no effect on Vd2+ T-cell cytotoxic capacity or cytokine production with immune checkpoint blockade. Conclusions: Our work suggests the expression of checkpoint receptors present on V?9Vd2 T-cells which may provide a mechanism with the potential to be utilised by tumour cells to subvert V?9Vd2 T-cell cytotoxicity. This work suggests important candidates for blockade by ICI therapy in order to increase the successful use of V?9Vd2 T-cells in immunotherapy. Overall design: Comparative gene expression profiling analysis of RNA-seq data for blood human gamma-delta t cells activated with either IL2 only, IL2+ZA, IL2+BCG, or IL2+KBCG for 13 days, and day 0 controls.