Iron Deprivation in Human T Cells Induces Nonproliferating ...€¦ · Iron Deprivation in Human T Cells Induces Nonproliferating Accessory Helper Cells Verena Berg, 1Madhura Modak,

Accessory Helper CellsIron Deprivation in Human T Cells Induces Nonproliferating

Peter Steinberger, Gerhard J. Zlabinger and Johannes StöcklVerena Berg, Madhura Modak, Jennifer Brell, Alexander Puck, Sarojinidevi Künig, Sabrina Jutz,

http://www.immunohorizons.org/content/4/4/165https://doi.org/10.4049/immunohorizons.2000003doi:

2020, 4 (4) 165-177ImmunoHorizons

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Iron Deprivation in Human T Cells Induces NonproliferatingAccessory Helper Cells

Verena Berg,1 Madhura Modak,1 Jennifer Brell, Alexander Puck, Sarojinidevi Kunig, Sabrina Jutz, Peter Steinberger,Gerhard J. Zlabinger, and Johannes StocklInstitute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, A-1090 Vienna, Austria

ABSTRACT

Iron uptake via the transferrin receptor (CD71) is a pivotal mechanism for T cell proliferation. Yet, it is incompletely understood if

targeting of CD71 also affects the differentiation and functional polarization of primary human T cells. In this study, we demonstrate

that inhibition of iron ingestion with blocking mAbs against CD71 induces nonproliferating T cells, which release high amounts of

IL-2. Targeting of CD71 with blocking or nonblocking mAbs did not alter major signaling pathways and the activation of the

transcription factors NF-kB, NFAT, or AP-1 as analyzed in Jurkat T cells. Growth arrest in iron-deficient (Fe-def) T cells was prevented

upon addition of exogenous iron in the form of ferric ammonium citrate but was not reversible by exogenous IL-2. Surprisingly,

protein synthesis was found to be intact in Fe-def T cells as demonstrated by comparable levels of CD69 upregulation and cytokine

production with iron-sufficient T cells upon stimulation with CD3 plus CD28 mAbs. Indeed, high amounts of IL-2 were detectable in

the supernatant of Fe-def T cells, which was accompanied with a reduced cell surface expression of IL-2R. When we used such Fe-

def T cells in allogeneic MLRs, we observed that these cells acquired an accessory cell function and stimulated the proliferation of

bystander T cells by providing IL-2. Thus, the results of our study demonstrate that iron deprivation causes nonproliferating, altruistic

T cells that can help and stimulate other immune cells by providing cytokines such as IL-2. ImmunoHorizons, 2020, 4: 165–177.

INTRODUCTION

Iron is essential formany biochemical processes in cells, includingthe regulation of proliferation (1, 2). The transferrin receptor(CD71) is critical for iron import into many cell types. Uptake ofironbound to transferrinviaCD71 is themajor route of ingestion inproliferating T cells (3, 4). Accordingly, CD71 cell surface expres-sion is low in nonproliferating cells but is upregulated upon T cellactivation. Iron deficiency is considered to be the most commonnutritional disorder worldwide but can also occur in a case ofmutatedCD71, leading to functionally impaired immuneresponses

due to unresponsiveness of T cells (5, 6). In addition, severalstudies have demonstrated that inhibition of iron uptake bychelators or targeting of CD71 with specific mAbs prevents T cellproliferation by inhibiting iron-containing enzymes such asribonucleotide reductase, which is critical for DNA-synthesis (7,8). In the absence of sufficient iron, T cells enter the cell cycle, butproliferation stops in the S or G2 phase (9).

Activation of T cells leading to proliferation is usually accom-panied with functional differentiation. Such differentiation getsobvious by polarization of T cells in the presence of appropriatesignals into well-established subsets of T cells. The classical

Received for publication January 8, 2020. Accepted for publication March 25, 2020.

Address correspondence and reprint requests to: Dr. Johannes Stockl, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, MedicalUniversity of Vienna, Lazarettgasse 19, A-1090 Vienna, Austria. E-mail address: [email protected]

ORCIDs: 0000-0003-3246-2317 (V.B.); 0000-0003-0079-6777 (M.M.); 0000-0001-5191-9031 (J.B.); 0000-0001-6848-4097 (P.S.); 0000-0002-7478-4173 (G.J.Z.); 0000-0002-3304-6975 (J.S.).1V.B. and M.M. contributed equally.

This work was supported by Austrian Science Fund Grant DK W1212.

Abbreviations used in this article: CB, cord blood; CBT, CB-derived naive T cell; DC, dendritic cell; FAC, ferric ammonium citrate; Fe-def, iron-deficient; Fe-suf, iron-sufficient; PBT, peripheral blood T; qPCR, real-time quantitative PCR.

The online version of this article contains supplemental material.

This article is distributed under the terms of the CC BY 4.0 Unported license.

Copyright © 2020 The Authors

https://doi.org/10.4049/immunohorizons.2000003 165

RESEARCH ARTICLE

Adaptive Immunity

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subsets of T cells are Th1, Th2, and Th17 cells, which are charac-terized by the production of distinct cytokine profiles and specifictranscription factors (10). Iron is important for DNA-synthesis inall types of T cells, but Th1-type T cells have been reported to bemore sensitive than Th2-type T cells for iron deficiency (11).Although the pivotal role of iron uptake via CD71 for T cellproliferation is well documented, it is incompletely understood iftargeting of CD71 also affects the functional polarization ofprimary human T cells.

This promptedus to analyze the impact ofCD71mAbsonTcellproliferation and functional differentiation. We observed thatmAbs, which inhibit uptake of iron, prevent the proliferation ofnaive and effector T cells. This nonproliferating state induced byanti-CD71 mAb was not reverted by adding exogenous IL-2 andwas not associated with an immediate cell death. Surprisingly,cytokine production was not reduced in iron-deficient (Fe-def )T cells comparedwithT cells cultured in the presence of sufficientiron, and the supernatant of Fe-def T cells contained high levels ofIL-2. When we used Fe-def T cells in allogeneic MLRs, weobserved that these cells can act as accessory cells for otherT cells.Depletion or neutralization of the prominent T cell growth factorIL-2 demonstrated that it was responsible for this stimulatoryfunction of Fe-def T cells. Thus, the results of our studydemonstrate that iron deficiency promotes “altruistic” T cells,which can help other immune cells by providing growth factors.

MATERIALS AND METHODS

Media, reagents, and chemicalsCells were cultured in RPMI 1640 and supplemented with 2 mML-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin(all fromLifeTechnologies, Paisley,U.K.), and 10%FCS (HyCloneLaboratories). PMA, ionomycin, and ferric ammonium citrate(FAC) were purchased from Sigma-Aldrich (St Louis, MO), andrecombinant human IL-2 was obtained from R&D Systems(Minneapolis,MN).RecombinanthumanGM-CSFand IL-4werekindly provided by Novo Nordisk (Bagsværd, Denmark).

AbsThe following murine mAbs against human cell surface receptorswere generated in our laboratory by Dr. Otto Majdic: negativecontrol mAb VIAP (against calf intestine alkaline phosphatase),VIP-1 (CD71, IgG1), 5-528 (CD71, IgG1), and CD25 (3G10). mAbVIP-1 was already characterized in the third human leucocytedifferentiation Ags “clusters of differentiation” workshop in 1987(12) and used in several publications (13, 14), whereas mAb 5-528was recently generated. The following mouse mAbs were pur-chased: mAb 15E8 (CD28) (Caltag Laboratories, Buckingham,U.K.), IL-4 (MP4-25d2-PE) (Invitrogen), IFN-g (clone no. 25723-allophycocyanin) and the neutralizing IL-2 mAb 5334 (R&DSystems), IL-2 (MQ1-17H12-PE-Cy7), and CD132 (TUG44-allophycocyanin) (BioLegend). CD122 (Mik-b3-BV421) and theCD71mAbM-A712were bought fromBDBiosciences. mAbOKT3(CD3) was obtained from Jansen-Cilag (Vienna, Austria).

Cell linesHuman T cell line Jurkat and the murine thymoma BW5417 cellswere obtained from American Tissue Culture Collection (Manas-sas, VA). CD71-transduced BW5417 as well as the multichannelreporter Jurkat T cells (from clone JE6) were generated at ourinstitute and have been previously described (15).

Isolation of primary T cells and generation ofmonocyte-derived dendritic cellsBuffy coats from healthy donors were purchased from eitherAustrian Red Cross or University Clinic for Blood Group Serologyand Transfusion Medicine, Medical University of Vienna (bothlocated in Vienna, Austria). Naive T cells were isolated from um-bilical cord blood (CB). CB samples from healthy donors werecollected during full-term deliveries. Ethical approval wasobtained from the Medical University of Vienna InstitutionalReviewBoard. Informedconsentwasprovided in accordancewiththe Declaration of Helsinki. Isolation of primary T cells fromperipheral blood and CB was performed as previously described(16). Purity of total T (peripheral blood T [PBT] cells and CB-derived naive T cells [CBT]) was checked routinely. Purity of eachcell population was found to be $97%. Monocyte-deriveddendritic cells (DCs) were generated by culturing purifiedmonocytes for 7 d with a combination of GM-CSF (50 ng/ml)and IL-4 (35 ng/ml) (17).

Flow cytometry analysisFormembrane staining, cells (23 105) were incubatedwith eitherunconjugated or conjugated mAbs for 30 min at 4°C. OregonGreen 488–conjugated goat anti-mouse IgG Ab (Life Technolo-gies, Carlsbad, CA) was used to detect unconjugated mAbs.

Intracellular cytokine productionwas determined by pretreat-ing the activated PBT cells, for 12 h with 5 mMmonensin (Sigma-Aldrich) and then by fixing cells in FIX-solution for 20 min atroom temperature before incubating with respective mAbs alongwith PERM-Solution (AN DER GRUB Bio Research, Kaumberg,Austria) for 20min at room temperature. Flow cytometry analyseswere performed using FACSCalibur or LSRFortessa and wereanalyzed by CellQuest or FlowJo Software (Becton Dickinson).

Transferrin-binding/-uptake assaysT cells (23 105 per condition) were activated with 100 nM PMA/ionomycin for 18 h. Afterwards, cells were resuspended in Fe-def DMEM (Life Technologies) and incubated with 10 mg/mlFITC-labeled holo-transferrin (Invitrogen) for 30 min at 0°C (forbinding assay) and at 37°C (for uptake assay). Flow cytometryanalysiswas performedusing FACSCalibur andCellQuest (BectonDickinson).

Downmodulation of CD71 upon engagement of CD71 withmAbs was performed by washing primary T cells preactivatedwith PMA/ionomycin or Jurkat T cells with cold PBS andincubatingwithmouseanti-humanCD71mAbs (20mg/ml)orwithisotype control mAb for 30 min at 4°C. Cells were then washedwith cold PBS and resuspended in cold PBS, and an aliquot wasstained using Oregon Green 488–conjugated goat anti-mouse IgG


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tomeasure the baseline for CD71 expression. The rest of themAb-loaded cells were resuspended in medium and incubated at 37°Cfor up to 18 h. Internalization was stopped by incubating the cellson ice and washing with cold PBS, followed by staining withOregon Green 488–conjugated goat anti-mouse IgG at 4°C. Flowcytometry analysis was performed using FACSCalibur andCellQuest (Becton Dickinson).

To analyze the downmodulation of CD71 using microscopy,Jurkat T cellswere stainedwith FITC-labeledVIP-1 for 30min onice, as described above. The cells were then incubated at 37°C for30 min. Images were taken prior to (binding) and after (internal-ization) incubationat37°CandwerecapturedusingNikonDS-Fi1cunder an inverted Nikon Eclipse Ti-S Fluorescence Microscope(Tokyo, Japan) with360 magnification.

T cell proliferation assayMAXISORP Nunc-Immuno Plates (Thermo Fisher Scientific)were coated overnight at 4°Cwith 5mg/ml CD3mAb (OKT3) andCD28 mAb (15E8). The plates were then washed to removeunboundmAbs, and purified T cells (23 105 per well) were addedalongwith solubleCD71mAbs (10mg/ml) to respectivewells. T cellproliferation was monitored by measuring [methyl-3H]-thymidine(PerkinElmer) incorporation at day 3, as described before (16).

Determination of cytokine productionPBT cells were activated via CD3 plus CD28 in the presence orabsence of CD71 mAbs, as described above. At day 3, supernatantswere harvested and pooled from triplicate wells. The cytokinesIL-2, IFN-g, IL-4, IL-17, IL-22, IL-10, and TNF-awere measuredby using Luminex100 System (R&D Systems), as described in themanufacturer’s protocol. All measurements were performed induplicates.

Restimulation of T cellsFe-def T, iron-sufficient (Fe-suf ) T, and mock T cells wereharvested at day 3 and were further cultured for another 4 d infresh media without stimulation. Cells were then restimulated(2 3 105 cells per well) via CD3 plus CD28 in the presence orabsence of exogenous IL-2 (20 U/ml). T cell proliferation wasanalyzed at day 3 by [methyl-3H]-thymidine incorporation. Assayswere performed in triplicates.

T cell coculture assayFe-def T, Fe-suf T, and mock T cells were harvested at day 3 andrested in fresh medium without any stimuli for 4 d, as describedabove. T cells were then irradiated (30 Gy, [137Cs] source) andcocultured in an allogeneic MLR at various numbers with DCs(53 104 cells per well) and responder PBT cells (13 105 cells perwell). Proliferation of responder T cells was measured at day 5 by[methyl-3H]-thymidine incorporation.

To analyze the effect of soluble factor on T cells proliferation,the supernatants from Fe-def T, Fe-suf T and mock T cells werecollected at day 3 and were added to responder T cells stimulatedbyallogeneicDCs. Proliferationof responderTcellswasmeasuredat day 5 by [methyl-3H]-thymidine incorporation.

Real-time quantitative PCRTotalcellularRNAwasisolatedusingpeqGOLDTriFast(Peqlab)withchloroform extraction, followed by isopropanol precipitation accord-ing to the manufacturer’s protocol. cDNA was generated using theRevert Aid MuLV-RT Kit (Fermentas) using Oligo(dT)18 Primeraccording to themanufacturer’sprotocol. cDNAwas storedat220°Cuntil further use. Real-time quantitative PCR (qPCR) was performedwith a CFX96 Real-Time PCR Detection System (Bio-Rad Labora-tories) usingSYBRGreenqPCRMasterMix (QuantaBiosciences) fordetection. CD3E was used as an endogenous reference gene (18).Specificprimers forhumanIFNG,IL4,IL2,EGR3,KLF2,CBLB,CD25,CD122, and CD132 were designed using software Primer 3 Plus (19)and were synthesized at Sigma-Aldrich (Table I). Data analysis wasperformed using CFX Manager Software (Bio-Rad Laboratories).Amplification specificity was determined via melting curve analysis,and relative expression was calculated using the 22ΔΔCt method (20).

Cell cycle analysisJurkat T cells were cultured in the presence or absence of CD71mAbs. After 3 d, cells were harvested and fixed using 70% ethanolat 4°C for 30min. Afterwashing, cellswere stainedwith 50mg/mlpropidium iodide in thepresenceof 100mg/mlRNAseA for45minat room temperature, as recently described (21). DNA content wasthen measured on a BD FACSCalibur Flow Cytometer and ana-lyzed via CellQuest (BD Biosciences).

IL-2 depletion and neutralization assayPrimary T cells (23 105 per well) were activated via plate-boundCD3 plus CD28 (coated at 5mg/ml, 4°C) in the presence of solublemAbs 5-528, VIP-1, or M-A712 (5 mg/ml each) or were left un-treated (mediumcontrol).The supernatants of these cultureswereharvested on day 3.

Supernatants were cleared from residual mAbs by overnightincubation (4°C) with Dynabeads M-280 sheep anti-mouse IgG(Thermo Fisher Scientific), followed by removal of complexes viacentrifugation, as previously described (21, 22). Part of the clearedsupernatant was then treated with anti–IL-2 mAb clone no. 5334(R&D Systems) at 2.5 mg/ml for 1 h (4°C) followed by addition ofDynabeads and incubated for another 2 h. Bead-Ab–IL-2 com-plexeswere thendepletedby centrifugation.Depletionof IL-2wascontrolled by Luminex.

AnallogeneicMLRwas establishedbymixingTcells (13 105 perwell) with DCs (2.5 3 104 per well). Cell suspensions were thendiluted with the supernatants at a 1:2 ratio (v/v). In addition to usingIL-2–depleted supernatants, IL-2 was also directly neutralized inuntreated supernatants from T cells by adding anti–IL-2 mAb(5 mg/ml) to the cultures, comparing it to IgG1 isotype control Ab(catalogueno.401408;BioLegend).Cellswerecultured for5dbeforethe addition of [methyl-3H]-thymidine andwere harvested on day 6.

Raw data obtained in MLRs was normalized to the meanproliferative responses in the presence of supernatants of donorT cells to account for the inhibition of VIP-1– or M-A712–treatedcells in the primary response. The cpm values obtained in theprimary proliferation were normalized to medium control, andthese values were then used as a normalizer for the MLR data.


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Statistical analysisStatistical analysiswas performed usingGraphPadPrism software(La Jolla, CA). Unpaired, two-tailed Student t test, followed byHolm–Sıdak test for multiple comparisons, was performed. Anydifferences in statistical significance are indicated. The p values,0.05 were considered significant and are represented as*p , 0.05, **p , 0.01, and ***p , 0.001.

RESULTS

Characterization of the CD71 mAbsTransferrin receptor (CD71)–mediated iron uptake is essential forT cell growth and expansion (23). Yet, the impact of iron-deprivation on T cell polarization has not been analyzed so far. Inthis study, we used two mAbs, namely VIP-1 and 5-528, produced

FIGURE 1. Characterization of CD71 mAbs VIP-1 and 5-528 used in this study.

(A) Reactivity of VIP-1 and 5-528 (filled histograms) with murine BW5417 cells transfected with human CD71 and with nontransfected cells. Open

histograms indicate the background reactivity of isotype control mAb VIAP. (B) Reactivity of CD71 mAbs VIP-1 and 5-528 (filled histograms) with

unstimulated T cells and T cells stimulated with PMA/ionomycin for 18 h. Open histograms indicate the reactivity of isotype control mAb VIAP. Data

are representative of four independent experiments (A and B). (C) The uptake (37°C) and binding (0°C) of FITC-labeled transferrin via CD71 in T cells

preactivated with PMA/ionomycin was assessed in the presence or absence of CD71 mAb VIP-1 or 5-528 by flow cytometry. Unstimulated T cells

served as control. Data are representative of three independent experiments. Results of three independent experiments are shown in (D). (E and F)

CD71R downmodulation is induced by VIP-1 or 5-528 (thick-lined, open histograms) in human primary T cells upon preactivation with PMA/

ionomycin at 37°C is compared with binding at 0°C (gray-filled histograms). Open histograms with the thin lines demonstrate the reactivity of a

nonbinding, isotype control mAb (n = 3). Results are shown as mean 6 SEM. *p , 0.05.




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in our laboratory to investigate the role of transferrin-dependentiron uptake on T cell differentiation and function. Resultspresented in Fig. 1A demonstrate that both mAbs specificallyrecognize human CD71. Both CD71 mAbs showed reactivity withmurine BW5417 cells retrovirally transduced to express humanCD71 (BW5417-CD71+) but not with parental BW5417 cells. Inaddition, VIP-1 and 5-528 did not recognize unstimulated PBTcells but reacted with stimulated PBT cells with similar bindingaffinities (Fig. 1B).Cross-inhibition studieswithmAbsVIP-1and5-528 on Jurkat T cells revealed that they recognize overlapping butdistinct epitopes on CD71 (Supplemental Fig. 1A).

Results presented in Fig. 1C, 1D demonstrate that targeting ofCD71 on PMA/ionomycin–stimulated PBT cells with VIP-1, butnot with 5-528, inhibits transferrin binding (0°C) and uptake(37°C). Next, we analyzedmodulation of cell surface expression ofCD71 by VIP-1 and 5-528 mAbs on PMA/ionomycin–activatedPBT cells. The addition of VIP-1 strongly diminished cell surfaceexpressionofCD71 on activated PBT cells at 37°CwhereasmAb 5-528 reduced expression to a lesser extent (Fig. 1E, 1F). A similareffect was also observed in Jurkat T cell line when soluble CD71mAbs were added. Binding of CD71 mAb VIP-1 led to in-ternalization of cell surface CD71, as analyzed by fluorescencemicroscopy and flow cytometry (Supplemental Fig. 1B, 1C).Commercially available CD71 mAb M-A712 has also been de-scribed before and was used to induce internalization of CD71 (6,24).However, cross-inhibition studiesperformedonJurkatTcellsrevealed that mAb M-A712 recognizes a distinct CD71 epitopecompared to VIP-1 or 5-528 (Supplemental Fig. 1D).

Inhibition of T cell proliferation by VIP-1 is mediated viairon deprivationNext, we tested whether the impact of mAb VIP-1 on transferrinbinding andmodulationof CD71 cell surface expression affects theproliferative response of T cells. Fig. 2A, 2B demonstrate that theproliferation of PBT cells as well as CBT was significantlydiminished in the presence of VIP-1, but not 5-528, uponstimulation with CD3 plus CD28 mAbs. Similar to VIP-1, theaddition of CD71 mAb M-A712 inhibited the proliferation of CD3plus CD28 stimulated PBT cells (Fig. 2C). Both inhibitory CD71mAbs, VIP-1, as well as M-A712 could also efficiently inhibit theproliferation of Jurkat T cells (Supplemental Fig. 1E).

To investigate if the inhibitory effect of VIP-1 was due to irondeprivation in cells upon CD71 targeting, we used FAC, which canpassively enter cells bypassing CD71-mediated uptake of iron (25).Results presented in Fig. 2C demonstrate that inhibition ofPBT cells triggered by mAb VIP-1 as well as mAb M-A712 wasnot detectable in the presence of sufficient iron in the form of FAC(Fig. 2C).Moreover, treatment ofT cellswithmAbVIP-1, 5-528, orM-A712 did not induce cell death in PBT stimulated via CD3 plusCD28 within 3 d (Fig. 2D). Likewise, we did not observe massivetoxic effects of the CD71mAbs used in this study on Jurkat T cellswithin 3 d of treatment (Supplemental Fig. 2A, 2B). Thus, theinhibitory effect of mAb VIP-1 was obviously caused by an irondeficiency in VIP-1–treated T cells (Fe-def T), which was not thecase upon targeting of CD71 on T cells with mAb 5-528 (Fe-sufT cells).

Fe-def T cells exhibit unaltered protein synthesis despite ofgrowth arrestInhibition of T cell proliferation is typically accompanied with areduced production and release of cytokines. So, we next analyzedif the production of various T cells cytokines is altered in Fe-defT cells compared with Fe-suf T cells. Results presented in Fig. 3demonstrate that IL2, IL4, and IFNG could be found at similarquantities atmRNA level by qPCR (Fig. 3A, Table I) and at proteinlevel by intracellular staining in Fe-def T and Fe-suf T cells (Fig.3B–D).Moreover, Fe-def T cells stimulatedwith PMA/ionomycinwere found to upregulate and express similar levels of theactivation marker CD69 on the cell surface as T cells activated inmediumwith sufficient iron (Supplemental Fig. 3A). Furthermore,CD3 plus CD28 stimulated Fe-def T cells re-expressed CD71 atlevels thatwere comparable to the levelsonFe-sufTcells analyzedafter 3 d of treatment followed by a resting phase in medium for4 d (Supplemental Fig. 3B). Thus, protein synthesis is intact inFe-def T cells.

Major T cell signaling pathways are operating uponiron deprivationSignificant proliferative unresponsiveness of Fe-def T cells raisedthe question of whether signaling pathways may be perturbed incells upon targeting of CD71 with VIP-1. In addition to its crucialrole in iron transport, CD71 is involved in immunological synapse

TABLE I. Real-time PCR primer sequences

Gene

National Center forBiotechnology Information

Identification No. Protein Forward Sequence Reverse Sequence

IL2RA 3559 CD25 59-GGCTGTGTTTTCCTGCTGAT-39 59-AAGCACAACGGATGTCTCCT-39IL2RB 3560 CD122 59-GCTGATCAACTGCAGGAACA-39 59-TGTCCCTCTCCAGCACTTCT-39IL2RG 3561 CD132 59-AATTCCCACCCTGAAGAACC-39 59-TTTGGGGGAATCTCACTGAC-39IFNG 3458 IFN-g 59-TTCAGCTCTGCATCGTTTTG-39 59-TCTTTTGGATGCTCTGGTCA-39IL2 3558 IL-2 59-CAAACCTCTGGAGGAAGTGC-39 59-ATGGTTGCTGTCTCATCAGC-39IL4 3565 IL-4 59-GCCACCATGAGAAGGACACT-39 59-ACTCTGGTTGGCTTCCTTCA-39KLF2 10365 KLF2 59-CTACACCAAGAGTTCGCATCTG-39 59-AGTTGCAGTGGTAGGGCTTC-39CBLB 868 CBL-B 59-ATGCTGAATGGAACACATGG-39 59-ACTATGCCTTGCAGGAGGTG-39EGR3 1960 EGR-3 59-CAACTGCCTGACAATCTGTACC-39 59-AGTAGGTCACGGTCTTGTTGC-39CD3E 916 CD3e 59-TGAGGGCAAGAGTGTGTGAG-39 59-TCCTTGTTTTGTCCCCTTTG-39




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formation (26). Furthermore, costimulation of T cells via specificCD71 mAbs induces protein kinase C activation, IL-2 secretion,and T cell proliferation (27). Therefore, by using the Jurkat E6

multichannel reporter cell line, we compared the activation ofdownstream signaling pathways in T cells upon ligation of CD71mAb VIP-1 and 5-528. Results presented in Fig. 4A demonstratethat inhibition of proliferation of Jurkat T cells by VIP-1 was alsodue to irondeprivation and couldbe overcomeby addingFAC, likein primary T cells (Fig. 2C). Activation of Jurkat T cells in thepresence of mAb 5-528 revealed a similar activation profile ofNFAT, AP-1, and NF-kB transcription factor as mock treated cells(Fig. 4B, 4C). Interestingly, Jurkat T cells treated with CD71 mAbVIP-1 showed an unaltered AP-1, NFAT, and NF-kB reporteractivity (Fig. 4B, 4C), implying that the three major signalingroutes analyzed in this study are intact in Fe-def cells. Similar tomAb VIP-1, treatment of Jurkat T cells with the inhibitory CD71mAb M-A712 did not show striking effects on the activation ofthe transcription factors AP-1, NFAT, andNF-kB (SupplementalFig. 2C).

Cell cycle analyses performedwith Jurkat T cells revealed thatcells were arrested in the S/G2 phase in the absence of thesufficient iron due (Fig. 4D). Interestingly, VIP-1–treated JurkatT cells could not overcome the growth arrestwhen cultured in theabsence of CD71 mAbs for next 14 d (Supplemental Fig. 2D).

Targeting of CD71 with mAb VIP-1 induces ahypoproliferative state in T cellsBecause Fe-def T cells re-expressed CD71 at levels that were com-parable to the levels in Fe-suf T cells (Supplemental Fig. 3B), wenext analyzed the ability of Fe-def T cells to respond torestimulation. As shown in Fig. 5A, Fe-def T cells proliferatepoorly to restimulation in contrast to Fe-suf T and mock T cells.This hypoproliferative state of Fe-def T cells could not beovercome by addition of exogenous IL-2 (Fig. 5A).

Next, we analyzed the expression levels of a regulator of T cellquiescence, KLF2 (28). Results presented in Fig. 5B demonstratethat KLF2 was downregulated in Fe-def T cells similar to Fe-sufT cells ormock T cells when compared with unstimulated T cells.We also analyzed the expression of negative regulators of T cellsactivation including the anergy-promoting factorsEGR3 andCBLB(29, 30). However, the expression levels of these mRNAs werefound to be not elevated in Fe-def T cells compared with Fe-sufTcellsormockTcells in thecaseofEGR3andratherreduced in thecase of CBLB (Fig. 5B). Thus, Fe-def T cells seemingly representnonproliferating, end-stage cells.

Fe-def T cells exert accessory function for responder T cellsLack of proliferation is typical for T cells with regulatory function(31). Therefore, we analyzedwhether Fe-def T cellsmay be able tosuppress the activation of bystander T cells. Results presented inFig. 6A demonstrate that Fe-def T cells, instead, supported theproliferation of cocultured T cells. Addition of Fe-def T to anallogeneic MLR containing responder T cells and DCs resulted inincreased proliferation of cocultured T cells, compared with bothFe-suf T cells and mock T cells (Fig. 6A).

To investigate if Fe-def T cells stimulate the proliferationof bystander T cells via soluble factor(s), we added the superna-tant Fe-def T cells to responder T cells stimulated by allogeneic

FIGURE 2. Inhibition of T cell proliferation by VIP-1 is mediated via

iron deprivation.

T cells were stimulated via plate-bound CD3 plus CD28 mAbs with or

without soluble CD71 mAbs. Proliferation was determined after 72 h via

[methyl-3H]-thymidine incorporation in (A) PBT cells (n = 6) and (B) CBT

(n = 3). (C) Primary T cells were cultivated in the presence of increasing

amounts of exogenous iron in the form of ferric ammonium citrate

(FAC) alone or in combination with the CD71 mAbs VIP-1, 5-528, or

M-A712. Data show mean 6 SEM of three independent experiments.

(D) Impact of CD71 mAbs on the viability of T cells preactivated via

CD3 plus CD28 was analyzed by propidium iodide staining and flow

cytometry (n = 2). Data show mean 6 SEM. *p , 0.05.




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DCs. Indeed, supernatants ofFe-defTcells exhibited a stimulatoryeffect on responder T cells, compared with the supernatant fromFe-suf T or mock T cells (Fig. 6B).

High levels of IL-2 are present in the supernatants ofFe-def T cellsThe above-mentioned observations suggested that Fe-def T cellsmay produce or release a different cytokinemilieu comparedwithFe-suf T and mock T cells. Therefore, we next analyzed thecytokine content of the supernatants of Fe-def T, Fe-suf T, andmockTcells.Weobserved that Fe-defT cells produce comparablelevels of various T cell cytokines, including IL-4, IL-10, IL-13,

IL-17A, IL-22, IFN-g, and TNF-a (Supplemental Fig. 3C). Surpris-ingly, the supernatant of Fe-def T cells contained significantly moreIL-2comparedwith the supernatant ofFe-sufTormockTcells (Fig.7A, Supplemental Fig. 3D). Likewith VIP-1, mAbM-A712 treatmentresulted in enhanced the levels of IL-2 in the supernatant of T cellsactivatedviaCD3plusCD28(SupplementalFig. 3D). Interestingly, inthe presence of sufficient iron in the form of FAC, similar levels ofIL-2were found in the supernatant of Fe-def T and Fe-suf T cells(Supplemental Fig. 4A).

To analyze whether higher amounts of IL-2 in the superna-tants of Fe-def T cells may account for the accessory function ofthese cells, we used a well-defined neutralizing anti–IL-2 mAb to

FIGURE 3. Cytokine production is not significantly altered in Fe-def T cells.

(A) Gene expression analysis of IL2, IL4, and IFNG normalized to CD3E in CD3 plus CD28–stimulated PBT cells in presence of mAbs VIP-1 (Fe-def

T cells) or 5-528 (Fe-suf T cells) after 24 h was performed via RT-qPCR (no. of experiments = 2, no. of donors = 2). Data show mean 6 SEM.

Cytoplasmic staining of IL-2, IL-4, and IFN-g in CD3 plus CD28–activated PBT cells in presence of CD71 mAbs was performed after 48 h and

analyzed by flow cytometry. Bar charts show the number of positive cells (B) and the mean fluorescence intensity (MFI) (C). Cumulative data are

shown as mean 6 SEM (n = 3). Representative histograms are shown in (D).




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block the function of this important T cell growth factor. Resultspresented inFig. 7Bdemonstrate that additionof anti–IL-2mAb tothe supernatant of Fe-def T cells reduced the stimulatory effect tobackground levels. In addition, depletion of IL-2 out of thesupernatant by immunoprecipitation abrogated the stimula-tory capacity of the supernatant from Fe-def T cells (Fig.7C, Supplemental Fig. 4B).

Surface expression of IL-2R chains is reduced onFe-def T cellsThe intracellular staining andmRNA analysis revealed that Fe-defT cells do not producemore IL-2 comparedwithFe-suf T ormockT cells (Fig. 3). Thus, the higher amounts of IL-2 in thesupernatants of Fe-def T cells seem to be due to less consumptionof IL-2 by Fe-def T cells. This promptedus to study the expressionof the IL-2R on Fe-def T cells. We analyzed the expression of theIL-2RchainsCD25 (commona-chain), CD122 (commonb-chain),and CD132 (common g-chain) (32). mRNA analysis of IL-2Rchains revealed thatFe-defTcells express comparable levels of IL-2R chains to Fe-suf T or mock T cells (Fig. 8A). Results presentedin Fig. 8B, 8C demonstrate that Fe-def T cells express low levels ofall three chains of the IL-2R, particularly of CD122, on the cellsurface. The lower expression levels of IL-2R in Fe-def T cells atprotein level may, therefore, result from perturbed endocytosisand the recycling of the receptor in Fe-def T cells, and high levelsof IL-2 found in the supernatants of Fe-def T cells may result fromlower consumption of IL-2 by Fe-def T cells.

DISCUSSION

Iron is an essential element of most living organisms. It is pivotalfor different biological processes in cells, including DNA rep-lication and cellular respiration (33). The transferrin receptor(CD71) is the primary receptor responsible for iron uptake inmostcells, including lymphocytes (34). Iron deficiency in humans andmice has been reported to result in reduced circulating T cellnumbers and thymic atrophy (35). Activation of T cells leading toproliferation is usually accompanied with functional differentia-tion. Such differentiation is apparent by polarization of T cells inthe presenceof appropriate signals intowell-established subsets ofT cells such as Th1, Th2, Th17 cells, and regulatory T cells (36).

FIGURE 4. Impact of iron deficiency on major signaling pathways.

(A) Proliferation of Jurkat T cells in the presence of CD71 mAb VIP-1 or

5-528 cultured with or without increasing amounts of exogenous iron

(FAC). Data represent mean 6 SEM of three independent experiments.

(B) VIP-1 or 5-528 mAb–pretreated (thick-lined, open histograms)

multichannel reporter Jurkat cells were analyzed after activation via

CD3 plus CD28 for the regulation of the respective transcription factors

(NFAT, NF-kB, and AP-1) via flow cytometry and compared with un-

treated (gray-filled histograms) and unstimulated (thin-lined, open

histograms) Jurkat T cells. Summarized data (mean 6 SEM) from three

independent experiments are shown in (C). (D) Jurkat T cells were

stimulated for 3 d via plate-bound CD3 plus CD28 Abs in the presence

or absence of VIP-1 or 5-528 mAbs. The cell cycle analysis of each

condition was determined via propidium iodide staining and measured

by flow cytometry. Numbers given indicate percentages of cells in the

respective phase of the cell cycle. Data are representative of three in-

dependent experiments.




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Although the central role of iron uptake via CD71 for T cellproliferation iswell documented, little is knownwhether targetingof CD71 also affects the functional polarization of primary humanT cells. In this study, we demonstrate that, although decreasedlevels of iron stop the proliferative response of primary humanTcells, suchFe-defTcellspromoteproliferationof otherTcells byprovidingmore IL-2. Taken together, Fe-def T cells can be seen asthe “end-stage type”ofTcells thatmayhelp other immunecells byproviding cytokines in an altruistic way.

Targeting of CD71 with mAb VIP-1 induced a proliferationarrest in primary T cells upon stimulation with CD3 plus CD28mAbs aswell as in immortalized Jurkat T cells. Because this effectwas not detectable in the presence of exogenous iron in form ofFACinbothcell types,weconclude that it is indeed thedeprivationof iron that is responsible for the lack of proliferation (Fig. 2C,Supplemental Fig. 1E). Other studies have raised the possibilitythat CD71 may play a role in cellular growth and activation that is

distinct from its transferrin-binding ability, including the modu-lation of signaling pathways (23, 26). To analyze if the treatmentof lymphocytes with our CD71 mAbs has an impact on classicalsignaling routes inTcells,weexamined theeffectofCD71mAbsoncellsof aJurkatTcell reporter line.Weobserved thatNF-kB,AP-1,or NFAT pathways were not altered upon targeting of CD71 withmAbs (Fig. 4). Although Jurkat T cells are a widely used modelsystem to study T cell biology, the results may not reflect thesituation in primary human T cells. So, we cannot exclude thatsignaling processes are altered inTcells uponCD71modulation, ashasbeenreported inother studies (26, 27).Thispossibilityneeds tobe analyzed in more detail in future studies in primary T cells.

FIGURE 5. Targeting of CD71 with mAb VIP-1 (Fe-def T cells) induces

a hypoproliferative state in T cells.

(A) Previously activated Fe-suf T and Fe-def T cells were restimulated

via plate-bound CD3 plus CD28 mAbs in the presence or absence of

exogenous IL-2. Cell proliferation was measured after 72 h via [methyl-3H]-thymidine uptake. Data are shown as mean 6 SD of duplicates and

are representative of two independent experiments. (B) Gene expres-

sion analysis of KLF2, EGR3, and CBLB relative to CD3E in CD3 plus

CD28–induced Fe-suf T and Fe-def T cells after 24 h was performed (n = 4).

Data show mean 6 SEM. *p , 0.05.

FIGURE 6. Fe-def T cells exert accessory function for T cells.

(A) Increasing numbers of preactivated and irradiated Fe-suf T and Fe-

def T cells or T cells preactivated via CD3 plus CD28 in the absence of

CD71 mAbs (mock) were added to an allogeneic MLR with a fixed

number of responder T cells (1 3 105) and DCs (5 3 104). Values are

displayed as mean 6 SEM. Dashed line indicates the proliferative re-

sponse of T cells stimulated with DCs in the absence of preactivated,

bystander T cells (n = 5).*p , 0.05). (B) T cells were stimulated with

allogeneic DCs in the absence (Medium ctrl) or in the presence of the

supernatants of preactivated Fe-suf T and Fe-def T cells or mock T cells

(n = 8). (A and B) Proliferation was measured via [methyl-3H]-thymidine

uptake, and data show mean 6 SEM. *p , 0.05, ***p , 0.0001.




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The proliferation arrest in Fe-def T cells was irreversible andcouldnot be overcomeuponadditionof exogenous IL-2 (Fig. 5). So,the functional state of Fe-def T cells is not classical hypores-ponsivenessoranergy.This is also reflectedby theexpression levelsof informative anergy factors such asEGR-3 or downmodulation ofquiescence factors such as KLF-2 in Fe-def T cells similar to Fe-sufT cells. Only CBL-b was found to be rather slightly reduced (Fig.5B). Interestingly, we observed that primary T cells and JurkatT cells temporarily lost CD71 cell surface expression upon mAbVIP-1 or 5-528 treatment but started to re-express CD71 and hadagain high levels of CD71 after a 4 d culture in the absence ofmAbs(Supplemental Fig. 3B). Nevertheless, despite sufficient iron in thecell culture medium, the stop of proliferation in these cells couldnot be hurdled (Supplemental Fig. 2C). This indicates that theimpact of iron deficiency on T cells during activation is deep andpermanent, and the provision of iron is not sufficient to restore aproliferative response.

Our findings are in linewith several studies that demonstratedthat CD71 mAbs, which block transferrin binding and/or down-modulate CD71, inhibit cell growth (Figs. 1, 2). How CD71 mAbsinhibit cell proliferation is incompletely understood. It is assumed

that it is a mixture of different processes (34). First, interferencewith transferrin binding and uptake is a potent mechanism. Inaddition, it has been reported that some CD71 Abs (IgM or IgA)may cross-link the receptors and thereby prohibit their in-ternalization required for transferrin import. Based on the resultsof our study, we propose that the inhibitory actions of VIP-1probably represent a composite effect of receptor downmodula-tion because of internalization and interference with transferrinbinding and uptake (Fig. 1C, 1D). Whether the receptor/mAbcomplex is then degraded or recycles to the surface needs to beinvestigated in future studies. It is also intriguing that a part of theCD71 molecules is proteolytically cleaved and the ectodomain ofCD71 is released from the surfaceuponVIP-1 engagement, therebyreducing the iron-uptake capacity (37).

The absence of T cell proliferation in the presence of appro-priate stimulatory signals is typical for T cells with immunosup-pressive abilities, such as regulatory T cells (31). However, weobserved that Fe-def T cells were rather stimulatory thaninhibitory for other T cells. Transfer of the supernatant of Fe-defT cells exerted a similar accessory function, indicating that solublefactor(s) are seemingly important (Fig. 6). The supernatants of Fe-defTcells containedhigh levels of IL-2,whereas the levels of othercytokines such as IL-10 or IFN-gwere slightly diminished (Fig. 7,Supplemental Fig. 3C). We could indeed demonstrate thatdepletion of IL-2 from the supernatant of Fe-def T cells orneutralization of the prominent T cell growth factor with ananti–IL-2 mAb abolished the accessory function of the superna-tant. Thus, elevated levels of IL-2 were critically responsible forthe stimulatory functionofFe-defTcells. Thisfinding is surprisingbecause Fe-def T cells express similar levels of IL-2 mRNA as Fe-suf T cells, and even intracellular amounts of IL-2 protein wereindistinguishable between Fe-suf T cells and Fe-def T cells.Interestingly, increased levels of IL-2 havebeen reported before inFe-def patients, whereas lower levels of IL-2 have been found inpatients with iron-overload (38, 39). In line with these findings,T cell activation in the presence of the iron chelator deferoxamineinhibited the proliferation, but the supernatants of deferoxamine-treated human lymphocytes contain equivalent IL-2 activity asuntreatedcells (40). So,what couldexplain thisphenomenon?Onepossibility was that Fe-def T cells express less IL-2Rs andtherefore consume less of the ligand. We, therefore, analyzed theexpression of the threemolecules of the IL-2R, CD25, CD122, andCD132. Indeed, all threeproteinswere transcribed inFe-defTcellsto thesameextentas inFe-sufTcells, butCD25,CD122,andCD132were lower on the surface of Fe-def T cells compared with Fe-sufT cells (Fig. 8). The mechanism for this observation needs to beinvestigated. Because both of our CD71 mAbs used in this studydownmodulate CD71, it is unlikely that the lower expression ofIL-2R subunits are the result of a cointernalization event inducedby mAb VIP-1 because this should have been also the case in5-528–treatedT cells (i.e., Fe-suf T cells). Therefore, it seems to bemore likely that cell surface expression of the IL-2R is regulated bythe intracellular levels of iron. Interestingly, the IL-2R is endocytosedvia a recently identified endophilin-dependent pathway and not viaclathrin-mediated endocytosis (41). It is, thus, tempting to speculate

FIGURE 7. High levels of IL-2 are present in the supernatants of

Fe-def T cells.

(A) Levels of IL-2 in supernatants of Fe-def T cells, Fe-suf T cells, and

mock T cells stimulated via CD3 plus CD28 for 72 h were measured

using a Luminex-based multiplex assay (no. of experiments = 6, no. of

donors = 6). (B) Analysis of the impact of the supernatant from Fe-suf T

and Fe-def T cells on T cell activation induced by allogeneic DCs in the

presence of a neutralizing IL-2 mAb (n = 5) or in (C) after depletion of

IL-2 from the supernatants of preactivated Fe-def T cells and Fe-suf

T cells (n = 8). Data show mean 6 SEM. *p , 0.05, ***p , 0.001.




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that the endothelin pathway may be dysregulated in the Fe-defcells leading to reduced surface expression of IL-2R but notof other cytokine receptors, which do not use this endocyticroute (42).

The availability of nutrients as well as cellular metabolism isemerging as a key determinant of T cell differentiation andfunction (43, 44). Iron is a decisive micronutrient for T cells.Thorson et al. (11) have demonstrated that cellular iron availabilitymodulates theproliferationofTh1andTh2cell subsets,whichmaypartly be related to the different dependence of these cells ontransferrin-mediated iron uptake. Whereas Th1 clones were verysensitive to treatment with anti-CD71, Th2 cells were almostresistant to this procedure in this study (11). It was considered thatthis difference was partly due to the fact that Th2 clones exhibitlarger cheatable iron storage pools than Th1 cells. However,children receiving the iron chelator deferoxamine had higherlevels of Th1 cytokines, whereas serum concentrations of IL-4

tended to be lower (45). This indicates that withdrawal of ironrather increases Th1-mediated immune function in vivo. More-over, although iron deficiency can be associatedwith an increasedrisk of infection, there is little evidence to suggest that irondeficiency causes a major, clinically relevant defect of immunefunctions in humans (46). So, what might be the biologicalrelevance of such IL-2–producing Fe-def T cells?

There are also other examples for “altruistic” lymphocytes.Starved T cells, which arise when glucose levels are down, stop toproliferate but still produce cytokines, including IL-2 (47), whichis reminiscent of the behavior of Fe-def T cells described in thisstudy. Apart from growth-promoting effects towards other im-mune cells, these cytokines stimulate the effector pathways. Forinstance, IL-2 is a potent stimulator of NK cells (48) andproinflammatory cytokines, such as TNF or IL-6, may recruitand activate the effector functions of monocytes or residentmacrophages, respectively (49).

FIGURE 8. The cell surface expression of the IL-2R is reduced in Fe-def T cells.

(A) Gene expression analysis of CD25, CD122, and CD132 normalized to CD3E in CD3 plus CD28–stimulated Fe-suf T and Fe-def T cells was

performed after 24 h (no. of experiments = 2, no. of donors = 2). (B and C) Surface expression of the IL-2R chains CD25, CD122, and CD132 in CD3

plus CD28–activated Fe-suf T and Fe-def T cells was analyzed by flow cytometry after 48 h. Representative histograms are shown in (C), gate was

set on live-cell population (data not shown). (B) Bar charts show the mean fluorescence intensity (MFI) and percentage of positive cells. Cumulative

data are shown as mean 6 SEM (n = 3 experiments, n = 3 donors). *p , 0.05, **p , 0.01.




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Theuse of such “end-stage–differentiatedcells” is an emergingtopic in cell biology. For instance, neutrophils entering a specialcell death program called NETosis, lose their classical phagocytefunctions but gain new properties, in that they release their DNAand play an important role in several diseases (50). Such a concepthas been recently defined also for human T cells in environmentswith metabolic restrictions or hypoxia in which T cells operatingin pathophysiological microenvironments may retain the capacityto produce effector cytokines (43, 47). For example, tumor-infiltrating lymphocytes are equally dependent on metabolicreprogramming like tumor cells to divert metabolites into bio-synthetic pathways to promote survival and proliferation and,ultimately, to mount a response against the tumor. The microen-vironment can contain conditions that will restrict the metabolismof the infiltratingTcells (44).Gaining control over ironhomeostasisis one of the central battlefields in deciding the fate of an infectionwith intracellular pathogens or a malignant disease. Nutritionalimmunity is the withdrawing or removal of iron from pathogens ortumors and mainly executed bymacrophages (51). It is tempting tospeculate that such Fe-def T cells may arise when microbes ortumors combat with immune cells nutritional iron. The results ofourstudydemonstrate that irondeprivationcausesnonproliferatingT cells, which can help and stimulate other immune cells byprovidingcytokines suchas IL-2. It is, therefore, tempting to suggestthat such iron-deprived T cells can fight back under adverseenvironmental conditions by gaining feeder cell function for otherimmune cells in nutritional immunity.

DISCLOSURES

The authors have no financial conflicts of interest.

ACKNOWLEDGMENTS

We thank Petra Waidhofer-Sollner, Petra Cejka, and Claus Wenhart forexpert technical assistance.

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Iron Deprivation in Human T Cells Induces Nonproliferating ...€¦ · Iron Deprivation in Human T Cells Induces Nonproliferating Accessory Helper Cells Verena Berg, 1Madhura Modak,