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Restoring oxidant signaling suppressesproarthritogenic T cell effector functionsin rheumatoid arthritisZhen Yang,1 Yi Shen,1 Hisashi Oishi,1* Eric L. Matteson,2 Lu Tian,3

Jörg J. Goronzy,1 Cornelia M. Weyand1†

In patients with rheumatoid arthritis (RA), CD4+ T cells hyperproliferate during clonal expansion, differentiating intocytokine-producing effector cells that contribute to disease pathology. However, the metabolic underpinnings ofthis hyperproliferation remain unclear. In contrast to healthy T cells, naïve RA T cells had a defect in glycolytic fluxdue to the up-regulation of glucose-6-phosphate dehydrogenase (G6PD). Excess G6PD shunted glucose into thepentose phosphate pathway, resulting in NADPH (reduced form of nicotinamide adenine dinucleotide phosphate)accumulation and reactive oxygen species (ROS) consumption. With surplus reductive equivalents, RA T cells insuf-ficiently activated the redox-sensitive kinase ataxia telangiectasia mutated (ATM), bypassed the G2/M cell cyclecheckpoint, and hyperproliferated. Moreover, insufficient ATM activation biased T cell differentiation toward theT helper 1 (TH1) and TH17 lineages, imposing a hyperinflammatory phenotype. We have identified several interven-tions that replenish intracellular ROS, which corrected the abnormal proliferative behavior of RA T cells and suc-cessfully suppressed synovial inflammation. Thus, rebalancing glucose utilization and restoring oxidant signalingmay provide a therapeutic strategy to prevent autoimmunity in RA.

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INTRODUCTION

The autoimmune disease rheumatoid arthritis (RA) damages tendons,cartilage, and bone and shortens life expectancy through accelerationof cardiovascular disease (1, 2). CD4 T cells in RA patients sustainsynovitis, promote autoantibody formation, facilitate osteoclast differ-entiation, and impose endothelial dysfunction (3). When activated,RA CD4 T cells insufficiently up-regulate the glycolytic enzymePFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3)and generate less ATP (adenosine 5′-triphosphate) and lactate (4).It is currently unknown whether and how metabolic abnormalitiesare mechanistically connected to their proinflammatory functions.

The cardinal feature of naïve CD4 T cells is the ability to massivelyproliferate when encountering antigen. When transitioning from naïveto effector status, T cells expand 40- to 100-fold within days (5), makingthem highly dependent on energy and biosynthetic precursors (6).Resting lymphocytes rely on oxidative phosphorylation and fatty acidbreakdown, but upon activation switch to aerobic glycolysis and tri-carboxylic acid flux, designating glucose as the primary source forATP generation in activated lymphocyte. Anabolic metabolism of glu-cose provides not only energy but also macromolecular buildingblocks for the exponentially expanding biomass, typically by shuntingglucose into the pentose phosphate pathway (PPP) (7). In the first rate-limiting step of the PPP, glucose-6-phosphate dehydrogenase (G6PD)oxidizes G6P to 6-phosphogluconolactone to generate five-carbonsugars (pentoses), ribose 5-phosphate, a precursor for nucleotide syn-thesis, and NADPH (reduced form of nicotinamide adenine di-nucleotide phosphate), one of the cell’s principal reductants. As an

1Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305,USA. 2Division of Rheumatology, Mayo Clinic College of Medicine, Rochester, MN55905, USA. 3Department of Health Research and Policy, Stanford University School ofMedicine, Stanford, CA 94305, USA.*Present address: Department of Anatomy and Embryology, University of Tsukuba,1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.†Corresponding author. E-mail: cweyand@stanford.edu

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electron carrier, NADPH provides reducing equivalents for biosyntheticreactions and by regenerating reduced glutathione, protects againstreactive oxygen species (ROS) toxicity. Cytoplasmic NADPH is anabsolute requirement to convert oxidized glutathione to its reducedform (GSH), which is converted back when hydrogen peroxide isreduced to water.

Oxidative stress results from the action of ROS, short-lived oxygen-containing molecules with high chemical reactivity toward lipids, pro-teins, and nucleic acids. Until recently, ROS were regarded as merelydamaging agents, but are now recognized as second messengers thatregulate cellular function through oxidant signaling (8, 9). Cells canproduce ROS in several of their organelles and possess specialized en-zymes, such as the family of NADPH oxidases (NOX), to supply fastand controlled access. Quantitatively, mitochondria stand out as per-sistent ROS suppliers, with the respiratory chain complexes I and IIIreleasing superoxide into the mitochondrial matrix and the inter-membrane space (9, 10). It is incompletely understood how redoxsignaling affects T cell proliferation and differentiation and how cell-internal ROS relate to pathogenic T cell functions.

The current study has investigated functional implications of meta-bolic and redox dysregulation in RA T cells. We find that RA T cells failto properly balance mitochondrial ROS production and the cellularantioxidant machinery. Molecular studies place excessive activity ofG6PD at the pinnacle of abnormal T cell regulation in RA and pro-vide a new paradigm for the connection between metabolic activities,abnormal proliferative behavior, and proinflammatory effector func-tions. Mechanistically, PPP hyperactivity oversupplies RA T cells withreducing equivalents, increasing NADPH, and depleting ROS. This in-sufficient oxidative signaling prevents sufficient activation of the cellcycle kinase ataxia telangiectasia mutated (ATM) and allows RA T cellsto bypass the G2/M cell cycle checkpoint. ATM deficiency shifts dif-ferentiation of naïve CD4 T cells toward the T helper 1 (TH1) andTH17 lineages, creating an inflammation-prone T cell pool. Severalmetabolic interventions are able to rebalance glucose utilization away

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from the PPP toward glycolytic breakdown, easing reductive stress andpreventing hyperproliferation and maldifferentiation of RA T cells.Such interventions represent possible drug candidates for anti-inflammatory therapy.

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RESULTS

Disproportionate PPP activation in RA T cellsCD4+CD45RO− T cells from RA patients have reduced glycolytic flux,generating lower ATP and lactate concentrations (4), while proliferat-ing vigorously (11), suggesting intactness of metabolic outputs thatsupport biomass generation. To examine competence of the PPP, wequantified gene and protein expression of the rate-limiting enzymeG6PD (Fig. 1, A and B). Compared to controls, RA T cells expressedhigher G6PD transcript and protein levels, and G6PD enzyme activitywas 30% increased (Fig. 1C), compatible with preferential PPPshunting in patient-derived T cells. The response of G6PD to T cellreceptor triggering was prompt and sustained (fig. S1), and RA T cellswere distinguishable from control T cells over the entire poststimulationperiod. The defect was disease-specific and was not present in T cellsfrom patients with psoriatic arthritis (PsA).

In a cohort of 31 patients and 32 age/gender-matched controls,G6PD overexpression coincided with PFKFB3 deficiency, and in indi-vidual patients, the ratio of G6PD/PFKFB3 was clearly shifted towardG6PD (Fig. 1D). To evaluate the relationship between shifted metabolicenzymes and RA inflammatory activity, we correlated transcript levelsin stimulated CD4+CD45RO− T cells with the composite disease ac-tivity measure DAS28. DAS28 scores were strongly correlated withG6PD/PFKFB3 ratio (R = 0.6, P < 0.001; Fig. 1E). To assess the impactof immunosuppressive therapy on G6PD induction, we comparedG6PD transcript levels in untreated patients and patients on differenttypes of medications (fig. S2). G6PD transcript levels were elevated inuntreated patients, and enzyme expression was similar in T cells frompatients on different types of medications.

To test whether RA T cells redirect glucose into the PPP and preferNADPH production over glycolytic breakdown, we quantified NADPHlevels and assessed the cells’ redox status via the quantification of re-duced glutathione (mBCI) and ROS (CellROX). RA T cells had higherNADPH levels, outperforming their healthy counterparts and T cellsfrom PsA patients by 40% (Fig. 1F). RA T cells, but not PsA T cells,contained significantly more reduced glutathione (Fig. 1, G to I). Ex-cess GSH generation was maintained in naïve T cells that converted tothe memory phenotype (Fig. 1G), but the bias toward reductive spe-cies in RA T cells became visible only after T cell stimulation and wasnot present in resting cells (fig. S3).

After T cell receptor (TCR) stimulation, intracellular ROS levelsdisplayed a characteristic kinetic with baseline levels maintained over24 hours, a steep increase to >12-fold higher levels over the subse-quent 48 hours, and a gradual decline between days 3 and 6. Thus,while transitioning from naïve to effector cell, T cells enter a periodof oxidative stress (Fig. 1J). RA T cells followed similar kinetics earlyafter stimulation, but ROS levels increased only eightfold to peak onday 3, suggesting surplus reducing equivalents. Between days 3 and 6,ROS levels in RA T cells were consistently lower. Compared to controland PsA, ROS levels in RA T cells were significantly reduced (Fig. 1K).Kinetics of ROS generation mirrored glycolytic activity, which alsopeaked after 72 hours (4).

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In essence, RA patients’ naïve CD4 T cells express an altered pat-tern of glucose-metabolizing enzymes, resulting in slowed glycolyticbreakdown and increased PPP shunting. The defect is specific for adiagnosis of RA and not present in PsA. Because of disproportionategain in NADPH and reduced glutathione, the cells consume ROS andare under reductive stress.

ROS reduction, hyperproliferation, and G2/M checkpointbypassing in RA T cellsThe PPP supplies reducing equivalents for macromolecule synthesis,the building blocks for new cells, rendering naïve CD4 T cells partic-ularly sensitive to changes in proliferative metabolism. To examinewhether excessive G6PD activity affects T cell proliferation, we treatedRA T cells with the G6PD inhibitor 6-aminonicotinamide (6-AN). Pre-venting glucose entry into the PPP profoundly reduced cellular pro-liferation (Fig. 2A) and also changed intracellular ROS levels (Fig. 2B).Upon 6-AN treatment, ROS levels doubled, and in parallel, prolifera-tive activity decreased. G6PD inhibition corrected the spontaneouslyelevated division indices of RA T cells (Fig. 2A). G6PD’s critical role inregulating T cell proliferation was confirmed by gene-specific RNAinterference. Transfection of two distinct small interfering RNAs (siRNAs)significantly reduced G6PD protein expression (fig. S4). G6PD knock-down in RA T cells reduced intracellular NADPH and GSH concentra-tions, increased ROS levels, and normalized division indices (Fig. 2C).

ROS reduction in RA T cells was associated with hyperprolifera-tion (Fig. 2A) and increased interleukin-2 (IL-2) production (Fig. 2D).On day 3, intracellular IL-2 was more than doubled in RA comparedto wild-type T cells. Higher division rates in RA T cells became func-tionally relevant in the conversion of naïve into memory T cells (Fig.2E). At the end of a 6-day stimulation period, 29% of control T cells,but only 18% of RA T cells, retained the naïve phenotype, demonstrat-ing that RA T cells converted to the memory phenotype at a morerapid pace.

To test the conversion of naïve into memory T cells in vivo, we useda human–severe combined immunodeficient (SCID) mouse chimericmodel (12). Human CD45RO− peripheral blood mononuclear cells(PBMCs) take residence in the spleen of reconstituted NSG (nonobesediabetic SCID gamma) mice and form organized T cell–B cell aggre-gates (fig. S5, A and B). Transfer of 10 million CD45RO− PBMCs intothe murine host prompted T cell proliferation and naïve-to-memoryconversion within 7 to 10 days (fig. S5C). Human naïve T cells requiredcotransfer of human APC (antigen-presenting cells), specifically mono-cytes, for optimal engraftment and expansion (fig. S6, A and B). Thus,all reconstitution experiments used memory T cell–depleted PBMCscomposed of naïve T cells, B cells, and monocytes. Transfer of suchpopulations from RA patients and control donors permitted the directcomparison of how naïve cells rapidly acquired a memory phenotype.RA-derived cells had a higher division index (Fig. 2F) and converted toa memory phenotype at a faster rate, as indicated by higher frequen-cies of CD4+CD95+ and CD8+CD95+ cells (Fig. 2G), confirming thatnaïve RA T cells are prone to faster cell cycle progression and fail tomaintain naïvety.

To understand functional consequences of ROS reduction for T cellproliferation, we suppressed ROS through the cell-permeable super-oxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl(Tempol) (Fig. 2, H and I) (13). TCR activation essentially moved allcells into the cell cycle. Six days after stimulation, two-thirds of theT cells had completed three to four doublings. Tempol accelerated

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proliferation and increased the proportion of T cells with >5 genera-tions from 15 to 26%. In RA T cells, proliferation was spontaneouslyhigher and further increased by ROS scavenging, from 22 to 32% ofcells. Proliferative rates in Tempol-treated wild-type T cells were simi-lar to the rates in untreated RA T cells.

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Cell cycle analysis revealed that Tempol treatment selectively fast-ened G2/M transit; progression through the G1 and S phases was un-affected (Fig. 2J). RA T cells effectively bypassed the G2/M checkpoint,even without treatment. After stimulation, 8% of control T cells werein the G2/M phase. Tempol reduced G2/M-retained T cells to 5%, similar

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in an accumulation ofNADPH and reduced glu-tathione and loss of ROS.CD4+CD45RO− T cells frompatients with RA, patientswith PsA, and age-matchedcontrols (Con) were stim-ulated for 72 hours. (A) Ex-pression of G6PD andPFKFB3 in 31 RA patients,14 PsA patients, and 32controls quantified by re-verse transcription polymer-ase chain reaction (RT-PCR).GAPDH, glyceraldehyde-3-phosphate dehydrogenase.(B) G6PD immunoblotsfrom four control and fourRA samples. Relative banddensities from eight RA-control pairs. (C) G6PD en-zyme activities quantifiedin 13 RA and 13 controlsamples. (D) Correlation ofG6PD and PFKFB3 mRNAexpression in individualpatients and controls. A.U.,arbitrary units. (E) Correla-tion of the disease activityDAS28 score with the ratioof G6PD and PFKFB3 tran-scripts. (F) NADPH levelsmeasured in T cell extractsof 11 RA patients, 8 PsApatients, and 14 controls.(G) Representative dot blotsofmonochlorobimane (mBCI)staining in control and RAT cells. (H) Intracellular glu-tathione levels quantified bymBCI fluorescence. Datafrom seven RA patients, sev-en PsA patients, and ninecontrols. MFI, mean fluo-rescence intensity. (I) Rep-resentative fluorescentimaging of mBCI stainingin normal and RA T cells.DAPI, 4 ′ ,6-diamidino-2-phenylindole. (J) Kinetics ofintracellular ROS over 6 daysafter stimulation measuredwith the fluorogenic probeCellROX in 11 RA patientsand 7 controls. (K) Intra- cellular ROS levels measured in T cell extracts of 15 RA patients, 8 PsA patients, and 14 controls. All data are means ± SEM.

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Fig. 2. ROS-depletedT cells hyperprolife-

rate and bypass theG2/M cell cycle check-point. (A) Proliferationo f CD4+CD45RO −

T cells with andwithoutthe G6PD inhibitor 6-ANmeasured by carboxy-fluorescein diacetatesuccinimidyl ester (CFSE)dilution 72 hours afterstimulation. Represent-ative histogram (left)and division indicesfrom seven experiments.Max, maximum. (B) In-tracellular ROS in RAT cells cultured with andwithout 6-AN. Repre-sentative histogram (left)andMFI from five experi-ments (right). (C) T cellsfrom four RA patientswere transfected withcontrol siRNA or two dif-ferent G6PD-targetingsiRNAs (si-1, si-2). NADPHlevels, GSH, intracellularROS, and division indicesweremeasured 72 hourslater. (D) MFI of intra-cellular IL-2 in four pa-tients and four controls.(E) Naïve-to-memoryconversion of CD4 T cellsafter TCR stimulationmonitored by flow cy-tometry of CD45RA.Data from four patient-control pairs. d0, day 0.(F) CFSE-labeled CD45RO−

PBMCs fromRApatientsand controls were in-jected intravenouslyintoNSGmice. Left: CFSEdilution in CD4 T cellsas a measure of in vivoproliferative activity.Right: Division indicesfrom 12 patients and 15controls. (G) Fluorescence-activated cell sorting(FACS) analysis of NSG

uman CD4 and CD8 T cells+CD95+ and CD8+CD95+

sults from 12 experiments.ultured with and withoutpol. Generational assign-E dilution. (H) Represent-

ative patient-control pairs. (I) Percentages of T cells that underwent >5 doublings from three patient-control pairs. (J) CD4 CD45RO− T cells cultured withand without the ROS scavenger Tempol. Assignment to the G1, S, and G2/M phases of the cell cycle by propidium iodide staining. Percentages in each cellcycle phase for 6 patients and 12 controls. (K) Representative scatter blots of cells in the G2/M phase identified with anti–phospho-histone H3 antibodystaining. Percentages of phospho-histone H3+ cells in seven patients and seven controls. All results are means ± SEM.

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to only 4% in RA T cells. Both RA origin and ROS scavenging re-duced the fraction of G2/M-specific phospho-histone H3–positivecells (Fig. 2K), confirming the role of ROS in regulating G2/Mtransition.

Together, the activation-induced elevation of intracellular ROSregulates cell cycle progression, proliferative efficiency, and naïve-to-memory conversion. ROS-deficient RA T cells hyperproliferate,fail to maintain the naïve phenotype, and bypass the G2/M cell cyclecheckpoint.

ATM insufficiency in RA T cellsThe cell cycle checkpoint leading to G2/M arrest is activated by theprotein kinase ATM (14). In healthy CD4+CD45RO− T cells, TCR

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stimulation induced a four- to fivefold rise in ATM transcripts. InRA T cells, ATM transcripts responded slower, increasing only two-to threefold (Fig. 3A). ATM protein concentrations followed a similarkinetic, with both ATMmonomers and dimers rising after stimulation(Fig. 3B). By 72 hours, most of the protein was in the active dimericstate. ATM protein was much less abundant in RA T cells, particularlythe dimeric form (Fig. 3, B and C). TCR stimulation resulted in ATMphosphorylation at serine 1981 (Fig. 3D), with maximal phosphoryl-ated ATM (pATM) concentrations recorded on day 3 (Fig. 3D). Incontrast, pATM was barely detectable in RA T cells (Fig. 3D).pATM kinetics paralleled cellular ROS dynamics, raising the questionwhether ATM activation deficiency was related to the ROSlow status ofRA T cells.

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Fig. 3. Insufficient activation of the ROS-sensitive cell cycle regulator ATMresults in T cell hyperproliferation. (A) ATM gene expression in activated

from one of four experiments are shown. (F) Cells were cultured with theATM inhibitor KU-55933, and proliferation was assessed by CFSE dilution. Fre-

CD4+CD45RO− T cells measured by RT-PCR in seven controls and six patients.(B) Quantification of ATM monomers (mATMs) and dimers (dATMs) by West-ern blotting. Poststimulation dynamics of protein expression for a represent-ative control and RA patient. (C) Relative band intensities for total ATMquantified at 72 hours. Results from eight patient-control pairs. (D) Kineticsof ATM phosphorylation on days 0, 1, 3, and 6 after T cell stimulation. Repre-sentative immunoblots (left) and results from four controls and four patients(right). (E) Healthy stimulated T cells were treated with H2O2 on day 3. Cellextracts were immunoblotted with anti-ATM and pATM (Ser1981). Results

quencies of proliferating T cells in five experiments. (G) Effect of the ATMinhibitor KU-55933 on naïve-to-memory conversion. KU-55933–treated T cellswere phenotyped as CD45RA+CD62L+ naïve, CD45RA−CD62L− effectormemory (EM), and CD45RA+CD62L− end-differentiated effector T cells(TEM) by flow cytometry. Results from six experiments. (H) Increasing cellularROS levels restore ATM activation. T cells were treated with menadione (Me)(3 mM) for 72 hours. ROS were measured with the fluorogenic probe CellROX(left). ATM and pATM were quantified by Western blotting; one of four ex-periments is shown (right). All results are means ± SEM. KU, KU-55933.

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Nuclear ATM is mostly activated by DNA fragments; the dimerassembly of cytoplasmic ATM is a redox-sensitive process (15). H2O2

rapidly induced dimer formation in CD4+CD45RO− T cells (Fig. 3E),with visible effects starting at doses as low as 10 mM and plateauing at20 mM. ROS-induced ATM dimerization promoted ATM Ser1981

phosphorylation (Fig. 3E). Pharmacologic inhibition of ATM revealedits role in T cell proliferation and naïve-to-memory conversion. KU-55933–treated T cells expanded more vigorously (Fig. 3F), and ATMimpairment accelerated the generation of effector memory and term-inally differentiated memory cells at the expense of naïve cells (Fig.3G). Thus, ATM inhibition in healthy T cells reproduced the abnor-mal proliferation behavior of RA T cells, implicating the kinase in clonalT cell expansion.

We examined whether replenishing ROS can restore ATM ac-tivation in RA T cells. Treatment with menadione, an analog of 1,4-naphthoquinone that generates intracellular ROS via redox cycling(16), increased ROS in RA T cells (Fig. 3H). Concomitantly, menadione-exposed T cells formed ATM dimers and accumulated pATM. ATMactivation could be blocked by KU-55933, known to interfere withATM phosphorylation at Ser1981 (15). KU-55933 effectively preventedmenadione-induced ATM phosphorylation (Fig. 3H), indicating thatthe ROS-inducing naphthoquinone analog restored physiologicATM activation.

Together, RA T cells fail to properly activate ATM, explaining the

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shortened G2/M phase as well as theDNA damage accumulation (17). Insuffi-cient ATM signaling is correctable byreplenishing intracellular ROS, mecha-nistically connecting G6PD overactivity,NADPH excess, reductive stress, andmalregulation of T cell proliferation.

Redox regulation of RAT cell differentiationProtective and pathogenic T cell functionsare closely linked to cytokine productionand thus to T cell differentiation.We exam-ined the impact of ROS scavenging ondifferentiating T cells (Fig. 4, A and B).Eighteen percent of healthy T cells and32% of patient-derived T cells committedto interferon-g (IFN-g) production. ROSscavenging via Tempol further enhancedthe commitment to the TH1 linage, to 33and 40%, respectively. Under cytokine-polarizing conditions, 1.8% of control T cellsand 4.2% of RA T cells stained positive forintracellular IL-17. ROS quenching withTempol increased TH17 cells to 5 and6%, respectively. There was a trend for low-er IL-4 and FoxP3 expression in the pres-ence of Tempol. Overall, ROS reductionswayed T cells to mature into proinflam-matory TH1 or TH17 cells.

To test whether ROS bias T cellstoward proinflammatory effector func-tions in vivo, we used the human-SCIDchimeric model (fig. S6). Mice reconsti-

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tuted with human T cells were injected daily with Tempol or vehicleto examine the in vivo behavior of ROS-depleted T cells. ROSscavenging resulted in a significant increase in TH1-committed T cells,whereas IL-4 producers remained unchanged (Fig. 4C).

Maldifferentiation and arthritogenic potential ofRA T cellsConsidering the effects of ROS depletion on T cell differentiation,we determined whether ROSlow RA T cells are spontaneously bi-ased to develop into IFN-g+ and IL-17+ effector cells (Fig. 5A).Without lineage-inducing cytokine cocktails, 10% of healthy naïveCD4 T cells differentiated into TH1 cells and 0.8% into TH17 cells.Corresponding frequencies in RA samples were 14% TH1 cells and1.7% TH17 cells.

To investigate the disease relevance of T cell maldifferentiation, wetested the cells’ arthritogenic potential in a human synovium–NSGchimeric model. Naïve CD4 T cells from either healthy subjectsor RA patients were adoptively transferred into human synovium–engrafted NSG mice. After 10 days, the synovial graft was analyzed forT cell infiltration and lineage commitment [TCR, T-bet, GATA-3,FoxP3, and RORg (RAR-related orphan receptor g), IFN-g, IL-17,and IL-4] (Fig. 5B). Inflammatory activity of synovial macrophagesand fibroblasts was assessed by profiling inflammation-associatedgenes (TNF-a, IL-1b, IL-6, CD16a/b, CD68, MMP3, RANKL, vimentin,

A

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Fig. 4. ROS scavenging mimics the maldifferentiation of RA T cells. (A and B) CD4+CD45RO− T cellswere cultured under T 1- and T 17-skewing conditions with or without the ROS scavenger Tempol, restimu-

H H

lated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, and stained for intracellular cytokines. (A)Representative dot plots. (B) Percentages of IFN-g–producing (left) and IL-17–producing (right) cells fromfour experiments. FoxP3, forkhead box P3. (C) Healthy PBMCs depleted of CD45RO+ cells were adop-tively transferred into NSG mice. On day 7, splenocytes were analyzed for human CD45+CD4+IFN-g+

cells by flow cytometry. Representative dot plots from one control-patient pair (left) and results from fourindependent experiments (right).

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Fig. 5. Arthritogenic effector func-tions in RAT cells. (A) CD4+CD45RO−

T cells were stimulated for 6 hours.IFN-g, IL-4, IL-17, and FoxP3 weredetected by intracellular stainingin six patients and six controls. Allresults are means ± SEM. (B) NSGmice were engrafted with humansynovium, and CD45RO-depeletedPBMCs from healthy controls orRA patients were adoptively trans-ferred into the chimeras. Synovialinflammation was assessed by RT-PCR analysis of 17 inflammation-related genes. Results from 8 to16 tissue grafts are shown as a heatmap. RANKL, receptor activator ofnuclear factor kB ligand; MMP3,matrix metalloproteinase-3; TNF-a,tumor necrosis factor–a. (C) Den-sities of synovial T cell infiltrateswere analyzed by immunostain-ing for human CD3. (D) T cells mi-grated into synovial tissue werequantified by RT-PCR for TCRtranscripts, and tissue-infiltratingT cells were enumerated by anti-CD3 staining per high-power field(HPF). (E) T cell mobility wasmeasured in Transwell migrationassays. Means ± SEM from ninepatient-control pairs.

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and cadherin 11) and by immunohistochemical analysis (Fig. 5C). RAT cells were prone to migrate into the synovial tissue and commit tothe TH1 and TH17 differentiation program (Fig. 5, B and C). Synovialmembranes infiltrated by RA T cells contained significant amounts ofIFN-g and IL-17. Conversely, much lower numbers of healthy CD4T cells were retained in the tissue and typically expressed GATA-3and IL-4. Inflammatory cytokines were abundant in the RA T cell–populated synovia. Evidence for synovial fibroblast activation camefrom the robust induction of vimentin and cadherin 11 (18). CD3-specific staining of tissue sections confirmed that RA T cells, butnot healthy T cells, differentiate into tissue-infiltrating effector cellsin vivo (Fig. 5, C and D). In a Transwell assay system, RA T cells werespontaneously hypermigratory, even in the absence of chemokinesignals (Fig. 5E).

ATM insufficiency and T cell maldifferentiationWe examined whether tissue invasiveness and proinflammatory effec-tor functions of RA T cells are mechanistically linked to ROS deficien-cy and ATM insufficiency. Under nonpolarizing conditions, 9% ofCD4 T cells committed to IFN-g production. Increasing doses of theATM inhibitor KU-55933 shifted T cell differentiation toward TH1ness,and IFN-g+CD4Tcellsmore thandoubled to as high as 20.0% (Fig. 6A),whereas IL-4+CD4 T cells were unchanged. TH1- or TH2-polarizingconditions drove 25% of cells toward TH1 differentiation and 13.5%to IL-4 production. ATM insufficiency markedly increased IFN-g–producing T cells but left IL-4 production unaffected (Fig. 6B).

Inhibiting ATM function by short hairpin RNA (shRNA)–mediatedsilencing confirmed that ATM regulates TH1 lineage commitment(Fig. 6, C and D). After transfection with pSuper-gfp/neo-shATMplasmids, frequencies of IFN-g–producing T cells in TH1-polarizingcultures were doubled from 14.7 to 32.4%. Effects of ATM insufficiencyon T cell function in vivo were explored in the human-SCID chimericmodel (fig. S6). Mice were reconstituted with CD45RO− PBMCs fromhealthy donors; chimeras were injected daily with the ATM inhibitorKU-55933 or vehicle. Human T cells from the chimeric spleen wereanalyzed for intracellular IFN-g, IL-4, IL-17, and FoxP3 expression(Fig. 6E). Suppressing ATM activity significantly increased TH1-and TH17-committed T cells, whereas IL-4 producers and FoxP3+

cells remained unchanged. To assess ATM’s effect on lineage-specifictranscription factors, we cultured T cells under TH1-, TH2-, TH17-,and Treg (regulatory T cell)–skewing conditions with and withoutthe ATM inhibitor KU-55933. Early during the differentiation process,expression of both T-bet and RORg was higher in ATM-insufficientT cells (Fig. 6F), suggesting that ATM-dependent signaling selec-tively regulates lineage-defining transcription factors.

To better understand the impact of ATM deficiency in arthritis de-velopment, we treated human synovium–NSG mice with the ATMinhibitor KU-55933 after the adoptive transfer of either controlor RA CD45RO− PBMCs. Inhibiting ATM kinase activity intensi-fied synovial inflammation induced by healthy and RA T cells (Fig.6G). ATM-impaired healthy T cells almost matched the proinflam-matory potential of RA T cells. ATM insufficiency further increasedinflammatory effector functions of RA T cells. KU-55933 treatmentstrongly up-regulated IL-17, RORg, TNF-a, IL-1b, and IL-6, and ex-pression of the osteoclastogenic ligand RANKL increased multifold(Fig. 6G). RANKL protein expression in tissue sections confirmedup-regulation of this osteoclastogenic ligand under ATM-deficientconditions (Fig. 6H).

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Functional consequences of prooxidant treatmentExcessive G6PD activity supplies undifferentiated RA T cells withreductive elements (Fig. 1). ROS shortage causes insufficient ATMactivation, and RA T cells prematurely exit the G2/M checkpoint (Figs.2 and 3), shifting their lineage commitment toward preferential dif-ferentiation of TH1 and TH17 cells (Figs. 4 to 6). To mechanisticallylink PPP hyperactivity with inflammatory maldifferentiation, weidentified two pharmacologic interventions that restore the redox bal-ance in naïve RA T cells and correct the overrepresentation of IFN-g+

T cells.We evaluated the synthetic naphthoquinone menadione, which is

reduced into an unstable semiquinone and generates ROSwhen formedinto a quinone. Treatment of T cells with menadione increased cellularROS levels (Fig. 3) and resulted in ATM dimerization and pATM forma-tion (Fig. 7, A and B). Combination of the ATM inhibitor KU-55933with menadione treatment did not prevent ATM dimer assembly(Fig. 7B), but, as expected (15), blocked ATM phosphorylation (Fig.7B). Menadione-induced restoration of ATM activation enabled pChk2accumulation; this effect was disrupted when ATM phosphorylation wasinhibited (Fig. 7B).

Naïve CD4 T cells from RA donors were differentiated in a polar-izing cytokine cocktail in the absence and presence of either menadioneor 6-AN, two interventions able to counteract the shift toward reductiveelements. Menadione corrected the bias of RA T cells to develop intoIFN-g producers (Fig. 7C). The G6PD inhibitor 6-AN provided an atleast equally successful intervention to down-regulate T cell IFN-g pro-duction (Fig. 7C). Blocking G6PD activity reduced the frequency ofIFN-g–producing T cells to less than 15%.

To evaluate the impact of ROS restoration on the arthritogenicpotential of RA T cells, we tested two ROS-inducing reagents inthe human synovium chimeras. Menadione raises ROS levels (Fig. 3I)through redox cycling. Buthionine sulfoximine (BSO) inhibits gamma-glutamylcysteine synthetase, lowers tissue glutathione (GSH) concen-tration, and consequently elevates intracellular ROS levels (fig. S7).Synovium-engrafted NSG mice were adoptively transferred with T cellsderived from untreated or high-disease activity RA patients, and micewere treated with optimized doses of either menadione or BSO. Treat-ment with both ROS inducers had a beneficial effect on synovitis (Fig.7D). Transcription factors (T-bet and RORg) driving proinflamma-tory T cells were effectively down-regulated, IFNg and IL-17 were re-duced, whereas FoxP3 was spared. RANKL expression responded toboth treatments (Fig. 7, D and E), as did the inflammatory cytokinesTNF-a, IL-1b, and IL-6. Menadione had more powerful effects thanBSO. Immunohistochemical analysis of RANKL expression confirmedthat tissue-infiltrating T cells were almost all RANKL+ in the controlarm but lost RANKL expression after menadione and BSO treatment.Bothmenadione and BSOwere able to correct the spontaneous hypermo-bility of RA T cells in Transwell migration assays (Fig. 7F). Overall, off-setting reductive stress in RA T cells effectively suppressed synovialinflammation.

DISCUSSION

CD4 effector T cells are major drivers of abnormal immunity in RAby sustaining chronic synovitis and supporting autoantibody pro-duction. Deriving from infrequent naïve precursor cells, such pathogen-ic T cells had to clonally expand and functionally differentiate. Here,

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KU-55933 (µM)

IFN-γ IL-4

% in

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H Healthy + KU RAHealthy RA + KU(F) Flow cytometric antranscription factors iTH1-, TH2-, TH17-, andwith or without KU-55from three experimenPBMCs from healthy i

were adoptively transferred into synoviuMice were treated with the ATM inhibitor Kexpression was quantified in explanted syMeans ± SEM from eight tissues. *P <0.001. (H) Immunohistochemistry of synosteoclastogenic ligand RANKL is visualize

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Fig. 6. The cell cycle ki-nase ATM regulates the

lineage commitmentand the arthritogenicpotential of T cells.(A) CD4+CD45RO− T cellswere cultured with theATM inhibitor KU-55933.Cytokine production pat-terns after nonpolariz-ing conditions from sixexperiments. (B) Cyto-kine production patternsafter culture under TH1-and TH2-skewing condi-tions with and withoutKU-55933. (C) T cellstransfected with con-trol or shATM (shRNAtargeting ATM) plas-mids were culturedunder TH0-, TH1-, and TH2-polarizing conditions.Intracellular cytokinestains from a represent-ative experiment. (D)Frequencies of cytokine-producing cells from fiveexperiments with ATM-silenced cells. (E) NSGmice were reconstitutedwith CD45RO-depletedPBMCs and injectedwithKU-55933 (0.5 mg/kgintraperitoneally) orvehicle daily. Cytokineproduction in spleno-cytes was measuredby intracellular cyto-kine staining in humancells. Left: Representativedot plots. Right: Per-centages of IFN-g, IL-4, IL-17, and FoxP3+

cells from four inde-pendent experiments.

alysis of lineage-definingn T cells cultured underTreg-skewing conditions

933. Means ± SEM of MFIts. (G) CD45RO+-depletedndividuals or RA patients

m-engrafted NSG mice.U-55933 for 9 days. Genenovial tissues by RT-PCR.0.05; **P < 0.01; ***P <ovial tissue sections. Thed by brown staining.

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36.7 23.8 20.6

Fig. 7. Replenishing intra-cellular ROS in RA T cells cor-rects ATM insufficiency, T cellmaldifferentiation, and ar-thritogenic effector functions.CD4+CD45RO− T cells from RApatients were stimulated asabove. (A) On day 3, T cellswere treated with menadioneor menadione plus KU-55933.Cell extracts were immuno-

www.ScienceTranslationalMedicine.org 23 March 2016

blotted with anti-ATM, pATM, Chk2 (checkpoint kinase 2), and pChk2 (phosphorylated Chk2). (B) Amounts of dATM, mATM, pdATM (phosphorylateddATM), pmATM (phosphorylated mATM), Chk2, and pChk2 were quantified in five experiments. (C) Effect of menadione and 6-AN treatment on IFN-gproduction under TH1-polarizing conditions. Representative dot plots (left) and results from five experiments (right). (D) CD45RO-depleted PBMCs from RApatients were adoptively transferred into NSG mice engrafted with human synovium. To increase intracellular ROS levels, mice were treated with dailyintraperitoneal injections of menadione or BSO for 9 days. T cell polarization and intensity of synovitis were analyzed as in Figs. 5 and 6. Means ± SEM from8 to 13 synovial tissues. *P < 0.05; **P < 0.01; ***P < 0.001. (E) Immunohistochemical analysis of synovial tissues for human CD3 (pink) and RANKL(brown). Double-positive cells are marked by a white arrow head, and CD3+RANKL− T cells by a black star. (F) Effects of menadione and BSO on T cellmobility measured in Transwell migration assays. Means ± SEM from nine experiments.

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we demonstrate that proliferative behavior and functional differentia-tion are critically determined by metabolic adaptations of the naïveprecursor cells. Specifically, naïve CD4 T cells from RA patients aremetabolically reprogrammed, favoring NADPH production overATP generation. Excess NADPH supplies the cell with excess reducedglutathione and depletes ROS, effectively exhausting the cell’s ROSpool and weakening ROS-dependent signaling. Such reductive stressfastens the T cells’ cell cycle progression, as they skip the G2/M cellcycle checkpoint because of insufficient ATM activation. ConstitutiveATM insufficiency in naïve RA T cells and pharmacologic ATM in-sufficiency in healthy T cells accelerate their conversion into effectormemory T cells. ROS loss and ATM insufficiency promote T cell mal-differentiation into IFN-g and IL-17 effector cells. These abnormalitiesare reversible by replenishing the ROS pool with the naphthoquinonemenadione, by disrupting synthesis of the ROS quencher glutathione,or by blocking glucose shunting into the PPP. These pharmacologicinterventions not only localize the pinnacle defect to excessive PPP uti-lization but also provide a framework for entirely new anti-inflammatorystrategies.

Effective T cell responses require the massive expansion of low-frequency naïve T cells into memory and effector T cells. To fulfill thedemands for energy, T cells, like malignant cells, depend on oxidativeglucose metabolism coupled with mitochondrial oxidative phospho-rylation to efficiently generate ATP (19, 20). However, they need morethan ATP to replicate single cells into thousands of copies. For bio-mass production, they require a carbon source and reducing power inthe form of NADPH. With excess NADPH, RA T cells are well pre-pared to replicate, as long as they have sufficient biosynthetic precur-sor molecules. In line with this metabolic state, RA T cells proliferatewell, in spite of telomeric features that identify them as pre-aged (11).

Current data pinpoint the source of excessiveNADPH in RAT cells:efficient shunting of glucose into the PPP (Fig. 1). Gain ofG6PDactivitycombined with the loss of PFKFB3 activity generates a metabolic mis-balance that promotes cellular proliferation. Biased glucose flux towardthe PPP, however, generates a ROS shortage, resetting the signalingma-chinery that requires ROS as a second messenger. Human T cells gen-erate an early ROS peak within minutes of TCR cross-linking (21).Cellular ROS levels rise again 2 to 3 days after activation, with sustainedelevation beyond day 6 (Fig. 1). Traditionally, joint inflammation hasbeen considered a consequence of excessive oxidative stress, but elegantwork by Perl et al. has demonstrated differential ROS production (22)and differential responsiveness to ROS scavenging therapy in lupus andRA (23). Also, positional cloning of genetic polymorphisms in arthritismodels identified theNADPHoxidaseNOX2 as a protective factor (24),with the oxidative burst suppressing autoimmune T cells (25). Mecha-nistically, ROS-producing macrophages suppress T cell immunity (26).NOX2 deficiency impacts arthritis susceptibility in aging-related mu-rine arthritis (27) by shifting T cell differentiation from Tregs towardTH1 cells. Also,N-acetyl cysteine treatment fosters TH17 cell generationby targeting pyruvate dehydrogenase kinase 1, which regulates theTH17/Treg balance (28). Further evidence for a detrimental role of ROSdeficiency stems from chronic granulomatous disease, a hereditary dis-order caused by NOXmutation. NOX-deficient patients have frequentinfections while requiring immunosuppression for granulomatous andautoimmune disorders (29).

ROS act as obligate second messengers by regulating kinases andphosphatases. In human T cells, ATM appears to be a critical target(Fig. 3), with an oxidation-sensitive site mapped to Cys2991 (15). RA T cells

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express low ATM levels, causing insufficient DNA damage repair (30).Deficient DNA repair may be related to bypassing the G2/M cell cyclecheckpoint (Fig. 2). Individuals born with mutated ATM genes sufferfrom ataxia telangiectasia (AT) and, like RA patients, have prematureimmune aging (31). AT patients and ATM-deficient mice have dys-functional mitochondria and heightened oxidative stress (32), con-trasting with the findings presented here. However, ATM insufficiencyin RA T cells is acquired, thus not affecting the developing immune sys-tem but rather the functionality of mature, peripheral T cells. Micelacking germline ATM have abnormal thymic T cell selection andrepertoire formation (33, 34). ATM-insufficient mice demonstratepersistent immune activation in a model of DNA damage–associatedcolitis (35) and effectively clear lymphocytic choriomeningitis virus infec-tion (36). ATMlow RA T cells are highly susceptible to apoptosis (17, 30),stressing ATM’s role in T cell maintenance.

Metabolic control of T cells impacts their development and theireffector functions, with a particular regulatory role for the energy sensoradenosine 5′-monophosphate (AMP)–activated protein kinase (AMPK)and the downstream signaling knot mammalian target of rapamycin(mTOR) (6, 37, 38). Checkpoint function has been assigned to mTOR,which appears to function through metabolic selection (39). Similarly,AMPK regulates TH1 and TH17 development and primary antimicrobialT cell responses (38). Functional intactness of these signaling networksin naïve RA T cells needs to be examined to search for further impli-cations of defective oxidant signaling beyond ATM. Also, the mechanis-tic causes underlying G6PD induction and PFKFB3 loss remain to beclarified. Correlation of the PFKFB3/G6PD ratio with RA inflammatoryactivity (Fig. 1E) suggests a formidable role of the metabolic defect inchronic inflammation. Vice versa, ATP and biomass generation are knownto be responsive to environmental conditions, such as oxygen availabil-ity. Naïve T cells live in secondary lymphoid organs, not in peripheralinflammatory lesions, raising the intriguing question: How could tissueinflammation regulate metabolic reprogramming in distant T cells?

An important notion of the current study is the reversibility of themetabolic wiring (Fig. 7), effectively preventing hyperproliferation andmaldifferentiation in vitro and in vivo. ROS induction via menadionerestored ATM signaling and suppressed IFN-g induction, shiftingT cell differentiation toward an anti-inflammatory phenotype. Menadione,known as vitamin K3, is used as a nutritional supplement (40). Largedoses can cause hemolytic anemia in G6PD-deficient individuals,emphasizing the mechanistic link between PPP utilization and redoxbalance. Interfering with production of the ROS generator BSO provedeffective in inhibiting synovial inflammation. Pharmacologic and genet-ic G6PD inhibition confirmed that the pinnacle defect lies in the exces-sive induction of this rate-limiting enzyme for the PPP. 6-AN treatmentwas even more effective in down-regulating proinflammatory cells,opening the door to targeting autoimmune T cells by metabolic inter-ference. Directing such intervention to naïve T cells promises a newconcept of preventing autoimmunity instead of blocking terminal in-flammatory pathways.

MATERIALS AND METHODS

Study designThis study explored functional implications of metabolic and redoxdysregulation in RA T cells and identified metabolic interventionsfor novel anti-inflammatory therapies. Table S1 presents demographic

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characteristics of 181 patients and 164 healthy control subjects. Allpatients fulfilled the diagnostic criteria for RA and were positive forrheumatoid factor and/or anti–CCP (cyclic citrullinated peptide) anti-bodies. Individuals with cancer, uncontrolled medical disease, or anyother inflammatory syndrome were excluded. Healthy individuals didnot have a personal or family history of autoimmune disease. The In-stitutional Review Board approved the study, and written informedconsent was obtained from all participants. Numbers of independentexperiments or individual patients and control donors are defined ineach figure legend. All statistical analyses were verified by L. Tian, De-partment of Health Research and Policy, Stanford University.

Cell preparation, cell culture, and cell transfersCD4 naïve (CD4+CD45RO−) T cells were purified by negative selec-tion with anti–human CD45RO microbeads followed by positive se-lection with anti–human CD4 microbeads (Miltenyi Biotec). Subsetpurity monitored by FACS routinely exceeded 95%. CD4+CD45RO−

T cells (1.0 × 105 per well) were stimulated with CD3/CD28-coatedbeads (Life Technologies; ratio 1:1) and cultured for 7 days in previ-ously described cytokine cocktails to induce T cell lineage commitment(41). Intracellular IFN-g, IL-4, IL-17, and FoxP3 were measured by flowcytometry after incubation with PMA/ionomycin in the presence ofbrefeldin A for 6 hours as described (42). For cell proliferation assays,naïve CD4 T cells were CFSE-labeled and stimulated with anti-CD3/CD28 beads with or without KU-55933, 6-AN, or Tempol (50 mM).T cell subpopulations were analyzed by surface staining for CD45RAand CD62L. For oxidative stress experiments, cells were stimulated(72 hours), pretreated with the ATM inhibitor KU-55933 (30 min), andincubated with increasing doses of H2O2 or menadione (3 mM, 30 min).

Ten million CD45RO− PBMCs were CFSE-labeled and adoptivelytransferred into irradiated (10.0 Gy) NSG mice by intravenous injec-tion. Spleens were collected on days 5, 7, 9, and 14 after transfer andanalyzed by flow cytometry.

Quantitative PCRRNA extraction and RT-PCR were performed as described (4). Primersequences are listed in table S2. Gene expression was normalized to18S ribosomal RNA.

Plasmid constructs and transfectionConstructs encoding shATM were purchased from Addgene (Addgeneplasmid 14581). An Eco RI/Kpn I shATM expression cassette was sub-cloned into the pSuper-gfp/neo vector for reconstructing pSuper-shATM-gfp plasmids (shATM plasmids). Naïve CD4 T cells were transfected usingAmaxa technology (4). Transfection efficiencies were monitored bymeasuring green fluorescent protein (GFP)–positive cells using flow cy-tometry. G6PD-targeting siRNAs were purchased from GE Healthcare(#L-008181-02-0005) and Thermo Fisher Scientific (#4390824).

Western blottingCellular proteins were extracted using kits from Active Motif. Expres-sion levels were examined after standard Western blotting protocols asdescribed (4). Primary antibodies used were as follows: anti-G6PD(#12263S, Cell Signaling Technology), anti-ATM (#NB100-306, GeneTex),and anti-pATM (#GTX70103, Novus Biologicals). Horseradish peroxidase–conjugated anti–GAPDH (glyceraldehyde-3-phosphate dehydrogenase)(#3683, Cell Signaling Technology) or anti–b-actin (#5125, Cell Sig-naling Technology) served as internal controls.

www.Scienc

NADPH measurementsCD4+CD45RO− T cells were stimulated for 72 hours and washed withcold phosphate-buffered saline (PBS), and NADPH levels were mea-sured with NADPH assay kits (Abnova).

Human synovial tissue–NSG chimerasNOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice (The Jackson Laborato-ry) were kept in pathogen-free facilities and used at the age of 10 to12 weeks as previously described (43, 44). Pieces of human synovialtissue free of inflammatory infiltrates were placed into a subcutaneouspocket. After engraftment, mice were injected with 10 million CD45RO−

PBMCs from untreated RA patients or patients with highly active dis-ease. Chimeric mice carrying the same synovial tissue were randomlyassigned to treatment arms: (i) vehicle control (PBS or dimethyl sulfoxide);(ii) treatment with KU-55933 (1 mg/kg per day), BSO (1000 mg/kg perday), or menadione (10 mg/kg per day). All treatments were deliveredby daily intraperitoneal injection over a period of 9 days. At completion,harvested synovial tissues were OCT (optimal cutting temperaturecompound)–embedded (Tissue-Tek, Sakura Finetek) for histology orshock-frozen for RNA extraction. All experiments were carried out inaccordance with guidelines required by the Institutional Animal Careand Use Committee.

Immunohistochemistry stainingHematoxylin-stained sections (5 mm) of explanted synovial tissueswere examined for inflammatory infiltrates, and synovial T cells wereidentified by immunohistochemical staining for human CD3 as de-scribed (43). Sections were analyzed by using an Olympus BX41 mi-croscope and cellSense software.

Statistical analysisAll data are presented as means ± SEM. Data were analyzed using SPSS10.0 software. Statistical significance was assessed by analysis of variance(ANOVA) and unpaired Student’s t test as appropriate. A P value of<0.05 was considered significant.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/8/331/331ra38/DC1Fig. S1. G6PD is regulated by T cell stimulation.Fig. S2. The impact of therapy on G6PD expression in RA T cells.Fig. S3. Intracellular GSH in resting naïve and memory T cells.Fig. S4. G6PD protein expression in RA T cells transfected with gene-specific siRNA.Fig. S5. Reconstitution of NSG mice with human T cells.Fig. S6. Autologous monocytes are required for the expansion of human T cells in NSG hosts.Fig. S7. BSO increases intracellular ROS in RA T cells.Table S1. Demographic and clinical characteristics of the study population.Table S2. List of primers.

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Funding: Supported by NIH (AR042527, AI044142, AI108906, HL058000, AI108891, andAG045779), the Govenar Discovery Fund, the Northern California Arthritis Foundation, and S. Cahill.Author contributions: Z.Y., Y.S., and H.O. planned and performed the experiments. E.L.M. re-cruited patients. Z.Y. and L.T. analyzed the data. C.M.W. and J.J.G. conceived the study, designedthe experiments, analyzed the data, and wrote the manuscript. Competing interests: The authorsdeclare that they have no competing interests.

Submitted 26 October 2015Accepted 9 February 2016Published 23 March 201610.1126/scitranslmed.aad7151

Citation: Z. Yang, Y. Shen, H. Oishi, E. L. Matteson, L. Tian, J. J. Goronzy, C. M. Weyand,Restoring oxidant signaling suppresses proarthritogenic T cell effector functions inrheumatoid arthritis. Sci. Transl. Med. 8, 331ra38 (2016).

eTranslationalMedicine.org 23 March 2016 Vol 8 Issue 331 331ra38 13

rheumatoid arthritisRestoring oxidant signaling suppresses proarthritogenic T cell effector functions in

Zhen Yang, Yi Shen, Hisashi Oishi, Eric L. Matteson, Lu Tian, Jörg J. Goronzy and Cornelia M. Weyand

DOI: 10.1126/scitranslmed.aad7151, 331ra38331ra38.8Sci Transl Med

rebalancing glucose utilization and restoring ROS may help treat rheumatoid arthritis.Thus,What's more, restoring intracellular ROS corrected this abnormal proliferation and suppressed inflammation.

bypassed a cell cycle checkpoint and contributed to hyperproliferation and proinflammatory cell differentiation.in rheumatoid arthritis. They found that a deviation in glycolytic flux led to increased ROS consumption, which

. report that a lack of reactive oxygen species (ROS) could boost proinflammatory T cellset alprocess. Now, Yang cytokines at greater rates than in healthy individuals. Yet little is known about the metabolic changes that fuel this

both proliferating and secreting inflammatory−−In autoimmune diseases, T cells engage their hyperdriveA glucose balancing act

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