Vitamin C enhances cytotoxicity of CAR-and εTRuC-engineered γδ T cells and modulates Ca2+, NFAT, NF-κB and ERK signaling in human γδ T cells.

γδ T cells have raised interest as effector cells in cancer immunotherapy, due to their broad and HLA-independent reactivity towards multiple tumor entities. Despite promising ongoing clinical studies, there is a need to improve the effector function and thereby the efficacy of γδ T cells. In addition to its role as anti-oxidant, Vitamin C is an epigenetic modifier and exerts multiple effects on T-cell differentiation. While Vitamin C enhances in vitro proliferation, cytokine production and cytotoxicity of γδ T cells, the associated signaling pathways have so far not been elucidated. Here we demonstrate that Vitamin C enhances the cytotoxic effector function of unmodified as well as anti-CD19 chimeric antigen receptor (CAR) and TCR fusion construct (εTRuC)-transduced zoledronate-expanded γδ T cells. This was associated with increased upregulation of CD25 and IFN-γ secretion. Vitamin C increased calcium influx and nuclear translocation of NFAT and NF-κB in short-term expanded γδ T-cell lines. Although Vitamin C did not alter CD3ζ phosphorylation following stimulation with anti-CD3 antibodies, it reduced phosphorylation of ERK1/2 without affecting p38 MAP kinases. Further analysis showed that Vitamin C did not modulate the expression of selected immune checkpoint molecules on γδ T cells. Our results identify important signaling events where Vitamin C enhances the effector activity of human γδ T cells.