of April 10, 2019.This information is current as

FunctionCells that Modulates Their Suppressive

Regulatory T−Program in Human CD45RA Regulated Cellular−BκTNF Activates a NF-

GoldsteinIlan Bank, Yoel Kloog, Gideon Rechavi and ItamarYackov Berkun, Shomron Ben-Horin, Ninette Amariglio, Meital Nagar, Jasmine Jacob-Hirsch, Helly Vernitsky,

http://www.jimmunol.org/content/184/7/3570doi: 10.4049/jimmunol.0902070February 2010;

2010; 184:3570-3581; Prepublished online 24J Immunol 

MaterialSupplementary

0.DC1http://www.jimmunol.org/content/suppl/2010/02/22/jimmunol.090207

Referenceshttp://www.jimmunol.org/content/184/7/3570.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2010 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Journal of Immunology

TNF Activates a NF-kB–Regulated Cellular Program inHuman CD45RA– Regulatory T Cells that Modulates TheirSuppressive Function

Meital Nagar,*,† Jasmine Jacob-Hirsch,* Helly Vernitsky,*,† Yackov Berkun,‡

Shomron Ben-Horin,x Ninette Amariglio,* Ilan Bank,x Yoel Kloog,† Gideon Rechavi,* and

Itamar Goldstein*,x

Emerging data suggest that regulatory T cell (Treg) dysfunction and consequent breakdown of immunological self-tolerance in

autoimmunity can bemediated by factors that are not Treg-intrinsic (e.g., cytokines). Indeed, recent studies show that in rheumatoid

arthritis the proinflammatory cytokine TNF reduces the suppressive function of Tregs, whereas in vivo TNF blockade restores this

function and accordingly self-tolerance. However, until now a coherent mechanism by which TNF regulates the Treg has not been

described. In this paper, we show that TNF induces preferential and significant activation of the canonical NF-kB pathway in human

Tregs as compared with CD25– conventional T cells. Furthermore, TNF induced primarily in CD45RA– Tregs a transcription

program highly enriched for typical NF-kB target genes, such as the cytokines lymphotoxin-a and TNF, the TNFR superfamily

members FAS, 4-1BB, and OX-40, various antiapoptotic genes, and other important immune-response genes. FACS analysis

revealed that TNF also induced upregulation of cell surface expression of 4-1BB and OX40 specifically in CD45RA–FOXP3+

Tregs. In contrast, TNF had only a minimal effect on the Treg’s core transcriptional signature or on the intracellular levels of the

FOXP3 protein in Tregs. Importantly, TNF treatment modulated the capacity of Tregs to suppress the proliferation and IFN-g

secretion by conventional T cells, an effect that was fully reversed by cotreatment with anti-TNFR2 mAbs. Our findings thus

provide new mechanistic insight into the role of TNF and TNFR2 in the pathogenesis of autoimmunity. The Journal of Immu-

nology, 2010, 184: 3570–3581.

Tumor necrosis factor (TNF-a) is a prototype member ofthe TNF superfamily (TNFSF) of ligands. It binds thecorresponding TNFR superfamily (TNFRSF) members

TNFR1 (TNFRSF1A; p55) and TNFR2 (TNFRSF1B; p75). TNF isgenerated and expressed by many types of cells, including lym-phocytes, and can induce a plethora of immune responses (1).Several lines of evidence suggest that TNF has a central role in thepathogenesis of a variety of human inflammatory disorders (2).Moreover, TNF blockade has a notable therapeutic efficacy ina number of T cell-dependent human autoimmune disorders, suchas inflammatory bowel disease, rheumatoid arthritis, juvenile id-

iopathic arthritis (JIA), ankylosing spondylitis, and psoriatic ar-thritis (3). Nevertheless, little is known at the molecular level

about the specific effects of TNF on the various T cell subsets, and

consequently the fine details of the mechanism of action of TNF

blockade, in vivo, remain somewhat obscure.Variousstudies inbothmiceandhumanssuggest thatTNFblockade

has the potential to augment regulatory T cell (Treg) numbers and

function. For example, in RA patients treated with anti-TNF mAbs

there is an increase in the quantity of circulatingCD25HITregs aswell

as restoration of their partly impaired suppressive function (4, 5).

Moreover, TNF blockade during the in vitro activation and expansion

of unselected human T cells results in augmented expansion of

FOXP3+CD4+ T cells with a regulatory phenotype (6, 7).It should be noted that studies in patients with various auto-

immune disorders and mice models show that Treg-type cells are

present at considerable numbers at the actual site of inflammation

(8, 9). Thus, a major challenge is to define the Treg extrinsic

factors that facilitate tissue inflammation even when Tregs are

present in abundance. In this regard, there is controversy as to

whether TNF—a cytokine abundant at sites of inflammation—can

interfere with the suppressive function of the Treg (4, 8–13).Tregs as opposed to CD25– conventional T cells (Tcons) consti-

tutively express high levels of TNFR2, but both subsets do not ex-

press TNFR1 (4, 12). Whereas the downstream molecular events

initiated followingTNFR1 ligation have been studied in great detail,

there is significantly less information about signal transduction via

TNFR2 that, as opposed to TNFR1, does not contain a death domain

(14, 15). Binding of TNF to TNFR1 activates at least three distinct

cellular pathways: IKK/NF-kB, JNK/c-Jun, and the proapoptotic

caspases 8 and 3 (1). At present, the specific cellular program in-

duced by TNFR2 stimulation in Tregs is practically unknown.

*Sheba Cancer Research Center, ‡Division of Pediatrics, and xDepartment of Med-icine, The Chaim Sheba Medical Center, Tel Aviv University, Sackler Faculty ofMedicine, Tel Hashomer; and †The George S. Wise Faculty of Life Sciences, TelAviv University, Tel Aviv, Israel

Received for publication June 29, 2009. Accepted for publication January 26, 2010.

This work was supported in part by an award from the Flight Attendant MedicalResearch Institute.

The microarray data presented in this article have been submitted to the NationalCenter for Biotechnology Information Gene Expression Omnibus under accessionnumber GSE18893.

Address correspondence and reprint requests to Dr. Itamar Goldstein, The ShebaCancer Research Center, Chaim Sheba Medical Center, Tel Hashomer 52621, Israel.E-mail address: itamar.goldstein@sheba.health.gov.il

The online version of this article contains supplemental material.

Abbreviations used in this paper: acTreg, activated regulatory T cell; DI, divisionindex; EASE, Expression Analysis Systematic Explorer; IPA, Ingenuity PathwaysAnalysis, JIA, juvenile idiopathic arthritis; MB, microbead; MFI, mean fluorescenceintensity; mTNF, membrane-bound TNF; nrTreg, naıve/resting regulatory T cell;PMA/Ion, PMA and ionomycin; rhIL-2, recombinant human IL-2; RTQPCR, real-time quantitative PCR; SF, synovial fluid; Tcon, conventional T cell; TNFRSF, TNFreceptor superfamily; TNFSF, TNF superfamily; Treg, regulatory T cell.

Copyright� 2010 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/10/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902070

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In this study, using various complementary methods, such as geneexpression microarrays, PCR-based assays, analysis of protein ex-pression and/or phosphorylation by FACS, and other relevant assaysto test suppressive functions, we now identify the cellular programthat mediates the inhibitory effect of TNF on the Treg suppressivefunction. Our data show a central role for activation of the canonicalNF-kB pathway—primarily in CD45RA– Tregs—that consequentlyinduces a distinctive proinflammatory cellular program.

Materials and MethodsHuman subjects

Blood samples were obtained from healthy adult blood donors. In-flammatory synovial fluid (SF) samples were obtained from JIA patients.Signed written informed consent or parental/guardian permission wasobtained from all of the study participants as appropriate. The InstitutionalEthics Committee at the Chaim Sheba Medical Center approved the study.

Reagents

Recombinant human IL-2 (rhIL-2) was obtained from Boeheringer-Man-nheim (Mannheim, Germany). Recombinant human TNF-a was purchasedfrom Biosource Invitrogen (Carlsbad, CA). The humanized chimeric anti-TNF mAb, Infliximab, was from Centocor (Horsham, PA). The mouse anti-hFOXP3 mAb 236A/E7 and Treg staining kit were obtained from eBio-science (SanDiego,CA).Thefluorochrome-conjugatedmousemAbs againsthuman CD4, CD134, CD137, CD95, CD69, CD25, CD45RA, CD120A,CD27, and CD127were all fromBDPharmingen (San Jose, CA). ThemousemAbs against TNFR2 (CD120B) were from R&D Systems (Minneapolis,MN). The fluorochrome-conjugated rabbit mAbs to phospho-IkB were fromCell Signaling Technology (Danvers,MA), and themousemAbs to phospho-NF–kB-p65 were from BD Biosciences (San Jose, CA).

FACS analysis

Cell samples were analyzed either on a FACSCalibur using the Cellquestsoftware or on a digital flow cytometer, FACSCanto II or FACSAria, usingthe FACSDiva 6.1.2 software (both from BD Biosciences). FACS dataanalysis was done with the FlowJo 7.2.5 software (Tree Star, Ashland, OR).Immunostaining of cell surface markers was carried out as previously de-scribed (16). For detection of FOXP3, the cells were fixed/permeabilizedusing the eBioscience FOXP3 staining buffer set, as previously described(17).

Treg and Tcon cell isolation

PBMCs and SF lymphocytes were obtained by density gradient centrifu-gation on Histopaque 1077 (Sigma-Aldrich, St. Louis, MO). Highly pureCD4+CD25HI Tregs were obtained as previously described (18). Briefly,CD4+ T cells were isolated from PBMCs by positive selection with CD4microbeads (MBs) from Miltenyi Biotec (Bergisch Gladbach, Germany)and further purified into CD4+CD25HI and CD25– T cells using a FACS-Aria cell sorter system (Fig. 2A). Less pure CD25++ and CD25– T cellpopulations were isolated using positive selection with CD25 MBs (Mil-tenyi Biotec). To obtain a population enriched for CD25++ T cells, wemodified the protocol recommended by the manufacturer and used 7.5 ml(instead of 10 ml) CD25 MBs per 1 3 107 PBMCs.

T cell cultures and activation

The cells were cultured in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (all from In-vitrogen Life Technologies, Carlsbad, CA) and maintained at 37˚C in a hu-midified 5% CO2 incubator. Unless otherwise specified, the cultures weresupplemented with rhIL-2 (100 IU/ml). Purified T cells were typically platedinto 24-well plates (Costar; Corning, Lowell, MA) at 2 3 106 cells per well.For TNF treatment, the various purified T cell populations were incubatedfor the indicated time with medium supplemented with 50 ng/ml TNF (orcontrol PBS). T cell activation and expansion were generally induced by im-mobilized plastic-boundOKT3 (2.5mg/ml), soluble anti-hCD28mAbs (1mg/ml), and rhIL-2 (100 IU/ml). Cell cultures were supplemented with freshcomplete medium plus 100 IU/ml rhIL-2 every 72 h until the end of the ex-periment.

Suppression assay and CFSE dilution

Tregs, isolated from PBMCs, were tested for their ability to suppress theproliferation of autologous CD4+CD25– Tcons, as previously described (6).

Briefly, CFSE-labeled CD25– T cells at 1 3 105 cells per well were platedinto 96-well plates (Costar) and activated by plate-bound immobilizedOKT3 (2.5 mg/ml) in the presence of graded amounts of CD25++ T cells.Selected cultures, as indicated in the text, were supplemented with TNF (50ng/ml), rhIL-2 (100 IU/ml), anti-TNFR2 mAbs (10 mg/ml), or Infliximab(50 mg/ml) at time 0 and 72 h later. As indicated, we also activated the Treg/Tcon cocultures with K562 artificial APCs, engineered to stably express theFcR CD32 alone (KT32), or with 4.1BBL (KT32/4.1BBL), kindly providedby Carl H. June (University of Pennsylvania Cancer Center, Philadelphia,PA). To induce potent TCR activation, the KT32 cells were preincubatedwith OKT3 and anti-CD28 mAbs at a concentration of 1 mg/ml for 10 minand then irradiated with 10,000 cGy. Regardless of the activation protocol, 5d later the T cells were harvested and analyzed by FACS. CD4+ Tconproliferation was determined using the proliferation platform of FlowJo7.2.5 to calculate the division index (DI) and draw a “fit model” of gen-erations onto the CFSE dilution histograms. The DI reflects the averagenumber of cell divisions that the cells underwent and is considered a goodobjective value to compare the rate of proliferation from sample to sample.

IFN-g analysis

To assess the capacity of Tregs to suppress the production of IFN-g byTcons, 100 ml supernatant samples were collected (on day 3) from thevarious Treg/Tcon cocultures. The supernatants were then analyzed by thehuman IFN-g cytoset ELISA kit (Biosource) according to the manu-facturer’s instructions. For intracellular cytokine detection, the T cellswere activated with 20 ng/ml PMA and 0.8 mM ionomycin (Sigma-Al-drich) in the presence of monensin (GolgiStop from BD Biosciences) ata concentration of 2 mg/ml for 5 h. Subsequently, the cells were fixed/permeabilized using the eBioscience FOXP3 staining buffer set and thenstained with anti–IFN-g–PE, CD4–FITC, and FOXP3–allophycocyaninmAbs (all from eBioscience), as previously described (17).

Isolation of RNA and real-time quantitative PCR

TotalRNAwas isolatedusing theRNeasymini kit (Qiagen,Valencia,CA), andcDNAwas synthesized using the RETROscript kit (Ambion,Austin, TX). Thereal-time quantitative PCR (RTQPCR) for mRNA levels of FOXP3, STAT1,NFKB2, TRAF3, LTA, and the housekeeping gene HPRT1 was carried outusing TaqMan gene expression kits supplied with predesigned optimizedprimers and probes (Applied Biosystems, Foster City, CA). The samples wererun according to the manufacturer’s instructions, in triplicates, using an ABIPrism 7900HT Sequence Detector (Applied Biosystems). The relative geneexpression was normalized to HPRT1 expression, and fold change was cal-culated using the 22DDCT comparative method.

Gene expression arrays, gene enrichment, and functionalanalyses

All of the experiments were performed using Affymetrix HU GENE 1.0 STArray (Affymetrix, Santa Clara, CA). Sample processing was performedaccording to the Affymetrix WT protocol (www.affymetrix.com/support).The gene-level log-scale robust multiarray analysis sketch algorithm(Affymetrix Expression Console and Partek Genomics Suite 6.2) was usedfor crude data analysis. Genes were filtered and analyzed using fold changecalculations and unsupervised hierarchical cluster analysis (Spotfire De-cisionSite for Functional Genomics). Further processing including func-tional gene networks analysis and gene set enrichment analysis wereperformed using DAVID Bioinformatics Resources (http://david.abcc.ncifcrf.gov/tools.jsp) or the Ingenuity Pathways Analysis (IPA) platform orbased on publication data sources as indicated. The primary microarraysdata from this research have been deposited in the NCBI Gene ExpressionOmnibus data repository under accession number GSE18893 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE18893).

Statistical analysis

The DAVID Expression Analysis Systematic Explorer (EASE) score (amodified Fisher exact p value) was used to determine gene-cluster over-representation, and p , 0.01 was considered significant. The p values forthe data in Fig. 5 were calculated by the paired Student t test with loga-rithmic transformation, and p , 0.05 was considered significant.

ResultsCD45RA–FOXP3+ Tregs express higher levels of TNFR2compared with other T cell subsets

Previous studies (4, 19) show that at homeostasis blood-derived CD4+

FOXP3+ T cells express as a whole higher levels of TNFR2/CD120B

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compared with CD25–FOXP3– T cells. It is also postulated that thehuman FOXP3+ Treg lineage is subdivided into two subsets based onCD45RA expression (20, 21). A recent study further suggests sub-dividing circulating human T cells into six subsets (22), based onCD45RA, CD25, and/or FOXP3 expression. As shown in Fig. 1A (leftpanel), this classification includes CD25+++/FOXP3HI/CD45RA– ac-tivatedTregs (acTregs) that are immediate effectors of suppression andCD25++/FOXP3lo/CD45RA+ naıve/resting Tregs (nrTregs) (R2 andR1, respectively). We now show that the highest expression ofCD120B is detected onCD45RA–CD25+++ acTregs (R2), followed byCD25++CD45RA+ nrTregs (R1) and CD4+CD25++CD45RA– (FOX-P3lo) recently activated Tcons (R3). By contrast, FOXP3–CD45RA–

Tcons (R4 and R5) express much lower levels of CD120B, and naiveCD25– CD45RA+ Tcons (R6) do not show meaningful expression ofthis receptor (Fig. 1A, right panel, numbers correspond to meanfluorescence intensity [MFI] of CD120B staining).Next, we purified by a FACSAria cell sorter only the CD4+

T cells with the top ∼5% of CD120B expression and found thatthe purified TNFR2HI population mostly contained FOXP3+

T cells with both low and high FOXP3 expression (Fig. 1B). Wealso found that freshly isolated circulating CD8+ T cells, regard-less of CD25 coexpression, rarely express CD120B. As previouslypublished (4), we also did not detect TNFR1/CD120A expressionon human CD4+ T cells (data not shown).To address the activation-dependent upregulation of CD120B,

we activated in vitro CD25+++ Tregs and naive CD25–CD4+

T cells. As shown in Fig. 1C, ∼24 h after ex vivo activation theTcons started to upregulate TNFR2 expression. Yet, throughoutthe culture period, CD120B levels remained significantly lower inactivated Tcons compared with those in the acTregs. No mean-ingful cell surface expression of CD120A was detected in bothT cell fractions following activation (data not shown).

To analyze the expression of CD120B on in vivo activated humanTregs versus Tcons, we isolated T cells from inflamed SF of JIApatients (Fig. 1D, left panel). Of note, in the patient samples tested(n. 5), nearly all of the synovial FOXP3+ T cells were CD45RA–

and thus were akin to acTregs. The expression of CD120B on thein vivo activated synovial CD4+CD25– Tcons was indeed upre-gulated compared with that on the isotype staining and circulatingTcons. However, CD120B expression on synovial CD45RA–

FOXP3+ was still significantly higher compared with that on theirFOXP3– Tcon counterparts (Fig. 1D, right panel).Thus, TNFR2—the main TNFR in human T cells—is generally

expressed at higher levels in FOXP3+ acTregs compared withthose in Tcons, in vivo at homeostasis and during inflammatoryresponses as well as upon ex vivo activation.

TNF induces preferential upregulation of multiple genetranscripts in acTregs

Because CD45RA– acTregs distinctively express high levels ofTNFR2 and, moreover, they are postulated to mediate the im-mediate effector–suppressor function (22), it was of interest tostudy the unique effects of TNF on acTregs. To obtain pure ac-Tregs, we isolated by FACSAria the CD4+CD25+++ T cells withthe top 3% of CD25 expression. This latter CD4+CD25HI pop-ulation was exceedingly enriched for FOXP3+ Tregs (.90%) thatwere also largely CD45RA–/CD120BHI (Fig. 2A).As an initial approach to examine the cellular program induced

by TNF in CD25HI acTregs versus Tcons, we employed micro-array gene expression analysis. Thus, the latter purified Treg andTcon populations were either treated with 50 ng/ml TNF or leftuntreated for 2 and 24 h to detect both early and late events. Thisrelatively high concentration of TNF was used because TNFR2has low affinity for soluble TNF, and indeed previous observations

FIGURE 1. TNFR2 is preferentially upregulated on CD45RA– Tregs at homeostasis and upon immune activation. A, CD4+ T cells isolated from PBMCs

were analyzed for surface expression of CD25, CD45RA, and CD120B. Left panel, Contour plot shows the subdivision of the CD4+ population into six

subsets, as defined by the expression of CD45RA and CD25 (R1 to R6). Right panel, The relative expression of CD120B in each region is shown in the

histogram overlays; numbers correspond to MFI of CD120B staining. B, CD4+ T cells were sorted by FACSAria into CD4+TNFR2HI (black line) and CD4+

TNFR2– (gray line) populations and immediately stained for FOXP3 (left panel) and CD120B (right panel) expression. C, Purified CD25– and CD25+

T cells were activated and cultured for 7 d as described in Materials and Methods. Plot (logarithmic scale) depicts the fold change in the MFI of CD120B

staining normalized to isotype staining at the indicated time points. D, Freshly isolated T cells from inflamed SF of a JIA patient were stained for cell

surface CD4 and intracellular FOXP3 (left panel). The histogram overlays show the cell surface CD120B expression for the FOXP3– gate (gray line) and

FOXP3+ gate (black line), and numbers indicate the MFI of the indicated population (right panel). Data were acquired with a FACSCalibur and FACSCanto

II instruments and analyzed with FlowJo 7.2.5 software. Data shown are representative of $3 experiments performed.

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imply that in vitro only high concentrations of soluble TNFmodulate the human Treg suppressive capacity (4).The microarray-based analysis of the mRNA transcripts induced

in acTregs by TNF showed that the transcription of 74 genes wasupregulated by $2-fold at 2 and/or 24 h compared with that inuntreated acTregs (Supplemental Table I). Importantly, the numberof gene transcripts induced by TNF in Tcons was much lower (total30 genes; Supplemental Table II), and this was evident at both timepoints as shown in the Venn diagram (Fig. 2B). Furthermore, moregene transcripts were upregulated in acTregs at 2 h compared withthose at 24 h. The transcription of five genes (NFKB2, NFKBIA,RELB, BIRC3, and SGK1) was consistently induced by TNF in thetwo T cell populations and at both time points.Of note, the number of gene transcripts downregulated by TNF in

both populations was very minimal (data not shown), an obser-vation that correlates with previous findings in other cell types (23).Thus, TNF treatment induces a more pronounced gene-transcrip-tion program in acTregs compared with that in Tcons.

TNF induces preferential upregulation of typical NF-kB targetgenes in Tregs

Studies analyzing the gene network induced by pure TNFR2stimulation in T cells that express TNFR2 as their sole TNFR havenot been published. To identify such functional networks within

our microarray gene expression data, we performed gene setenrichment analysis using the online available DAVID Bio-informatics Resources (http://david.abcc.ncifcrf.gov/tools.jsp) thatuse multiple heterogeneous functional gene annotation databasesources (24).We first performed the analysis for the list of gene transcripts

induced by TNF in Tregs at both time points (Supplemental TableI). In the enrichment analysis report (Supplemental Table III), weincluded only annotation source terms enriched .2-fold with athreshold of minimum five counts and an EASE score (a modifiedFisher exact p value) ,0.01. This analysis revealed significantenrichment for three main clusters of functional gene networks, asfollows: 1) IKK/NF-kB cascade and its related TNFR2 signalingpathway (NFKB1, NFKB2, RELB, NFKBIA, TNFAIP3, TANK,IKBKE, LTA, etc.); 2) immune response activation and responseto stimulus (TNFRSF9, FAS, LTA, IL27RA, CD74, CD69, STAT1,IRAK2, CD83, CCR8, etc.); and 3) negative and positive regula-tion of apoptosis (NFKB1, TNFAIP3, BIRC3, IFIH1, CD74,TNFRSF9, FAS, etc.). Notably, the highest-ranking terms in thisDAVID-based enrichment analysis were the TNFR2 signalingpathway (BIOCARTA) and I-kB kinase/NF-kB cascade (GO),indicating that TNF induces in acTregs robust transcription ofgenes that are directly involved in feedback regulation of signaltransduction downstream of TNFR2.Next, we performed gene set enrichment analysis against a list of

TNF-regulated genes published by Tian et al. (23). In a series ofworks, these investigators generated a high-quality list of 50unique annotated genes regulated by TNF in HeLa cells. Usinga Tet-regulated system for the expression of a nondegradable NF-kB inhibitor (FLAG-I–kB-Mut), they further identified within theformer list a subgroup of 28 genes that were both TNF-regulatedand NF-kB–dependent, and importantly all contained at least onefunctional NF-kB binding site within their promoter. The en-richment analysis revealed (Supplemental Table III, last row) 10hits with a high fold enrichment score (59.8) and an extremelysignificant p value of 5.74 3 10217 (EASE score). Of note, nogenes were common between our gene list and the related list ofTNF-regulated but NF-kB–independent genes also reported in thisstudy (e.g., JUN, JUNB, FZD2, GATA2, etc.).Given the lower number of gene transcripts induced by TNF

treatment in Tcons (Supplemental Table II), the gene set enrich-ment analysis for the latter gene list was of a lower yield. The twomost relevant GO terms significantly enriched were as follows:immune system process (nine hits, p = 2.623 1024) and apoptosis(six hits, p = 6.46 3 1023). Moreover, a much smaller percentagewithin the genes induced by TNF treatment in Tcons, were alsocommon to the gene list reported by Tian et al. (23) (five hits, p =1.39 3 1026), and notably these genes were all essential membersof the IKK/NF-kB cascade itself (NFKB1, NFKB2, RELB,NFKBIA, and TNFAIP3).To further illustrate the intricate gene–gene interactions within

the unique gene network induced by TNF in Tregs, we used theIPA platform’s neighborhood explorer feature, filtered to includeonly transcription- or expression-related gene–gene interactions inthe context of the NF-kB pathway and the list of transcripts induced$1.5-fold by TNF in acTregs (Supplemental Table III). This anal-ysis (Fig. 3) illustrates an elaborate NF-kB–dependent geneticnetwork regulating the transcription or expression ofmultiple genes,including genes involved in inflammation (e.g., LTA, TNF, ICAM1,TNFRSF4, TNFRSF9, and IRF1) and the inhibition of apoptosis(e.g., TNFAIP3, BIRC3,NFKB1, and BCL2L1/Bcl-xL). Importantly,within this genetic network, several gene products (marked by redrectangles) have been previously associated with the down-modulation of Treg-mediated suppression (4, 10, 13, 25–30).

FIGURE 2. TNF induces distinct gene transcripts in CD25HI Tregs

versus Tcons. A, CD4+ T cells were isolated by a FACSAria instrument

into CD4+CD25HI T cells with the top 3% of CD25 expression and CD25–

Tcons. The purified CD4+CD25HI (right panels) and CD25– (left panels)

subsets were then analyzed for cell surface CD25, CD45RA, CD120B, and

intracellular FOXP3. B, Venn diagrams of TNF-induced gene transcription

in CD25HI Tregs and CD25– Tcons. Data were obtained after either 2 (left

panel) or 24 h (right panel) of TNF treatment. Only genes with increased

expression $2-fold in TNF treated versus control media cultures were

included. Data shown are representative of two experiments using T cells

from two different healthy blood donors.

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TNF preferentially activates the canonical NF-kB pathway inTregs

Previous studies have yielded conflicting results as to whether pureTNFR2 signaling activates the canonical NF-kB pathway (14). Thehallmark of activation of this pathway is phosphorylation anddepletion of I-kB followed by phosphorylation and nucleartranslocation of NF-kB p65 (31).To detect changes in p65 and I-kB phosphorylation in Tregs

versus Tcons, we used phospho-protein–specific multiparametricflow cytometric analysis. Thus, by costaining for surface CD4 andCD25, we could specifically follow the changes in phosphoryla-tion (at single-cell resolution) in CD25HI and CD25–CD4+ T cellswithin a rather heterogeneous population of T cells.Freshly isolated CD25+ and CD25– T cells were rested over-

night in the presence of rhIL-2 (100 IU) and then treated for 15min with TNF, PMA and ionomycin (PMA/Ion) or vehicle only.As shown in Fig. 4A, TNF treatment (red heavy line) induced a 2-fold increase in the MFI of phospho-p65 in CD4+CD25HI Tregscompared with that in untreated cells (green line), whereas thesame TNF treatment induced a smaller (1.3-fold) increase inphospho-p65 levels in CD25– Tcons (Fig. 4A, lower panel). Aspredicted, treating the cells with the potent mitogen PMA/Ion(blue line) led to a robust and rapid increase in phospho-p65 levelsof a similar magnitude in both cell types. Furthermore, plotting thechanges in the MFI of phospho-p65 against time (5, 10, 15, and 30

min) revealed that the area under the curve for TNF treatment(Fig. 4B) was ∼3-fold larger in Tregs (upper panel) comparedwith that in Tcons (lower panel). This measure for PMA/Iontreatment was, in contrast, slightly larger in Tcons compared withthat in Tregs. In parallel experiments, we found that TNF alsoinduced within 10–15 min a moderate, but reproducible, increasein the intracellular level of phospho-I–kB within CD4+CD25HI

Tregs as compared with that in untreated cultures or TNF-treatedTcons (data not shown).Thus, taken together the data presented above imply that at

homeostasis TNF induces activation of the canonical NF-kBpathway in CD25HI Tregs and that the activation was as a rule lessmarked in unactivated CD25– (CD120Bdim) Tcons.

Analysis of TNF effect on functional pathways involved ininflammation

To facilitate comprehensive inflammation-focused associationanalyses, Loza et al. (32) assembled a list of key inflammation-related genes and further divided them into 17 functionally definedsubpathways. Thus, we sorted the genes that were induced $1.5-fold by TNF in Tregs (Supplemental Data Set 1) according to thisfunctional classification.We found that our gene list was significantly enriched for two

subpathways: NF-kB and TNFSF signaling (Tables I, II, re-spectively). Careful inspection of the TNF-induced changes in the

FIGURE 3. TNF induces a prominent NF-kB–dependent gene network in Tregs. The IPANeighborhood Explorer featurewas used to depict knownmolecular

interactions between theNF-kBcomplex and the different annotatedgenes upregulated$1.5-foldbyTNF.Thegene–gene interactionswere filtered to includeonly

transcription- or expression-associated connections. The upregulated genes are depicted in shades of red, with red indicating the highest and light pink the lowest

fold change. The arrows indicate acts on [i.e., induces directly (solid line) or indirectly (dashed line) transcription and/or expression], and each gene or protein is

shown in its most likely subcellular location. Gene products within red rectangles have been previously shown to downmodulate Treg function (4, 10, 13, 25–30).

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transcription of genes associated with the two latter subpathwaysunderlines that their upregulation generally peaked at 2 h and wasmore pronounced in acTregs. Moreover, TNF induced the tran-scription of LTA and the TNF gene itself (both specific TNFR2ligands), signifying the induction of a positive autocrine feedbackloop. Interestingly, within the large list of TNFRSF genes (TableII), TNF selectively induced the transcription of only threemembers: 4-1BB (CD137, TNFRSF9), FAS (CD95, TNFRSF6),and OX40 (CD134, TNFRSF4).Next, we confirmed by RTQPCR that TNF indeed upregulated

the mRNA abundance for four selected genes within these two

subpathways: NFKB2, STAT1, LTA, and TRAF3. In agreementwith the microarray data, we found that TNF treatment primarilyupregulated the transcription of these genes in Tregs (Fig. 5A, foldchange in parentheses). For instance, the transcription of NFKB2and LTAwas particularly increased in Tregs compared with that inTcons (19.2- and 4.8-fold versus 4.6- and 1.6-fold, respectively).Next, we analyzed in highly purified naive CD45RA+ Tcons,CD45RA– acTregs, and CD45RA+ nrTregs (sorted by FACSAria)the different effects of TNF treatment on the transcription ofrelevant genes. As shown in a typical experiment (Table IV), theinduction of transcription of the proinflammatory genes NFKB2,STAT1, and LTA by TNF was significantly higher within the ac-Treg population as compared with those within the other twoT cell subsets.Next, we asked whether on top of inducing transcription of the

TNF gene its translation into protein product was also enhanced inTregs following exogenous TNF treatment. To address this issue,we isolated CD25+ and CD25– T cells from PBMCs and treatedpart of them with TNF for 3 d. The various cultures were thenactivated with PMA/Ion in the presence of a Golgi transport in-hibitor and immediately analyzed for FOXP3 expression and in-tracellular TNF production. Our data show that the CD4+FOXP3+

T cells within the CD25++ cultures were more strongly induced toproduce intracellular TNF following TNF treatment as comparedwith FOXP3–CD25++ or CD25– T cells (Supplemental Fig. 1). Toverify that the latter increase in TNF signal detected in Tregs didnot originate from exogenous TNF bound to the membrane (viaTNFR2, for example), we also stained separately unpermeabilizedcells, where indeed no meaningful staining for membrane-boundTNF (mTNF) was detected.

Minimal effect of TNF treatment on the Treg transcriptionalsignature

To identify the effect of TNF on the major genes that distinguish thehuman Treg lineage, we first scanned our data for transcripts thatwere .3-fold increased in freshly isolated acTregs compared withthose in Tcons. The gene list thus generated (Table III) indeedincluded many of the canonical Treg genes that shape the Tregtranscriptional signature (33, 34). Importantly, we found thatoverall TNF did not significantly change (.1.5- or ,0.5-foldchange) the transcription of this gene list, including FOXP3,CTLA4, and IL2RA. Likewise, the transcription of TNFR2(TNFRSF1B) that ranked fifth among the Treg signature genes wasonly minimally affected.Because FOXP3 is the master regulator of the Treg lineage (35,

36) and influences its transcriptional signature, we measured theeffect of TNF treatment on its mRNA and protein expression. ByRTQPCR, we did not detect significant inhibition of FOXP3transcription by TNF treatment (Fig. 5B). In parallel, by flowcytometry, we also analyzed the changes in FOXP3 protein levels.We found that TNF in the context of IL-2 cotreatment did reduceby ∼30% the IL-2–dependent upregulation of FOXP3, but im-portantly TNF treatment per se did not significantly changebaseline FOXP3 expression (Fig. 5C).

TNF specifically induces cell surface expression of 4-1BB, OX40,and FAS in acTregs

As shown in preceding experiments, mRNA levels of threeTNFRSF members were preferentially induced by TNF in acTregs.Thus, we tested whether this effect was also translated into anincrease at the protein-product level at 24 h after TNF treatment. Byimmunostaining and subsequent FACS, analysis we found that both4-1BB and OX40 were robustly induced by TNF in FOXP3+ Tregsbut not in Tcons. As seen in a typical healthy donor (Fig. 6A), in

FIGURE 4. TNF induces significant phosphorylation of p65 in Tregs.

CD25++ and CD25– T cells obtained from healthy blood donors were

treated with TNF (red line), PMA/Ion (blue), or vehicle only (green). CD4+

T cells were analyzed for cell surface CD25 and intracellular phospho-p65

levels. A, Histogram overlays show an example of phospho-p65 staining in

the CD25HI (upper panel) and CD25– (lower panel) subsets. B, Plots depict

the fold change in the MFI of intracellular phospho-p65 staining in re-

sponse to TNF or PMA/Ion treatment at the indicated time points (from 5–

30 min), as normalized to the appropriate isotype staining. The plots show

phospho-p65 kinetics within the CD4+CD25HI Treg (upper panel) or CD4+

CD25– Tcon populations (lower panel). Data shown are representative of

three independent experiments.

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Tregs treated with TNF the cell surface levels of 4-1BB and OX-40 were upregulated 3.4- and 3.2-fold, respectively, as comparedwith untreated cells. This increase was abrogated when the cellswere pretreated with anti-TNFR2 mAbs (data not shown). Theexpression of FAS that was initially much higher in Tregs wasfurther enhanced by TNF, whereas FAS expression was onlymarginally increased by TNF in Tcons. In contrast, the expressionof CD27, another TNFRSF member, used as a negative control,was practically unaffected. Inconsistent with the gene microarraydata, the cell surface expression of CD69 and CD127 was onlymarginally increased in Tregs or Tcons following TNF treatment(Fig. 6A, lower panels).To further answer whether these three TNFRSF members are

regulated differently by TNF in acTregs versus nrTregs, we treatedwith TNF blood-derived CD25++/HI T cells, a cell population thatcontains a variable (age- and donor-dependent) percentage ofacTregs and nrTregs. The subsequent FACS analysis of a typicalexperiment revealed that 4-1BB and OX40 were mostly inducedby TNF in CD45RA– FOXP3+ Tregs but to a much lesser extent inCD45RA+ Tregs (Fig. 6B, Table IV).Because increased TNF levels normally characterize the

inflamed synovium, we also compared FOXP3+ and FOXP32

T cells isolated from SF with their counterparts isolated from theblood of the same JIA patient for the cell-surface expression of 4-1BB, OX40, and FAS (Fig. 6C). Consistent with the data obtainedfrom in vitro TNF treatment, 4-1BB and OX40 were particularlyupregulated on synovial FOXP3+ acTregs compared with theircirculating counterparts (5.4- and 4.6-fold versus 2.1- and 1.7-fold, respectively; values correspond to the ratio of MFI of

FOXP3+ to FOXP3– events). FAS surface expression was alsoincreased on circulating Tregs compared with that on Tcons andfurther upregulated by 6.8-fold on synovial acTregs.

TNF/TNFR2 signaling modulates the Treg suppressive function

Previous studies using the in vitro coculture suppression assaysuggest that exogenous soluble TNF can inhibit the human Tregsuppressive function (4, 19). To further investigate the functionalconsequences of TNF treatment on Tregs in our system, we co-cultured freshly isolated CD25+ Tregs with CFSE-labeled CD25–

Tcons at various ratios. The cocultures were activated by immo-bilized OKT3 mAbs and treated, as indicated, with TNF and/orrhIL-2, TNF and anti-TNFR2 mAbs, or anti-TNF mAb (In-fliximab), or untreated. Tcon proliferation in the various co-cultures was assessed on day 5 by the proliferation analysis tool ofthe FlowJo 7.2.5 software to calculate the DI and draw a fit modelof generations onto the relevant CFSE dilution histograms (Fig.7A). We found that excess of either exogenous rhIL-2 (100 IU/ml),TNF (50 ng/ml), or both modulated the Treg suppressive function,which was otherwise very evident and Treg-ratio–dependent (Fig.7B, left panel). Importantly, the TNF-dependent inhibition ofsuppression was fully reversed by cotreatment with anti-TNFR2blocking mAbs. Infliximab treatment resulted in augmented sup-pression, and as previously shown (6, 7), it moreover directlyreduced Tcon proliferation independent of Treg function.Next, we analyzed by ELISA the effect of TNF treatment on the

Treg capacity to suppress IFN-g secretion by Tcons (Fig. 7B, rightpanel). Using a similar suppression assay scheme, as above, wefound that TNF or rhIL-2 treatment alone had a moderate in-hibitory effect on the robust Treg-dependent suppression of IFN-gsecretion. Importantly, cotreatment with both rhIL-2 and TNFalmost completely inhibited this Treg-dependent suppression. Bycontrast, the blockade of TNFR2 by mAbs restored suppression tobaseline. Infliximab was very potent in reducing overall IFN-gsecretion in a manner that was quite Treg-independent.Given that TNF consistently induced significant upregulation of

4-1BB on acTregs and signaling via the latter receptor has beenstrongly implicated in downmodulation of Treg-dependent sup-pression in mice, we next asked whether 4-1BB signaling by ex-ogenous 4-1BB ligand has a similar effect in human T cells. Toaddress this issue, we performed in vitro suppression assays wherewe activated the Treg/Tcon cocultures with artificial APCs eitherKT32 or KT32/4.1BBL, which were preincubated with OKT3/anti-CD28 mAbs as previously detailed (37). In addition, we also testedthe effect of cotreatment with TNF on suppression in this system.The data show, as predicted, that either TNF or 4-1BBL alonesignificantly inhibited suppression but more importantly that TNFtreatment—a strong inducer of 4-1BB in acTregs—in the presenceof KT32/4.1BBL almost completely abolished suppression (Fig. 8).

DiscussionThis study reveals in detail the mechanism by which TNF mod-ulates the function of human CD45RA– acTregs and consequentlysheds new light on how TNF blockade restores immunologic self-tolerance. Our data show that TNF via TNFR2, constitutivelyexpressed in Tregs and particularly at higher levels in theCD45RA– subset, induces the activation of the canonical NF-kBcascade. Moreover, we describe for the first time the distinctiveproinflammatory NF-kB–regulated transcription program induced,preferentially, in acTregs by TNF that functions to modulate theirsuppressive capacity. It should be pointed out that the variousexperiments analyzing gene expression patterns within CD45RA–

Tregs are based on a somewhat heterogeneous CD4+CD25HI

T cell population usually containing ∼90% FOXP3+ cells.

Table I. List of NF-kB signaling subpathway genes induced by TNF$1.5-fold

Gene Symbol Treg 2 h Treg 24 h Tcon 2 h Tcon 24 h

NFKB2 8.3 4.9 4.1 2.9NFKBIA 4.8 4.3 3.6 2.8RELB 4.6 4.7 2.7 2.5TANK 2.7 2.1 2.3 1.8IRAK2 2.6 1.8 1.8 1.6NFKB1 2.3 NSa 2.7 NSNFKBIE 2.2 1.8 NS NSNFKBIZ 2.0 NS NS NSREL 2.0 1.5 1.9 1.6BCL3 1.8 NS NS NSSTAT1 1.6 2.1 NS NSBCL6 1.6 1.7 1.7 1.6

Numbers represent fold change in gene expression induced by TNF treatment ineither Tregs or Tcons at the indicated time point.

aA ,1.5-fold change.

Table II. List of TNF(R)SF signaling subpathway genes induced byTNF $1.5-fold

Gene Symbol Treg 2 h Treg 24 h Tcon 2 h Tcon 24 h

TNFAIP3 3.4 3.1 1.8 2.2TNFRSF9 3.2 2.1 NS NSLTA 2.4 1.6 1.9 NSFAS 2.5 2.3 NS 1.7TRAF3 2.0 1.9 1.7 1.7TNFRSF4 1.8 NSa NS 1.7TRAF4 1.6 1.7 NS NSTNF 1.6 NS NS NSTRAF1 1.6 NS NS NSTRAF2 1.5 1.7 NS NS

Numbers represent fold change in gene expression induced by TNF treatment ineither Tregs or Tcons at the indicated time point.

aA ,1.5-fold change.

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Previous studies offer conflicting results regarding the effect ofTNF on Treg functions. Two reports based on human data agree thatTNF directly downmodulates Treg suppressive function (4, 5). Incontrast, a study in mice implies that TNF may actually augmentTreg suppressive function in vitro and probably in vivo as well(12). This discrepancy may represent an example of cross-speciesdifferences between human and mice T cells. Regarding this itshould be noted that multiple studies in both humans and miceimply that in vivo TNF blockade augments Treg function (2, 4, 5,

38, 39). Interestingly, another recent study proposes that the sol-uble (cleaved) form of TNFR2 is of prime importance in medi-ating the Treg suppressive function, by quenching surplus TNF atsites of inflammation (19).Our findings that TNF/TNFR2 signaling inhibits the Treg sup-

pressive function via activation of the NF-kB cascade are the firstto experimentally demonstrate such a function for TNF. Whereasthere is ample evidence that TNFR1 signaling in many cell typesother than T cells activates the NF-kB cascade, studies in Tregsshowing a link between TNFR2 and NF-kB activation have notbeen reported (1, 14). A previous study by Valencia et al. (4) thatshowed an inhibitory effect of TNF/TNFR2 signaling in Tregsalso concluded that TNF inhibited of FOXP3 transcription, thoughNF-kB activation was not addressed in this study. By contrast, ourdata based on various complementary modalities do not showsignificant inhibition of FOXP3 transcription during the first 24 hafter TNF treatment, while demonstrating a central role for theactivation of the canonical NF-kB pathway. Moreover, previousstudies (6, 11) and our present data show that CD4+ T cells iso-lated from inflamed SFs of JIA patients—a milieu where TNF isabundant—are actually enriched for T cells expressing high levelsof FOXP3 (Fig. 1D). Studies that have addressed the transcrip-tional regulation of FOXP3 also do not report that NF-kB/RELfamily members bind conserved DNA motifs within the FOXP3promoter or its other regulatory regions (40, 41).Data from a number of studies imply, even if indirectly, that TNF

via TNFR2 activates a NF-kB pathway-dependent transcriptionprogram in T cells. In mice lacking TNFR2, stimulation of CD8+

T cells via TCR and CD28 results in reduction in the time span of

FIGURE 5. TNF induces NFKB2, LTA, TRAF3, and STAT1 but does not change FOXP3 transcription. A and B, RTQPCR analysis of indicated gene

transcription in human CD4+CD25++ and CD25– T cells treated for 24 h with TNF (50 ng/ml) or left untreated, relative to expression in untreated Tregs and

normalized to HPRT1. Numbers in parentheses show the fold change induced by TNF. The y-axis is in log scale, and data (mean 6 1 SD of duplicates) are

representative of three independent experiments in different donors. pp , 0.05; ppp , 0.01. C, CD25+ and CD25– T cells were treated for 24 h with IL-2,

TNF, both, or media alone and then stained for cell surface CD4 and intracellular FOXP3 (dot plot). The histogram overlays show FOXP3 expression (CD4+

gate) in the various cultures, and numbers represent their respective MFIs. The data represent three independent experiments.

Table III. TNF has a minimal effect on the highest-ranking Tregsignature genes

Gene SymbolTreg versus

Tcona Treg 2 h Treg 24 h Tcon 2 h Tcon 24 h

IL2RA 27.2 1.2 1.0 0.9 1.4ZNFN1A2 13.5 1.1 1.1 1.2 0.9HLA-DRA 12.9 0.7 0.7 1.5 1.1FOXP3 9.3 0.9 1.0 1.0 1.0TNFRSF1B 7.2 1.0 1.0 1.2 0.8VAV3 5.6 1.1 1.1 0.9 1.0BCL2 4.4 0.8 0.9 1.1 1.2HLA-DRB1 4.4 0.8 0.9 1.2 0.9CCR4 4.4 1.0 0.8 0.8 0.8CTLA4 4.2 1.0 0.9 0.5 0.8ZNFN1A4 4.1 1.1 1.0 1.2 1.1IL2RB 3.8 1.1 1.0 1.0 0.8

aFold change in gene transcript abundance (numbers in boldface) in untreatedTregs versus Tcons at 2 h ranked in descending order. Numbers in nonboldfacerepresent fold change in the indicated gene transcript induced by TNF treatment ineither Tregs or Tcons at the specified time point.

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activation of the I-kB/NF-kB cascade (42). Transgenic mice in-duced to express human p75-TNFR2 develop severe multiorganinflammatory syndrome associated with constitutive NF-kB cas-cade activation in the PBMC compartment. Moreover, studies inprimary cortical neurons from TNFR12/2 mice show that pureTNFR2 stimulation induces significant phosphorylation and de-pletion of I-kB followed by nuclear translocation of p65/RelA (43).Our data further establish the link between TNF and the human

Treg lineage by showing that at homeostasis TNFR2 expression isparticularly increased on circulating acTregs and moreover thatFOXP3+ T cells can be sorted out with good purity based on theexpression of TNFR2. In addition, in vivo induced FOXP3+ Tregsisolated from inflamed SFs of arthritis patients express signifi-cantly higher levels of TNFR2 compared with their counterpart

CD4+FOXP3– effectors. These data support a view that the up-regulation of TNFR2 on acTregs is not merely a reflection of theiractivated state but is more likely a subset-specific characteristic.This latter point is further underlined by the observation thatTNFRSF1B (TNFR2) ranked among the five top genes that definethe human Treg transcriptional signature (Table III), a list that alsoincludes IL2RA, ZNFN1A2, and FOXP3. Consequently, it may beenvisaged that TNFR2 is important for Treg homeostasis in vivo,as it is indeed postulated for the other three genes (44).It can be hypothesized that at homeostasis low physiological

levels of mTNF expressed by various cells may support Tregmaintenance by, for example, delivering NF-kB–dependent anti-apoptotic signals. However, persistently high levels of mTNF—asoccurs during autoimmune joint inflammation—directly inhibit

FIGURE 6. TNF selectively upregulates expression of OX40, 4-1BB, and FAS on CD45RA– Tregs. A, CD25++ and CD25– T cells isolated from a healthy

blood donor were treated with TNF (50 ng/ml) or untreated. Twenty-four hours later, the CD4+ T cells were analyzed for CD25 and one of the following

markers: CD95/FAS, CD134/OX40, CD27/TNFRSF7, CD137/4-1BB, CD127/IL7R, or CD69. Numbers represents the MFI of untreated (green) or TNF-

treated (red) cells for CD4+ gate. B, In parallel, the CD4+CD25++ T cell subset was analyzed for cell surface coexpression of CD45RA and one of CD137,

CD134, or CD95. The contour plots depict either TNF-treated (right panels) or untreated (left panels) cultures, and numbers in parentheses correspond to

MFI of CD45RA– events. C, Freshly isolated CD4+ T cells from PBMCs and inflamed SF of a typical JIA patient were analyzed for expression of in-

tracellular FOXP3 and cell surface CD137, CD134, or CD95. Left panels, FOXP3+ gate. Right panels, FOXP3– gate. Color-coded numbers correspond to

the MFIs. All data represent $3 independent experiments.

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Treg function as shown in this study and very likely also inducesome level of resistance in activated TNFR2+ effector Tcons to-ward suppression. This scenario is supported by observationsmade in the transmembrane TNF transgenic mouse model en-gineered to overexpress transmembrane forms of TNF. The

transmembrane TNF transgenic mice invariantly develop aggres-sive inflammatory arthritis that indeed can be ameliorated bygenetic deletion of p75 or treatment with recombinant solublehuman TNFR2 (45, 46).Among the proinflammatory immune response genes induced by

TNF specifically in acTregs, we have identified two members of theTNFSF (LTA and TNF) and three members of the TNFRSF (FAS,TNFRSF4, and TNFRSF9). Evidence is moreover provided thatthis may occur in situ, because FOXP3+ synovial T cells mostlikely exposed in vivo to elevated TNF levels significantly upre-gulate the expression of these TNFRSF members as comparedwith their blood-circulating counterparts. Regarding the role ofTNFRSF4 and TNFRSF9 in Treg physiology, although it seemscomplex- and context-dependent, the prevailing view [as recentlyreviewed by Croft et al. (30)] is that signaling via OX40 or 4-1BBduring immune activation can reversibly inhibit Treg suppressivefunctions. This occurs by both a direct effect on Tregs and anindirect effect on Tcons boosting their resistance to suppression.Indeed, our data from suppression assays using KT32 transfectedto express human 4-1BBL suggest that 4-1BB signaling furtherenhances the inhibitory effects of TNF on acTregs. Thus, wepropose that paracrine TNF (soluble or membrane-bound) fromTcons and/or APCs induces a NF-kB–dependent positive feed-back loop in acTregs that includes expression of cell surface 4-1BB plus OX-40. The latter TNFRSF members subsequently(upon ligation) further activate the NF-kB cascade to promote

FIGURE 7. TNF and/or IL-2 modulate the Treg suppressive function.

CD252 Tcons were labeled with CFSE, plated into 96-well plates, and

activated by plastic-bound OKT3 alone or in the presence of graded

amounts of CD25++ Tregs. A, Five days later, the CD4+ Tcons were ana-

lyzed for CFSE dilution by FACS. We used the Proliferation Platform

(FlowJo 7.2.5) to calculate the DI and draw a fit model of generations onto

the CFSE dilution histograms. Shown are examples of proliferation anal-

ysis results for untreated dividing Tcons (right panel) or at a 1:1 ratio of

Tcons to Tregs (left panel). B, The plot depicts the DI values of Tcons at

different Treg ratios. The different cocultures were treated (as indicated in

the embedded legend) with TNF, IL-2, TNF and IL-2, TNF and anti-

TNFR2 mAbs, or Infliximab or left untreated. C, The plot depicts IFN-g

levels in the supernatants of the same cocultures collected 3 d after acti-

vation (logarithmic scale; values are mean of duplicates). Data represent

three independent experiments.

FIGURE 8. TNF and 4-1BB signaling cooperate to abrogate in vitro

suppression. In this suppression assay KT32 or KT32/4.1BBL preincubated

with OKT/CD28.2 mAbs were used as artificial APCs to activate the co-

cultures. Where indicated the cocultures were also treated with TNF-a

(50 ng/ml). Five days later, CD4+ Tcons were analyzed for CFSE dilution, as

detailed in Fig. 7. A, Examples of the analysis of activated dividing Tcons

alone (right panel) or at 1:1 ratio with Tregs (left panel). B, The plot depicts

DI values of Tcons at different Tcon to Treg ratios. The type of APC and

treatment used are indicated in the embedded legend. Data represent three

independent experiments.

Table IV. TNF primarily induces NF-kB–dependent gene transcriptionin CD45RA– acTregs

T Cell SubsetGene

SymbolFold

Changea CD No.Fold

Changeb

STAT1 CD95CD25++ RA+ 2.1 0.9CD25+++ RA2 4.6 1.3CD252 RA+ 1.6 1.3

NFKB2 CD134CD25++ RA+ 2.8 1.3CD25+++ RA2 14.1 2.8CD252 RA+ 2.4 1.0

LTA CD137CD25++ RA+ 1.1 1.3CD25+++ RA2 19.2 3.4CD252 RA+ 1.9 1.1

Boldface numbers indicate acTreg population.aFold change in gene transcription, as measured by RTQPCR, induced by TNF

treatment (relative to untreated cells and normalized to HPRT1) in the indicated Tregsubset or Tcons.

bFold change in MFI staining of the specified CD marker induced by TNF treat-ment (relative to untreated cells).

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transient Treg dysfunction. Preliminary in vitro experiments sug-gest that TNF pretreatment, apart from inducing FAS upregulation,specifically increases the susceptibility of acTregs to undergo ap-optosis upon exposure to agonistic mAbs to FAS, yet because thissystem is rather artificial, it is difficult to determine whether TNFactually renders acTregs susceptible to apoptosis in vivo.In summary, we show that TNF induces a TNFR2/NF-kB–

dependent proinflammatory program predominantly in CD45RA–

acTregs that counters their innate suppressive capacity but withoutdrastically altering their core transcriptional signature. It can beenvisioned that when TNF levels drop, for example, during anti-TNF therapy, the Treg suppressive function can be reinstatedrather rapidly, a mechanism that may explain the remarkableclinical efficacy of TNF blockade in restoring immunological self-tolerance in many treated arthritis patients (47).

AcknowledgmentsThis work was performed in partial fulfillment of the requirements for

a Ph.D. degree of M.N. and H.V. (The George S. Wise Faculty of Life Sci-

ences, Tel Aviv University, Israel). G.R. holds the Djerassi Chair in Oncol-

ogy at the Tel Aviv University Sackler Faculty of Medicine, Israel.

DisclosuresThe authors have no financial conflicts of interest.

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