A comparative view on vitamin C effects on αβ- versus γδ T-cell activation and differentiation

Abstract

Vitamin C (VitC) is an essential vitamin that needs to be provided through exogenous sources. It is a potent anti-oxidant, and an essential cofactor for many enzymes including a group of enzymes that modulate epigenetic regulation of gene expression. Moreover, VitC has a significant influence on T-cell differentiation, and can directly interfere with T-cell signaling. Conventional CD4 and CD8 T cells express the αβ TCR and recognize peptide antigens in the context of MHC presentation. The numerically small population of γδ T cells recognizes antigens in an MHC-independent manner. γδ T cells kill a broad variety of malignant cells, and because of their unique features, are interesting candidates for cancer immunotherapy. In this review, we summarize what is known about the influence of VitC on T-cell activation and differentiation with a special focus on γδ T cells. The known mechanisms of action of VitC on αβ T cells are discussed and extrapolated to the effects observed on γδ T-cell activation and differentiation. Overall, VitC enhances proliferation and effector functions of γδ T cells and thus may help to increase the efficacy of γδ T cells applied as cancer immunotherapy in adoptive cell transfer.

INTRODUCTION

The functional properties of T cells can not only be influenced by cytokines, but also by a variety of other immunomodulatory substances, which may have the potential for improving immunotherapy by governing T-cell differentiation to the desired functional status. Vitamin C (VitC), known for some time to have an activating effect on immune cells, is such a substance. Yet, the underlying mechanisms of how VitC modulates T-cell activation and differentiation have been investigated only recently. The VitC-dependent modulation of αβ T cells has been attributed to effects on T-cell activation as well as to epigenetic processes, because VitC represents a cofactor for DNA- and histone demethylating enzymes. Depending on the T-cell stimulation context, VitC not only enhances T-cell effector functions, but also potently promotes T-cell differentiation into proinflammatory or regulatory phenotypes. Therefore, VitC might be a useful tool in equipping in vitro expanded αβ and γδ T cells with the desired features for different types of immunotherapy.

THE ROLE OF VITAMIN C IN PHYSIOLOGY

VitC or L-ascorbic acid is a 6-carbon ketolactone, which is synthesized in a 2-step reaction from L-galactose in green plants or from glucose in most animals.¹ However, humans (as well as other primates, guinea pigs, and fruit bats) are unable to synthesize VitC, because of a deficiency in the L-gulono-gamma-lactone oxidase (GULO), the enzyme necessary for catalyzing the last step of the VitC synthesis.² Insufficient VitC-uptake results in scurvy, a VitC-deficiency disease, characterized by bleeding gums, impaired wound healing, anemia, fatigue, and depression.¹

Under physiologic conditions VitC-levels are maintained in a range between micromolar concentrations in the blood plasma (∼50 μM) and millimolar concentrations (∼1–10 mM) inside cells.³ VitC can be either transported by hexose transporters, when present in its oxidized form dehydroascorbate (DHA),⁴ or more specifically by the sodium-dependent VitC transporters (SVCT) 1 and 2 (SLC23A1 and SLC23A2), by which intracellular accumulation of VitC can be achieved.⁵ Highest intracellular levels are found in the brain, the adrenal gland, and lymphocytes, where 10–100-fold higher VitC-concentrations are reached compared to plasma levels.⁶ The high VitC levels in lymphocytes suggest a functional role for VitC in these cells. VitC is an important anti-oxidant, radical scavenger, cofactor for many enzymes and plays an important role in many physiologic processes. Therefore, not surprisingly, different mechanisms of its influence on the immune system have been described. VitC can augment immune functions and has been shown to modulate differentiation, proliferation, and function of different lymphocyte populations.

VitC has been considered as an anti-cancer agent since the 1970s,⁷ and has been demonstrated to slow down tumor growth in some animal models. In human clinical studies with cancer patients using high oral doses of VitC, however, no clear effect could be demonstrated, so far.^8,9 VitC plasma concentrations reached by oral uptake seem to be limited compared to high dose i.v. application.^10,11,12 Different mechanisms have been shown to contribute to the direct anti-cancer effects of VitC. At high concentrations VitC acts as a pro-oxidant and can induce the generation of H₂O₂, which is cytotoxic. Colon cancer cells with a KRAS or BRAF mutation often have elevated amounts of the GLUT1 transporter. DHA can compete with glucose for the GLUT1-mediated transport and thereby interferes with tumor-cell glucose metabolism. In addition, the increased DHA uptake can lead to intracellular glutathione (GSH) depletion and thus favor the accumulation of reactive oxygen species (ROS).^13,14 Moreover, VitC negatively affects tumor growth by promoting the function of 2-oxoglutarate-dependent dioxygenase (2-OGDD) family enzymes, leading to hypoxia-inducible factor 1 (HIF-1) -instability and hence to the incapacity of tumor cells to adapt to hypoxic conditions.¹²

αβ and γδ T cells

Two types of T cells using V(D)J recombination of germline-encoded TCR genes exist throughout all jawed vertebrates. αβ T cells, which represent the blueprint of adaptive immunity, and γδ T cells, which share features of the adaptive and innate immunity. The αβ TCR binds to a peptide-MHC complex. Unlike αβ T cells, γδ T cells recognize antigens independently of HLA-presentation, but require self-molecules for their activation. In this respect, members of the extended butyrophilin family (which belong to the B7 molecule superfamily), including Skint-1 in mice, play important roles. As an example, human intestinal Vγ4 γδ T cells interact with BTNL3 molecules on intestinal epithelial cells.¹⁵ This interaction might be relevant for tissue-specific tonic signals and homing, whereby in addition MHC- and CD1-related molecules might represent the nominal antigens for the γδ TCR.¹⁶ In humans, γδ T cells can be discriminated into two major subpopulations according to their expressed Vδ element, namely, Vδ1 T cells, which are more abundant in tissues and Vδ2 T cells, which are the predominant subpopulation in the peripheral blood.¹⁷ Vδ1 T cells have been shown to interact in a TCR-dependent way with CD1d and with the stress-induced MHC class I polypeptide-related sequence A (MICA) molecule.^18,19,20 Virtually all Vδ2 T cells in the peripheral human blood co-express the Vγ9 element. Interestingly, there is no γδ T-cell population homologous to Vγ9Vδ2 T cells in the mouse. Human Vδ2Vγ9 T cells are activated by small pyrophosphate antigens, also referred to as phosphoantigens (pAg), which are intermediates of the isoprenoid biosynthesis pathway, which is involved in cholesterol synthesis and protein prenylation. These pAg have been shown to bind to the intracellular part of the butyrophilin molecule BTN3A1, thereby inducing a conformational change of the extracellular BTN3A domain, which in turn is recognized by the γδ TCR.²¹ For the activation of the Vγ9Vδ2 T cells the heterodimeric interaction of BTN3A1 and BTN3A2 seems to be essential.²² pAg, intermediates of the microbial nonmevalonate pathway, such (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), have a 1000-fold higher Vγ9Vδ2 T-cell stimulatory potential compared to intermediates of the eukaryotic mevalonate pathway, such as isopentenyl pyrophosphate (IPP).²³ Also synthetic pAg, such as bromohydrin pyrophosphate (BrHPP), can be used to selectively activate Vδ2 T cells.²⁴ Even though, endogenous eukaryotic pAg have a poorer Vδ2 T-cell activating capacity, a dysregulated mevalonate pathway, often found in tumor cells, can lead to the accumulation of IPP and in consequence to the activation of Vδ2 T cells.²⁵ The pharmacologic inhibition of the mevalonate pathway, by blockade of the farnesyl pyrophosphate synthase using the aminobisphosphonate zoledronate, leads to the upstream accumulation of IPP and subsequent Vδ2 T-cell activation. In line with this, zoledronate-treatment of tumor cells has been shown to enhance the cytotoxicity of Vδ2 T cells against tumor cells.²⁶ Because of their tumor reactivity and HLA-independent antigen recognition, γδ T cells are interesting candidates for immunotherapeutic approaches.²⁷ In initial clinical trials, γδ T cells have been shown to be also suitable for allogeneic transfer without inducing graft vs. host disease.²⁸

Overall, human γδ T cells display high functional plasticity and a differentiation potential, which is comparable to CD4 αβ T cells. This high functional plasticity of Vδ2 T cells could be harnessed for in vitro fine tuning of their desired features according to the therapeutic application. By manipulation of the local cytokine milieu, Vδ2 T cells can differentiate into subpopulations which resemble Th1, Th2,²⁹ Th9,³⁰ Th17,^31,32 and Tfh phenotypes.^33,34

In the following we discuss the effects of VitC on T-cell differentiation with a focus on γδ T cells. In this context, it is important to consider that some of the γδ T-cell populations, intensively studied in one species, do not necessarily have a homologue in other species, as for instance pAg-reactive human Vγ9Vδ2 T cells, which do not exist in mice. On the other hand, a particular IL-17-producing γδ T-cell population, which is already imprinted in the thymus, is only found in mice. This IL-17-poducing murine γδ T-cell population is characterized by the absence of CD27, whereas murine CD27-positive γδ T cells produce IFN-γ.^35,36 Moreover, only mice harbor an IFN-γ-producing Vγ5Vδ1 T-cell population of dendritic epidermal T cells (DETC), which requires the butyrophilin family molecule Skint-1 for their development.^37,38

The TCRs of αβ- and γδ T cells in general fulfill similar signaling functions, although the signals transduced by the two TCRs seem to be slightly different. The CD3 complex of both T-cell populations is composed of similar components, but their combinations differ. In αβ T cells CD3 is composed of two heterodimers, CD3δ/CD3ϵ and CD3γ/CD3ϵ, and a ζ-chain homodimer.^39,40 In γδ T cells the CD3 complex consists of two CD3ε/CD3γ heterodimers and a CD3ζ homodimer,⁴¹ but lacks CD3δ which is always present in αβ T cells. In activated γδ T cells, the FCRγ can be incorporated into the TCR/CD3 complex, thereby replacing one CD3ζ chain.⁴² Moreover, the conformational change of CD3 upon ligand binding differs between αβ and γδ T cells.⁴³ The signal transduced by the γδ TCR was found to be stronger compared to the αβ TCR signal, measured by the induction of calcium mobilization and ERK phosphorylation upon CD3 crosslinking.⁴²

For the initiation of TCR signal transduction, the phosphorylation of ITAMs is required, which is mediated by the coreceptor associated tyrosine Src-family kinases Lck or Fyn, which are recruited together with the coreceptors (CD4, CD8) to the TCR/CD3 complex in αβ T cells.⁴⁴ γδ T cells also require Src-kinase activity for efficient TCR signaling.⁴⁵ Because most γδ T cells do not express the coreceptors CD4 or CD8 on their surface, the mechanism of the Src-kinase recruitment to the CD3/TCR complex remains elusive.⁴⁴ Moreover, a recent study using a Lck-Cre mouse model found, that unlike in αβ T cells, which use the proximal Lck-promoter at the double negative (CD4⁻CD8⁻; DN) and double positive (CD4⁺CD8⁺; DP) stage of thymic development,⁴⁶ in murine γδ T cells it was used during fetal but not during adult thymic development and not in differentiated cells.⁴⁷ In both T-cell populations the tyrosine kinase ζ-associated protein of 70 kDa (Zap70) is recruited upon phosphorylation of the TCR-associated ITAMs, where it contributes to the phosphorylation of the scaffold proteins SLP76 and LAT. This induces the formation of the TCR-signaling complex and the recruitment of PLCγ1, from which most downstream signaling events originate.⁴⁸ PLCγ1 converts phosphatidylinositol-4, 5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol-1, 4, 5-trisphosphate (IP3). DAG in turn initiates the NF-κB- as well as the ERK1/2 signaling pathway, whereas PIP2 initiates Ca²⁺ signaling.⁴⁹ In mice harboring a mutation in LAT at the PLCγ1 binding site the CD8 αβ and normal γδ T-cell development was abrogated, whereas CD4 αβ and CD4 γδ T cells were hyper-proliferative. Therefore, the LAT/PLCγ1-mediated signaling seems to play different roles in the distinct γδ T-cell subpopulations as well as in CD4 and CD8 αβ T cells.^50,51 However, pAg-stimulation or CD3-crosslinking of human Vγ9Vδ2 T cells led to a phosphorylation pattern of the downstream signaling molecules Zap70, phosphatidyl-inositol-3 kinase (PI3K), LAT, Lck, Erk1/2, and p38 MAPK,^52,53,54 as well as an induction of Ca²⁺ signaling,⁵⁵ similar to what is found in αβ T cells.

IMMUNE MODULATION BY THE ANTI-OXIDANT ACTIVITY OF VITAMIN C—THE INFLUENCE ON SIGNAL TRANSDUCTION

VitC exists in different redox states and is considered the most relevant naturally occurring reducing substance.⁵⁶ Fully reduced VitC (ascorbate or ascorbic acid) can be oxidized both intracellularly or extracellularly. As an anti-oxidant, VitC (at micromolar concentrations) can react with ROS and thereby neutralize detrimental amounts of ROS.⁵⁷ Ascorbate oxidization, by free radicals or ROS, results in the less reactive ascorbate radical (Asc•−), which is subsequently fully oxidized to DHA.⁵⁸ DHA has a short half-life (less than 1 min) and therefore only makes up about 1–5% of the total VitC present in the human body. If DHA is not transported inside of cells, it becomes irreversibly hydrolyzed into 2, 3-L-diketoglutonate (2, 3-DKG), which is further degraded into oxalic acid and threonic acid. Within the cells, DHA is rapidly reduced by reacting with reduced GSH, whereby VitC (ascorbate) is regenerated. It is noteworthy that VitC can also act as a pro-oxidant, when present at very high (millimolar) plasma concentrations.¹

The most obvious mechanism by which VitC can modulate T-cell activation and function is its anti-oxidant activity. Different stages of the T-cell activation signaling cascade are sensitive to the redox conditions, which in turn can be influenced by VitC. Intermediate concentrations of ROS are part of the T-cell activation signaling process and modify protein function and structure by oxidation, whereas very high concentrations of ROS lead to cell death.⁵⁹ Upon TCR stimulation the production of mitochondrial ROS is induced,⁶⁰ which influences T-cell activation, proliferation, as well as effector functions.⁵⁹

Zap70 is a crucial molecule for the initial steps of proximal TCR signaling. Zap70 signaling can be induced by the ROS H₂O₂.⁶¹ The stability of Zap70 is influenced by an intrinsic cysteine-based redox-active motif, which stabilizes the protein when oxidized. Vice versa, deletion of this motif or treatment with the anti-oxidant N-acetylcysteine (NAC), leads to destabilization of Zap70.⁶² A similar influence on protein stability through cysteine motifs is also suggested for other tyrosine kinases, which can integrate signals into the TCR signaling such as the Src-kinase Lck.^63,64 On the other hand, ROS can negatively influence function or stability of phosphatases or kinases involved in T-cell activation. The stability of the SHP2 protein tyrosine phosphatase can be compromised by oxidization,⁶⁵ whereby negative signals from inhibitory receptors can be overcome. Therefore, the possible implications of the anti-oxidant activity of VitC on the proximal TCR signaling are similar in αβ and γδ T cells. Even though Lck appears to be differently regulated in γδ T cells,⁴⁷ Src kinases as such are generally required for (αβ and γδ) T-cell activation⁴⁵ and VitC might enhance Zap70 as well as Src-kinase signaling in αβ as well as in γδ T cells.

ROS are known to be important for the activation of NF-κB, whereby low concentrations of ROS can promote and higher concentrations can negatively influence NF-κB signaling.^66,67 I-κB prevents NF-κB activation and nuclear translocation. When I-κB is phosphorylated by Iκ B kinase (IKK), it is targeted to proteasomal degradation and NF-κB is released. ROS can negatively influence NF-κB activation by inhibiting IKK function, because the cysteine residue in the reactive center of IKK-β has been found to be sensitive to oxidization by ROS, but also to directly react with oxidized GSH.^68,69 For stimulation with TNF-α it has been shown that VitC impairs the NF-κB activation and nuclear translocation of NF-κB. This effect was also attributed to inhibition of IKK-β activity. In presence of VitC, IKK-β failed to induce I-κBα-phosphorylation and subsequent degradation and therefore did not activate NF-κB.^70,71 VitC can inhibit NF-κB when oxidized by ROS to DHA by directly inhibiting the kinase activity of IKK-α and IKK-β as well as of p38 in vitro.⁷²

Upon T-cell stimulation, Ca²⁺ influx into the cytoplasm triggers the activation of calcineurin, which in turn dephosphorylates nuclear factor of activated T cells (NFAT) and thereby initiates the NFAT translocation from the cytosol into the nucleus. Calcineurin contains iron and zinc in its reactive site, which are sensitive to high ROS levels.⁷³ By using purified calcineurin for in vitro activity assays, it was demonstrated that oxidants (hydrogen peroxide, superoxide) inhibit the phosphatase activity of calcineurin, whereas its activity was enhanced by anti-oxidants such as VitC, L-ascorbic acid 2-phosphate (pVC), NAC, or GSH.⁷⁴ In a mouse model, the CD4 T-cell specific absence of the cell-endogenous anti-oxidant GSH (CD4cre-GCLC^fl/fl) resulted in elevated intracellular ROS levels. Due to the elevated ROS levels the NFAT activation was found to be compromised, which resulted in impaired mTOR activation and reduced MYC expression.⁷⁵

Different signaling pathways, including TCR- and costimulatory- as well as metabolic signals, culminate in mTOR activation. mTOR can form two different multiprotein complexes, mTOR-C1 and mTOR-C2, in which mTOR is associated with Raptor or Rictor, respectively. mTOR-C1 mediated phosphorylation promotes S6K function and disables the 4E-BP1-mediated inhibition of EIF4E, whereas mTOR-C2 promotes Akt, SGK1, and PKC signaling.⁷⁶ The PI3K-Akt-mTOR-C1 axis connects T-cell activation with metabolic cell programming by the induction of Myc and switching the energy balance to glycolysis and oxidative phosphorylation.^77,78 This axis, or more specifically, the activation of Akt, is negatively influenced by the protein tyrosine phosphatase PTEN. The function of PTEN has been demonstrated to be reversibly inactivated by oxidizing agents.⁷⁹ Oxidation of PTEN by ROS leads to the attenuation of its activity, and thereby promotes the activation of the PI3K pathway and thus induces mTOR activity.⁸⁰ The ROS-dependent inhibition of mTOR reported by Mak et al. and the ROS-dependent activation of mTOR described by Kim et al. stand in contrast to each other and may result from different experimental approaches. However, mTOR function has been shown to govern the balance between regulatory- and IL-17-producting αβ and γδ T cells,^81,82 which therefore might be modulated by anti-oxidant treatment.

HIF-1 is a transcriptional master regulator of the hypoxia response. Under normal oxygen conditions the HIF-1 subunit HIF-1α is quickly hydrolyzed by HIF-hydroxylases and thereby subjected to proteasomal degradation. Under hypoxic conditions, due to the lack of O₂, HIF-1α-hydroxylation is inhibited and HIF-1α is stabilized.⁸³ ROS can also lead to deactivation of HIF hydroxylases, as has been shown for the hydroxylase PHD2.⁸⁴ In activated T cells high levels of ROS are produced in the mitochondria⁷⁷ and HIF-1 is found to be more abundant in activated T cells in response to TCR and CD28 costimulatory signaling, independent of the oxygen conditions.⁸⁵ This increase of HIF-1α was shown to be independent of protein stabilization, but depended on de novo protein synthesis most probably induced by mTOR signaling, as the induction was found to be sensitive to rapamycin treatment.⁸⁶ The HIF-hydroxylases PHD1–3 and FIH all belong to the family of 2-OGDD^87,88; therefore it is not surprising, that for optimal HIF-1-hydroxylase activity VitC is required as a cofactor (which cannot be replaced by other reducing agents).⁸⁹ In line with this, it was shown, that VitC enhances the activity of PHD2 in a dose-dependent manner,⁹⁰ and that low VitC levels result in impaired HIF-1α hydroxylation under normally HIF-1α stabilizing hypoxia conditions.⁹¹ Moreover, VitC was shown to directly inhibit HIF-1α activity in cell culture and to prevent HIF-1α-dependent gene activation.^91,92 HIF-1 plays an important role in the switch from oxidative phosphorylation to glycolysis following T-cell activation,⁸⁵ and HIF-1α favors Th17 differentiation over Treg differentiation.⁹³ Treg vs. effector cell differentiation strongly depends on the metabolic programming, for which the signals are (partially) transduced by HIF-1. Therefore, the negative influence of VitC on HIF-1α could also contribute to the VitC-dependent promotion of the Treg differentiation. The evident effects of VitC on the epigenetic programming of Treg are discussed in more detail below.

In summary, VitC can affect T-cell activation on several levels. By reducing ROS, VitC can interfere with proximal TCR signaling, but it also promotes signaling through NFAT and PI3K-AKT-mTor pathways. Through more direct interactions with IKK and HIF-hydroxylases, VitC inhibits NF-κB activation and promotes HIF-1α degradation, respectively. An overview of the various possible VitC-dependent modulations of T-cell signaling is provided in Figure 1.

VITAMIN C ENHANCES LYMPHOCYTE ACTIVATION AND EFFECTOR FUNCTIONS

The effects of VitC on lymphocyte proliferation, reported so far, are not completely consistent, because positive, negative as well as no effects of VitC treatment have been reported. Especially high doses of VitC have been found to have a negative effect on cell proliferation and viability in vitro, which most probably can be attributed to the pro-oxidant capacity of VitC,¹ leading to the acidification of the cell culture medium. Therefore, the phospho-modified VitC derivative, pVC, is often used instead of VitC, because it is more stable and less cytotoxic at higher concentrations.⁹⁴

In streptozotocin-induced diabetic rats, the degree of the induced diabetes was alleviated by oral treatment with VitC (100 mg/kg body weight). Moreover, isolated T cells from VitC-treated rats displayed restored surface levels of CD28 as well as a restored proliferative response and cytokine production upon CD3/-28 mAb- or ConA stimulation.⁹⁵ In another study with in vitro activated murine T cells, no effect of low VitC doses (62.5–125 μM) was found, but higher doses (250–500 μM) decreased the viability and proliferation of the T cells.⁹⁶ In elderly humans, generally suffering from low VitC levels in serum and tissues, VitC supplementation (500 mg/d) enhanced the in vitro proliferation of isolated T cells,⁹⁷ whereas other studies with VitC-treated scurvy patients did not reveal enhanced T-cell responsiveness to the mitogen phytohemagglutinin.⁹⁸

VitC is known to influence the expression of cell cycle and cell repair genes in other cell types (fibroblasts).⁹⁹ The ex vivo expansion of NK cells within human PBMCs was enhanced in the presence of VitC (95 μM).¹⁰⁰ Normal doses of VitC (100 μM) also enhanced the proliferation of human lymphocytes upon concanavalin A or LPS stimulation.¹⁰¹ Isolated human T cells stimulated in vitro by phorbol-2-myristate-13-acetate (PMA)/ionomycin or anti-CD3/-28 mAb did not show a clearly enhanced proliferation in the presence of normal doses of VitC (62.5 μM), but at higher doses (500–1000 μM) the proliferation decreased, whereas apoptosis increased, when VitC was applied before stimulation.¹⁰²

In our studies on the modulation of Vδ2 T-cell activation by VitC, we found a negative effect of high doses of VitC on the in vitro proliferation,¹⁰³ as has been described before for PHA stimulated T cells.¹⁰² Low doses of VitC (57 μM) had no effect on the proliferation of BrHPP- or zoledronate stimulated γδ T cells within the PBMC, but higher doses of VitC (284–1136 μM) almost completely abrogated the proliferation, most likely due to cytotoxic effects. Interestingly, the more stable VitC derivate pVC did not have such negative effects at high concentrations (173–692 μM), but clearly enhanced the proliferation of isolated γδ T cells upon BrHPP or HMBPP stimulation (as shown for 173 μM pVC). Moreover, we found for zoledronate-expanded Vδ2 T cells that VitC decreased the cell death during the late expansion phase (days 14–21), but had no effect on activation-induced cell death (AICD) upon restimulation with the pAg BrHPP.¹⁰³

With regard to lymphocyte effector functions, both positive and negative effects of VitC on NK cells have been described.¹⁰⁴ The oral treatment of humans with a single high dose of VitC (60 mg/kg), for instance, enhanced the cytotoxic antitumor activity of their PBMC in vitro, which in this study was attributed to NK-cell mediated cytotoxicity.¹⁰⁵ Mice deficient for GULO (the enzyme required for the biosynthesis of VitC) survived shorter time when transplanted with ovarian cancer cells and their NK cells displayed a strongly impaired in vitro cytotoxicity against the cancer cells, when VitC was not supplemented exogenously. These VitC-depleted NK cells displayed reduced NKG2D surface expression and secreted reduced levels of IFN-γ in coculture with the cancer cells.¹⁰⁶ In a mouse model using murine dendritic cells (DC) activated ex vivo in the presence of VitC, the DC secreted elevated amounts of IL-12 and IL-15. When VitC-treated DC were loaded with tumor-antigens and transferred back into the mouse, they generated IFN-γ-producing effector and effector memory CD8 T cells, which had superior antitumor effector activity, and the mice survived for longer time.¹⁰⁷

Our group also observed a remarkable impact of VitC on the cytotoxic activity of human γδ T cells. We found, that γδ T cells, activated by zoledronate and expanded in the presence of VitC, exerted increased cytotoxicity toward different types of tumor cells. The same was true when VitC was directly added to the coculture of expanded Vδ2 T cells and tumor cells during the cytotoxicity assay. The enhanced cytotoxic effector functions were accompanied by the enhanced release of effector molecules (IFN-γ, granzyme B). Whereas VitC treatment seems to promote a proinflammatory Th1/Th17 phenotype in αβ T cells,^108,109,110 VitC treatment of pAg (BrHPP) restimulated γδ T cells did not induce a clear bias toward a certain differentiation pathway (in the presence of IL-2). Instead, the secretion of Th1 cytokines (IFN-γ) and Th2 related cytokines (IL-5 and IL-13) was increased simultaneously, which correlated with an enhanced co-induction of T-bet and Gata-3.¹⁰³

Taken together, VitC can influence both the T-cell expansion and the direct cytotoxic effector functions of NK cells and γδ T cells. Overall, the experimental evidence regarding the pro-proliferative effect is more conclusive at this point.

VITAMIN C ACTS AS A COFACTOR FOR 2-OGDD FAMILY ENZYMES

DNA and histone modifications, commonly referred to as epigenetic modifications, represent reversible marks that play an important role in regulating gene expression without altering the genetic code. Epigenetic processes are important for cellular differentiation, because they can define the transcriptional profile and cell fate. DNA methylation as well as histone acetylation and methylation are the functionally most important and best characterized epigenetic modifications.

DNA methylation occurs in CpG regions as 5-methyl-cytosine (5mC), which leads to transcriptional silencing. Thereby, genes and gene-specific enhancers can be controlled without altering the genetic code. A widespread remodeling of the DNA methylation occurs for instance during CD4 T-cell differentiation^111,112,113 and also γδ T-cell differentiation is most likely governed by identical processes.¹¹⁴

Modifications on histones can have different functional implications depending on the position and type of the methylation. A di- or tri-methylation mark on lysine 9 of histone 3 (H3K4me2/3) or on H3K79me3 are normally associated with transcriptional activity, whereas methylation on H3K9me2/3, H3K27me2/3, and H4K20me3 are associated with transcriptional inactive chromatin.¹¹⁵ VitC is a critical cofactor for numerous enzymes, donating its electron (functioning as a reducing agent) to metal ions bound to the enzymes, acting as a cofactor to enable enzyme activity, which in most cases is catalyzing the hydroxylation of their substrates.¹ VitC-utilizing enzymes can be categorized into two families, the copper-containing monooxygenases and the Fe²⁺- and 2-OGDD (also known as α-ketoglutarate -dependent dioxygenases [αKGDDs]). Most of the VitC-utilizing enzymes belong to the 2-OGDD family. Functionally both atoms of molecular oxygen are integrated into the reaction products, whereby one oxygen atom is used for the hydroxylation of the substrate and the other reacts with αKG. This results in the decarboxylation of αKG and the subsequent formation of succinate and CO₂. VitC contributes to the reaction by reducing the oxidized cofactor Fe(III) back to Fe(II), thereby restoring enzyme activity.¹¹⁶

In mammalian cells, enzymes of the 2-OGDD family contribute to many biologic functions, such as collagen synthesis, transcriptional inactivation and degradation of HIF-1α,¹¹⁷ carnitine synthesis, tyrosine catabolism, and the demethylation of proteins (e.g., histones) as well as of DNA and RNA.¹

Ten-eleven translocation (Tet) enzymes and lysine demethylase (KDM), mediating DNA demethylation, and histone lysine demethylation, respectively, both belong to the 2-OGDD enzyme family, where VitC is utilized as a cofactor. Therefore, it is not surprising, that increased VitC-levels have been found to augment the activity of enzymes from this family. Ten-eleven translocation enzymes (Tet enzymes) catalyze the DNA demethylation, by initially converting 5mC to 5-hydroxymethylcytosine (5hmC) and into 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in subsequent reactions. The role of VitC as a cofactor for the Tet-enzyme-mediated 5mC hydroxylation, has been demonstrated in in vitro experiments showing that addition of VitC enhances the generation of 5hmC, which is followed by full cytosine demethylation.¹¹⁸ The demethylation-promoting effect of VitC cannot be replaced by other reducing agents, as has been shown for the capability of the isolated C-terminal catalytic domain of Tet2 to oxidize 5mC to 5hmC and 5fC.¹¹⁹ Moreover, only VitC (ascorbic acid), but not DHA, enhances the DNA demethylation, which has been shown by blockade of VitC (SVCTs) and DHA transport (glucose transporter, GLUTs).¹²⁰

Likewise, VitC is also required for the optimal catalytic activity of JmjC domain-containing histone lysine demethylases (JmjC-KDM). Withdrawal of VitC from the in vitro cell culture resulted in the abrogation of histone demethylation.^121,122

Being a cofactor for JmjC-KDM and Tet enzymes, it is reasonable that VitC can influence various cell differentiation processes, in which epigenetic modification is known to play an important role.

VITAMIN C INFLUENCES T-CELL DIFFERENTIATION

Vitamin C promotes hematopoiesis

VitC is widely used in cell culture protocols, because it has been shown to promote the propagation and generation of different types of stem cells. In human embryonic stem cells VitC induces a widespread demethylation of specific genes,¹²³ and enhances the generation of induced pluripotent stem cells (iPSC) by attenuating the hypermethylation of a gene cluster, characteristically hypermethylated in differentiated cells.^124,125 The JmjC-KDMs KDM2A/B (Jhdm1a/1b) have been found to strongly contribute to the VitC-dependent promotion of iPSC reprogramming.¹²⁶ Furthermore, VitC is important for the differentiation of stem cells. Mice transplanted with hematopoietic stem cells (HSC) deficient for Slc23a2 (Solute Carrier Family 23 [Ascorbic Acid Transporter], Member 2) are virtually devoid of T-cell maturation, as the HSC are not able to accumulate VitC intracellularly.¹²⁷ Therefore, VitC is required for the differentiation of HSC.

αβ T cells and γδ T cells develop from the same thymocyte precursors. However, during the thymic development γδ T cells leave the thymus after the DN3 stage, whereas αβ T cells proceed to the DN4 stage.^128,129 Therefore, γδ- unlike αβ T cells, do not undergo thymic ligand-driven positive- or negative selection.¹³⁰

VitC might influence the thymic development by functioning as an anti-oxidant. During αβ T-cell development NF-κB signaling induced by the pre-TCR is necessary for the survival signal at DN3 and transition to DN4 stage.^131,132 γδ T cells, which leave the thymus at the DN3 stage, do not seem to require NF-κB signaling during thymic development.¹³³ However, this has been established using an Lck-Cre mouse model, which might not be fully suitable to study γδ T-cell development, as the proximal Lck-promoter is not used in most γδ T cells.⁴⁷ If really only αβ T-cells require NF-κB-signaling for their thymic development, VitC might favor γδ- over αβ T-cell development, because NF-κB activation can be negatively influenced by VitC.^68,69,72

Cellular metabolism can also influence thymocyte development, and mTOR-C1 and -C2 are of special importance in this respect.^134,135 Raptor-deficiency in murine thymocytes resulted in instability of the cyclin D2/D3-cyclin-dependent kinase 6 complexes and impaired development of the DN2 and DN3 stages.¹³⁴ Yang et al. demonstrated that in a Raptor- (and therefore mTor-C1) deficient mouse model, the transition from the DN3 to the DN4 stage of thymic αβ T-cell development was impaired and, as a consequence, more γδ T cells were generated. This was partially attributed to the inability of the Raptor ^−/− thymocytes to switch efficiently to glycolytic metabolism. Moreover, DN3 thymocytes and γδ T cells from Raptor ^−/− mice displayed elevated ROS levels. By using the anti-oxidants GSH and NAC, the defective αβ T-cell development in the Raptor-deficient mice was partially restored and fewer γδ T cells were generated.⁸² High ROS levels are a hallmark of T-cell activation,⁶⁰ and can further enhance the TCR signal strength.^61,62,63,64 A stronger (γδ) TCR signal in turn is known to promote γδ T-cell over αβ T-cell development.^136,137,138 In mice, unlike in humans, IL-17-poducing γδ T cells represent a distinct γδ T-cell lineage, which already develops in the thymus. The intra-thymic decision between the γδ T(1) and γδ T(17) lineage is again based on the strength of the γδ TCR signal, whereby a stronger signal favors γδ T(1) cell differentiation.³⁵ The anti-oxidant-mediated (e.g., GSH or VitC) reduction of the TCR-signal strength therefore might bias thymocyte development towards αβ T-cell lineage commitment. Moreover, an attenuated TCR-signal also might promote the intra-thymic (murine) γδ T(17)-cell differentiation.

Finally, VitC can also promote thymocytes maturation more directly, by functioning as a cofactor for Tet- and KDM enzymes. For certain γδ T-cell populations it has been shown that their phenotype is imprinted on the epigenetic level. In murine γδ T(17) (CD27⁻CCR6⁺) cells the activating H3K4me2 marks are found on lineage characteristic genes such as IL17, BLK and the corresponding transcription factor RORC. Interestingly, on IFNG and TBX21, activating histone marks were not only found in the IFN-γ-producing γδ T(1) (CD27⁺) subset, but also in the in γδ T(17) subset.¹³⁹ The transition of murine bone marrow derived progenitor cells to functional T cells was enhanced by VitC by supporting the selection of a functional αβTCR after the β-selection. This effect was functionally attributed to the VitC-induced up-regulation of genes involved in the TCR signaling (Zap70, CD8a/b, NFAT; measured 72 h after activation).¹²⁷ By using inhibitors for histone lysine methyl transferases (HMT) (BIX01294) or DNA methyl transferases (DMT) (RG108) it was demonstrated, that in combination with these inhibitors, VitC further strongly increased the number of DP T cells, supporting a role for VitC in promoting the DNA and histone demethylation. The direct effect of VitC on the decreased overall histone methylation was specifically demonstrated for the CD8a gene.¹²⁷ In line with this, VitC also promotes the generation of human T cells from HSC by enhancing the DN1 to DN2- (pro-T1 to pro-T2 transition), as well as the DN to DP progression.¹⁴⁰

Epigenetic effects of vitamin C on the post-thymic differentiation

Th1/Th17 differentiation promoting effects of vitamin C

Several reports indicate that VitC can also affect post-thymic differentiation of T cells. A polarizing influence on T-cell differentiation with a bias toward Th1 or Th17 commitment has been found in several studies. In a mouse model of OVA-induced allergic asthma, it was found, that dietary treatment with high doses of VitC (130 mg/kg BW/d) attenuated the induction of allergic inflammation and reduced the infiltration of eosinophils. Upon VitC treatment, the cytokine profile within the bronchoalveolar lavage fluid was also shifted from Th2 toward Th1 cytokines, determined by the ratio of IL-5 and IFN-γ production.¹⁰⁸

In a mouse model of delayed type hyper-responsiveness to skin application of DNFB (2, 4-dinitro-1-fluorobenzene), mice were treated i.p. with high doses of VitC (0.625 mg/animal/d) during sensitization, challenge, and the following experiment. The VitC-treated animals displayed a less severe inflammation (as measured by ear swelling). The T cells from these VitC-treated animals exhibited a higher proliferative potential ex vivo, as well as a shift of their cytokine profile from a Th2- toward a Th1 phenotype, with reduced IL-4 and enhanced IFN-γ and TNF-α production.¹⁰⁹ One possible way by which VitC might polarize the differentiation toward a Th1 phenotype is through promoting IL-12 secretion by DC and thereby indirectly affecting T-cell differentiation.¹⁴¹

However, a direct effect of VitC seems to be most relevant for T-cell differentiation. Because VitC is a cofactor for JmjC-KDM, differentiation-processes in which these enzymes are involved are likely to be modulated by VitC. Histone methylation patterns have been demonstrated to be important for the stability of functionally different CD4 T-cell subsets.¹⁴² JmjC domain-containing histone demethylase (Jmjd)3 (KDM6B)-deficient CD4 T cells, differentiated in vitro into Th1, Th2, Th17, or Treg cells, displayed a clearly impaired plasticity, especially with respect to Th1 differentiation. The global histone H3K27me2 and H3K27me3 methylation levels (associated with inactive chromatin) were increased in Jmjd3-deficient thymic CD4 SP T cells, whereas H3K4 levels were only slightly affected.¹⁴³ The Jmjd3-mediated H3K27 demethylation seems to be important for Th17 differentiation. In a murine model of experimental encephalomyelitis (EAE), CD4 T cells with a CD4-specific Jmjd3-deletion displayed a clearly impaired Th17 differentiation in vitro and in vivo. Furthermore, Jmjd3 was demonstrated to directly interact with the RORC gene locus, coding for the master transcription factor of Th17 differentiation, ROR-γT, and to reduce H3K27 tri-methylation levels at the RORC and IL17 genes.¹⁴⁴ Song et al. reported that VitC enhanced the Th17 differentiation of FACS-sorted murine naïve CD4 T cells upon anti-CD3/-28 mAb activation in presence of IL6 and TGF-β plus anti-IL2 (which was crucial to prevent FoxP3-induction). Mechanistically, this effect was attributed to the VitC-induced enhanced histone-demethylating activity of the Jmjd2 (KDM4A). The VitC-promoted Jmjd2-mediated reduction of H3K9me3 (transcriptional inactivating histone mark) within the IL17 gene promoter correlated with enhanced IL-17 production.¹¹⁰ As a cofactor, VitC thus influences JmjC-KDM activity, which seems to be most relevant for the Th1 and Th17 differentiation.

Vitamin C promotes regulatory T-cell (Treg) differentiation

CD4⁺CD25^high Tregs are important to maintain the immunologic self-tolerance. They express FoxP3 as a key transcription factor, which is required for stable Treg function.^145,146 Within the FOXP3 gene, there are CpG-island-containing conserved noncoding sequences (CNS), which are highly methylated in conventional T cells, but almost fully demethylated in functional Treg cells. The Treg CNS2, also known as Treg-specific demethylated region (TSDR), is regarded as the most reliable marker for a stable Treg lineage commitment in mice and humans.^147,148 The demethylation of the FOXP3 TSDR is a prerequisite for a stable FoxP3 gene expression.¹⁴⁹

Treg are classified as naturally occurring thymic-derived tTreg (which do not depend on TGF-β signaling for their differentiation) and peripheral-induced iTreg, which require TGF-β to acquire FoxP3 expression and suppressive activity. Therefore, in TGF-β-deficient mice, the development of peripherally induced Treg is impaired, whereas tTreg develop normally.¹⁵⁰ Ex vivo isolated Treg from both mouse and humans have an unmethylated TDSR, whereas the TDSR in conventional T cells and in in vitro TGF-β-induced Treg is highly methylated.^149,151 Tet enzymes are important for the induction and stability of FoxP3 expression. During the thymic development of murine tTreg, Tet2/Tet3 are required for the demethylation of the CNS1 and CNS2.¹⁵² In the periphery IL-2-induced Tet2 has been shown to protect CNS2 in murine Treg from being remethylated by DMT and from losing FoxP3 expression under inflammatory conditions.¹⁵³ In comparison to FoxP3⁻ murine CD4 T cells, the VitC-transporter SVCT2 is expressed at higher levels by FoxP3⁺ Treg, and moreover is specifically up-regulated upon activation.¹⁵⁴ The enhanced expression of SVCT2 and the role of VitC as a cofactor for Tet enzymes, suggests that VitC and Tet enzymes cooperate in stabilizing the FoxP3 expression in Treg. Several studies have described effects of VitC on the FoxP3 expression and Treg function. In a mouse model of ex vivo induced alloantigen-tolerant Treg, peripheral FoxP3⁻ CD4 T cells were cocultured with alloantigen-presenting DC in the presence of IL-2, TGF-β, and retinoic acid. When VitC was additionally added to the cell culture only a slight increase in iTreg numbers was found when compared to conventional iTreg. The VitC-induced iTreg displayed nonetheless a more stable FoxP3 expression and promoted prolonged survival of a skin allograft in vivo. Moreover, unlike iTreg generated in the absence of VitC, the CNS2 (TSDR) of VitC-treated iTreg was strongly demethylated, comparable to the degree of demethylation present in naturally occurring Treg.¹⁵⁵ Another study on murine Treg also found that higher numbers of TGF-β-induced iTreg, with increased FoxP3 protein-expression, were generated upon VitC supplementation, in vitro. Nonetheless, the VitC-treated Treg were less efficient in suppressing the proliferation of cocultured CD4 T cells. When naive T cells were transferred into T-cell deficient (RAG-KO) mice with an allogeneic skin transplant and VitC was applied orally in combination, once again FoxP3⁺ T cells were found in higher numbers. However, this had no positive impact on the skin-graft survival.¹⁵⁴

The effect of VitC on the generation of Treg mainly depends on Tet activity even though the role of the different Tet family members is not completely clear. Nair et al. found that the VitC effect, leading to the CNS2 demethylation during the induction of iTreg, was no longer present in mice lacking Tet2 (Tet2 −/−).¹⁵⁶ Yue et al. found by using a murine knock out model for Tet2, Tet3, and Tet2/3, that VitC enhances both Tet2 and Tet3 activity and thereby enhances the demethylation of the CNS2, by which the FoxP3 expression was stabilized in TGF-β-induced murine Treg, in vitro and in vivo. Moreover, the VitC-treated iTreg displayed superior suppressive activity on CD8⁺ T-cell proliferation, in vitro.¹⁵² Taken together, in presence of TGF-β, VitC augments the generation of a stable αβ iTreg phenotype by enhancing the activity of Tet enzymes, even though observations differ with respect to their functionallity. A scheme of the VitC-supported epigenetic activities and their influence on T-cell differentiation is depicted in Figure 2.

As described for αβ T cells, TGF-β also can induce a regulatory phenotype in γδ T cells,^157,158 as well as an IL-17-,^31,159,160 or IL-9-producing phenotype.³⁰ Whereas TGF-β can promote the generation of Vδ2 T cells with high cytotoxic potential in absence of VitC,¹⁶¹ our unpublished data indicate that VitC (pVC) in combination with TGF-β promotes FoxP3-induction and a Vδ2 T(reg) phenotype. This effect is in line with studies in αβ T cells.^152,154,155 Moreover, we found that in VitC-treated Vδ2 T cells the FoxP3 protein-expression could be detected for six more days after TGF-β-withdrawal, with the most stable expression in anti-CD3/-28 mAb-expanded Vδ2 T cells. Strikingly, the robust induction and enhanced stability of FoxP3 correlated with a pronounced CNS2 demethylation. The CNS2 remained methylated in FoxP3-negative or in TGF-β-only treated FoxP3-positive Vδ2 T cells, but was strongly demethylated exclusively in FoxP3-positive Vδ2 T cells from VitC- plus TGF-β-treated cell cultures. Our observations suggest that Tet enzyme-dependent mechanisms, similar to those described for αβ T cells,^152,156 are also involved in the generation of VitC- plus TGF-β-induced regulatory Vδ2 T(reg) cells. VitC- plus TGF-β-induced αβ iTreg, even though they displayed a more stable and more pronounced FoxP3-expression, did not necessarily have a higher suppressive capacity in vitro and in vivo.^152,154,155 In contrast, we observed that VitC-treated Vδ2 T(reg) cells exerted enhanced suppressive activity on autologous CD4 T-cell proliferation.

Because γδ T cells have been implicated in various autoimmune diseases, such as autoimmune diabetes, multiple sclerosis/experimental autoimmune encephalitis (EAE), inflammatory bowel disease, and rheumatoid arthritis,¹⁶² the ex vivo generation of functional regulatory γδ T(reg) cells with stable FoxP3 protein expression might provide an interesting tool to suppress the inflammatory immune response in the context of γδ T-cell driven autoimmunity.

Perspectives for the use of vitamin C in γδ T-cell based immunotherapy: concluding remarks

VitC exhibits pleiotropic effects on the immune system and on T cells in particular. Many of the VitC-dependent effects on αβ T cells have also been observed for γδ T cells, albeit in most cases it is not clear, if the mechanisms underlying these effects are the same as in αβ T cells. The effects of VitC on γδ T cells have not been extensively studied so far, and more detailed studies are needed to allow precise statements about the molecular basis of the VitC-mediated effects on γδ T cells.

Due to its high safety profile, VitC represents an ideal tool to condition T-cell differentiation and effector functions and could be used to shape the T-cell response in vitro as well as in vivo. γδ T cells can be easily expanded by pAg stimulation or using aminobisphosphonates in vitro, also at clinical scales and under GMP conditions. Due to their unique features (HLA-independency, recognition of phosphorylated metabolites rather than antigenic peptide/HLA complexes, potent effector function), γδ T cells are promising candidates for immunotherapeutic approaches by adoptive transfer.^27,163 VitC might be of special interest for optimizing the in vitro generation of γδ T cells for their subsequent adoptive transfer, because the desired effects could be specifically restricted to the cell population of interest, thereby avoiding the other countless effects of VitC on other cell populations. VitC can enhance the effector functions and proliferation of Vδ2 T cells or, in the presence of TGF-β, promote the differentiation of Vδ2 T(reg) (Fig. 3).

Because γδ T cells have been implicated in different auto-immune driven diseases,¹⁶² studies on the in vivo function of VitC-induced Vδ2 T(reg) in adequate animal models are needed to address the question whether their more stable phenotype and stronger suppressive activity, observed in in vitro, really has in vivo relevance. Moreover, the role of (Tet) enzymes regulating this process in γδ T cells has to be formally proven. Also, the possible influence of VitC on JmjC-KDM in γδ T-cell differentiation processes has not yet been addressed, but might be relevant.

On the other hand, Vδ2 T cells are known to exhibit potent antitumor activity. In the absence of TGF-β, VitC enhances the proliferation and cytotoxic effector functions of Vδ2 T cells. Therefore, the efficacy of adoptive transfer of VitC-treated Vδ2 T cells has to be tested in suitable preclinical animal models for autoimmunity and cancer immunotherapy. Finally, the in vivo treatment with high doses of (i.v.) VitC, which can exert direct cytotoxic effects on tumor cells, should also be tested in combination with in vivo activation of Vδ2 T cells using aminobisphosphonates, or with adoptive transfer of in vitro expanded γδ T cells.

ACKNOWLEDGMENTS

The authors’ work on the in vitro effects of VitC on the γδ T-cell differentiation was supported by the Deutsche Forschungsgemeinschaft (Ka 502/19-1, D.K.), the German Academic Exchange Service (DAAD, L.K.), and the Werner-and-Klara Kreitz Foundation (L.K.).

AUTHORSHIP

C.P. wrote the draft of the manuscript and created the figures, L.K. and D.K. contributed to the discussion and the finalization of the manuscript.

DISCLOSURES

D.K. is a member of the Scientific Advisory Boards of Imcheck Therapeutics, Incysus Therapeutics, Lava Therapeutics, and Qu Biologics. The other authors declare no conflicts of interest.

REFERENCES