Plasticity of human gd T cells: Modulation by Vitamin C
During the first funding period, we demonstrated that Vitamin C (VitC) increases significantly the activation and effector functions of human gamma/delta T cells. VitC increases the proliferative activity, the metabolic activity, and the cytokine production in gamma/delta T cells. However, we also showed that VitC, in the additional presence of transforming growth factor-beta (TGF-beta), upregulates the expression of the transcription factor FOXP3 and induces suppressive activity in human gamma/delta T cells, associated with strong hypomethylation in specific regions of the FOXP3gene. Transcriptome analysis performed by RNAseq and genome-wide methylation studies identified several candidates of VitC-regulated genes in human gamma/delta T cells. We also contributed to a clinical phase I study where allogeneic gamma/delta T cells expanded in presence of VitC were adoptively transferred into cancer patients. Based on these results, the goals of the second funding period are to investigate further the influence of VitC on the plasticity of human gamma/delta T cells. We will focus on the following questions: (1) How does VitC modulate T-cell receptor signaling in gamma/delta T cells and what is the significance of some of the VitC-regulated genes identified in our previous studies; (2) how does VitC increase the cytotoxic effector function of gamma/delta T cells? (3) How does VitC in combination with other, clinically relevant epigenetic modifiers such as histone deacetylase inhibitors modulate the activation and effector functions of human gamma/delta T cells? The overall goal of this project is to optimize the effector function of human gamma/delta T cells for application in cellular immunotherapy.
Plasticity of human gamma/delta T cells: (Epigenetic) Modulation by Vitamin C
Gamma/delta T cells account for 3-5% of human peripheral blood CD3+ T cells. The majority of peripheral blood gamma/delta T cells expresses a Vgamma9Vdelta2-encoded T-cell receptor (Vg9Vd2 T cells). In contrast to conventional alpha/beta T cells, Vg9Vd2 T cells recognize microbial and eukaryoric pyrophosphate molecules in a butyrophilin 3A/CD277-dependent manner. Vg9Vd2 T cells exert multiple effector functions including cytokine production, cytotoxic activity, but also regulatory activity (including expression of transcription factor FoxP3) and professional antigen presenting capacity. Activated Vg9Vd2 T cells can pick up exogenous antigen and deliver peptides into the MHC class I presentation pathway for cross-presentation to CD8 T cells. Cellular differentiation is strongly influenced by epigenetic regulation, primarily at the level of histone acetylation and DNA methylation. Vitamin C is an essential cofactor for many enzymatic reactions and needs to be taken up through appropriate nutrition. Recently, it has been discovered that vitamin C – in addtion to its role as anti-oxidants – also acts as epigenetic modifier, mainly via the regulation of so-called „Ten-eleven translocation“ (Tet) dioxygenases. Tet dioxygenases regulate DNA methylation (and thereby gene activation) by oxidizing 5-methyl cytosine to 5-hydroxymethyl cytosine. In this project we plan to investigate in detail the influence of vitamin C on the functional plasticity of human gamma/delta T cells. To this end, we plan to stimulate purified gamma/delta T cells with Vg9Vd2-selective ligands in the absence or presence of vitamin C. Phenotye and effector function will be analyzed at the level of cytokine production, transcription factor expression and cytotoxic activity. The potential role of Tet dioxygenases will be studied with inhibitors such as 2-HG and RNAi-mediated down-regulation. In a second work package we will investigate the influence of vitamin C on human gamma/delta T cells at the level of transcriptome (RNAseq) and DNA hydroxymethylation by Reduced Representation Bisulfite Sequencing (RRBS). In a third work package we will perform a small proof-of-principle study to analyze the impact of high dose oral uptake of vitamin C on the functional activity, transcriptome and DNA hydroxymethylation of gamma/delta T cells. Based on our previous work on epigenetic modulation of human gamma/delta T cells by inhibitors of histone acetylation and preliminary results on modulation of gamma/delta T cells by vitamin C, this project will help to understand in more detail the epigenetic regulation of the plasticity of human gamma/delta T cells.