Vitamin C promotes the proliferation and effector functions of human γδ T cells

Abstract

γδ T cells are of interest as effector cells for cellular immunotherapy due to their HLA-non-restricted lysis of many different tumor cell types. Potential applications include the adoptive transfer of in vitro-expanded γδ T cells. Therefore, it is important to optimize the culture conditions to enable maximal proliferative and functional activity. Vitamin C (L-ascorbic acid) is an essential vitamin with multiple effects on immune cells. It is a cofactor for several enzymes, has antioxidant activity, and is an epigenetic modifier. Here, we investigated the effects of vitamin C (VC) and its more stable derivative, L-ascorbic acid 2-phosphate (pVC), on the proliferation and effector function of human γδ T cells stimulated with zoledronate (ZOL) or synthetic phosphoantigens (pAgs). VC and pVC did not increase γδ T-cell expansion within ZOL-or pAg-stimulated PBMCs, but increased the proliferation of purified γδ T cells and 14-day-expanded γδ T-cell lines in response to γδ T-cell-specific pAgs. VC reduced the apoptosis of γδ T cells during primary stimulation. While pVC did not prevent activation-induced death of pAg-restimulated γδ T cells, it enhanced the cell cycle progression and cellular expansion. Furthermore, VC and pVC enhanced cytokine production during primary activation, as well as upon pAg restimulation of 14-day-expanded γδ T cells. VC and pVC also increased the oxidative respiration and glycolysis of γδ T cells, but stimulus-dependent differences were observed. The modulatory activity of VC and pVC might help to increase the efficacy of γδ T-cell expansion for adoptive immunotherapy.

Vitamin C supports conversion of human γδ T cells into FOXP3-expressing regulatory cells by epigenetic regulation

“…5g). Overall, the RRBS analysis may support our previous report on stimulatory effects of pVC on γδ T-cell proliferation and effector functions in the absence of TGF-β 22 . In addition, the RRBS analysis revealed an effect of pVC on TGF-β-treated γδ T cells at the level of gene regulation which may add a regulatory component to the FOXP3 demethylation and cellular functional properties, consistent with our functional results.…”

“…We have recently investigated the effects of VC and pVC on the in vitro activation and effector functions of human γδ T cells. We found that VC and pVC significantly enhanced their proliferative and metabolic activity as well as their cytokine production 22 . We also observed that γδ T cells expanded in the presence of pVC exerted stronger cytotoxic capacity.…”

“…Furthermore, our study has implications for the optimization of γδ T-cell based immunotherapy, notably for the adoptive transfer of in vitro expanded γδ T cells. When starting with purified γδ T cells (i.e., not with PBMC where TGF-β has a negative impact on the proliferative activity), both TGF-β and VC/pVC, when added independently, can actually increase the expansion and functional activity as measured by cytokine production and cytotoxic effector activity 11,22,43 . However, in combination, TGF-β and pVC increase the regulatory activity of Vγ9 Vδ2 T cells by enhancing and stabilizing FOXP3 protein expression as a consequence of the demethylation of the FOXP3 TSDR, which is a known pre-requisite for functionally active Tregs 54 .…”

Cancer immunotherapy with γδ T cells: many paths ahead of us

“…To conclude, the effect of Vit C on γδ T cell plasticity depends on the overall environmental signals. 166 , 167 As discussed above, Vit C actually conveys a regulatory phenotype and induces FOXP3 hypomethylation in the additional presence of TGF-β. 112 However, in the absence of TGF-β during the expansion phase, Vit C substantially enhances effector functions desired in the context of cancer immunotherapy.…”

“…Further studies showed that pVC reduced intracellular ROS levels and increased cell cycle progression and Ki-67 expression in surviving γδ T cells, thereby promoting the expansion of surviving cells rather than preventing cell death. 167 Vδ2T cells expanded in the presence of pVC also displayed stronger cytotoxicity against tumor cells in vitro and were more active upon transfer into immunodeficient mice transplanted with a human lung tumor cell line (Yu et al, unpublished results). To conclude, the effect of Vit C on γδ T cell plasticity depends on the overall environmental signals.…”