The differentiation and functional plasticity of T cells are transcriptionally and epigenetically regulated by signals imposed by the local cytokine milieu and a variety of additional factors, including vitamins. Vitamin C has pleiotropic functions in the immune system. It exerts antioxidant activity, can directly kill selected tumor targets, promotes early T-cell differentiation, and enhances Th1 cytokine production in mature T cells

Kabelitz, Dieter

Klein, Katharina

Peters, Christian

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Vitamin C and Vitamin D—friends or foes in modulating γδ T-cell differentiation?

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COMMENT OPENVitamin C and Vitamin D—friends or foes in modulatingγδ T-cell differentiation?Christian Peters 1, Katharina Klein 1 and Dieter Kabelitz 1✉© The Author(s) 2022Cellular & Molecular Immunology (2022) 19:1198–1200; https://doi.org/10.1038/s41423-022-00895-wThe differentiation and functional plasticity of T cells aretranscriptionally and epigenetically regulated by signals imposedby the local cytokine milieu and a variety of additional factors,including vitamins. Vitamin C has pleiotropic functions in theimmune system. It exerts antioxidant activity, can directly killselected tumor targets, promotes early T-cell differentiation, andenhances Th1 cytokine production in mature T cells [1, 2]. Vitamin Cis also an epigenetic modifier that acts on ten-eleven translocation(TET) enzymes to demethylate FOXP3 and stabilize FOXP3 proteinexpression and regulatory T-cell (Treg) function [3]. We previouslyexplored the role of vitamin C in the modulation of the activationand differentiation of human γδ T cells. γδ T cells have recentlyattracted substantial interest as effector cells in cell-based cancerimmunotherapy due to their potent capacity to kill a varietyof different cancer cell types in the absence of HLA restriction.While the available data are promising, there is a clear need tooptimize the efficacy of γδ T cells in clinical applications [4]. Formany potential strategies, including the use of γδ T-cell-selectiveactivating antibodies or γδ T-cell-targeting bispecific antibodyconstructs and the development of drug-resistant γδ T cells, wereasoned that vitamin C might also be considered to boost theeffector functions of γδ T cells Fig. 1.In a previous study published in this journal, we reported thatL-ascorbic acid-2-phosphate (“phospho-vitamin C”, pVC), which ismore stable than unmodified vitamin C and does not acidify thecell culture medium, enhances the proliferative expansion andcytokine production of human γδ T cells activated by γδ T-cell-selective pyrophosphates (“phosphoantigens”). Importantly, pVCreduced the proportion of apoptotic cells during the in vitroexpansion of γδ T cells. pVC also strongly promoted the cellularexpansion of surviving γδ T cells after T-cell receptor restimulationof short-term expanded γδ T-cell lines [5]. When expanded in thepresence of pVC, γδ T cells also displayed enhanced metabolicactivity and increased killing capacity as measured against severaltumor cell targets [5, 6]. Our discovery of the enhancing effect ofpVC on the effector functions of γδ T cells led to the first adoptivetransfer of allogeneic γδ T cells expanded in vitro in the presenceof vitamin C into patients with solid cancers [6].In view of the known effect of vitamin C on the activation ofTET enzymes and thus on the demethylation in Treg-specificdemethylated regions (TSDRs) of the FOXP3 locus and on Tregfunction, we more recently extended our studies to FOXP3regulation in human γδ T cells. We observed that pVC actuallyincreased FOXP3 expression in γδ T cells and induceddemethylation in FOXP3 TSDRs but only in the presence of TGF-β, which is involved in Treg induction. In the absence of pVC butpresence of TGF-β, cell sorter-purified FOXP3-expressing γδT cells did not show any hypomethylation in FOXP3 TSDRs [7].Taken together, our previous work established that vitamin C andpVC have strong potential to enhance the effector functions of γδT cells that could likely be extended to other antitumor effectorcells such as CAR T cells or NK cells [8]. Importantly, however, thepresence of additional factors such as TGF-β (which is frequentlyencountered in the local tumor microenvironment) could deviatethe desired effects of γδ T cells into tumor-promoting effects, e.g.,by inducing an “active” Treg phenotype.In addition to vitamin C, vitamin D is another vitamin withmany immunomodulatory effects. Vitamin D plays an importantrole in bone mineralization and calcium homeostasis. The activeform 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) binds to thenuclear vitamin D receptor (VDR), which acts as a transcriptionfactor regulating a variety of target genes. There is growingevidence that vitamin D exerts a protective role against severaltypes of cancer[9]. Similar to vitamin C, 1,25(OH)2D3 has also beenfound to impact T-cell differentiation at multiple levels. In humanT cells, it induces FOXP3 protein expression and a suppressivephenotype but – in contrast to vitamin C – does not inducedemethylation of TSDRs of the FOXP3 locus [10]. Moreover,1,25(OH)2D3 suppresses interferon-γ (IFN-γ) and promotesinterleukin-10 (IL-10) production and thus downregulates T-cell-mediated inflammation [11]. In support of this finding, clinicalstudies indicate that oral vitamin D supplementation hasbeneficial effects in patients with autoimmune diseases such asmultiple sclerosis [12].To date, the available information on the potential modula-tion of γδ T-cell activation by 1,25(OH)2D3 is limited. It has beenreported that 1,25(OH)2D3 suppresses the in vitro proliferationand IFN-γ production of human phosphoantigen-reactive γδT cells [13]. Similarly, we also observed reduced productionof IFN-γ by γδ T cells in the presence of 1,25(OH)2D3 [14].Moreover, we found that 1,25(OH)2D3 also inhibited γδT-cell expansion when γδ T cells within PBMCs were stimulatedwith phosphoantigen or aminobisphosphonate zoledronicacid. In these experiments, the presence of monocytes withinPBMCs played a significant role. Furthermore, 1,25(OH)2D3 alsoreduced the killing of selected tumor target cells by expandedγδ T cells. Overall, our and previous studies indicate that1,25(OH)2D3 downregulates γδ T-cell function [13, 14]. InReceived: 14 June 2022 Accepted: 16 June 2022Published online: 7 July 20221Institute of Immunology, Christian-Albrechts University, Kiel, Germany. ✉email: dietrich.kabelitz@uksh.dewww.nature.com/cmi1234567890();,:striking contrast, a recent report by Li et al. actually demon-strated costimulatory activity of 1,25(OH)2D3 on the IFN-γ andTNF-α production of human γδ T cells and CD8 T cells activatedby anti-CD3/CD28 antibody stimulation [15]. The authorsalso observed that 1,25(OH)2D3 increased CD28 expressionbut reduced PD-1, TIGIT and Tim-3 expression on CD8 T cellsand γδ T cells and thus reverted their exhausted phenotype.Furthermore, γδ T cells pretreated with 1,25(OH)2D3 exertedenhanced antitumor activity in vitro and in vivo upon transferinto immunodeficient mice transplanted with human tumorcells. Importantly, the results also indicated that therapeuticsupplementation with calcitriol (1,25(OH)2D3) can revert theexhausted phenotype of CD8 and γδ T cells in cancer patientsand enhance their functional activity [15]. Currently, it isdifficult to reconcile the mechanistic basis for the discrepancybetween published studies regarding the inhibitory [13, 14]and stimulatory [15] effects of 1,25(OH)2D3 on IFN-γ productionin vitro.However, there is evidence that both vitamin C and vitamin Dmight exert beneficial effects in cancer patients [1, 8, 9, 15].Therefore, how the combination of these two vitaminsmodulates the immune system should be considered. Specifi-cally, we suggest investigating how the combination of vitaminC and 1,25(OH)2D3 modulates the activity of immune cellsimplicated in antitumor defense, including γδ T cells. Werecently initiated such experiments and analyzed the regulatoryinteraction between the two vitamins in modulating the IFN-γ production of human CD4 and γδ T cells. Purified CD4 and γδT cells were preactivated for 2 h with their respective T-cellreceptor ligands (staphylococcal superantigens SEA/B for CD4T cells, phosphoantigen bromohydrin pyrophosphate [BrHPP]for γδ T cells) to allow responsiveness to 1,25(OH)2D3 byupregulation of VDR before adding pVC, 1,25(OH)2D3, or thecombination of both. As expected, pVC increased the secretionof IFN-γ by CD4 T cells, while 1,25(OH)2D3 completelysuppressed IFN-γ secretion. Our unpublished results indicatethat the suppressive effect of 1,25(OH)2D3 dominated overthe stimulatory effect of pVC, as there was still completeinhibition of IFN-γ secretion in the presence of the combinationof 1,25(OH)2D3 and pVC. In line with our previous reports [5, 14],we also found that pVC enhanced and the reduced IFN-γ secretion induced by 1,25(OH)2D3 in the supernatants ofγδ T cells activated for 7 days with BrHPP. In contrast to thecase for CD4 T cells, inhibition by 1,25(OH)2D3 was not complete,and in fact, inhibition was reversed in the additional presenceof pVC.We aimed to extend the findings of our original paper on theeffects of vitamin C published in this journal [5], and ourunpublished preliminary work indicates that 1,25(OH)2D3 over-rides the costimulatory effect of pVC by completely shutting downIFN-γ secretion in CD4 T cells but much less stringently in γδT cells. Obviously, such studies need to be expanded to othercytokines and effector functions. Further detailed in vitro experi-ments and preclinical studies in appropriate mouse models will berequired to delineate if and how the two vitamins can becombined to achieve the best antitumor efficacy.REFERENCES1. Yun J, Mullarky E, Lu C, Bosch KN, Kavalier A, Rivera K, et al. Vitamin C selectivelykills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH. Science.2015;350:1391–6. https://doi.org/10.1126/science.aaa5004.2. Peters C, Kouakanou L, Kabelitz D. A comparative view on vitamin C effects onαβ- versus γδ T-cell activation and differentiation. J Leukoc Biol. 2020;107:1009–22. https://doi.org/10.1002/JLB.1MR1219-245R.3. Yue X, Samaniego-Castruita D, González-Avalos E, Li X, Barwick BG, Rao A. Whole-genome analysis of TET dioxygenase function in regulatory T cells. EMBO Rep.2021;22:e52716. https://doi.org/10.15252/embr.202152716.4. Kabelitz D, Serrano R, Kouakanou L, Peters C, Kalyan S. Cancer immunotherapywith γδ T cells: many paths ahead of us. Cell Mol Immunol. 2020;17:925–39.https://doi.org/10.1038/s41423-020-0504-x.5. Kouakanou L, Xu Y, Peters C, He J, Wu Y, Yin Z, et al. Vitamin C promotes theproliferation and effector functions of human γδ T cells. Cell Mol Immunol.2020;17:462–73. https://doi.org/10.1038/s41423-019-0247-8.6. Xu Y, Xiang Z, Alnaggar M, Kouakanou L, Li J, He J, et al. Allogeneic Vγ9Vδ2 T-cellimmunotherapy exhibits promising clinical safety and prolongs the survival ofpatients with late-stage lung or liver cancer. Cell Mol Immunol. 2021;18:427–39.https://doi.org/10.1038/s41423-020-0515-7.7. Kouakanou L, Peters C, Sun Q, Floess S, Bhat J, Huehn J, et al. Vitamin Csupports conversion of human γδ T cells into FOXP3-expressing regulatory cellsby epigenetic regulation. Sci Rep.2020;10:6550. https://doi.org/10.1038/s41598-020-63572-w.8. Kouakanou L, Peters C, Brown CE, Kabelitz D, Wang LD. Vitamin C, from sup-plement to treatment: a re-emerging adjunct for cancer immunotherapy?. FrontImmunol. 2021;12:765906. https://doi.org/10.3389/fimmu.2021.765906.9. Carlberg C, Muñoz A. An update on vitamin D signaling and cancer. Semin CancerBiol. 2022;79:217–30. https://doi.org/10.1016/j.semcancer.2020.05.018.10. Matos C, Renner K, Peuker A, Schoenhammer G, Schreiber L, Bruss C, et al.Physiological levels of 25-hydroxyvitamin D3 induce a suppressive CD4+ T cellphenotype not reflected in the epigenetic landscape. Scand J Immunol.2022;95:e13146. https://doi.org/10.1111/sji.13146.11. Chauss D, Freiwald T, McGregor R, Yan B, Wang L, Nova-Lamperti E, et al.Autocrine vitamin D signaling switches off pro-inflammatory programs ofTH1 cells. Nat Immunol. 2022;23:62–74. https://doi.org/10.1038/s41590-021-01080-3.12. Yeh WZ, Gresle M, Jokubaitis V, Stankovich J, van der Walt A, Butzkueven H.Immunoregulatory effects and therapeutic potential of vitamin D in multiplesclerosis. Br J Pharmacol. 2020;177:4113–33. https://doi.org/10.1111/bph.15201.13. Chen L, Cencioni MT, Angelini DF, Borsellino G, Battistini L, Brosnan CF. Transcriptionalprofiling of gamma delta T cells identifies a role for vitamin D in the immunor-egulation of the Vgamma9 Vdelta2 response to phosphate-containing ligands. JImmunol. 2005;174:6144–52. https://doi.org/10.4049/jimmunol.174.10.6144.14. Bernicke B, Engelbogen N, Klein K, Franzenburg J, Borzikowsky C, Peters C, et al.Analysis of the seasonal fluctuation of γδ T cells and its potential relation withvitamin D3. Cells. 2022;11:1460. https://doi.org/10.3390/cells11091460.Fig. 1 Modulation of human γδ T-cell activation by vitamin Cand vitamin D. Vitamin C enhances the cytokine production(notably IFN-γ), proliferative expansion and cytotoxic activity ofphosphoantigen-reactive human γδ T cells. In the additionalpresence of TGF-β, vitamin C induces hypomethylation of TSDRs inthe FOXP3 locus and stabilizes FOXP3 expression in γδ T cells [5–7].Vitamin D also modulates the IFN-γ secretion, proliferation andcytotoxic activity of human γδ T cells, but the reported effectsare controversial. We and others have observed inhibition ofproliferation, IFN-γ production and cytotoxicity [13, 14], in contrastto a recent report where the effector functions of γδ T cells werefound to be enhanced by vitamin D [15]C. Peters et al.1199Cellular & Molecular Immunology (2022) 19:1198 – 120015. Li P, Zhu X, Cao G, Wu R, Li K, Yuan W, et al. 1α,25(OH)2D3 reverses exhaustionand enhances antitumor immunity of human cytotoxic T cells. J ImmunotherCancer. 2022;10:e003477. https://doi.org/10.1136/jitc-2021-003477.ACKNOWLEDGEMENTSThis work was supported by a grant from the Deutsche Forschungsgemeinschaft(Ka502/19-3 to DK).AUTHOR CONTRIBUTIONSDK wrote the manuscript, and CP and KK performed the experiments mentioned inthe text. All authors contributed to the finalization of the manuscript.FUNDINGOpen Access funding enabled and organized by Projekt DEAL.COMPETING INTERESTSThe authors declare no competing interests.ADDITIONAL INFORMATIONCorrespondence and requests for materials should be addressed to Dieter Kabelitz.Reprints and permission information is available at http://www.nature.com/reprintsOpen Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in anymedium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the CreativeCommons license, and indicate if changes were made. The images or other third partymaterial in this article are included in the article’s Creative Commons license, unlessindicated otherwise in a credit line to the material. If material is not included in thearticle’s Creative Commons license and your intended use is not permitted bystatutory regulation or exceeds the permitted use, you will need to obtain permissiondirectly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.© The Author(s) 2022C. Peters et al.1200Cellular & Molecular Immunology (2022) 19:1198 – 1200

Vitamin C and Vitamin D-friends or foes in modulating γδ T-cell differentiation?

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Vitamin C and Vitamin D-friends or foes in modulating γδ T-cell differentiation?

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