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.
Highlights
Vitamin C (L-ascorbic acid) is an essential vitamin that must be obtained through appropriate nutrition or dietary sources
In the first set of experiments, we compared the effects of vitamin C (VC) and phospho-Vitamin C (pVC) over wide concentration ranges on the selective activation and short-term expansion of Vγ9Vδ2 T cells within Peripheral blood mononuclear cells (PBMCs)
Despite the lack of a significant growth-promoting effect of VC on γδ T-cell expansion within ZOL-stimulated PBMCs at day 7 (Fig. 1a), VC increased the intensity of Ki-67 expression measured at later time points on γδ T cells within PBMCs stimulated with ZOL and pAgs (HMBPP and bromohydrin pyrophosphate (BrHPP)) (Fig. 2a)
Summary
Vitamin C (L-ascorbic acid) is an essential vitamin that must be obtained through appropriate nutrition or dietary sources. VC augments the in vitro proliferative capacity of human NK cells.[11] it has been reported that VC increases the stability and function of regulatory T cells through Tet-dependent demethylation of the FoxP3 regulatory elements.[12,13] Overall, it is clear that VC can modulate multiple lymphocyte functions and might exert beneficial effects in the context of cellular immunotherapy.[14] γδ T cells have recently attracted substantial interest as potential effector cells in cancer immunotherapy, mainly because of their potent cytotoxicity toward tumor cells and their MHC/HLA-independent mode of action.[15] The dominant subset of γδ T cells in human peripheral blood expresses a Vγ9Vδ2-encoded T-cell receptor (TCR), while Vδ1 γδ T cells are more abundant in (mucosal) tissues.[15] Both subsets can efficiently kill tumor cells and may play a role in antitumor immunity.[16] Vγ9Vδ2 γδ T cells recognize microbial pyrophosphate molecules (“phosphoantigens”, pAgs), which are intermediates of the prokaryotic non-mevalonate pathway of isoprenoid biosynthesis secreted by many bacteria and some parasites. The mevalonate pathway is frequently dysregulated in tumor cells, resulting in Received: 6 December 2018 Accepted: 18 May 2019 Published online: 6 June 2019
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