Influence of Vitamin C on the differentiation and functional plasticity of human gamma delta T cells
Gamma delta (gd) T cells are a numerically small subset of CD3+ T cells in human peripheral blood. They have recently attracted substantial interest as effector cells for cellular immunotherapy, due to their MHC-independent recognition and lysis of many solid tumor and leukemia/lymphoma cells. Depending on the micro-environmental cues, gd T cells also produce Th1/Th2/Th9/Th17-type cytokines, process and present antigen to conventional T cells, and can acquire regulatory activity. Potential clinical applications of in vitro expanded human gd T cells include the adoptive transfer into cancer patients. gdT cells can use different mechanisms to exert their effector functions. However, a better understanding of how their functional plasticity can be modulated is required to improve their translational efficacy. In this regard, it is important to develop novel strategies to optimize the culture conditions enabling maximal proliferative and functional activity. Vitamin C (L-ascorbic acid) is an essential vitamin that has to be supplied through appropriate nutrition or dietary sources. Vitamin C plays important roles in many different biological processes, spanning from stem cell differentiation to cancer cell biology. Moreover, Vitamin C has multiple effects on immune cells, acts as cofactor for several enzymes, has antioxidant activity and is known as facilitator of DNA hydroxymethylation through its capacity to enhance the capacity of Ten-eleven-translocation enzymes. The goal of the present thesis was to analyze the influence of the more stable derivative of Vitamin C, L-ascorbic 2-phosphate (pVC) on the differentiation and effector functions of human gd T cells. Moreover, the mechanisms sustaining pVC-mediated gd T-cell differentiation were also investigated. The first part of this thesis studied the effect of pVC on the proliferation, differentiation and cytotoxic effector function. The results demonstrated that supplementation of pVC to the in vitro expanded human gd T cells significantly enhances their proliferative expansion. Further investigations demonstrated that pVC does not prevent activation-induced death of (phospho)antigen-re-stimulated gd T cells but rather enhances cell cycle progression and thereby cellular expansion. Moreover, pVC sustained the polarization of expanded gd T cells towards a mixed Th1+2-like phenotype along with the expression of Th1 and Th2 signature cytokines. Finally, we observed that the cytotoxicity of the in vitro expanded gd T cells against pancreatic cancer cell lines was enhanced in the presence of pVC and was associated with an increased release of IFN-gamma. In the second part, this thesis investigated the effect of pVC on the TGF-beta-induced expression of the regulatory T-cell-specific transcription factor Foxp3 and the resulting regulatory activity of purified human Vg9Vd2 T cells. pVC induced a significant increase of TGF-beta-induced Foxp3 expression (both at protein and transcriptional levels) and stability as well as an increase in suppressive activity in vitro. Methylation analysis of the Treg-specific demethylated region (TSDR) revealed that the TGF-b-expanded gd T cells treated with pVC showed a more pronounced demethylation of FOXP3. The third pillar of this thesis addressed the influence of pVC on the genome-wide gene expression and DNA methylation of human gd T cells activated for eight days in vitro. At this time point, pVC had only minor effects on the transcriptome and genome-wide DNA methylation of the expanded gd T cells. Taken together, the results of this thesis demonstrated that Vitamin C has a significant influence on the functional activity of human gd T cells and thus appears to be suitable for enhancing the effector function of gd T cells for adoptive cell transfer in cancer patients. In addition, Vitamin C may also enhance the regulatory activity of gd T cells (in the presence of TGF-beta), which may be of interest in the context of autoimmune diseases.
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