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

Abstract

Human γδ T cells are potent cytotoxic effector cells, produce a variety of cytokines, and can acquire regulatory activity. Induction of FOXP3, the key transcription factor of regulatory T cells (Treg), by TGF-β in human Vγ9 Vδ2 T cells has been previously reported. Vitamin C is an antioxidant and acts as multiplier of DNA hydroxymethylation. Here we have investigated the effect of the more stable phospho-modified Vitamin C (pVC) on TGF-β-induced FOXP3 expression and the resulting regulatory activity of highly purified human Vγ9 Vδ2 T cells. pVC significantly increased the TGF-β-induced FOXP3 expression and stability and also increased the suppressive activity of Vγ9 Vδ2 T cells. Importantly, pVC induced hypomethylation of the Treg-specific demethylated region (TSDR) in the FOXP3 gene. Genome-wide methylation analysis by Reduced Representation Bisulfite Sequencing additionally revealed differentially methylated regions in several important genes upon pVC treatment of γδ t cells. While Vitamin C also enhances effector functions of Vγ9 Vδ2 T cells in the absence of TGF-β, our results demonstrate that pVC potently increases the suppressive activity and FOXP3 expression in TGF-βtreated Vγ9 Vδ2 T cells by epigenetic modification of the FOXP3 gene. Most T cells express the αβ T-cell receptor (TCR) which serves to recognize peptides presented by MHC/HLA class I or class II molecules to CD8 T cells or CD4 T cells, respectively. The germline TCR repertoire of αβ T cells is highly diverse, due to the large number of available variable (V) Vα and Vβ elements that can be selected during TCR gene rearrangement. By contrast, only a few Vγ and Vδ germline gene elements are available for the recombination of functional human γδ TCR 1. γδ T cells comprise 2-5% of peripheral blood T cells but are enriched in mucosal tissue. In human peripheral blood, the majority of γδ T cells expresses a Vγ9 Vδ2 TCR, whereas Vδ1 associated with various Vγ elements is predominantly expressed by intestinal γδ T cells 2,3. Vγ9 Vδ2 T cells recognize pyrophosphate molecules ("phosphoantigens", pAg) which are intermediates of the eukaryotic mevalonate or the prokaryotic non-mevalonate pathway of isoprenoid biosynthesis 4. Prototypes of such pAg are the eukaryotic isopentenyl pyrophosphate (IPP) and the prokaryotic (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) which selectively activate Vγ9 Vδ2 T cells at micro-and nanomolar concentrations, respectively 4,5. Synthetic analogs of naturally occurring pAg like bromohydrin pyrophosphate (BrHPP) have been described, exerting potent and selective stimulation of human Vγ9 Vδ2 T cells 6. While activation of Vγ9 Vδ2 T cells by pAg does not require HLA class I or class II molecules, there is an indispensable requirement for the transmembrane protein butyrophilin 3 A (BTN3A/CD277) 7. The current model implies that pAg bind to the intracellular B30.2 domain of BTN3A, thereby initiating a conformational change of the extracellular domain which is then selectively sensed by the Vγ9 Vδ2 TCR 8. Activated Vγ9 V...

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“…We observed a strong enhancement of FOXP3 expression and regulatory activity of purified γδ T cells stimulated with phosphoantigen and TGF-β in the presence of Vit C. More importantly, strong hypomethylation of FOXP3 TSDRs was observed only in the presence of Vit C, suggesting that TGF-β frequently expressed in the tumor microenvironment might prime local γδ T cells for suppressive activity if additional epigenetically active signals are present. 112 In some circumstances, however, it appears that γδ T cells can also downregulate αβ T cell responses independent of FOXP3 expression. Upon activation, γδ T cells transiently upregulate various inhibitory receptors and costimulatory molecules, including PD-1, PD-L1, CTLA4, and CD80/CD86.…”

“… 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. This may also include the potent production of IL-13, 167 which is known to contribute to antitumor immunity.…”

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“…Indeed, Vδ2 cell activation using anti-CD3 and anti-CD28 antibodies instead of phosphoantigens leads to transient FOXP3 expression that does not correlate with the regulatory phenotype (47,48). Interestingly, vitamin C increases the stability of TGF-β-induced FOXP3 expression in Vδ2 cells through an epigenetic modification of the FOXP3 gene, and enhances their suppressive capacities (49). Li et al demonstrated that upon TCR stimulation Vδ1 T cells can be polarized toward a suppressive phenotype in the presence of IL-2 and TGF-β.…”

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“…Th3 cells can be FoxP3 − and shown to suppress autoimmune colitis [ 40 , 41 ]. Furthermore, it was noted that stable expression of FoxP3 was essential for Treg function and was sustained through epigenetic regulation of both in the FoxP3 gene locus and Treg-specific demethylated region (TSDR) [ 42 , 43 ]. Naïve FoxP3 − CD4 + T cells in the mouse can express FoxP3 in the presence of transforming growth factor β (TGF-β) or retinoic acid, which promotes the development of peripherally induced Tregs.…”