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SYSTEM IC LUPUS ERYTHEMATOSUS

Normalization of CD4+ T cell metabolism reverses lupusYiming Yin,1 Seung-Chul Choi,1 Zhiwei Xu,1 Daniel J. Perry,1 Howard Seay,1 Byron P. Croker,1

Eric S. Sobel,2 Todd M. Brusko,1 Laurence Morel1*

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Systemic lupus erythematosus (SLE) is an autoimmune disease in which autoreactive CD4+ T cells play an essen-tial role. CD4+ T cells rely on glycolysis for inflammatory effector functions, but recent studies have shown thatmitochondrial metabolism supports their chronic activation. How these processes contribute to lupus is unclear.We show that both glycolysis and mitochondrial oxidative metabolism are elevated in CD4+ T cells from lupus-prone B6.Sle1.Sle2.Sle3 (TC) mice as compared to non-autoimmune controls. In vitro, both the mitochondrial me-tabolism inhibitor metformin and the glucose metabolism inhibitor 2-deoxy-D-glucose (2DG) reduced interferon-g(IFN-g) production, although at different stages of activation. Metformin also restored the defective interleukin-2(IL-2) production by TC CD4+ T cells. In vivo, treatment of TC mice and other lupus models with a combination ofmetformin and 2DG normalized T cell metabolism and reversed disease biomarkers. Further, CD4+ T cells fromSLE patients also exhibited enhanced glycolysis and mitochondrial metabolism that correlated with their activationstatus, and their excessive IFN-g production was significantly reduced by metformin in vitro. These results suggestthat normalization of T cell metabolism through the dual inhibition of glycolysis and mitochondrial metabolism isa promising therapeutic venue for SLE.

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INTRODUCTION
CD4+ T cells are active mediators of systemic lupus erythematosus(SLE) pathogenesis. Autoreactive nucleosome-specific T cells providehelp to anti-DNA–specific B cells in mice (1) and SLE patients (2).Lupus-prone mice carry large numbers of activated T cells (3), andsignaling defects have been found in SLE CD4+ T cells (4). In addition,SLE patients and lupus-prone mice show an increase in T helper 1(TH1) and TH17 subsets (5), as well as a dysregulated expression ofinterferon-g (IFN-g) and interleukin-2 (IL-2) (6–8).

The activation, proliferation, and differentiation of CD4+ T cellsare tightly regulated by cellular metabolism. Quiescent T cells have alow energy demand and can interchangeably use glucose, lipid, andamino acids to fuel oxidative metabolism (9, 10). Activation throughthe T cell receptor increases both glycolysis and mitochondrial metab-olism (11–14). The up-regulation of glycolysis results from a transcrip-tional reprogramming regulated by c-MYC, HIF1a (hypoxia-induciblefactor 1a), and ERRa (estrogen-related receptor a) (12, 15, 16). The im-portance of glucose metabolism in autoimmunity was demonstratedby the overexpression of the glucose transporter Glut1 in mice that ledto CD4+ T cell hyperactivation, hypergammaglobulinemia, and im-mune complex–mediated nephritis (17). Cellular metabolism alsoregulates effector T cell differentiation and memory cell formation.TH1, TH2, and TH17 cells are highly glycolytic, whereas Foxp3

+ reg-ulatory T (Treg) cells and long-lived memory T cells have high lipidoxidation rates (18, 19).

Several studies have suggested that SLE CD4+ T cells present de-fective mitochondrial functions. Splenocytes from (NZB × NZW)F1(NZB/W) mice showed an enhanced pyruvate oxidation (20). More-over, mitochondrial membrane hyperpolarization was associatedwith an elevated mammalian target of rapamycin (mTOR) activityin CD4+ T cells from SLE patients (21). Accordingly, SLE patients treatedwith rapamycin showed reduced disease manifestations (22). Further-

1Department of Pathology, Immunology, and Laboratory Medicine, University of Florida,Gainesville, FL 32610, USA. 2Division of Rheumatology and Clinical Immunology,Department of Medicine, University of Florida, Gainesville, FL 32610, USA.*Corresponding author. E-mail:[email protected]

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more, CD4+ T cells use oxidative metabolism for chronic activation inresponse to alloantigens (23), and adenosine triphosphate (ATP) syn-thase inhibitors eliminated pathogenic T cells (23, 24). Given theseconflicting results between the preponderance of either glycolysis ormitochondrial metabolism to support the function of activated effectorT cells, themetabolism of SLECD4+ T cells remains poorly understood.

The B6.Sle1.Sle2.Sle3 lupus-prone mouse model (a triple congenicstrain hereafter called TC) contains three NZM2410-derived lupussusceptibility loci—Sle1, Sle2, and Sle3—on a non-autoimmune C57BL/6(B6) background (25).TCmice spontaneously develop symptoms simi-lar to SLE patients, including the production of anti–double-strandedDNA (dsDNA) immunoglobulin G (IgG) and a high penetrance of im-mune complex–mediated fatal glomerulonephritis (GN). The Sle1c2susceptibility locus corresponds to the reduced expression of the Esrrggene, which contributes to CD4+ T cell activation and increased IFN-gsecretion (26). Esrrg controls cellular metabolism by up-regulatingmitochondrial oxidative phosphorylation (OXPHOS) (27, 28). Thisfinding led us to hypothesize that cellular metabolism contributes to lu-pus pathogenesis through T cell activation and that metabolism mod-ulators could be used to reduce or revert disease.We tested this hypothesisin TC mice, which express Sle1c2, and CD4+ T cells from SLE patients.Here, we show that both glycolysis andmitochondrial oxygen consump-tion are elevated in CD4+ T cells from TC mice. This enhanced metabo-lism was observed in naïve T (Tn) cells and was amplified with age andactivation. In vitro, treatment with mitochondrial electron transportchain complex I inhibitor metformin (Met) or glucose metabolism in-hibitor 2-deoxy-D-glucose (2DG) normalized IFN-g production by TCCD4+ T cells. In vivo, treatment of TCmice and other lupus models withthe combination of Met and 2DG normalized T cell metabolism andreversed disease phenotypes. We also demonstrated that CD4+ T cellsfrom SLE patients exhibited both a higher glycolysis and mitochondrialmetabolism as compared to healthy controls (HCs), which correlatedwithT cell activation and subset distribution. Moreover, their excessive IFN-gproduction was significantly reduced by Met in vitro. These results sug-gest that targeting T cell metabolism represents a promising therapeuticstrategy for SLE.

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RESULTS

CD4+ T cells from TC mice exhibited elevatedT cell metabolismTC CD4+ T cells present with several immune abnormalities that aretypical of lupus pathogenesis (25, 29), including T cell hyperactivation(fig. S1AandFig. 5B), accumulation ofCD44+CD62L− effectormemory(Tem) and CD44+CD62L+ central memory T (Tcm) cells (fig. S1B andFig. 5C), as well as increased IFN-g production (fig. S1C and Fig. 3H).To test whether these CD4+ T cell phenotypes were associated withalterations in cellular metabolism, we measured their extracellular acid-ification rate (ECAR), which is primarily attributed to glycolysis, andthe oxygen consumption rate (OCR), which corresponds to OXPHOS.CD4+ T cells from 2-month-old predisease TC mice showed enhancedECAR and OCR compared to age-matched B6 counterparts. Thisdifference in CD4+ T cell metabolism became more pronounced in9-month-old TC mice, which have developed clinical disease (Fig. 1,A toC). CD4+T cells from9-month-oldTCmice also showed a higherspare respiratory capacity (SRC) (Fig. 1D), an indication of cellularenergy reserve that is essential for memory T cell formation and func-tion (30). The enhanced glycolysis of TC CD4+ T cells was confirmedby increased extracellular lactate production (Fig. 1E).Despite increasedglycolysis and OXPHOS, TC CD4+ T cells presented intracellular ATPlevels comparable to B6, both ex vivo and after activation (Fig. 1F). Thisresult suggests that the increased metabolism leads to ATP consump-tion by TC CD4+ T cells to support elevated effector functions. Overall,CD4+ T cells from TC mice present with an enhanced cellular metab-

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olism that precedes disease manifestation and increases as T cells be-come more activated and disease progresses.

Naïve and activated CD4+ T cell subsets have different metabolicprofiles (31). The expansion of Tem cells in TCmice (fig. S1B) could bethe source of the elevated metabolism observed in total CD4+ T cells.Tem cells showed a significantly higher ECAR and, to a lesser extent,OCR than did Tn cells in both B6 and TCmice (Fig. 1, G andH). Thisresult implies that the higher percentage of Tem cells inTCmice contrib-utes to the higher metabolism of total CD4+ T cells. In addition, TC Tn

cells also showed a higher ECAR and OCR than did B6 Tn cells. Todirectly characterize the metabolism of TC CD4+ T cells after activa-tion, we compared the metabolic profiles of B6 and TC-sorted Tn cellsafter stimulation with anti-CD3 and anti-CD28 for 24 hours. In vitrostimulatedTCTn cells exhibited significantly higher ECAR andOCR ascomparedtoB6(Fig.1, I andJ).TheactivityofmTORcomplex1(mTORC1),a sensor for cellular energy (32), was elevated in TC CD4+ T cells, asshown by an increased phosphorylation of ribosomal protein S6 and4E-BP1 (eukaryotic translation initiation factor 4E–binding protein1) and the increased expression of CD98 and CD71 (Fig. 2A and fig.S2). The mTORC1 inhibitor rapamycin reduced glycolysis and mito-chondrial oxygen consumption in anti-CD3/CD28–activated B6 CD4+

T cells (Fig. 2, B and C), linking mTORC1 activity to both glycolysisand mitochondrial metabolism. These results indicate that naïve TCCD4+ T cells exhibit both an increased glycolysis and mitochondrialoxygen consumption ex vivo and in response to in vitro activation.

TC CD4+ T cells showed an elevated expression of glycolytic genesHif1a,Hk2, and Slc16a3 (fig. S3A). On the other hand, the level of Pdk1,

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Fig. 1. CD4+ T cells from TC mice show an enhanced metabolism.(A toD) ECAR (A),OCR (BandC), andSRC (D)measured in total CD4+Tcells from

(F) ATP production by B6 and TC CD4+ T cells stimulatedwith PMA/ionomycin(IO) or anti-CD3/CD28. RQ, relative quantity. (G and H) ECAR (G) and OCR

2- and 9-month-old B6 and TCmice. (B) Representative OCR in 9-month-old B6and TCCD4+ T cells. FCCP, carbonyl cyanidep-trifluoromethoxyphenylhydrazone.(E) Extracellular lactate production from 3-month-old B6 and TC CD4+ T cells.

(H) in Tn and Tem from 9-month-old B6 and TCmice. (I and J) ECAR (I) andOCR (J) in B6 and TC Tn after 24-hour stimulation with anti-CD3/CD28.n = 3 to 6.




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which inhibits pyruvate oxidation, was significantly lower in TC than inB6 CD4+ T cells (fig. S3B). The expression ofCpt1a, a key fatty acid oxi-dation regulator (30), was higher inTCCD4+T cells than inB6 (fig. S3C).Consistently, TC CD4+ T cells showed an enhanced uptake of fattyacids (fig. S3D). Finally, TCCD4+ T cells showed a higher expressionof Gls2 and Odc (fig. S3E), two genes involved in amino acid metab-olism (12). In summary, CD4+T cells from lupus-prone TCmice presentan enhanced metabolism fueled through both glycolysis and mitochon-drial metabolism, with evidence that glucose, fatty acids, and glutaminemay all contribute to the latter.

TC CD4+ T cell dysfunction was normalized by metabolicmodulators in vitroWe tested whether targeting glycolysis and mitochondrial metabolismcould normalize TC CD4+ T cell functions in vitro. As expected, 2DGdose-dependently inhibited CD4+ T cell ECAR (Fig. 3A) but also de-creased OCR (Fig. 3B), presumably by decreasing glucose oxidation.Consistent with its inhibition of mitochondrial complex I (33), Metdose-dependently decreased OCR (Fig. 3C) and increased ECAR ata high concentration (Fig. 3D), likely as an alternative pathway to gen-erate ATP. We examined whether IFN-g production could be modu-lated byMet or 2DG in vitro. TCCD4+T cells producedmore IFN-g thandid B6 T cells after a short stimulationwith PMA (phorbol 12-myristate13-acetate)/ionomycin, which was decreased by treatment with Met(Fig. 3E). The ATPase inhibitor oligomycin and the combination ofcomplex I/III inhibitors rotenone/antimycin A also inhibited IFN-gproduction, indicating that mitochondrial metabolism is required forIFN-g production during early CD4+ T cell activation. 2DG, however,

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had no effect, indicating that IFN-g production does not rely on gly-colysis at that stage. Under TH1 polarizing condition, TC CD4+ Tcells also generatedmore IFN-g than did B6 T cells (Fig. 3F).Met addedat the beginningof the culture dose-dependently decreased IFN-g produc-tion (Fig. 3F), which could be observed as early as day 1 and throughoutthe 3-day culture (Fig. 3G). When added at day 2 of polarization, Methad no effect, whereas 2DG markedly reduced IFN-g production inboth strains (Fig. 3H). These results show that, in both normal and lu-pusmice, IFN-g production by CD4+ T cells depends onmitochondrialmetabolism in the activation phase when it can be reduced by Met,whereas its production during the proliferation phase depends onglycolysis and can be reduced by 2DG.

IL-2 production is defective in lupus T cells (7). Anti-CD3/CD28–stimulated TC CD4+ T cells produced less IL-2 than did B6 T cells,and Met treatment increased IL-2 production in both strains in a dose-dependent manner (Fig. 3, I and J, and fig. S4). In summary, TCCD4+

T cells display excessive IFN-g as well as a defective IL-2 secretion.Both Met and 2DG inhibited their capacity to produce IFN-g, but atdifferent stages of activation, and Met promoted IL-2 production.This resulted in an overall normalization of TC CD4+ T cell func-tions in vitro.

A treatment combining Met and 2DG reversed diseasein TC miceTo target glucose metabolism and mitochondrial metabolism simulta-neously, we treated 7-month-old TC mice with a combination of 2DGand Met (Met + 2DG) in drinking water for 3 months. At that age, TCmice are at the early stage of clinical disease that includes splenomegaly,

Fig. 2. CD4+ T cells fromTCmice show an increasedmTORC1 activity. (A)S6 and 4E-BP1 phosphorylation and expression of CD98 and CD71 in totalCD4+ T cells as well as Tn, Tem, and Tcm subsets from 2-month-oldmice. n= 3to 4. (B and C) ECAR (B) and OCR (C) in B6 CD4+ T cells stimulated with anti-CD3/CD28with orwithout rapamycin (Rapa) (100 nM) for 24 hours. Represent-ative graphs of two independent assays each performed with n = 7 technicalreplicates.





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anti-dsDNA IgGproduction, and accumulation of activatedT (fig. S1) andB cells, as well as splenic plasma cells (25). The Met + 2DG treatment sig-nificantly decreased ECAR (Fig. 4A) and OCR (Fig. 4B) in TC CD4+

T cells to levels similar to that of B6 T cells, indicating that the treat-ment effectively targetedCD4+T cellmetabolism in vivo. B6CD4+T cellswere, however, not affected, suggesting that the treatment is selective tothemoremetabolically active TC cells. A further characterization showedthat the in vivo treatment selectively down-regulatedmetabolism in TCeffector T cells (fig. S5). TheMet + 2DG treatment significantly reducedsplenomegaly in TC mice but had little effect in B6 mice (Fig. 4C). Thetreatment significantly decreased the production of anti-dsDNA IgGand anti-nuclear autoantibodies (ANAs), whereas it increased in un-

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treated TCmice (Fig. 4, D and E). The inhibitory effect of the treatmenton autoantibody production was confirmed with antigens arrays, inwhich four of five treated TC mice showed a profile similar to B6 mice(Fig. 4F). The Met + 2DG treatment also reduced the level of C3 andIgG2a immune complex deposition in TC kidneys (Fig. 4G). Further-more, GNwas less severe in treated TCmice, assessed either by severityrank or by score distribution (Fig. 4, H and I).

Met + 2DG decreased the percentage of total splenic CD4+ T cellsin TCmice (Fig. 5A), as well as activationmeasured as the percentagesof CD69+ expression (Fig. 5B) and Tem (Fig. 5, C and D). Globally, Met +2DG decreased the expression of markers associated with activationand effector functions in TC CD4+ T cells and increased their expression

Fig. 3. MetabolicmodulatorsnormalizedTCCD4+Tcelleffector functionsin vitro. (A to D) ECAR (A and C) and OCR (B and D) in B6 CD4+ T cells stim-

A/rotenone (A&R) (both 0.5 mM), or oligomycin (Olig) (1 mM). (F toH) IFN-g pro-duction in CD4+ T cells cultured under TH1 condition for 3 days withMet [0

ulated with anti-CD3/CD8 for 24 hours in the presence of 2DG or Met. Rep-resentative graphs of three assays each performed with technical replicates.(E) IFN-g production in CD4+ T cells stimulatedwith PMA/ionomycin/GolgiPlugfor 6 hours (Ctrl), in the presence of Met (2 mM), 2DG (5 mM), antimycin

to 5mM in (F) or 1mM in (G)] or 2DG (1mM) added fromday 0 (d0) (F andG)or day 2 (d2) (H). (I and J) IL-2 production in CD4+ T cells stimulatedwith anti-CD3/CD28 for 3 dayswith 0 to 1mM (I) or 1mM (J). (G) Statistical significanceof a two-way analysis of variance (ANOVA) between B6 and TC. n = 3 to 8.





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of the inhibitory molecule CTLA-4 (cytotoxic T lymphocyte–associatedprotein 4) (fig. S6). For most markers, the expression level on CD4+ Tcells from treated TC mice was similar to that of age-matched B6 mice.Both follicular helper (Tfh) and follicular regulatory (Tfr) T cell subsetswere reduced in TC spleens after Met + 2DG treatment (Fig. 5, E toG), which reflects the overall decrease in FO T cells. The expression lev-

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els of PD-1, CXCR5, and BCL6 were drastically reduced by the treat-ment (fig. S6).Within the FO T cells, the treatment expanded Tfr at theexpense of Tfh population (P = 0.04). The high frequency of germinalcenter (GC) B cells in TCmice (34) was also reduced after treatment (Fig.5H). Finally, Met + 2DG restored the impaired production of IL-2 byTC CD4+ T cells (Fig. 5I), and reducedmTORC1 activity (Fig. 5, J toM).

Fig. 4. Treatment with Met + 2DG for 3 months reversed disease in 7- histone 2B. (G) Immune complex deposition in TC glomeruli. Representative
month-old B6 and TC mice. (A and B) ECAR (A) and OCR (B) in CD4+ T cells.(C) Spleenweight (representative spleens on the right of 14 for TC and 5 for B6).(D) Serumanti-dsDNA IgG inTCmice (two-wayANOVA). (E) Initial (I) and terminal(T) serum ANA from TC mice. Representative images of 14 sera per group andANA intensity quantification,with each linked symbol representing amouse. Un-treatedB6miceare shownas control (paired t tests). (F) Autoantibodymicroarrayanalysis of terminal sera (IgG). ssDNA, single-strandedDNA;H2A,histone2A;H2B,
imagesof14kidneyspergroupwithC3and IgG2adeposits (left) andC3 intensity(three to six glomeruli permouse). (H) Renal pathology assessedby severity rank(median and interquartile range, two-tailed Mann-Whitney test) and GN scoredistribution (c2 test). (I) Representative glomeruli (periodic acid–Schiff stain) fromuntreated mice (left) showing large subendothelial deposits (arrows) and fromtreatedmice (right) showing open capillaries and reduced hypercellularity (stars)(scale bars,25 mm). n = 14 TC and 5 B6 mice per group.





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In conclusion, Met + 2DG treatment resulted in a profound and globalreversal of T cell activation in TC mice, whereas it had minimal effectson B6 T cells.

Met + 2DG–treated TC and B6 mice maintained their body weightand blood glucose (fig. S7). The treatment did not result in a generalhumoral suppression because total serum IgMand IgGwere unchangedin TC mice (fig. S8A). The reason for the increased total IgG in treatedB6 mice is not clear at this point. Furthermore, Met + 2DG treatmentdid not impair the humoral response in response to a foreign antigen

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(fig. S8B). Met + 2DG–treated B6mice immunizedwith nitrophenyl –keyhole limpet hemocyanin (NP-KLH) produced the same amount ofanti-NP IgM and IgG1 with no difference in affinity or kinetics. Thenumber of B cells, GC B cells, and plasma cells (fig. S8C), Tem CD4+ Tcells, Tfh cells, and Tfr cells, as well as their expression of effector surfacemarkerswere similar between treated and untreatedmice (fig. S8,D andE). A 1-month treatment also decreased the metabolism of TC CD4+ Tcells (fig. S9, A and B), and there was a trend for the reduction of spleensize (fig. S9C). The treatment reduced the production of anti-dsDNA

Fig. 5. A 3-month Met + 2DG treatment normalized CD4+ T cell phe-notypes in aged TCmice. (A to C) Percentage of total splenic CD4+ T cells

CD4+-gated FACS plots showing the PD-1hiCXCR5hiBCL6+Foxp3−Tfh andPD-1hiCXCR5hiBCL6+Foxp3+ T subsets (G). (H) Frequency of GC CD19+ B

(A), CD69+ (B), and Tem (C) CD4+ T cells in B6 and TC-treated and control(Ctrl) mice. (D) Representative CD4+-gated FACS (fluorescence-activatedcell sorting) plots showing the CD62L+CD44− Tn and CD62−CD44+ Tem sub-sets. (E to G) Frequency of Tfh (E) and Tfr (F) CD4

+ T cells and representative

fr

cells. (I) IL-2 production in CD4+ T cells stimulated with anti-CD3/CD28 for24 hours. (J toM) Effect of treatment on phosphorylation of S6 (pS6) (J) and4E-BP1 (p4E-BP) (K), as well as expression of CD98 (L) and CD71 (M) in total,Tn, and Tem T cells. n = 4 to 14. MFI, mean fluorescence intensity.




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IgG (fig. S9D) and ANA (fig. S9, E and F), the percentage of CD4+ Tcells (fig. S9G), CD4+ T cell activation (fig. S9, H and I), as well as thepercentage of GC B cells (fig. S9J). The short-term treatment also de-creased glomerular IgG2a immune complexes (fig. S7, K and L) andimproved renal pathology (fig. S9, M and N). Thus, 1 month of Met +2DG treatment reversed disease biomarkers in TC mice.

Met + 2DG treatment reversed disease phenotypes in othermouse models of SLEWe tested howMet + 2DG treatment affects disease phenotypes in twoadditionalmodels of SLE. A 1-monthMet + 2DG treatment of 7-month-old NZB/W mice reduced their CD4+ T cell metabolism, although thedifference was significant only for OCR (Fig. 6, A and B). It also de-creased the production of anti-dsDNA IgG (Fig. 6C) and tended to re-duce serum ANA levels, despite an increase in control mice (Fig. 6D).Contrary to TCmice, Met + 2DG significantly decreased total IgG (Fig.6F), and there was a trend in the same direction for IgM (Fig. 6E). Thetotal CD4+ T cell percentage was unaffected by treatment (Fig. 6G), butCD4+T cell activation (Fig. 6H), the percentage of Tem (Fig. 6I), Tfh, andTfr CD4

+ T cells (Fig. 6, J and K) as well as GC B cells (Fig. 6L), and

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mTROC1 activity (Fig. 6, M to P) were all decreased in treated NZB/Wmice. Renal pathology was, however, unchanged (Fig. 6, Q and R).Thus, a 1-month Met + 2DG treatment reversed the immunopheno-types of NZB/W mice.

The impact ofMet + 2DG treatment was also assessed in the chronicgraft-versus-host disease (cGVHD)–induced model of lupus (35). Treat-ment did not affect the induced splenomegaly (fig. S10A), but it reducedthe production of anti-dsDNA IgG (fig. S10B), as well as the percentageofTcm, but notTem,CD4

+Tcells (fig. S10,C andD).Therefore,Met+2DGprevented the induction of T cell–dependent production of autoanti-bodies. Met monotherapy reduced splenomegaly (fig. S10A) and thesize of thememory T cell subset (fig. S10, C andD) in thismodel, but ithad no effect on the induction of anti-dsDNA IgG (fig. S10B). Thissuggested that the combination of the two metabolic inhibitors is re-quired for the effective suppression of autoimmunity.

Met and 2DG showed a synergistic effect in vivoWe evaluated the relative contributions of 2DG and Met to disease re-versal in 7-month-old TCmice treated for 1 month at the same doseused for the combination treatment. 2DG decreased ECAR but had no


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Fig. 6. Met +2DG treatment for 1month reversed immunophenotypesin NZB/W mice. (A and B) ECAR (A) and OCR (B) in splenic CD4+ T cells. (C)

CD4+ T cells (G); CD69+ (H), Tem (I), Tfh (J), and Tfr (K) CD4+ T cells; and GC B

cells (L). (M to P) Effect of treatment of mTORC1 targets: phosphorylation of
Change between the terminal and the initial serum anti-dsDNA IgG for eachmouse. (D) Serum ANA intensity, with each linked symbol representing theinitial and final values for each mouse. (E and F) Change between the ter-minal and initial total serum IgM (E) and IgG (F). (G to L) Frequency of total
S6 (M) and 4E-BP1 (N) and expression of CD98 (O) and CD71 (P) in total CD4+

T cells. (Q and R) Renal pathology assessed by severity rank (median andinterquartile range) (Q) and distribution of GN mesangial (Mn 2–3 and 4)scores (R). n = 4 to 5.




effect onOCR inTCCD4+T cells (fig. S11, A andB).Wedid not observeany effect on spleenweight (fig. S11C), autoantibody production (fig. S11,D to F), CD4+ T frequency (fig. S11G), CD4+ T and B cell activation,or effector subset distribution in these treated mice (fig. S11, H to L).Although 2DG lowered immune complex deposition in the kidneys(fig. S11, M and N), renal pathology was not ameliorated (fig. S11, OandP).Metmonotherapy did not alter either ECARorOCR inTCCD4+

T cells (fig. S12, A and B) or any of the biomarkers that were affectedby the combination treatment (fig. S12, C toN). Therefore, 1-month treat-ment of neither 2DG nor Met alone had an effect on the establishedautoimmune pathology of TCmice. Although it is possible thatmono-therapy for a longer time could have stronger effect on the disease, it isclear that Met + 2DG treatment is superior to Met or 2DG alone,

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which strongly suggests a synergistic effect between 2DG and Met inreversing lupus phenotypes.

CD4+ T cells from SLE patients exhibited elevatedcellular metabolismSLE patients present with a reduced percentage of CD4+CD45RA+

CCR7+ Tn cells and a corresponding increase in CD4+CD45RA−CCR7+

Tcm cells, leading to an altered ratio of Tn/Tcm subsets as comparedto HCs (fig. S13, A to D and F). No significant difference was foundin the percentage of circulating CD4+CD127−/loCD25+ Treg cells (fig. S11,E andG).We compared ECAR,OCR, and SRC inCD4+T cells purifiedfrom peripheral blood of SLE patients and HCs, with and without anti-CD3 and anti-CD28 activation (Fig. 7, A and B). Given that the SLE


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Fig. 7. CD4+ T cells from SLE patients have an enhancedmetabolism.(A andB) Representative ECAR (A) andOCR (B) graphs of humanCD4+ T cells

patients. (F to H) Correlations between Tn percentages and activatedECAR (F) or basal OCR (G) and between Treg percentages and activated

during a mitochondrial stress test. Anti-CD3/CD28 or isotope controls, oligo-mycin, FCCP, and antimycin A/rotenone (Rote + Anti) were added to thecells as indicated. (C to E) ECAR (C), OCR (D), and SRC (E) inHC and SLE CD4+

T cells, with and without anti-CD3/CD28 activation. n = 19 HCs and 20 SLE

ECAR (H). The significance and correlation coefficient of Pearson testsare shown. (I) IFN-g production in TH1-polarized CD4+ T cells with or with-outMet. Two-tailed paired t tests compared the effect of treatment withineach cohort. n = 6.




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patients were treated with at least one immunosuppressive drug (table S1),we tested whether three of the most commonly used drugs could affectCD4+ T cell metabolism. HC CD4+ T cells were treated for 24 hourswith either dexamethasone (Dex), hydroxychloroquine (HCQ), or my-cophenolate mofetil (MMF) at a concentration that did not affect cellviability. Dex inhibited ECAR but not OCR, whereas HCQ and espe-cially MMF reduced both ECAR and OCR (fig. S13, H and I). Despitethe decreased metabolism induced by these treatments, activated CD4+

T cells showed a higher ECAR in SLE than inHC samples (Fig. 7C), andboth resting and activated SLE CD4+ T cells showed a higher OCR thandidHCTcells (Fig. 7D).Notably, anti-CD3/CD28activation induceda rap-id increase in both ECAR and OCR (Fig. 7, A and B), suggesting that bothpathways are important for the early activation of human CD4+ T cells.Moreover, both resting and activated SLE CD4+ T cells have a signifi-cantly higher SRC (Fig. 7, B and E). These metabolic parameters werecorrelated with the distribution of circulating CD4+ T cell subsets. Thepercentage of Tn was inversely correlated with both activated ECAR(Fig. 7F) and basal OCR (Fig. 7G) levels, whereas that of Treg was pos-itively correlated with activated ECAR (Fig. 7H). These results stronglysuggest that, as shown in the mouse, cellular metabolism regulates hu-man CD4+ T cell activation and effector functions. Further, our resultsshow a strong association between a dysregulated homeostasis in SLECD4+ T cells and an elevated cellular metabolism. Consistent with ourfindings in mice (Fig. 3), SLE CD4+ T cells produced more IFN-g thandid HCs after TH1 induction (Fig. 7I). Met decreased IFN-g productionfrom SLE and HC CD4+ T cells to comparable levels between the twocohorts (Fig. 7I). Overall, these results showed that Met treatment nor-malized the in vitro IFN-g production by SLE CD4+ T cells.

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DISCUSSION
CD4+ T cells up-regulate glycolysis after activation, a phenomenon verysimilar to theWarburg effect described in cancer cells (36). Glycolysisis critical for T cell effector functions, and increased glycolysis leads toautoimmunity (13, 17).More recently, the field has gained a better ap-preciation for mitochondrial oxidativemetabolism inmediating T cellactivation and proliferation (12, 14, 23). Here, we showed that CD4+ Tcells from the TC lupus model as well as from SLE patients exhibitelevated glycolysis and mitochondrial oxidative metabolism as com-pared to non-autoimmune controls, both ex vivo and after in vitroactivation. We also showed that the enhanced oxidative metabolismis attributed to a combination of higher glucose, fatty acid, and gluta-mine oxidation. Moreover, the enhancedmetabolism in CD4+ T cells isassociated with an increasedmTORC1 activity. These findings led us tohypothesize that lupus could be treated with metabolic modulators thattarget both glycolysis and mitochondrial metabolism. We found thattreating TC mice with a combination of Met and 2DG normalized Tcell metabolism and reversed disease phenotypes, including T cell acti-vation, autoantibody production, and renal disease. The combinationtreatment was also effective on the NZB/W spontaneous lupus modeland the cGVHD-induced model. This result suggests that dysregulatedT cell metabolism may be underlying SLE pathogenesis and that T cellmetabolism can be a therapeutic target.

Metabolic modulators have been evaluated for a number of dis-eases. Rapamycin reduced disease activity in MRL/lpr mice (37) andin SLE patients (22). Treatment with N-acetylcysteine, a precursor ofglutathione that blocks mTOR, improved disease outcomes in NZB/W

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mice (38) and SLE patients (39). A clinically approved inhibitor of gly-cosphingolipid biosynthesis, N-butyldeoxynojirimycin, normalizedthe functions of CD4+ T cells from SLE patients (40). Met is common-ly used to treat type 2 diabetes, and 2DGhas been tested to treat cancer(41) and Alzheimer’s disease (42). Either 2DG orMet alone inhibited thedevelopment of experimental autoimmune encephalomyelitis, a mousemodel of multiple sclerosis (13, 43). 3PO, a small-molecule inhibitorof PFKFB3, an enzyme that controls a rate-limiting step of glycolysis, pre-vented the development of T cell–mediated delayed hypersensitivity andimiquimod-induced psoriasis in the mouse (44). To the contrary, weshowed in this study that a dual inhibition of glycolysis and mitochon-drial metabolism synergizes for the reversal of lupus in the TC model,which highlighted the complexity of this disease. A synergistic effect ofMet and 2DG has also been shown in cancer models (45). Because re-cent studies have revealed the importance of glycolysis and mitochon-drial metabolism in controlling T cell effector functions, dual inhibitionof glycolysis and mitochondrial metabolismmay have broader applica-tions to other immune-mediated diseases besides lupus.

Our study provides possible mechanisms by which Met and 2DGreversed immunophenotypes in vivo.Met regulates cellularmetabolismthrough complex mechanisms (46), including the inhibition of themitochondrial respiratory chain complex I (33). Here, the Met + 2DGtreatment decreased T cell mitochondrial oxygen consumption as wellas mTORC1 signaling in both the TC and NZB/W models. Therefore,inhibiting mitochondrial metabolism and antagonizing mTOR signalingare the likely mechanisms by which Met normalized TC CD4+ T cellmetabolism in vivo. We also observed a decrease in glycolysis after theMet + 2DG treatment, which is likely the mechanism by which 2DGnormalized TC CD4+ T cell metabolism in vivo.

In summary, we have identified defective CD4+ T cell metabolismas a therapeutic target in bothmurine and human SLE. Besides CD4+

T cells, Met + 2DG may also target directly other immune cell typesin vivo. Glucose metabolism is essential for B cell functions (47). Bothglycolysis and mitochondrial metabolism are important for the activa-tion and maturation of dendritic cells (48, 49), which can indirectly af-fect T cells. It will be important to find out whether lupus mice haveabnormalmetabolism in other immune cell compartments andwheth-er the metabolism of those cells is altered after Met + 2DG treatmentin vivo. Although Met + 2DG was effective at reversing disease pheno-types, whereas the monotherapies at the same doses were not, it ispossible that different doses or a longer duration would be effectivefor eitherMet or 2DG alone.We have also not yet investigated whetherthe treatment needs to be maintained to prevent flares or whether itresets the immune system for an extended period of time. Another lim-itation of the present study is that 2DG blocks the entire glucose meta-bolic pathway and does not discriminate between the contribution ofglycolysis and glucose oxidation. Finally, a comprehensive evaluationof the effect of Met, with and without glucose inhibitors, on thefunctions of human CD4+ T cells should be performed.

MATERIALS AND METHODS

Study designThe objective of the study was to define the metabolism of mouse andhuman SLE CD4+ T cells and to explore potential metabolism-basedtherapeutic strategies. Themetabolism ofmouseCD4+ T cells was firstevaluated ex vivo and after in vitro stimulation in the presence ofmetabolic





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inhibitors. Six- to 7-month-old anti-dsDNA IgG-positive lupusmice andage-matched B6 controls were randomized to treatment and control groups.Treatment was performed for 1 or 3 months withMet (3 mg/ml) or 2DG(5 mg/ml), or a combination of the two, dissolved in drinking water. Age-matched control mice received plain drinking water. Peripheral bloodwas collected biweekly to analyze autoantibody production; bodyweight and blood sugar levels were monitored weekly and biweekly, re-spectively. At the end of the treatment, spleens were collected for flowcytometry and metabolic analysis of CD4+ T cells, and kidneys were eval-uated for renal pathology in a blinded fashion. All experiments wereconducted according to protocols approved by theUniversity of Florida(UF) Institutional Animal Care and Use Committee. For human subjects,at least 20 ml of peripheral blood was obtained after signed informed con-sent in accordance with UF Institutional Review Board. Female SLE pa-tients fulfilled at least four of the revised SLE criteria of the AmericanCollegeofRheumatology.Healthy female volunteerswithno familyhistoryof autoimmune disease served as age- and ethnicity-matched HCs. Allpatients were treated with at least one medication, none of them with abiologic treatment. The demographics and treatment regimens of thepatients and HCs are summarized in table S1.

MiceTC mice have been described previously (18). C57BL/6J (B6), B6(C)-H2-Ab1bm12/KhEgJ, and (NZB×NZW)F1micewere purchased fromThe Jackson Laboratory. Only female mice were used in this study.cGVHD was induced as previously described (29).

Metabolic measurementsSplenocyte suspensions were enriched for CD4+ T cells by negativeselection with magnetic beads (Miltenyi). Naïve (Tn, CD4

+CD44−

CD62L+) and effector memory (Tem, CD4+CD44+CD62L−) subsets

were sortedwith a FACSAria cell sorter (BDBiosciences). SortedmouseTn were activated with plate-bound anti-CD3e (145-2C11, 2 mg/ml)and soluble anti-CD28 (37.51, 1 mg/ml) for 24 hours. ECAR andOCR were measured using either an XFe24 or an XFe96 ExtracellularFlux Analyzers under mitochondrial stress test conditions (Seahorse).Assay bufferwasmade of nonbufferedRPMImedium (Sigma) supple-mented with 2.5 mMdextrose, 2 mM glutamine, and 1 mM sodium pyr-uvate. Baseline ECAR andOCRvalueswere averaged between technicalreplicates for these first three successive time intervals. Extracellularlactate production was measured using L-Lactate Assay Kit (Abcam).Intracellular ATPwasmeasured using the ATPDeterminationKit (LifeTechnologies). Fatty acid uptake by CD4+ T cells was measured by flowcytometry with the Bodipy dye (Life Technologies).

Gene expression analysisSYBRGreen (Applied Biosystems)–based qPCR (quantitative polymer-ase chain reaction)was performed on complementaryDNA frombead-enrichedCD4+ T cells with primer sequences shown in table S2.mRNAlevels were expressed as relative quantities to Ppia (cyclophilin A).

Flow cytometryThe immunophenotypes of human CD4+ T cells were determined withantibody panels emulated by the Human Immunophenotyping Con-sortium (table S4). The other antibodies used in this study are listed intable S3. Cytokine production was analyzed in cells treated with the leuko-cyte activation cocktail (BDBiosciences) and the Fixation/Permeabilizationkit (eBioscience).

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Cell cultureMouse splenic CD4+ T cells were stimulated by anti-CD3e and anti-CD28 for 3 days and IL-12 (10 ng/ml) for TH1 differentiation. IL-2 wasmeasured in CD4+ T cells stimulated by anti-CD3e and anti-CD28.Human CD4+ T cells were enriched from peripheral blood using theRosetteSep Enrichment Cocktail (StemCell Technologies). For TH1polarization, cells were stimulated in complete RPMI with beads coatedwith anti-CD3 and anti-CD28 (Dynabeads Human T-Activator, LifeTechnologies) for 6 days with IL-12 (10 ng/ml), IL-2 (20 U), and anti–IL-4 (1 mg/ml). In some assays, Dex, HCQ, or MMF (1 mM, Sigma) wasadded, and ECAR and OCR were measured 24 hours later.

Antibody measurementSerum anti-dsDNA, anti-chromatin IgG, and ANA were measured aspreviously described (25). Autoantibody microarray analysis was per-formed at the Microarray Core Facility in University of Texas South-western Medical Center.

Renal pathologyGN was scored, and the glomerular deposition of C3 and IgG2a im-mune complexes was evaluated on frozen kidney sections as previ-ously described (50).

Statistical analysisStatistical analyses were performed using the GraphPad Prism 6.0software. Unless indicated, datawere normally distributed, graphs showmeans and SEM for each group, and results were compared with two-tailed t tests with a level of significance of P < 0.05. Bonferroni correc-tions were applied for multiple comparisons. Each in vitro experimentwas performed at least twice with reproducible results.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/7/274/274ra18/DC1Fig. S1. TC mice showed enhanced CD4+ T cell activation.Fig. S2. Representative FACS plots of ex vivo CD4+-gated T cells from B6 (black) and TC (red)mice for pS6, p4E-PB1, CD98, and CD71.Fig. S3. CD4+ T cells from TC mice showed an altered expression of metabolic genes.Fig. S4. IL-2 gating in CD4+ T cells.Fig. S5. The Met + 2DG treatment normalized the metabolism of effector CD4+ T cells.Fig. S6. A 3-month Met + 2DG in vivo treatment resulted in a global normalization of TC CD4+ Tcell effector phenotypes.Fig. S7. The Met + 2DG treatment did not have adverse effects on body weight and blood sugar.Fig. S8. Met + 2DG treatment did not affect normal humoral response.Fig. S9. One-month Met + 2DG treatment reduced disease severity in TC mice.Fig. S10. Met + 2DG treatment prevented autoantibody production in the cGVHD model.Fig. S11. 2DG treatment failed to reverse immunophenotypes in TC mice.Fig. S12. Met treatment failed to reverse immunophenotypes in TC mice.Fig. S13. CD4+ T cell immunophenotypes in SLE patients.Table S1. Human subject demographics and disease parameters.Table S2. Primers for qPCR assays.Table S3. Antibodies used in this study.Table S4. Source data (Excel file).

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Acknowledgments: We thank C. Mathews for helpful suggestions and N. Kanda and L. Zeumerfor expert technical help. Funding: Supported by NIH grants R01 AI045050 and ALR-TIL0000075018 (to L.M.), P01 AI42288 (to T.M.B.), and a JDRF grant (CDA 2-2012-280) (to T.M.B.).Author contributions: Y.Y., T.M.B., and L.M. conceived and designed the experiments; Y.Y., S.-C.C., Z.X., D.J.P., and H.S. performed the experiments; Y.Y., S.-C.C., D.J.P., B.P.C., T.M.B., and L.M. ana-lyzed data; E.S.S. contributed materials/samples; and Y.Y. and L.M. wrote the paper. Competinginterests: Y.Y. and L.M. have filed a patent application entitled “Treatment of lupus using meta-

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bolic modulators” (U.S. Provisional Application No.: 62/107,160). The other authors declare nocompeting interests.

Submitted 14 October 2014Accepted 6 January 2015Published 11 February 201510.1126/scitranslmed.aaa0835

Citation: Y. Yin, S.-C. Choi, Z. Xu, D. J. Perry, H. Seay, B. P. Croker, E. S. Sobel, T. M. Brusko,L. Morel, Normalization of CD4+ T cell metabolism reverses lupus. Sci. Transl. Med. 7,274ra18 (2015).



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T cell metabolism reverses lupus+Normalization of CD4

and Laurence MorelYiming Yin, Seung-Chul Choi, Zhiwei Xu, Daniel J. Perry, Howard Seay, Byron P. Croker, Eric S. Sobel, Todd M. Brusko

DOI: 10.1126/scitranslmed.aaa0835, 274ra18274ra18.7Sci Transl Med

treating SLE.suggest that inhibiting both glycolysis and mitochondrial metabolism could be a new therapeutic strategy for

datacell metabolism and decreased markers of SLE in animal models as well as in cells from SLE patients. These normalized T−−2-deoxy-d-glucose (2DG) and metformin−−more, inhibitors of these pathways currently in the clinic

are elevated in cells from SLE patients as well as in mouse models of disease. What's−−oxidative metabolism glycolysis and mitochondrial−− report that two metabolic pathwayset al.cells contributes to disease. Now, Yin

T cells are critical to SLE pathogenesis, but it has remained unclear if metabolism in these+healthy tissues. CD4Systemic lupus erythematosus (SLE) is an autoimmune disease where the immune system attacks normal,

Normalizing immune cell metabolism treats lupus

ARTICLE TOOLS http://stm.sciencemag.org/content/7/274/274ra18

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