Alansary, Dalia

Niemeyer, Barbara A.

Qu, Bin

Schwarz, Eva C.

Schwär, Gertrud

Yang, Wenjuan

Yin, Deling

Zhao, Renping

Zou, Huajiao

English

East Tennessee State University

East Tennessee State University Digital Commons @ East Tennessee State University ETSU Faculty Works Faculty Works 12-1-2020 High Glucose Distinctively Regulates Ca2+ Influx in Cytotoxic T Lymphocytes Upon Target Recognition and Thapsigargin Stimulation Huajiao Zou Central South University Wenjuan Yang Universitätsklinikum des Saarlandes Medizinische Fakultät der Universität des Saarlandes Gertrud Schwär Universitätsklinikum des Saarlandes Medizinische Fakultät der Universität des Saarlandes Renping Zhao Universitätsklinikum des Saarlandes Medizinische Fakultät der Universität des Saarlandes Dalia Alansary Universitätsklinikum des Saarlandes Medizinische Fakultät der Universität des Saarlandes See next page for additional authors Follow this and additional works at: https://dc.etsu.edu/etsu-works Citation Information Zou, Huajiao; Yang, Wenjuan; Schwär, Gertrud; Zhao, Renping; Alansary, Dalia; Yin, Deling; Schwarz, Eva C.; Niemeyer, Barbara A.; and Qu, Bin. 2020. High Glucose Distinctively Regulates Ca2+ Influx in Cytotoxic T Lymphocytes Upon Target Recognition and Thapsigargin Stimulation. European Journal of Immunology. Vol.50(12). 2095-2098. https://doi.org/10.1002/eji.202048577 PMID: 32697355 ISSN: 0014-2980 This Letter is brought to you for free and open access by the Faculty Works at Digital Commons @ East Tennessee State University. It has been accepted for inclusion in ETSU Faculty Works by an authorized administrator of Digital Commons @ East Tennessee State University. For more information, please contact digilib@etsu.edu. High Glucose Distinctively Regulates Ca2+ Influx in Cytotoxic T Lymphocytes Upon Target Recognition and Thapsigargin Stimulation Copyright Statement This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License Creator(s) Huajiao Zou, Wenjuan Yang, Gertrud Schwär, Renping Zhao, Dalia Alansary, Deling Yin, Eva C. Schwarz, Barbara A. Niemeyer, and Bin Qu This letter is available at Digital Commons @ East Tennessee State University: https://dc.etsu.edu/etsu-works/9073 Eur. J. Immunol. 2020. 50: 2095–2098 DOI: 10.1002/eji.202048577 2095Letter to the Editor[DOI: 10.1002/eji.202048577]High glucosedistinctively regulatesCa2+ influx incytotoxic Tlymphocytes upontarget recognition andthapsigarginstimulationThe killing processes of CD8+ cytotoxic Tlymphocytes (CTLs) are highly dependenton calcium, both extracellular and cytoso-lic [1]. Engagement of TCRs triggers store-operated calcium entry (SOCE) mediatedby stromal interaction molecules (STIM) totrap and activate ORAI channels [2]. Glu-cose is essential for effector functions ofCTLs [3]. Elevated level of blood glucose isone of the leading symptoms of diabetes.Yet, the impact of high glucose (HG) onCTL function is not well understood.To investigate the impact of HGon Ca2+ influx in CTLs, we used pri-mary human CD8+ T cells, stimulatedwith anti-CD3/anti-CD28 antibody-coatedbeads (CD3/CD28 beads) and cultured innormal glucose medium (NG, 5.6 mM) orHG medium (25 mM). To elicit SOCE, weused thapsigargin (Tg) to deplete Ca2+from the ER [4]. For Tg-induced SOCE, wefound no difference between NG- and HG-condition for unstimulated CD8+ T cells(Fig. 1A-D) or at day 1.5 poststimulation(Fig. 1E-H). Remarkably, at days 3 and 7after bead stimulation, Tg-induced SOCEwas higher in HG-cultured CTLs comparedto their NG counterparts (Fig. 1I-P). Uponbead stimulation, CTLs exhibited a sig-nificantly enhanced SOCE, which startedto decline in NG condition but not inHG condition from day 3 (Fig. 1Q, R).These results suggest that Tg-elicited SOCEin CTLs is elevated by prolonged treatmentof HG.Target cell recognition, a more physio-logical stimulus than Tg, also elicits SOCEin CTLs. We used staphylococcal entero-toxin A and B (SEA/SEB)-pulsed Raji cellsas target cells, which induce CTL-mediatedcytotoxicity [1, 5]. We observed that at day3 poststimulation, in HG-cultured CTLs,target recognition-induced Ca2+ influx wassignificantly reduced relative to their NGcounterparts, despite of the same basallevel of intracellular Ca2+ (Fig. 2A-D).At day 7, upon target cell engagement,only a moderate difference in the Ca2+influx rate and the Ca2+ peak was identi-fied between NG- and HG-cultured CTLs,whereas no difference in Ca2+ plateau(Fig. 2E-H). Furthermore, the Raji cellswithout SEA/SEB-pulsing did not elicitCa2+ influx in CTLs (Fig. 2I). Together, weconclude that in CTLs, target recognition-induced Ca2+ influx is reduced by HG-culture.Next, we investigated how HG coulddifferently regulate SOCE induced by Tg ortarget recognition in CTLs. We first exam-ined glucose uptake using a fluorescentanalog of glucose 2-NBDG, and found nodifference between HG- and NG-culturedCTLs (Fig. 2J, K). Subsequently, we ana-lyzed the expression of STIMs and ORAIs.At mRNA level, in HG-cultured CTLs,ORAI1 stayed unchanged, while ORAI2and ORAI3 were slightly upregulated; bothSTIM1 and STIM2 were downregulated(Fig. 2L, M). Interestingly, at protein level,STIM1, STIM2, and ORAI1 in HG-CTLswere upregulated in Donor 1 and 4, butremained unchanged in Donor 2 and 3(Fig. 2N, O). Due to lack of reliable anti-body against ORAI2 and ORAI3, their pro-tein levels could not be determined. Ourfindings suggest that the increase in Tg-induced SOCE in CTLs by HG could bemediated by upregulation of STIM1 andSTIM2. Since ORAI2 and ORAI3 mainlyfunction as negative regulators of Ca2+entry in T cells [6, 7], upregulation ofORAI2 and ORAI3 by HG could contributeto the reduction of target recognition-induced Ca2+ influx. Inhibitory splice vari-ants of STIMs, for example, STIM2.1 orphosphorylation of STIMs could also playa role.After glucose loading, proportion ofCTLs is reduced in humans [8]. Interest-ingly, we found that after killing started,the fraction of necrotic CTLs was higherin HG-condition after 16 h but not after 2h (Fig. 2P, Q), suggesting that in a timescale of several days in HG, CTLs are moreprone toward necrosis after execution oftheir killing function.One main difference between Tg andtarget-recognition is that engagement ofTCRs upon target-recognition activatesmany signaling cascades, whereas Tg onlyblocks the sarco/endoplasmic reticulumCa2+-ATPase, leading to passive deple-tion. In addition, CTLs are stimulatedglobally with Tg and locally with tar-get cells at the immunological synapse(IS). Along this line, other factors couldalso be involved, for example, mitochon-dria or PMCA (plasma membrane Ca2+ATPase). Both are enriched at the IS [9, 10]but scarcely distributed in Tg-stimulation.Considering that decreased intracellularC© 2020 The Authors. European Journal of Immunology published by Wiley-VCH GmbH www.eji-journal.euThis is an open access article under the terms of the Creative Commons Attribution-NonCommercialLicense, which permits use, distribution and reproduction in any medium, provided the original workis properly cited and is not used for commercial purposes.2096 Huajiao Zou et al. Eur. J. Immunol. 2020. 50: 2095–2098Figure 1. SOCE in CTLs is enhanced by HG-culture. Ca2+ imaging was conducted (20 × objective). Primary human CD8+ T cells were eitherunstimulated or stimulated with CD3/CD28 beads in NG medium (5.6 mM) or HG (25 mM) medium and then loaded with Fura-2-AM. Exemplarycells are shown in A, E, I, and M. Calcium influx curves are shown in B, F, J, N (n [independent experiments/donors] = 4/6 [Day 0/Day 1.5], 5/7[Day3], or 5/8 [Day7]). Paired t-test was used except for Q and R (unpaired t-test). Results are presented as mean ± SEM. Scale bars are 5 μm.Ca2+ concentration in CTLs is correlatedwith enhanced CTL-mediated cytotoxicity[1], our findings suggest that increase theenvironmental glucose for a short period oftime might be beneficial for CTLs to fightaberrant cells more efficiently. However,considering our result that HG-treatedCTLs tend to die more after killing, in dia-betic patients, it might lead to a reductionin CTL numbers, resulting in a comprisedimmune response against pathogens in along run.C© 2020 The Authors. European Journal of Immunology published byWiley-VCH GmbHwww.eji-journal.euEur. J. Immunol. 2020. 50: 2095–2098 2097Figure 2. HG-culture reduces Ca2+ influx in CTLs upon target recognition and impairs CTL viability after killing. (A-I) Ca2+ imaging was conducted.CD3/CD28 bead-stimulated CTLs were loaded with Fura-2-AM (20 × objective, Scale bars: 5 μm). Exemplary cells are shown in A and E. Raji cellspulsed with (A-H) or without (I) SEA/SEB. n (independent experiments/donors) = 5/7 (A-D), 3/6 (E-H), 3/5 (I, Day3), or 3/6 (I, Day7) (J, K) Glucoseuptake in CTLs. Day 3 CD3/CD28 bead-stimulated CTLs were loaded with 2-NBDG without target cells. Light gray: unstained cells. n = 5 independentexperiments/8 donors. (L, M) Expression of ORAIs and STIMs in CTLs. The mRNA level was determined using qRT-PCR and either normalized toORAI1 in NG-cultured CTLs (L) or normalized to NG condition as fold change (M). n = 2 independent experiments/6 donors. (N, O) Protein levelsof STIMs and ORAI1. Quantification by densitometry analysis following Western blotting (N) and normalization to β-actin (Q). n = 4 donors. (P, Q)Necrosis of CTLs after killing. Bead-stimulated CTLs were co-incubated with indicated target cells for 2 h (P, Raji n = 2 independent experiments/3donors, Raji-SEA/SEB n = 4 independent experiments/5 donors.) or 16 h (Q, Raji n = 3 independent experiments/4 donors, Raji-SEA/SEB n =5 independent experiments/6 donors). Propidium iodide (PI) was used to determine necrotic CTLs death. Gating strategy shown in Supportinginformation Fig. 1. Gray: no PI-staning. Paired t-test was used for all statistical analysis. Data shown as mean ± SEM.C© 2020 The Authors. European Journal of Immunology published byWiley-VCH GmbHwww.eji-journal.eu2098 Huajiao Zou et al. Eur. J. Immunol. 2020. 50: 2095–2098Huajiao Zou1,2, Wenjuan Yang2,Gertrud Schwär2, Renping Zhao2,Dalia Alansary3, Deling Yin1,4,Eva C. Schwarz2, Barbara A. Niemeyer3and Bin Qu2,51 Xiangya School of Pharmaceutical Sciences,Central South University, Changsha, China2 Biophysics, Center for Integrative Physiology andMolecular Medicine (CIPMM), School ofMedicine, Saarland University, Homburg,Germany3 Molecular Biophysics, CIPMM, School ofMedicine, Saarland University, Homburg,Germany4 Department of Internal Medicine, College ofMedicine, East Tennessee State University,Johnson City, TN, USA5 INM-Leibniz Institute for New Materials,Saarbrücken, GermanyAcknowledgments: We thank the Insti-tute for Clinical Hemostaseology andTransfusion Medicine for providing donormaterials; Carmen Hässig and SusanneRenno for excellent technical help; GirishRamesh for Western blotting; SandraJanku for her careful and critical read-ing. We are grateful to Markus Hothfor constant support, inspiring discus-sions, and advice regarding writing of themanuscript. This project was funded by theDeutsche Forschungsgemeinschaft (SFB1027, A2 to B.Q., C4 to B.N.; Forschungs-großgeräte (GZ: INST 256/423-1 FUGG)for the flow cytometer), and by the Leibniz-Gemeinschaft (INM Fellow Program, toB.Q.).Open access funding enabled and orga-nized by Projekt DEAL.Ethical Considerations: Research car-ried out for this study with human mate-rial (leukocyte reduction system chambersfrom human blood donors) is authorizedby the local ethic committee (declarationfrom 16.4.2015 [84/15; Prof. Dr. Rettig-Stürmer]).Conflict of Interest: The authors declareno commercial or financial conflict of inter-est.References1 Zhou, X. et al., J. Physiol. 2018. 596: 2681–2698.2 Amcheslavsky, A. et al., Biophy. J. 2015. 108:237–246.3 Geltink, R. I. K. et al., Annu. Rev. Immunol. 2018.36: 461–488.4 Parekh, A. B. and Putney, J. W., Jr., Physiol.Rev. 2005. 85: 757–810.5 Qu, B. et al., J. Immunol. 2011. 186: 6894–6904.6 Vaeth, M. et al., Nat. Commun. 2017. 8: 14714.7 Alansary, D. et al., Biochim. Biophys. Acta 2015.1853: 1541–1550.8 Miya, A. et al., Front. Endocrinol. 2018. 9.9 Quintana, A. et al., EMBO J. 2011. 30: 3895–3912.10 Korthals, M. et al., Sci. Rep. 2017. 7: 8358.Keywords: cytotoxic T cells  glucose metabolism  calcium  SOCEFull correspondence: Dr. Bin Qu, Biophysics,CIPMM, School of Medicine, SaarlandUniversity, 66421 Homburg, Germany.INM-Leibniz Institute for New Materials66123 Saarbrücken, Germany.e-mail: bin.qu@uks.euThe peer review history for this article isavailable at https://publons.com/publon/10.1002/eji.202048577Received: 6/2/2020Revised: 11/6/2020Accepted: 21/7/2020Accepted article online: 22/7/2020The detailed Materials and methodsfor Letters to the Editor are availableonline in the SupportinginformationC© 2020 The Authors. European Journal of Immunology published byWiley-VCH GmbHwww.eji-journal.eu

High Glucose Distinctively Regulates Ca\u3csup\u3e2+\u3c/sup\u3e Influx in Cytotoxic T Lymphocytes Upon Target Recognition and Thapsigargin Stimulation

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High Glucose Distinctively Regulates Ca\u3csup\u3e2+\u3c/sup\u3e Influx in Cytotoxic T Lymphocytes Upon Target Recognition and Thapsigargin Stimulation

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