Transmembrane TNF−TNFR2 Impairs Th17 Differentiation by Promoting Il2 Expression Patrick G. Miller, Michael B. Bonn and Susan C. McKarns This information is current as J Immunol 2015; 195:2633-2647; Prepublished online 12 of October 3, 2021. August 2015; doi: 10.4049/jimmunol.1500286 http://www.jimmunol.org/content/195/6/2633 Downloaded from References This article cites 76 articles, 37 of which you can access for free at: http://www.jimmunol.org/content/195/6/2633.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Transmembrane TNF–TNFR2 Impairs Th17 Differentiation by Promoting Il2 Expression

Patrick G. Miller,* Michael B. Bonn,* and Susan C. McKarns*,†

The double-edged sword nature by which IL-2 regulates autoimmunity and the unpredictable outcomes of anti-TNF therapy in autoimmunity highlight the importance for understanding how TNF regulates IL-2. Transmembrane TNF (tmTNF) preferentially binds TNFR2, whereas soluble TNF (sTNF) binds TNFR1. We previously showed reduced IL-2 production in TNFR12/2 TNFR22/2 CD4+ T cells. In this study, we generated TNFR12/2, TNFR22/2, or TNFR12/2 TNFR22/2 5C.C7 TCR Il2-GFP mice and report that CD4+ –intrinsic tmTNF/TNFR2 stimulates Il2 promoter activity and Il2 mRNA stability. We further used tmTNF Foxp3 gfp reporter mice and pharmacological TNF blockade in wild-type mice to report a tmTNF/TNFR2 interac- tion for Il2 expression. IL-17 is critical for host defense, but its overabundance promotes autoimmunity. IL-2 represses Th17 differentiation, but the role for TNFR2 in this process is not well understood. We report elevated expression of TNFR2 under

Th17-polarization conditions. Genetic loss-of-function experimental models, as well as selective TNF blockade by and Downloaded from XPro1595 in wild-type mice, demonstrate that impaired tmTNF/TNFR2, but not sTNF/TNFR1, promotes Th17 differentiation in vivo and in vitro. Under Th17-polarizing conditions, elevated IL-17 production by TNFR2-knockout CD4+ T cells was asso- ciated with increased STAT3 activity and decreased STAT5 activity. Increased IL-17 production in TNFR2-knockout T cells was prevented by adding exogenous IL-2. We conclude that CD4+ T cell–intrinsic tmTNF/TNFR2 promotes IL-2 production that inhibits the generation of Th17 cells in a Foxp3-independent manner. Moreover, under Th17-polarizing conditions, selective blockade of CD4+ T cell–intrinsic TNFR2 appears to be sufficient to promote Th17 differentiation. The Journal of Immunology, http://www.jimmunol.org/ 2015, 195: 2633–2647.

nterleukin-2 is essential for tolerance against self-Ags, as il- IL-2 that are produced impact the outcome of immune responses. lustrated by the autoimmune phenotype of the IL-2 knockout In naive cells, the Il2 promoter is silent, but an integrated assembly I (KO) mouse (1–3). IL-2 is produced mainly by activated CD4+ of chromatin-remodeling complexes, histone modifications, and T cells. This pleotropic signals through intermediate transcription factors, including AP-1, NF-kB, NFAT, and OCT-1, 29 affinity (KD ∼ 10 M) CD122 (IL-2Rb)/CD132 (gc) and ligand- facilitate a rapid and transient onset of Il2 promoter activity. Il2 211 specific high-affinity (KD ∼ 10 M) CD122/CD132/CD25 (IL- expression is controlled by the strength and duration of TCR sig- by guest on October 3, 2021 2Ra) receptor complexes that respond to ∼1 nM and ∼10 pM doses naling, costimulation, and rapid Il2 mRNA degradation (8, 10, 11). of IL-2, respectively (4), to activate downstream STAT5, MAPK, The CD28 response element, located 2164 to 2152 bp immedi- and PI3K signaling cascades (5). Consumption of IL-2 in the pe- ately upstream of the transcriptional start site, is especially impor- riphery by CD4+ effector T cells or regulatory T cells (Tregs) con- tant for Il2 transcription and posttranscriptional regulation tributes to the control of Th cell differentiation and suppressor Treg of Il2 mRNA stability. function, respectively (6–9). Given the dichotomous role of IL-2 Our knowledge of how different ligand–receptor interactions on immune regulation and the nature by which multiple cell pop- contribute to T cell activation and differentiation has steadily ulations compete for a common pool of IL-2, the rate and amount of grown to include a host of costimulatory molecules. In addition to signal 1 through the TCR and signal 2 (costimulation), we (12) and other investigators (13, 14) have shown that TNFRs promote IL-2 *Laboratory of TGF-b Biology, Epigenetics, and Cytokine Regulation, Center for Cellular and Molecular Immunology, Department of Surgery, University of Missouri production. TNF, similar to other TNF family members (e.g., LIGHT, School of Medicine, Columbia, MO 65212; and †Department of Molecular Micro- FasL, and TRAIL), exists in membrane-bound and soluble forms. biology and Immunology, University of Missouri School of Medicine, Columbia, MO The matrix metalloprotease TNF-converting enzyme cleaves trans- 65212 membrane TNF (tmTNF) from the cell surface to generate a 17-kDa Received for publication February 6, 2015. Accepted for publication July 14, 2015. soluble TNF (sTNF) (15). sTNF and tmTNF preferentially signal This work was supported by National Institutes of Health Grant R01 ES022966 and a University of Missouri Richard Wallace Faculty Incentive Grant Award (both to through TNFR1 (CD120a, p55) and TNFR2 (CD120b, p75), re- S.C.M.). spectively (16, 17). In contrast to the ubiquitous expression of Address correspondence and reprint requests to Dr. Susan C. McKarns, Department TNFR1, TNFR2 is restricted mostly to hematopoietic cells, endo- of Surgery, University of Missouri School of Medicine, M616 Medical Sciences thelium, microglia, and oligodendrocytes. Signaling downstream Building, Columbia, MO 65212. E-mail address: [email protected] of TNFR1 and TNFR2 is distinct, yet overlapping, and is mediated Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; AHR, aryl hydro- by the recruitment of adaptor and the activation of down- carbon receptor; ARNT, AHR nuclear translocator; cRPMI, complete RPMI; CsA, cyclosporin A; CT, cycle threshold; dKO, double knockout; DN-TNF, dominant- stream transcription factors, including NF-kBandJNK.Incontrast negative TNF; KO, knockout; LAP, latency-associated peptide; LN, lymph node; LT, to TNFR2, TNFR1 contains an intracellular death domain and ; MCC, moth cytochrome c; MFI, mean fluorescence intensity; MLN, mesenteric LN; MOG, myelin oligodendrocyte glycoprotein; pY, tyrosine phosphor- promotes caspase-mediated apoptosis (18, 19). TNFR2 contains ylation; qRT-PCR, quantitative RT-PCR; ROR, retinoic acid–related orphan receptor; intracellular TNFR-associated factor binding domains. We pre- SPLN, spleen; sTNF, soluble TNF; Tg, transgenic; tmTNF, transmembrane TNF; Treg, viously associated TNFR1/TNFR2 double deficiency with im- regulatory T cell; UTR, untranslated region; WT, wild type. paired IL-2 production (20), but the individual contribution of Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 each of these receptors remains undefined. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500286 2634 TNFR2 REGULATES IL-2 TO CONTROL IL-17

Following activation, CD4+ T cells differentiate into distinct Abs and reagents effector subpopulations characterized by unique , tran- + TCRVb11 (KT11), CD120a (55R-286), CD120b (TR75-89), CD26 (H194- scription factors, and immune-regulatory properties. CD4 Th17 112), CD278 (C398.4A), and CD62L (MEL-14) were purchased from Bio- T cells are characterized by the expression of retinoic acid–related Legend (San Diego, CA). TCRb (H57-597), CD4 (RM4-5), CD4 (GK1.5), orphan receptor (ROR)-gt and the production of two related CD8a (Ly-2), CD120a (55R-286), CD69 (H1.2F3), CD25 (PC61), TNF effector cytokines, IL-17 and IL-17F. Th17 cells are essential (MP6-XT22), CD45Rb (16A), and CD16/CD32 (2.4 G2) were purchased from BD Biosciences (San Jose, CA). Foxp3 (FJK-16s), CD28 (37.51), and for host protection against bacterial and fungal infections, but latency-associated peptide (LAP; TW7-16B4) were purchased from eBio- too much IL-17 can promote inflammation or autoimmunity (21). science (San Diego, CA). Annexin V and 7-aminoactinomycin D (7-AAD) How TNF regulates Th17 cells is poorly understood. Given the were purchased from BD Biosciences. Lyophilized recombinant mouse 77 233 recent interest in selective activation of TNFR2 as a therapeutic TNF (aa Val –Leu , expressed in Escherichia coli) was purchased from R&D Systems (Minneapolis, MN) as a lyophilized powder, 0.2 mm filtered target, a better understanding of the selective roles of TNFR1 and 2 + in PBS without a carrier , stored at 80˚C, and reconstituted in TNFR2 on cytokine production by CD4 T cells is needed. The sterile PBS with 0.01% BSA (Fraction V; Sigma-Aldrich, St. Louis, MO) objective of this study was three-fold: to determine the individual as a carrier protein just prior to use. MCC aa 88–103 and myelin oligo- contribution of TNFR1 and TNFR2 to IL-2 expression, to deter- dendrocyte glycoprotein (MOG) aa 35–55 were purchased from AnaSpec minewhetherregulationofIL-2expressionbyTNFR1orTNFR2 (Fremont, CA). PMA, ionomycin, and cyclosporin A (CsA) were pur- + + chased from Calbiochem-EMD Millipore (Dormstadt, Germany). CFA is CD4 effector T cell specific, and to determine whether CD4 was purchased from Sigma-Aldrich. effector T cell–specific ablation of TNFR2 influences Th17 cell differentiation. Pharmacological TNF blockade To investigate the individual contribution of TNFR1 and TNFR2 A novel dominant-negative TNF (DN-TNF) analog XPro1595 (Xencor, 2 2 Downloaded from IL-2 expression, we generated B10.A (H-2a) 5C.C7 Rag2 / TCR Monrovia, CA), engineered for selective sTNF inhibition, was a gift from Rag22/2 Il2-GFP reporter mice that are singly deficient for either Dr. David Szymkowski (Xencor). XPro1595 was administered at concen- ∼ TNFR1 or TNFR2 or doubly deficient for both TNFR1 and TNFR2 trations 10-fold higher than native sTNF. XPro1595 rapidly (within minutes) neutralizes .99% of sTNF while sparing tmTNF activity (29, 30). to examine IL-2 production at the level of promoter activity. We Etanercept (Enbrel; Amgen, Thousand Oaks, CA), a decoy TNFR2 that + report that an interaction between CD4 T cell–intrinsic TNFR2 nonselectively blocks sTNF, tmTNF, and LT (31, 32), was purchased from and tmTNF, but not TNFR1/sTNF, costimulates Il2 expression to a pharmacy. For invivo TNF neutralization, groups of mice were treated with fine-tune the generation of CD4+ IL-2 producers. Although TNF twice-weekly s.c. injections of XPro1595 (10 mg/kg) (29, 30), etanercept http://www.jimmunol.org/ (10 mg/kg) (33), or saline vehicle as control starting on the day of immuni- has been implicated in Th17 differentiation (22, 23), not much is zation. Etanercept was previously shown to antagonize IL-2 production (34). known about the generation of Th17 cells in response to TNFR2 signaling. In this article, we show that, in addition to promoting Measurement of TNF bioactivity + the generation of Foxp3 Tregs, TNFR2 inhibits Th17 differen- The manufacturer (R&D Systems) used lysis of murine L929 cells in the tiation by promoting Il2 expression. Lastly, we show that blockade presence of actinomycin D to establish an ED50 value 0.1 ng/ml, corre- of CD4+ T cell–intrinsic TNFR2 is sufficient to promote Th17 dif- sponding to a sp. act. $ 1 3 107 U/mg. Given that sTNF is bioactive only as ferentiation under Th17-polarizing conditions. a homotrimer of noncovalently linked 17-kDa monomers, and disinte- gration of the recombinant homotrimer complex is possible, we further

tested the bioactivity of recombinant sTNF in our culture system. For these by guest on October 3, 2021 Materials and Methods analyses, pooled lymph nodes (LNs; axillary, brachial, and mesenteric) and splenocytes (5 3 106 cells/ml in complete RPMI [cRPMI]) were Mice stimulated with soluble anti–TCR-b (0.5 mg/ml), with increasing con- All animals were bred and housed under specific pathogen–free conditions centrations of reconstituted murine rTNF, and incubated at 37˚C and 5% in University of Missouri facilities that are accredited by the Association CO2. NO, released from cells and converted to NO2 in the culture medium, for Assessment and Accreditation of Laboratory Animal Care Interna- was determined using the Griess reaction, as previously described (35). tional. All experimental procedures using animals were approved by the Aliquots (100 ml) of supernatants were incubated with an equal volume University of Missouri Institutional Animal Care and Use Committee of Reagent A (1% sulfanilamide in 5% phosphoric acid) plus Reagent B and were performed in accordance with the Guide for the Use and Care (0.1%, w/v; N-1-napthylethylenediamine dihydrochloride), and absor- of Laboratory Animals. 5C.C7 mice are specific for moth cytochrome bance was measured at 543 nm using a BioTek Synergy 4 spectropho- k c (MCC) aa 88–103 and pigeon cytochrome c aa 81–104 bound to I-E tometry microplate reader (BioTek; Winooski, VT). NO2 was quantified +/+ (24). 5C.C7, 5C.C7 Il2-GFP (referred to as wild-type [WT]) (25), and using serially diluted (0–100 mM) NaNO2 as a standard (36). The data are 5C.C7 TNFR1/2 double-KO (dKO) (14, 26) mice were obtained from the expressed as mMNO2,withalimitofdetectionof0.78mM. National Institute of Allergy and Infectious Diseases contract facility + (Taconic Farms, Germantown, NY). WT mice were crossed with 5C.C7 CD4 T cell purification TNFR1/2-dKO mice to generate the following mouse models that are 2/2 +/+ +/2 LN T cells from 5–16-wk-old 5C.C7 Rag2 mice were, at times, sorted either homozygous ( ) or heterozygous ( ) for GFP: 5C.C7 TNFR1/2- for purity of CD4+CD45high or CD4+CD44lowCD62high cells (.95%) using dKO Rag22/2 Il2-GFP+/2 (Il2+/2)and5C.C7TNFR1/2-dKORag22/2 +/+ 2/2 a MoFlo XDP flow cytometer (Beckman Coulter, Pasadena, CA). T cells Il2-GFP (Il2 ) reporter mice (referred to as TNFR1/2 dKO); 5C.C7 purified from the spleens (SPLNs) of 5C.C7 mice and the LNs and SPLNs TNFR1-KO Rag22/2 Il2-GFP+/2 (Il2+/2) and 5C.C7 TNFR1-KO Rag22/2 +/+ 2/2 of C57BL/6J mice were enriched using EasySep CD4 negative selection Il2-GFP (Il2 ) reporter mice (referred to as TNFR1 KO); and 5C.C7 (STEMCELLTechnologies, Vancouver, BC, Canada), prior to MoFlo XDP TNFR2-KO Rag22/2 Il2-GFP+/2 (Il2+/2) and 5C.C7 TNFR2-KO Rag22/2 +/+ 2/2 sorting. T cells from C57BL/6J Foxp3-GFP reporter mice were further Il2-GFP (Il2 ) reporter mice (referred to as TNFR2 KO). Heterozy- + D1–12 sorted to exclude Foxp3-GFP cells. Postsort cell viability was assessed by gous TNF mutant mice, generated by G. Kollias (27), were purchased trypan blue exclusion. MoFlo XDP flow cytometer sorts typically yielded from the Italian National Research Council European Mouse Mutant Ar- .99% purity. chive at the International Campus in Monterotondo (Rome, Italy). TNFD1–12 mice were generated by replacing the endogenous TNF alleles with mutant CD4+ T cell activation tmTNF alleles that have a deletion (D) of aa 1–12 to eliminate the TNF- converting enzyme cleavage site. Lymphotoxin (LT)a gene expression in Naive T cells were isolated from cervical, axillary, brachial, and inguinal homozygous TNFD1–12 mice is comparative to that of Tnftm/wt mice (27). LNs, as well as mesenteric LNs (MLNs), of 5C.C7 Rag22/2 mice. Greater Homozygous TNFD1–12 mice were backcrossed onto the C57BL/6J back- than 90% of the freshly isolated cells from the LNs were CD4+ Tcells. ground for eight generations and then cross-bred with C57BL/6J 2D2 TCR Unless otherwise indicated, no further CD4+ T cell purification was per- Foxp3-GFP reporter mice, carrying the Va3.2/Vb11 TCR transgene (28). formed. Freshly isolated CD4+ T cells were washed in cRPMI, collected by C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, centrifugation, and plated at a density of 1 3 105 cells/ml in round-bottom ME). The Foxp3-GFP reporter mice were a gift from H. Zaghouani (Uni- 96-well plates (Corning, Corning, NY). Direct addition of soluble MCC88–103 versity of Missouri). to cell cultures was used to study Ag-induced responses. Alternatively, The Journal of Immunology 2635

plates precoated with anti-TCRb (10 mg/ml or as otherwise indicated) plus the ΔΔCT value for group i of mouse j at time k, m is the overall mean DDCT anti-CD28 or cocultures using MCC88–103 in combination with irradiated value, ti denotes the group effect, Sij denotes the effect of mouse j in group WT splenocytes as APCs were used. Cell cultures were maintained in i, gk denotes the effect of time k,(tigk) denotes the interaction between RPMI 1640 (Invitrogen Life Technologies, Grand Island, NY), supple- group i and time k,and«ijk denotes the experimental error. An F-test was mented with 10% FCS, 2 mM L-glutamine, 0.1 mM nonessential amino used to determine differences in the degradation of Il2 mRNA between acids, 100 U/ml penicillin, 100 g/ml streptomycin, 40 mg/ml gentamicin, WT and TNFR2-KO T cells. 10 mM HEPES, 1 mM sodium pyruvate, and 50 mM 2-ME. Cultures were supplemented with anti-CD28 (37.51 ascites fluid [a gift from J. Allison, Flow cytometry MD Anderson Cancer Center, Houston, TX] at a final dilution of 1/1000) for costimulation. Alternatively, cells were activated in response to PMA Cells were incubated with anti-CD16/32 prior to staining to prevent (10 ng/ml) and ionomycin (1 mM). Foxp3-GFP reporter and tmTNF- nonspecific Ab capture by FcRs. Cells were then stained directly with transgenic (Tg) mice were on the H-2b–restricted C57BL/6J background, conjugated Abs in FACS buffer (PBS containing 1% FCS, 0.05% EDTA, and some of these mice were 2D2 TCR Tg. T cells from these mice were and0.02%sodiumazide).7-AADand,insomecases,annexinVwereused isolated and purified as described above but activated in response to plate- to exclude dead cells and quantify cell viability. A FACSCalibur (BD Biosciences) and FlowJo v8.8.7 software (TreeStar, Ashland, OR) were bound TCRb and anti-CD28 or MOG35–55. used. Cytokine secretion NF-kB and AP-1 activity IL-2, IL-17A, IFN-g, and TNF were quantified by mouse Ready-SET-Go! ELISA kits (eBioscience). Cell-free culture supernatants and plasma NF-kB/Rel and JNK transcriptional activity was determined by the samples were stored at 280˚C until use. The absorbance (450 nm) minus oligonucleotide-binding capability of p50, p52, RelA (p65), RelB, Rel C, background (570 nm) of the colorimetric reactions was quantified using c-Jun, and c-Fos. The NFkB Family EZ-TFA Transcription Factor Assay a BioTek Synergy 4 spectrophotometry microplate reader (BioTek). Chemiluminescent Kit (EMD Millipore) and the TransAM AP-1 DNA-

The lower limit of sensitivity of the TNF assay was 8 pg/ml. The recogni- Binding Kit (Active Motif, Carlsbad, CA) were used. Nuclear extracts Downloaded from tion of TNF by this assay is not interfered with by 10003 excess soluble were prepared, according to the manufacturer’s recommended protocols, TNFR1 or TNFR2. from FACS-purified WT or TNFR2-KO CD4+ T cells after 2 and 40 h of stimulation with plate-bound anti-TCRb plus soluble anti-CD28. Th17 cell differentiation The Coomassie (Bradford) protein assay (Thermo Pierce, Rockford, In vivo. FACS-purified CD4+CD62highCD44low T effector cells (1 3 106) IL) was used to quantify nuclear protein. Nuclear extracts (2.5 mg) were from WT or 5C.C7 TNFR2-KO Il2-GFP+/2 and Il2-GFP+/2 mice were incubated with a biotinylated double-stranded oligonucleotide probe transferred into B10.A Rag22/2 mice. The host mice were immunized with containing either the NF-kB/Rel consensus sequence, 59-GGGACTT- http://www.jimmunol.org/ MCC88–103 (20 mg) emulsified in CFA (200 ml), as previously described TCC-39, or the AP-1 consensus sequence, 59-TGACGTCA-39,forAP-1 (37). LNs and SPLNs were harvested 4 d after immunization. CD4+ T on streptavidin-coated 96-well plates. Captured complexes, including cells were enriched (.90% purity) using EasySep CD4+ negative enrich- positive-control NF-kB or AP-1 proteins from Raji nuclear extracts or ment kits and restimulated with PMA and ionomycin for 12 h. Cell-free TNF-treated HeLa whole-cell extracts (supplied by manufacturers), were supernatants were collected and stored at 280˚C until use. IL-17A and incubated with the primary anti-rabbit Abs, followed by HRP-conjugated IFN-g cytokine secretion was quantified by ELISA. secondary Ab and tetramethylbenzidine substrate. The absorbance of the In vitro. For Th0 polarization, naive CD4+ T cells (1 3 106 cells/ml), colorimetric (450 nm) and chemiluminescence reactions were quantified purified from WT or TNFR2-KO B10.A 5C.C7 Rag22/2 Il2-GFP+/2 using a BioTek Synergy 4 spectrophotometry microplate reader. All Abs, (Il2+/2)orIl2-GFP+/+ (Il22/2) mice were stimulated for 3 d with plate- excluding c-Rel (Santa Cruz), were provided with the EZ-TFA and bound anti-TCRb (H57; 5 mg/ml) and soluble anti-CD28, with no added TransAM kits.

cytokines. For Th17 polarization, cells were polarized with IL-6 (50 ng/ml; by guest on October 3, 2021 PeproTech), TGF-b (2.5 ng/ml; R&D Systems), anti–IFN-g (XMG1.2; Quantitative real-time RT-PCR 10 mg/ml; Bio X Cell), and anti–IL-4 (11B11; 10 mg/ml; Bio X Cell), in Real time quantification of IL-17A, IL-17F, IFN-g, Foxp3, retinoic acid addition to the plate-bound anti-TCRb and soluble anti-CD28. When in- receptor-RORgt, RORa, aryl hydrocarbon receptor (AHR), and aryl hy- dicated, IL-2 (100 U/ml; Invitrogen Life Technologies) was added at the beginning of stimulation. Cells were collected at 72 h for RNA, intracel- drocarbon receptor nuclear translocator (ARNT) was performed using lular cytokine staining, flow cytometry, and ELISA. SYBRGreenPCRMasterMix(AppliedBiosystems)andaStepOneReal- Time PCR System (Invitrogen Life Technologies). Total RNAwas isolated Stability of mRNA using the RNeasy Mini Kit and reverse transcribed into cDNA using the

+ 6 High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and Naive CD4 T cells (1 3 10 ) were stimulated with anti-TCRb plus plate- random hexamer primers, according to the manufacturer’s instructions. bound anti-CD28 for 20 h. Further Il2 transcription was blocked by the Purity and concentration of RNA was determined by UV spectroscopy addition of CsA to the cell cultures to a final concentration of 200 ng/ml, using a NanoDrop 1000 Spectrophotometer (Thermo Scientific). Target without removal of the original stimulant. Cells were collected at 0, 1, 2, gene mRNA was determined by qRT-PCR following normalization to 4,6,and8haftertheadditionofCsA.TotalRNAwasisolatedusingthe b-actin using the ΔΔCT method for relative quantitation, with target gene RNeasy Mini (QIAGEN, Venlo, Limberg, The Netherlands) and re- mRNA levels in unstimulated WT cells as the calibrator (38). PCR was per- verse transcribed into cDNA using Superscript III reverse transcriptase formed using the following primer sets: AHR,59-CTGGTTGTGACAG- and random hexamer primers, according to the manufacturer’s recom- CAGATGCCT-39 (forward) and 59-CGGTCTTCTGTATGGATGAGCTC-39 mended protocol (Invitrogen Life Technologies). The level of Il2 mRNA (reverse); ARNT,59-CTCACGAAGGTCGTTCATCTGC-39 (forward) and was determined by quantitative RT-PCR (qRT-PCR) following normal- 59-CCACAAAGTGAGGTTCTCCTTCC-39 (reverse); b-actin,59-ATGGT- ΔΔ ization to the T cell–specific gene, CD3d,usingthe cycle threshold GGGAATGGGTCAGAA-39 (forward) and 59-CCATGTCGTCCCAGTTGG- (CT) method for relative quantitation with Il2 mRNA levels in unstimu- TAA-39 (reverse); Foxp3,59-CAAGGGCTCAGAACTTCTAG-39 (forward) lated cells as the calibrator (38). TaqMan probe and primers for Il2 and and 59-GGTTCAAGGAAGAAGAGGTG-39 (reverse); IFN-g,59-ACTGG- CD3d were used (Applied Biosystems, Foster City, CA). The addition of GAAAAGGATGGTGAC-39 (forward) and 59-GACCTGTGGGTTGTTGA- the general inhibitor of transcription, actinomycin D, was shown previ- CCT-39 (reverse); IL-17A,59-TCCAGAAGGCCCTCAGACTA-39 (forward) ously to stabilize Il2 mRNA degradation (39, 40). Therefore, we decided and 59-AGCATCTTCTCGACCCTGAA-39 (reverse); IL-17F,59-CCCAGG- to use CsA-induced blockage of Il2 transcription (41, 42). AAGACATACTTAGAAGAAA-39 (forward) and 59-CAACAGTAGCAAA- The exact, nonparametric Wilcoxon–Mann–Whitney U test was used to GACTTGACCA-39 (reverse); RORa,59-TTGGTCGGATGTCCAAGAAG-39 determine differences in mRNA at 0 h between WT and TNFR2-KO T cells, (forward) and 59-TGGCTGAGATGTTGTAGGTG-39 (reverse); and RORgt, using the DDCT values from qRT-PCR. To determine whether Il2 mRNA 59-CAGTCTACATGCAGAAGTGC-39 (forward) and 59-ATGTAAGTGT- degradation differed between WT and TNFRs, a generalized linear mixed GTCTGCTCCG-39 (reverse). model was used to model DDCT values obtained from qRT-PCR for the WT and TNFR2-KO T cells (n = 4, each group). The model is of the form, Statistical analyses

DDCtijk = m + tI + Sij + gk +(tigk)+«ijk Unless otherwise indicated, statistical analyses used GraphPad Instat 3 (GraphPad, La Jolla, CA). Differences were determined using the two- where i denotes the group (WT or TNFR2 KO), j denotes the mouse tailed Student t test or the nonparametric Wilcoxon–Mann–Whitney U (j =1,2,3,4,5,6,7,8), and k denotes the time (k = 0,1,2,3,6,8). DDCtijk denotes test. 2636 TNFR2 REGULATES IL-2 TO CONTROL IL-17

Results in the 39-untranslated region (UTR) (43). In addition, GFP has a tmTNF, but not sTNF, promotes Il2 transcription during CD4+ considerably longer half-life than Il2. Therefore, this genetic model T cell priming provides an ideal system to study the role of TNFR in Il2 promoter activity. In an initial set of experiments, single-cell suspensions, We previously showed a lower frequency of IL-2–producing CD4+ consisting of .90% CD4+ T cells, from pooled LNs isolated from T cells in TNFR1/2-dKO mice (14). To directly study the effect of 2 2 homozygous Il2-GFP reporter (IL-2 / ) mice were activated with T cell–intrinsic TNFR1 and TNFR2 on Il2 transcription, we now plate-bound anti-TCRb plus anti-CD28 in the presence or absence use Il2-GFP reporter mice (25). These mice were originally gen- of increasing concentrations (0–300 ng/ml) of recombinant sTNF erated by replacing the Il2 gene with a single copy of a cDNA en- (Fig. 1). Under these conditions, ∼ 75% of all CD4+ T cells tran- coding GFP and subsequent breeding onto the B10.A 5C.C7 TCR scribed the Il2 gene, as reflected by GFP expression. The addition of 2 2 Rag2 / background (14). The gfp transcripts in these Tg mice exogenous sTNF to the cell cultures did not influence Il2 promoter lack the UA-rich sequence instability elements that normally reside activity, as determined by the time of onset of GFP expression, the Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 1. Increased IL-2 production by CD4+ T cells in response to tmTNF but not sTNF. (A) The addition of sTNF during primary activation did not alter the frequency of IL-2 producers or the magnitude of Il2 promoter activity in Vb3+CD4+7-AAD2-gated T cells from B10.A 5C.C7 TCR-Tg Il2-GFP 2 2 homozygous (Il2 / ) reporter mice. Freshly isolated LN cells (1 3 106 cells/ml; 200 ml/well; .90% Vb3+CD4+ T cells) were stimulated with plate-bound anti-TCRb plus soluble anti-CD28 supplemented with increasing concentrations of sTNF (0, 3, 10, 30, 100, or 300 ng/ml) for 12, 24, 36, 48, 60, and 72 h. TNF was added directly to the cell cultures at the time of activation. (B) The experiments in (A) were repeated, with the exception that sTNF (0, 10, or 100 ng/ml) was added to the cell cultures 24 h after the T cells were stimulated. The cells were harvested 36 h thereafter and stained for CD4 and Vb3; 7-AAD2Vb3+CD4+ cells were gated for analyses by flow cytometry. Values shown represent the mean 6 SEM of three independent experiments in triplicate (three mice/group/experiment). Addition of sTNF did not yield a statistically significant difference. (C) Culture supernatants were tested for levels of NO to validate sTNF bioactivity. Values shown represent the mean 6 SEM of three experiments, performed in triplicate, of total splenocytes (106 cells/ml; 200 ml/plate) or pooled axillary and brachial LNs (106 cells/ml; 200 ml/plate) from two 5C.C7 Il2-GFP+/2 mice/experiment stimulated with plate-bound anti-TCRb plus anti-CD28 in the presence or absence of increasing concentrations of sTNF (0, 3, 10, 30, 100, or 300 ng/ml) for 48 h. The percentages (D)and total numbers (E)ofCD4+Foxp32,CD8+,andCD4+Foxp3+ T cells from freshly isolated cervical, axillary, brachial, and inguinal LNs, MLNs, and SPLNs from C57BL/6 WT and tmTNF Foxp3-GFP reporter mice were compared. 7-AAD2 lymphocytes were gated for analyses by flow cytometry. (F)IL-2 2 secretion differs between WT and tmTNF CD4+ T cells. FACS-purified CD4+CD45RbhighFoxp3 T effector cells (1 3 105 cells/well) from 2D2 WT 6 or tmTNF mice were stimulated with irradiated splenocytes (1 2 10 cells/well) from WT C57BL/6J mice and cognate MOG35–55 (10 mM). IL-2 secretion from cell-free culture supernatants was quantified by ELISA at times up to 72 h. Line graphs depict the results of three independent experiments performed in triplicate (mean 6 SEM) (G) Representative staining patterns of CD4+Foxp32 and Foxp3+CD4+ T cells among total peripheral LNs from WTand tmTNF mice are shown, with the frequencies (mean 6 SEM) of CD4+Foxp32 T cells from four mice of each strain indicated (left panels). Viable (7-AAD2)CD4+ T cells were gated for tmTNF expression and analyzed by flow cytometry (right panel). The bar graph summarizes the tmTNF MFI (mean 6 SEM) on CD4+ Foxp32 cells from WT and tmTNF mice before and after stimulation for the times indicated. The results from two independent experiments (three mice/ experiment; cultured separately) are shown. (H) Representative staining patterns of surface TNFR2 expression on FACS-purified CD4+Foxp32 T cells from (G) following stimulation for 48 h. The MFIs (mean 6 SEM) of TNFR2 from two independent experiments (three mice/experiment; cultured separately) are indicated. (I) Representation of CD69 expression patterns on the FACS-purified CD4+Foxp32 T cells from (G)stimulatedfor48h.TheMFIs(mean6 SEM) of CD69 from two independent experiments (three mice/experiment; cultured separately) are shown. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 2637 frequency of IL-2 producers, and the mean fluorescence intensity draining LNs and SPLNs 24 h following immunization with (MFI) of individual IL-2 producers (Fig. 1A). Although slight MCC (300 mg) and LPS (10 mg). Viable CD4+ T cells were gated shifts in the frequency and MFI of GFP+ CD4+ T cells were ob- to analyze IL-2–GFP and cell surface CD69 expression by flow served at the later times following TNF treatment, these effects cytometry (Fig. 2A). The percentage of IL-2–producing CD4+ were without dose response or statistical significance (p . 0.05) T cells was reduced following total, but not soluble, TNF block- (Fig. 1A). Given that CD4+ T cells upregulate TNFR1 and TNFR2 ade (p , 0.05) (Fig. 2B). Neither of the TNF-blocking treatments following activation (14), we next determined whether the addi- inhibited the magnitude of IL-2 production per cell, as determined tion of exogenous TNF subsequent to, rather than at the time of, by Il2-GFP MFI levels (p . 0.05) (Fig. 2C). Despite normal levels T cell activation was more effective in promoting Il2 promoter of IL-2 production, CD25 expression on CD4+-gated cells was re- activity. Accordingly, the first set of experiments was repeated, duced by both total TNF– and sTNF-selective blockade (p , 0.05) with the exception that sTNF was added 24 h following T cell (Fig. 2D). CD4+ T cell expression of CD69 also was unaffected activation, coinciding with elevated surface TNFR2 expression on by TNF blockade (p . 0.05) (Fig. 2E). Inhibition of IL-2 pro- CD4+ T cells (14). Cells were collected 64 h after stimulation. duction by TNFR2:Fc, but not XPro1595, was further revealed Similar to the first set of experiments, no change in IL-2 production by reduced circulating levels of IL-2 (Fig. 2F). Overall, these in response to sTNF was observed (Fig. 1B). Increased NO pro- studies suggest that tmTNF, but not sTNF, promotes IL-2 produc- duction in splenic cultures validated bioactivity of the sTNF in our tion. These observations are consistent with a model whereby culture conditions (Fig. 1C). In contrast to the splenocyte cultures, tmTNF/TNFR2 provides a third signal to costimulate Il2 promoter little NO was produced by LN cultures (Fig. 1C). These results are activity. consistent with previous reports demonstrating NO production in TNFR2, but not TNFR1, augments Il2 promoter activity during Downloaded from SPLNs but not LNs (44). + Although tmTNF preferentially binds to TNFR2, sTNF pri- the priming of CD4 T cells marily binds to TNFR1. Further, these two TNFRs activate distinct tmTNF is more effective in activating TNFR2 than is sTNF (16, downstream signaling cascades. To test the hypothesis that tmTNF, 17, 45). TNFR1 and TNFR2 activate distinct, albeit sometimes but not sTNF, selectively costimulates Il2 expression in CD4+ convergent, downstream signaling (17). Therefore, we evaluated T cells, we crossed memTNFD1–12 mice, encoding an uncleavable the role of these two TNFRs in Il2 transcription. New mouse mod- tmTNF (27), with Foxp3-GFP mice (28) to generate tmTNF els were generated by crossing 5C.C7 Il2-GFP mice to 5C.C7 http://www.jimmunol.org/ Foxp3-GFP reporter (tmTNF) mice. These tmTNF mice were then TNFR1/2-dKO mice to generate Ag-specific Il2-GFP mice that bred onto the MOG35–55-specific 2D2 TCR background to provide were deficient for either TNFR1 and/or TNFR2, as described an Ag-specific model to assess the role of tmTNF in IL-2 pro- in Materials and Methods. Lymphocytes (.90% Vb3+CD4+ duction. The percentage of Foxp32CD4+ T cells in LN (p , 0.05) T cells) from the LNs or SPLNs of Il2-GFP (WT), TNFR1-KO and SPLN (p , 0.001) were reduced in tmTNF mice relative to Il2-GFP (referred to as TNFR1-KO), TNFR2-KO Il2-GFP (TNFR2- age- and gender-matched WT cohorts. The frequencies of LN and KO), or TNFR1/2-dKO Il2-GFP (referred to as TNFR-dKO) mice + + MLN, but not SPLN, Foxp3 CD4 Tregs also were reduced in were stimulated with increasing concentrations of MCC88–103 tmTNF mice compared with WT mice (p , 0.05) (Fig. 1D). In (Fig. 3). Mice that are homozygous for the GFP allele do not by guest on October 3, 2021 contrast to CD4+ T cells, the frequencies of MLN and SPLN, but transcribe Il2 mRNA (25). Further, IL-2 signaling may indirectly not LN, CD8+ T cells were lower in tmTNF mice compared with impact GFP expression. Therefore, for these studies, homozygous WT mice (p , 0.05) (Fig. 1D). These changes coincided with and heterozygous Il2-GFP mice were compared. The frequency a decrease in the total number of Foxp32CD4+ T cells in the SPLN of IL-2 producers (percentage of CD4+ Il2-GFP+) (Fig. 3A) and (p , 0.05), as well as the total number of Foxp3+CD4+ Tregs in the the magnitude of Il2 transcription (Il2-GFP MFI of the CD4+ Il2- SPLN and MLN (p , 0.05). The number of MLN or SPLN CD8+ GFP+ cells) (Fig. 3B) were quantified by flow cytometry from T cells did not differ between WT and tmTNF mice (p . 0.05) 7-AAD2 CD4+-gated cells. These studies revealed similar Il2 pro- (Fig. 1E). Collectively, these observations indicate that selective moter activity in GFP homozygous and heterozygous WT mice, blockade of sTNF is sufficient to perturb T cell homeostasis, in- suggesting IL-2R signaling–independent Il2 promoter activity. cluding Tregs, in secondary lymphoid organs but does not induce The reason for the decline in GFP MFI after 48 h is not completely overt inflammation or spontaneous autoimmunity. clear; however, cell division and GFP degradation may be con- To determine whether CD4+ T cell–intrinsic tmTNF plays tributory factors. In comparing homozygous and heterozygous a regulatory role in IL-2 production, naive CD4+CD45Rbhigh Il2-GFP cells at 48 h, the MFI of homozygous Il2-GFP producers Foxp3-GFP2 T cells from 2D2 WT or tmTNF mice were FACS (Fig. 3B, upper panel) differed from heterozygous Il2-GFP cells purified and activated with MOG35–55 in the presence of irradi- (Fig. 3B, lower panel) at the highest Ag dose (p , 0.05). Neither ated T cell–depleted WT C57BL/6J splenocytes. IL-2 secretion the absence of Il2 promoter activity in unstimulated T cells nor the was quantified by ELISA at time points up to 72 h. CD4+ T cells rate of onset of Il2 induction in response to T cell activation was from tmTNF mice produced more IL-2 (Fig. 1F) and expressed markedly affected by TNFR1 or TNFR2 deficiency. Moreover, more cell surface TNF (Fig. 1G), but not TNFR2 (Fig. 1H), rel- consistent with the two-step quantitative biallelic model of Il2 ative to WT CD4+ T cells. CD69 expression did not differ be- promoter activity (14, 46), the number of IL-2 producers was equiv- tween WT and tmTNF CD4+ T cells (p . 0.05), implying that alent for homozygous and heterozygous Il2-GFP mice. However, in increasedproductionofIL-2did not positively correlate with contrast to heterozygous cells, the MFI of individual cells with two TCR signaling (Fig. 1I). The absence of sTNF secretion by tmTNF GFP alleles increased with the strength of Ag stimulation. Sub- CD4+ T cells following T cell activation was confirmed by ELISA optimal T cell stimulation, delayed kinetics of GFP production (data not shown). (14), and cell division are possible contributors to the ,2-fold in- To further investigate differential regulation of IL-2 production crease in GFP MFI in homozygous versus heterozygous CD4+ Il2- by sTNF and tmTNF, 5C.C7 Il2-GFP+/2 (Il2+/2) mice were admin- GFP+ Tcells. istered XPro1595 (10 mg/g) or TNFR2:Fc (etanercept; 10 mg/g) Comparison of TNFR-sufficient (WT) CD4+ T cells with s.c. prior to immunization to pharmacologically block sTNF or TNFR1-KO, TNFR2-KO, and TNFR1/2-dKO CD4+ T cells iden- total TNF, respectively. CD4+ T cells were collected from the tified TNFR2, but not TNFR1, as a major contributor to IL-2 pro- 2638 TNFR2 REGULATES IL-2 TO CONTROL IL-17

A

FIGURE 2. Blockade of tmTNF inhibits IL-2 production in vivo. (A) Draining LNs, SPLNs, B D and sera collected from PBS (VEH)-treated, XPro1595 (10 mg/g, s.c.)-treated, or TNFR2:Fc (10 mg/g, s.c.)-treated B10.A 5C.C7 Il2-GFP+/2 (Il2+/2) mice 20 h following immunization with

MCC88–103 and LPS (10 mg/g) associate a block- ade of tmTNF with impaired IL-2 production. (B– D) Each symbol represents an individual mouse. d, live CD4+-gated cells isolated from draining LNs; ♦, live CD4+-gated cells isolated from C E SPLNs. Horizontal lines represent the geometric B + means. ( ) The percentage of Il2-GFP cells of Downloaded from total live CD4+ T cells was determined by flow cytometry. (C) The MFI of Il2-GFP was deter- mined from live GFP+CD4+-gated T cells in (B). (D and E) All (100%) of the live LN and splenic CD4+ T cells from VEH-pretreated mice were CD25+CD69+, as determined by flow cytometry.

Therefore, the MFI of CD25 (D) and CD69 (E) for http://www.jimmunol.org/ all treatment groups was determined by gating on total live CD4+ T cells. (F)Serumwascol- lected from the retro-orbital sinus at necropsy. The concentration of secreted IL-2 protein was determined by ELISA. Each symbol represents F an individual mouse; the mean concentrations of IL-2 (pg/ml) are indicated by horizontal lines. VEH, n = 4 mice; TNFR2:Fc, n =5mice, XPro1595, n =4mice. by guest on October 3, 2021

duction (Fig. 3C). Regardless of the Ag strength, TNFR2-KO tween WT LNs (60.3 6 3.87%, mean 6 SEM) and WT SPLNs cells yielded nearly 1.8–1.9–fold fewer IL-2 producers relative (66.1 6 8.75%, mean 6 SEM) (p . 0.05) (Fig. 3E, middle to WT cells (p , 0.05). Although the deficiency in TNFR1 alone panels),butitwasreducedinTNFR2-KOCD4+ T cells (Fig. 3E, resulted in a modest reduction (p . 0.05)ofIL-2producersat right panels)(p , 0.001). ELISA assays further revealed reduced the early 24-h time point, regulation by TNFR1 was not evident IL-2 protein secretion by TNFR2-KO CD4+ T cells compared at later time points during maximal IL-2 production (Fig. 3C). with WT cells (Fig. 3F). Cell survival, as determined by 7-AAD In contrast to TNF blockade in vivo, Il2-GFP was reduced in and annexin V staining, did not differ between WT and TNFR2- TNFR2-KO and TNFR1/2-dKO CD4+ T cells following stimula- KO T cells (data not shown). Collectively, these results suggest tion with 1 mM, but not 10 mM or 0.1 mM, MCC (Fig. 3D). Together, that CD4+ T cell–intrinsic TNFR2, but not TNFR1, signaling con- these studies identify TNFR2 as a regulator of Il2 transcription tributes to maximal Il2 promoter activity. in CD4+ T cells. TNFR signaling in CD4+ T cells: a role for NF-kB + CD4 T cell–intrinsic TNFR2 augments IL-2 production Although the integration of signaling cascades controlling Il2 It is plausible that TNFR2 ligands, including TNF-a,LT-a,and induction remains enigmatic, NF-kB/Rel and JNK protein bind- PGRN, bind to TNFRs located on non-T cells to promote IL-2 ing has been mapped to the first 300-bp minimal essential reg- production (47–49). Therefore, we asked whether TNFR2 func- ulatoryregionoftheIl2 promoter (14, 50–52). NF-kBandJNK tions in a CD4+ T cell–intrinsic manner to regulate Il2 promoter cascades are activated downstream of TNFR2 activation, and im- activity. For these experiments, FACS-purified naive CD4+ T paired c-Rel binding at the proximal Il2 promoter was reported cells from LNs or SPLNs of WT or TNFR2-KO 5C.C7 Il2-GFP+/2 for TNFR1/2-dKO T cells (14). Therefore, we postulated that mice were stimulated with anti-TCRb and anti-CD28. The fre- TNFR1 and TNFR2 may regulate different NF-kB/Rel and JNK quency of IL-2 producers (CD4+GFP+ T cells) was similar be- signaling pathways in CD4+ T cells. To assess NF-kB/Rel activity, The Journal of Immunology 2639

A B

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D http://www.jimmunol.org/ by guest on October 3, 2021 E F

FIGURE 3. TNFR2 costimulates Il2 promoter activity during priming of CD4+ T cells. (A and B) The kinetics of Il2 promoter activation during the priming is Ag-concentration dependent. Naive CD4+ T cells were isolated from 5C.C7 Il2-GFP+/2 (Il2+/2)or5C.C7Il2-GFP2/2 (Il22/2)miceand 5 + activated in vitro (2 3 10 cells/well, .90% CD4 T cells; 96-well round-bottom plate) in response to increasing concentrations of MCC88–103 (0,0.1,1,and 10 mM). Cells were harvested prior to (0 h) or 24, 36, 48, and 64 h after T cell activation. (A) The percentage of Il2-GFP+ cells of total live (7-AAD2)CD4+ T cells was determined by flow cytometry. The mean 6 SEM of three independent experiments (three mice/group/experiment) is shown. 10.01 mM . 0 mM, p , 0.05; 21 mM . 0 mM, p , 0.05; 310 mM . 0 mM, p , 0.05. (B) The MFI of Il2-GFP was determined from live GFP+CD4+-gated T cells in (A). 310 mM . 0.01 mM, p , 0.05. (C and D) Genetic deficiency in TNFR2, but not TNFR1, impairs Il2 promoter activity. Naive CD4+ T cells were obtained from TNFR1- and TNFR2-sufficient ( WT), TNFR1-deficient (TNFR1 KO), TNFR2-deficient (TNFR2 KO), or TNFR1/2-dKO (TNFR dKO) 5C.C7 Il2-GFP2/2 (Il22/2) mice. Single-cell suspensions (1 3 105 cells/ml; 200 ml/well; .90% CD4+ T cells) were either left unstimulated (0 mM) or stimulated with 10 mMMCC(left panels), 1 mMMCC(center panels), or 0.1 mMMCC(right panels) for 24, 36, 48, and 64 h. Cells were stained for CD4 and 7-AAD and 7-AAD2CD4+ cells were gated for analyses. (C) The percentage of Il2-GFP+ cells of total live (7-AAD2)CD4+ T cells was determined by flow cytometry. The mean 6 SEM of four independent experiments (three mice/group/experiment) is shown. 2TNFR2 KO , WT, p , 0.05; 3TNFR dKO , WT, p , 0.05. (D) The MFI of Il2-GFP was determined from live GFP+CD4+-gated T cells in (C). 2TNFR2 KO , WT, p , 0.05; 3TNFR dKO , WT, p , 0.05. (E and F)CD4+ T cell–intrinsic TNFR2 costimulates maximal IL-2 production. Naive CD4+CD45Rbhigh T cells were FACS purified from LNs and SPLNs of TNFR2-sufficient (WT) or 2 2 TNFR2-deficient (TNFR2-KO) 5C.C7 Il2-GFP+/ (Il2+/ ) mice and then stimulated in vitro (2 3 105 cells/well; 96-well round-bottom plate) with plate- bound anti-TCRb plus anti-CD28 for 64 h. The results of three independent experiments (three mice/group/experiment) are shown. (E) The percentage of Il2-GFP+ cells of total live CD4+ T cells was determined by flow cytometry. The mean (6 SEM) frequency of Il2-GFP+ cells is indicated. (F) The con- centration of IL-2 was determined by ELISA from tissue culture supernatants of the samples collected in (E). *p , 0.05. 2640 TNFR2 REGULATES IL-2 TO CONTROL IL-17 the binding of RelA (p65), RelB, c-Rel, p50 (NF-kB1), and p52 canonical and noncanonical NF-kB pathways to promote Il2 in- (NF-kB2) to the kB consensus oligonucleotide, 59-GGGAC- duction in CD4+ T cells.

TTTCC-39, was determined. In a similar manner, JNK/MAPK + activity was determined by recruitment of c-Fos and c-Jun to the CD4 T cell–intrinsic TNFR2 controls the stability of Il2 AP-1 consensus sequence, 59-TGACGTCA-39. For these studies, mRNA FACS-purified CD62highCD44lowCD4+ T cells (purity . 96%) In activated CD4+ T cells, transcriptional mechanisms operate in from 5C.C7 WT, TNFR1-KO, and TNFR2-KO mice were stim- parallel with posttranscriptional mRNA stabilization to control ulated with anti-TCRb plus anti-CD28 (14). Prior to T cell stim- IL-2 production. JNK pathways target 39-and592UTRs of the ulation, constitutive NF-kB and AP-1 DNA binding did not differ Il2 locus to control Il2 mRNA turnover (53). Therefore, we next among WT, TNFR1-KO, and TNFR2-KO T cells (Fig. 4A, 4B). determined whether TNFR2 regulates Il2 mRNA stability. For In all cases, with the exception of c-Rel and p52 in TNFR2-KO these studies, naive WTor 5C.C7 TNFR2-KO CD4+ T cells were T cells, DNA binding increased following T cell stimulation stimulated with anti-TCRb plus anti-CD28 for 20 h. Further (Fig. 4B). Although DNA binding of RelA increased following transcription was blocked by the addition of CsA, a pharmaco- T cell stimulation in all three cell types, the magnitude of bind- logical inhibitor of the Ca2+-sensitive phosphatase calcineurin, ing was substantially reduced in TNFR2-KO cells compared with and Il2 mRNA levels were determined over time by qRT-PCR. WT and TNFR1-KO T cells (p , 0.05). These observations are Tcellactivationresultedina1.63-foldincreaseinsteady-state consistent with our previous results showing impaired c-Rel bind- Il2 mRNA in WT T cells compared with TNFR2-KO T cells prior ing to the proximal Il2 promoter in TNFR1/2-dKO T cells (14). to the addition of CsA. After blocking transcription, the decay Together, these results suggest that TNFR2 signaling activates of steady-state Il2 mRNA was more prominent in TNFR2-KO Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 4. DNA-binding activity of NF-kB/Rel proteins and stability of Il2 mRNA differs between TNFR1-KO and TNFR2-KO CD4+ T cells. (A and B) Oligonucleotide-binding assays were used to determine the influence of TNFR1 and TNFR2 on NF-kB/Rel and JNK activity in CD4+ T cells. CD4+ Tcells from 5C.C7 Il2-GFP+/2 (Il2+/2) WT, TNFR1-KO, or TNFR2-KO mice were left unstimulated or stimulated with anti-TCRb plus soluble anti-CD28 for 4 h or 36 h. Nuclear extracts were incubated with a biotinylated double-stranded oligonucleotide probe containing the NF-kB/Rel consensus sequence, 59- GGGACTTTCC-39, or the AP-1 consensus sequence, 59-TGACGTCA-39, on streptavidin-coated plates. Captured complexes were incubated with the primary anti-rabbit Abs for c-Fos and c-Jun (A) or p50, RelA, c-Rel, RelB, and p52 (B). Primary Ab capture complexes were incubated with HRP-conjugated secondary anti-rabbit Ab. The c-Jun– and c-Fos–containing complexes were detected by colorimetric absorbance (450 nm), and the NF-kB/Rel complexes were detected by chemiluminescence. Results are depicted as fold increase in relative absorbance normalized to unstimulated WT controls. The data are expressed as the mean 6 SEM of triplicate experiments, with duplicate reactions in each experiment. (C) CD4+ T cell–intrinsic TNFR2 stabilizes Il2 mRNA. Naive CD4+ T cells were purified (.95% Vb3+CD4+ T cells) from 5C.C7 Il2-GFP+/2 (Il2+/2) WTor TNFR2-KO mice using EasySep mouse CD4+ T cell negative isolation kits (STEMCELL Technologies). Cells (1 3 106 cells/well) were activated in anti–TCRb-coated (10 mg/ml) six-well plates in 2 ml of cRPMI containing soluble anti-CD28. After16 h,CsAwas added ata final concentration of200ng/ml. Cells were harvestedformRNA isolationat the timeofCsAaddition (0h)andat

1, 2, 4, 6, and 8 h after CsA addition. The ΔΔCT method was used for relative quantitation of steady-state mRNA. Values are expressed as log10 relative units following normalization to CD3d using the Il2 mRNA levels in unstimulated cells as the calibrator. Determination of the half-life for mRNA decay is described in Materials and Methods. Values shown represent mean 6 SEM of four independent experiments using two mice/group/experiment. *p , 0.05. The Journal of Immunology 2641

T cells (T1/2 =19min,48s)thaninWTTcells(T1/2 =28min,33s) A (Fig. 4C). Overall, these results reveal that T cell–intrinsic TNFR2 signaling controls IL-2 production through transcriptional and posttranscriptional mechanisms. Regulation of in vivo IL-2 production by TNFR2 To further study the regulation of Il2 promoter activity in vivo, WT or TNFR2-KO Il2-GFP+/2 mice were immunized with MCC plus LPS, a potent activator of TNF. Both naive WT and TNFR2-KO CD4+ T cells required stimulation for Il2 transcrip- tion to occur (Fig. 5A). At 8 h, 33 6 5% (mean 6 SEM) of SPLN and 21 6 2% of LN CD4+ T cells from WT mice expressed GFP, and these values were lower in TNFR2-KO T cells (p , 0.05). By 36 h, the frequency of Il2-GFP–expressing CD4+ T cells declined in the WT mice (19 6 2%, SPLN; 16 6 2%,LN),andthisre- duction was even greater in TNFR2-KO mice (12 6 1%, SPLN; 8 6 1%, LN, p , 0.001). Similar to the in vitro experiments, TNFR2 deficiency did not alter the MFI of GFP+ cells (Fig. 5A). The reduced frequency of Il2-GFP+ cells in TNFR2-KO mice compared with WT mice did not correlate with a change in the Downloaded from number of CD4+ T cells (data not shown) or robustness of stim- ulation through the TCR, as determined by CD69 expression (Fig. 5B). In fact, CD69 expression following T cell activation was enhanced in TNFR2-KO T cells relative to WT T cells (Fig. 5B). CD69 expression did not differ (p . 0.05) between WT and TNFR2-KO CD4+ T cells prior to T cell activation (data http://www.jimmunol.org/ not shown). Although most commonly viewed as a stimulatory molecule that is expressed upon T cell activation, CD69 may also participate in feedback suppression of an immune response B through the upregulation of TGF-b.TGF-b can inhibit IL-2 production (54). Furthermore, TNF antagonizes TGF-b signal- ing (55), and TNFR2 regulates Il2 expression in a CD4+ T cell– autonomous manner. However, in our studies, TGF-b1(LAP) expression did not differ between TNFR2-KO and WT CD4+ by guest on October 3, 2021 T cells. These results are consistent with a T cell–intrinsic TGF- b–independent mechanism for suppression of IL-2 production in TNFR2-KO T cells. Additional surface molecules, which are also upregulated by NF-kB signaling and costimulate IL-2 produc- tion, were examined (Fig. 6B–E). These studies revealed de- creased expression of CD25, CD26, and CD28 by LN, but not splenic, TNFR2-KO T cells in comparison with WT T cells. In- terestingly, elevated LAP expression by splenic T cells correlated with decreased CD25, CD26, and ICOS expression. Taken to- FIGURE 5. Regulation of in vivo IL-2 production by TNFR2. 5C.C7 gether, these data imply that TNFR2 deficiency is sufficient to Il2-GFP+/2 (Il2+/2) WT or TNFR2-KO mice were immunized with MCC disrupt Il2 promoter activity. These results further suggest pos- (300 mg) plus LPS (10 mg). (A) Representative line graphs depicting GFP 2 sible regulatory cross-talk between T cell–intrinsic TGF-b and expression in 7-AAD CD4+-gated T cells recovered from LN (left panels) TNFR2onCD25,CD26,CD28,andICOSexpressioninacti- and SPLN (right panels) following no immunization (top panels)or8h vated CD4+ T cells. It remains to be determined whether TNFR2 (middle panels)or36h(bottom panels) of immunization. The first number in each panel represents the percentage (mean 6 SEM) of Il2-GFP+ cells directly or indirectly regulates Il2 induction through modulation from three separate experiments performed in triplicate. The second number of these, or other, costimulatory molecules. in each panel indicates the MFI (mean 6 SEM) of the Il2-GFP+ cells. WT + + Impaired Il2 expression in TNFR2-KO CD4+ T cells promotes CD4 T cells are indicated by solid lines, and TNFR2-KO CD4 T cells are indicated by dashed lines. Results represent three separate experiments IL-17 production performed in triplicate. (B)CD4+ T cells recovered from the LN and SPLN of IL-2 inhibits the generation of Th17 cells, which secrete IL-17 immunized mice in (A), stained for CD69 expression. 7-AAD2CD4+GFP2 2 and express the transcription factor ROR-gt (37, 56). Therefore and 7-AAD CD4+GFP+ cells were gated for analyses. MFI (mean 6 SEM) we asked whether the deficiency of IL-2 production in TNFR2 of CD69 expression after 36 h of stimulation. KO CD4+ T cells was reciprocated by increased IL-17 produc- tion. For these experiments, CD4+ T cells from WT or TNFR2-KO supernatants by ELISA. In support of our hypothesis, CD4+ T 5C.C7 TCR Il2-GFP+/2 (Il2+/2)orIl2-GFP+/2 (Il2+/2) donors were cells from TNFR2 Il2+/2 mice had a marked increase in IL-17 pro- adoptively transferred to B10.A Rag22/2 recipient mice. Four duction compared with WT Il2+/2 cells (p , 0.05) (Fig. 7A). The days after immunization of the recipient mice with MCC emul- increase in IL-17 production by TNFR2-KO Il2+/2 T cells in- sified in CFA, CD4+ T cells were isolated from draining LNs versely correlated with reduced IFN-g production compared with and SPLNs and restimulated in vitro with PMA and ionomycin. WT Il2+/2 cells (p , 0.05) (Fig. 7B). To more clearly determine IL-17 and IFN-g protein secretion was determined from culture whether IL-2 was responsible for the impaired IL-17 production, 2642 TNFR2 REGULATES IL-2 TO CONTROL IL-17

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FIGURE 6. Expression of T cell surface co- stimulatory proteins in response to CD4+ T cell autonomous TNFR2 deficiency. CD4+ T cells recovered from the draining LNs and C SPLNs of the immunized 5C.C7 Il2-GFP+/2 (Il2+/2) WT or TNFR2-KO mice described in Fig. 5 were stained for cell surface CD4 Downloaded from and LAP (A), CD25 (B), CD26 (C), CD28 (D), or ICOS (E). 7-AAD2CD4+GFP2-gated (left panels) and 7-AAD2CD4+GFP+-gated (middle panels) cells were analyzed for comparison. Representative flow cytometric line graphs of D thedataareshown.MFI(mean6 SEM) of

costimulatory molecules from three individ- http://www.jimmunol.org/ ual mice are depicted in the bar graphs (right panels).

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we compared the differentiation of naive CD4+ T cells from mented IL-17 production, whereas blockade by XPro1595 re- WTand TNFR2-KO Il22/2 mice. In contrast to CD4+ T cells from flected an overexpression of noncleavable TNF in Tg mice (Fig. 7D). TNFR2-KO Il2+/2 mice, T cells from TNFR2-KO Il22/2 mice did In contrast, neither TNF-blocking treatment markedly altered not produce increased amounts of IL-17. Instead IL-17 production IFN-g production upon restimulation (p . 0.05) (Fig. 7E). by TNFR2-KO Il22/2 T cells was reduced compared with WT To extend our in vivo studies, we explored a causal relationship Il22/2 T cells (Fig. 7A). In contrast to TNFR2-KO Il2+/2 T cells, between impaired IL-2 production and Th17 development in re- reduced IL-17 production by TNFR2-KO Il22/2 T cells was not as- sponse to TNFR2 blockade by determining whether increased sociated with increased IFN-g production compared with WT IL-17 production in TNFR2-KO CD4+ T cells was reversed with Il22/2 CD4+ T cells (Fig. 7B). IL-2 was barely detectable in the the addition of exogenous IL-2. For these studies, naive CD4+ supernatants of IL-2+/2 cells following restimulation and was not T cells isolated from 5C.C7 Il2+/2, Il22/2,TNFR2-KOIl2+/2,or detected in IL-22/2 cultures (data not shown). Overall, these results TNFR2-KO Il22/2 mice were stimulated for 4 d with plate-bound suggest that tmTNF and TNFR2, but not sTNF or TNFR1, nega- anti-TCRb and anti-CD28 under Th17-polarizing conditions tively regulate Th17 differentiation, at least in part, through IL- (TGF-b1, IL-6, anti–IFN-g, and anti–IL-4) in the presence or ab- 2–dependent mechanisms. To further test our hypothesis that sence of exogenous IL-2 (100 U). Consistent with previous reports, TNFR2 costimulation negatively regulates Th17 differentiation, cell surface expression of TNFR2 was upregulated on live-gated we investigated IL-17 production following TNFR2 blockade in CD4+ T cells cultured under Th0 conditions (anti–TCRb +anti– WT mice. For these studies, B10.A Rag22/2 recipients were pre- CD28) but the magnitude of TNFR2 expression was markedly el- treated with TNFR2:Fc (10 mg/g) or XPro1595 (10 mg/g) to block evated on cells cultured under Th17 conditions (Fig. 7F). Neither total TNF or only sTNF, respectively. Following adoptive transfer genetic Il2 deficiency nor addition of exogenous IL-2 affected the of naive WT or TNFR2-KO Il2+/2 CD4+ T cells, the recipients were frequency of CD4+ T cells that upregulated TNFR2. The MFI of immunizedwithMCCemulsifiedinCFA(Fig.7C).Fourdays TNFR2 expression was also unaffected. These results reveal that, later, CD4+ T cells were isolated from the SPLNs and draining LNs in addition to CD4+ Foxp3 Tregs (57), TNFR2 is highly expressed and restimulated in vitro. As expected, TNFR2:Fc treatment aug- by Th17 cells. Further, the regulation of TNFR2 on Th17 cells does The Journal of Immunology 2643

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FIGURE 7. CD4+ T cell–intrinsic TNFR2 promotes Il2 expression to inhibit Th17 differentiation. B10.A Rag22/2 recipient mice were reconstituted 2 2 2 2 2 2 with 1 3 106 CD4+CD45Rbhigh cells from WT and TNFR2-KO 5C.C7 Il2-GFP+/ (Il2+/ ) or WT and TNFR2-KO 5C.C7 Il2-GFP / (Il2 / ) donors. + The recipients were immunized with 20 mgMCC88–103 emulsifiedin200mlofCFA.CD4 T cells from the SPLNs and draining LNs were harvested 4 d after immunization and rechallenged with PMA + ionomycin in vitro for 12 h. IL-17A (A)andIFN-g (B) were quantified from cell-free culture supernatants using ELISA. The results shown represent the mean 6 SEM of three separate experiments performed in duplicate. (C) Experimental design used to assess the role of TNF in wild-type mice using pharmacological blockade. For the results in (D)and(E), B10.A Rag22/2 mice were administered (s.c.) 10 mg/g XPro1595, 10 mg/g etanercept (TNFR2:Fc), or 200 mlPBS(VEH)1and4dpriortoadoptive transfer of naive CD4+ T cells from WT 5C.C7 Il2-GFP+/2 +/2 (Il2 ) donors. The recipients were immunized (20 mgMCC88–103 emulsified in 200 ml of CFA) the following day and received a third treatment (TNF blockade or VEH control) 48 h thereafter. CD4+ T cells were collected from the draining LNs and SPLNs 4 d following immunization and re- challenged with PMA + ionomycin in vitro for 12 h. IL-17 (D)andIFN-g (E) were quantified from the culture supernatants by ELISA. Each symbol represents an individual mouse. Horizontal lines represent the geometric means. (F–I) To determine whether enhanced production of IL-17 by TNFR2-KO CD4+ T cells could be reversed by the addition of exogenous IL-2, naive CD4+ T cells isolated from 5C.C7 Il2+/2, Il22/2, TNFR2-KO Il2+/2,orTNFR2-KO Il22/2 mice were stimulated for 4 d with plate-bound anti-TCRb (5 mg/ml) and anti-CD28 under Th17-polarizing conditions (2.5 ng/ml TGF-b1, 50 ng/ml IL-6, 10 mg/ml anti–IFN-g, and 10 mg/ml anti–IL-4) in the presence or absence of exogenous IL-2 (100 U). For comparison, CD4+ T cells were also cultured under Th0 conditions (plate-bound anti-TCRb + anti-CD28). (F) Following stimulation, cells were harvested and stained for CD4, TNFR2, and 7-AAD. The MFI of cell surface TNFR2 was determined on 7-AAD2CD4+-gated cells by flow cytometry. Representative flow cytometric line graphs (left panel) and TNFR2 MFI (mean 6 SEM) of cells isolated from three independent experiments (right panel). *p , 0.01, Th17 or Th17 + IL-2–polarized cells versus Th0-polarized counterparts. (G and H) Cell culture supernatants were collected, and ELISA was performed to quantify IL-2 or IL-17 and IFN-g. Each symbol represents an individual mouse. Horizontal lines represent the geometric means. ♦,7-AAD2CD4+-gated cells from unstimulated Il2+/2 mice; 7-AAD2CD4+-gated cells isolated from WT mice (d)orTNFR2KOIl2+/2 mice (s) stimulated under Th0- or Th17-polarizing conditions; 7-AAD2CD4+-gated cells isolated from WT mice (n)orTNFR2KOIl22/2 mice (N) stimulated under Th0- or Th17- polarizing conditions. *p , 0.05. (I)TotalRNA was extracted from gradient (Ficoll-Plaque)-purified viable cells, and IL-17A, IL-17F, IFN-g, and Foxp3 steady-state mRNA expression was determined by qRT-PCR. The ΔΔCT method was used for relative quantitation of steady-state mRNA. Values are expressed as relative expressions (fold change) fol- lowing normalization to b-actin using the mRNA levels in WT Th0 cells as the calibrator. The error bars, which are visible only if they are bigger than the diameters of the symbols, indicate the SEM of three independent experiments. 2644 TNFR2 REGULATES IL-2 TO CONTROL IL-17 not appear to be influenced by T cell–intrinsic or -extrinsic IL-2. (again, this was not exacerbated in TNFR2-KO Il22/2 T cells), As expected, the amount of IL-2 produced by TNFR2-KO CD4+ and this was reversed by the addition of IL-2, with the exception T cells was greatly reduced compared with WT T cells stimulated of TNFR2-KO Il22/2 T cells. In comparison, under Th17 condi- under Th0 culture conditions. In comparison, neither WT nor tions, the frequency of pY STAT3–expressing cells was increased TNFR2 KO T cells produced very much IL-2 when cultured under by the absence of IL-2 or TNFR2 but not by a combined defi- optimal Th17 conditions (Fig. 7G). ciency in IL-2 and TNFR2, and this was reversed by the addition of As shown in Fig. 7H, the generation of IL-17 under Th17- IL-2. Together, these data show altered expression of ROR-gt, polarizing conditions in vitro was enhanced by a deficiency in STAT3, and STAT5 in TNFR2-KO CD4+ T cells. either IL-2 or TNFR2 (p , 0.05) but not by a deficiency in both. Supplementation of Th17-polarized cultures with IL-2 greatly Discussion reduced IL-17 production by WT Il2+/2,WTIl22/2,andTNFR2- In this study, we show that CD4+ T cell–intrinsic tmTNF/TNFR2 KO Il2+/2 CD4+ T cells. In contrast to Th17 conditions, cells promotes Il2 expression to fine-tune the proportion of IL-2–pro- cultured under Th0 conditions yielded limited IL-17. Impaired ducing CD4+ T cells. Further, under Th17-polarizing conditions, IL-17 production by the addition of IL-2 under Th17-polarizing TNFR2 blockade is sufficient to promote Th17 differentiation, conditions was associated with increased IFN-g production by and this can be prevented by the addition of exogenous IL-2. WT, but not TNFR2-KO, CD4+ T cells. These in vitro results Overall, we conclude that tmTNF activation of TNFR2 expressed corroborate increased Th17 differentiation with TNFR2 defi- on CD4+ T cells stimulates Il2 expression and Il2 mRNA stability. ciency invivo. Consistent with these observations, when cultured During Th17 differentiation, TNFR2 is increased. When tmTNF/ under Th17-polarizing conditions in the absence of IL-2, TNFR2- TNFR2 signaling is blocked or impaired, Th17 differentiation is KO CD4+ T cells expressed elevated steady-state Il17A and Il17F promoted, and IL-17 activates STAT3 and STAT6 (Fig. 8C). We Downloaded from mRNA compared with WT T cells (Fig. 7I). Conversely, the addi- propose that decreased STAT5 activity, in concert with increased tion of IL-2 during Th17 polarization suppressed Il17A and Il17F STAT3 activity, is at least one mechanism by which TNFR2 block- mRNA in both WT and TNFR2-KO T cells and elevated Foxp3 ade promotes Th17 development. Our findings are consistent with mRNA in WT T cells. Consistent with our earlier observation of previous reports demonstrating displaced STAT3 binding at the impaired IFN-g secretion by TNFR2-KO T cells, the induction of Il17a–Il17f locus by IL-2/IL-2R signaling (56, 60, 61). Our findings IFN-g mRNA by TNFR2-KO CD4+ T cells was reduced compared contrast a previous report suggesting that TNF promotes Th17 dif- http://www.jimmunol.org/ with WT T cells cultured under Th0 conditions (p , 0.05). Col- ferentiation (23). In these earlier studies, cell cultures were sup- lectively, these results suggest that TNFR2 blockade promotes plemented with a combination of TNF and IL-1b. The individual Th17 differentiation independently of Foxp3 or IFN-g. contribution of TNF or IL-1b was not studied. IL-1b was since shown to promote Th17 differentiation by suppressing Socs3,an TNFR2 targets ROR-gt expression and tyrosine inhibitor of STAT3 (62). It remains to be determined whether phosphorylation of STAT3 and STAT5 to modulate Th17 IL-1b was, in fact, the key promoter of Th17 differentiation in differentiation the earlier studies. Although ROR-gt orchestrates Th17 differentiation, other tran- In mice, the transcription factors ROR-gt, ROR-a, and AHR by guest on October 3, 2021 scription factors, including RORa and AHR, also regulate Il17 are important regulators of IL-17–producing Th17 cells. During promoter activity (58, 59). We compared the expression of these Th17 differentiation, Rorgt expression is upregulated in TNFR2- transcription factors in WT and TNFR2-KO CD4+ T cells cul- KO T cells, but the expression of Ahr and Rora is not. Therefore, it tured under Th0- and Th17-polarizing conditions in the absence is not likely that AHR or ROR-a, by itself, causes the difference in or presence of IL-2. The expression of Rorgt, Rora,andAhr was IL-7 production between WTand TNFR2-KO T cells. CD4+CD25+ elevated by Th17 conditions for WT and TNFR2-KO T cells, and, Foxp3+ Tregs also were demonstrated to promote Th17 differenti- uniquely, Rorgt mRNA was more highly expressed in TNFR2- ation through the consumption of IL-2 (37). However, Foxp3 ex- KO CD4+ T cells compared with WT T cells (Fig. 8A). In con- pression is decreased in TNFR2-KO T cells. Therefore, IL-17 trast, the expression of ARNT, a binding partner for AHR, was production in response to TNFR2 blockade appears to be Treg in- comparable between WT and TNFR2-KO CD4+ T cells and was dependent. Overall, our observations are consistent with an in- unaffected by Th17 polarization or the addition of IL-2. IL-2 creasing number of studies demonstrating immune modulation by substantially inhibited Rorgt and Rora, but not Ahr,inTNFR2- TNF. There is little doubt that TNF plays a major role in the path- KO, but not WT, CD4+ T cells. Collectively, these results suggest ogenesis of autoimmune disorders, yet anti-TNF therapies remain that ROR-gt may contribute to the increased IL-17 production unpredictable. For example, etanercept, a TNF antagonist, often observed in TNFR2-KO CD4+ T cells. triggers an unexpected onset or exacerbation of CNS demyelin- The relative balance of STAT3 and STAT5 activity is a key ating autoimmune disorders in patients with inflammatory bowel determinant of Il17 promoter activity. Therefore, we assessed disease or rheumatoid arthritis (63). TNFR2 and IL-2 both pro- tyrosine phosphorylation (pY) of STAT5 and STAT3. WT and mote the expansion of Tregs, a promising therapy for multiple TNFR2-KO T cells were stimulated under Th17-polarizing con- disorders, including cancer, autoimmunity, and transplant rejec- ditions, with or without the addition of IL-2, and the results were tion (11, 61, 64, 65). Therefore, strategies aimed to mimic the compared with cells stimulated under Th0 culture conditions stimulatory actions of TNFR2 on Il2 expression while bypassing (Fig. 8B). Under Th0 conditions, the frequency of CD4+ T cells its cytotoxic effects have therapeutic potential. expressing elevated levels of pY STAT5 was unaffected; how- Although naive and previously activated CD4+ cells respond ever, IL-2 and TNFR2 deficiency each reduced pY STAT5 MFI, very differently to TNF, little is known about TNFR1 or TNFR2 and this was not exacerbated in TNFR2-KO Il22/2 T cells. In signaling cascades in these cells upon T cell activation. We now comparison, the frequency of cells expressing elevated levels establish that TNFR2, but not TNFR1, promotes Il2 promoter of pY STAT3 was reduced in TNFR2-KO Il22/2 T cells compared activity and Il2 mRNA stability to increase IL-2 production. with Il2+/2 T cells, and pY STAT3 MFI was unaffected. Under These observations are consistent with previous reports of im- Th17 conditions, the frequency of pY STAT5–expressing cells paired IL-2 production in TNFR1/2-dKO mice and reduced IL-2 and pY STAT5 MFI were reduced by IL-2 or TNFR2 deficiency production by TNFR22/2 CD8+ T cells (12–14). So, why the di- The Journal of Immunology 2645

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FIGURE 8. TNFR2 targets ROR-gt expression and pY of STAT3 and STAT5 to modulate Th17 differentiation. (A) Steady-state ROR-gt mRNA expression is elevated in TNFR2-KO CD4+ T cells compared with WT T cells. The cDNA used in these assays was obtained from WT and TNFR2- KO Il2+/2 cells stimulated under Th17-polarizing conditions in the presence or absence of exogenous IL-2 or under Th0 conditions from Fig. 7F–I. Values for steady-state ROR-gt, ROR-a, AHR, and ARNT are expressed as relative expression (fold change) following normalization to b-actin using the mRNA levels in WT Th0 cells as the calibrator. (B) Intracellular tyrosine-phosphorylated STAT5 and tyrosine-phosphorylated STAT3 were deter- 2 2 B mined for 5C.C7+/ Il2-GFP+/ WT or TNFR2-KO CD4+ T cells that were left unstimulated, activated for 4 d under Th17-polarizing conditions with or Downloaded from without the addition of IL-2 (100 U/ml), or acti- vated for 4 d under Th0 conditions. IL-6 (50 ng/ml) and IL-2 (100 U/ml) were added for the final 20 min of cell culture. Representative flow cy- tometry line graphs for pY STAT5 and pY STAT3 using 7-AAD2CD4+-gated T cells for analyses

(upper panels). The dashed vertical line indicates http://www.jimmunol.org/ pY STAT3 or pY STAT5 staining in unstimulated cells. The mean 6 SEM of pY STAT+ cells (i.e., those to the right of the dashed line) from three independent experiments are shown. Bar graphs show the mean 6 SEMoftheMFIofpYSTAT3+ or pY STAT5+ CD4+ T cells from the cells acquired and analyzed above (lower panels). *p , 0.05. (C) Overall, we conclude that tmTNF activation of TNFR2 expressed on CD4+ T cells stimulates Il2 expression and Il2 mRNA stability. During Th17 by guest on October 3, 2021 differentiation, TNFR2 is increased. When tmTNF/ TNFR2 signaling is blocked or impaired, Th17 dif- ferentiation is promoted, and IL-17 activates STAT3 and STAT6.

vergence between TNFR1 and TNFR2 signaling? One possi- or to a signaling cascade that is distinct from tmTNF. We con- bility is ligand specificity for receptor activation. sTNF activity sidered the possibility that decreased Il2 expression in TNFR2- is largely restricted to TNFR1, whereas tmTNF preferentially acti- KO T cells is consequential to elevated TNFR1 signaling as a result vates TNFR2. Although our results suggest tmTNF/TNFR2 in- of increased ligand availability, as opposed to a loss of stimulatory teractions, other TNFR2 ligands, including LT-a (LT-a3,TNFb) TNFR2 signaling. However, this is in contrast to our observation and membrane-bound LT-a1b2, were described (66, 67). In con- that Il2 expression in TNFR2-KO T cells parallels Il2 expression trast to LT-a1b2, which binds to a distinct receptor LTbR (68), in TNFR1/2-dKO T cells. If TNFR1 contributed to the silencing of secreted LT-a3 binds to both TNFR1 and TNFR2. Although, not the Il2 promoter, we would have expected even less Il2 expression without controversy, the PGRN also was proposed in TNFR2-KO T cells compared with TNFR1/2-dKO T cells. Sur- to bind TNFR2 (49, 69). Although TNF and TNFR2 are highly prisingly few studies have addressed signal transduction through expressed by Th17 cells, not much is known about the production TNFR1andTNFR2inCD4+ T cells. In principle, activation of and function of these alternative TNFR2 ligands in Th17 cells. TNFR1 rapidly recruits the adapter molecules TRADD, RIP1, and Therefore, it is not possible to exclude the possibility that alter- TRAF2 (70) to initiate a cascade of phosphorylation and ubiq- native TNFR2 ligands contribute to the regulation of IL-2. uitination events that activate MAPK and NF-kB/Rel pathways A second possibility involves receptor density. TNFR2, not to induce the expression of proinflammatory and antiapoptotic TNFR1, is the predominant TNFR that is upregulated upon ac- survival . In contrast, TNFR2 recruits TRAF2 to activate tivation of naive CD4+ T cells under Th0 and Th17 conditions NF-kB/Rel and MAPK pathways. Upon CD4+ T cell activation, (14). An alternative possibility is that TNFR1 and TNFR2 simply NF-kB/Rel proteins, normally sequestered within the cytoplasm, regulate different downstream signaling cascades, as reported translocate into the nucleus following the degradation of inhibi- in other cell types. It is not completely clear whether the failure tory proteins to activate canonical and noncanonical NF-kB/Rel of sTNF to modulate Il2 expression is due to low receptor density signaling. The importance of NF-kBandMAPKinIl2 expression 2646 TNFR2 REGULATES IL-2 TO CONTROL IL-17 is well established (51). Our present data, acquired from oligo- activation and autoimmunity in mice lacking -2 receptor beta. Science 268: 1472–1476. nucleotide DNA binding, associate silencing of the Il2 promoter 4. Liao, W., J. X. Lin, and W. J. Leonard. 2011. IL-2 family cytokines: new insights + in TNFR2-KO CD4 T cells with reduced cRel and p52 DNA into the complex roles of IL-2 as a broad regulator of differenti- binding. Similar changes were not evident in TNFR1-KO CD4+ ation. Curr. Opin. Immunol. 23: 598–604. 5. Cheng, G., A. Yu, and T. R. Malek. 2011. T-cell tolerance and the multi-functional T cells to suggest receptor signaling specificity. role of IL-2R signaling in T-regulatory cells. Immunol. Rev. 241: 63–76. Overall, we propose that canonical and noncanonical NF-kB 6. Shevach, E. M. 2009. Mechanisms of foxp3+ T regulatory cell-mediated sup- signaling participate in the regulation of Il2 expression by TNFR2. pression. Immunity 30: 636–645. 7. Pipkin, M. E., J. A. Sacks, F. Cruz-Guilloty, M. G. Lichtenheld, M. J. Bevan, and Our data are consistent with a requirement for cytokine-dependent A. Rao. 2010. Interleukin-2 and inflammation induce distinct transcriptional degradation of cytosolic IkBb to permit c-Rel nuclear transloca- programs that promote the differentiation of effector cytolytic T cells. Immunity tion (71, 72), as well as a requirement of c-Rel for chromatin 32: 79–90. 8. Liao, W., J.-X. Lin, and W. J. Leonard. 2013. Interleukin-2 at the crossroads of remodeling of the Il2 locus (52). Future studies will address epi- effector responses, tolerance, and immunotherapy. Immunity 38: 13–25. genetic modifications associated with silencing of the Il2 promoter 9. Ballesteros-Tato, A., B. Leo´n, B. A. Graf, A. Moquin, P. S. Adams, F. E. Lund, Il2 and T. D. Randall. 2012. Interleukin-2 inhibits germinal center formation by in response to TNFR2 deficiency. Similar to other cytokines, limiting T follicular helper cell differentiation. Immunity 36: 847–856. expression is also regulated by posttranscriptional mRNA stabi- 10. Wells, A. D. 2009. New insights into the molecular basis of T cell anergy: lization. The ability of TNFR2 to regulate Il2 stability is consis- anergy factors, avoidance sensors, and epigenetic imprinting. J. Immunol. 182: 7331–7341. tent with NF-kB– and JNK-dependent recruitment of AU-binding 11. Malek, T. R., and I. Castro. 2010. Interleukin-2 receptor signaling: at the in- proteins to AU-rich elements located within the 39-UTR, 59-UTR, terface between tolerance and immunity. Immunity 33: 153–165. andcodingregionoftheIl2 gene (40, 53). The precise signaling 12. McKarns, S. C., and R. H. Schwartz. 2008. Biphasic regulation of Il2 tran- scription in CD4+ T cells: roles for TNF-alpha receptor signaling and chromatin pathways by which TNFR2 regulates IL-2 expression remain im- structure. J. Immunol. 181: 1272–1281. Downloaded from portant areas for future studies. Importantly, not all members of 13. Kim, E. Y., and H. S. Teh. 2004. Critical role of TNF receptor type-2 (p75) as the superfamily of TNFRs regulate IL-2 production (73), po- a costimulator for IL-2 induction and T cell survival: a functional link to CD28. J. Immunol. 173: 4500–4509. tentially making TNFR2 the critical signaling pathway neces- 14. Kim, E. Y., and H. S. Teh. 2001. TNF type 2 receptor (p75) lowers the threshold sary to achieve the threshold level of NF-kB activity required of T cell activation. J. Immunol. 167: 6812–6820. 15. Black, R. A., C. T. Rauch, C. J. Kozlosky, J. J. Peschon, J. L. Slack, for Il2 promoter activity. M. F. Wolfson, B. J. Castner, K. L. Stocking, P. Reddy, S. Srinivasan, et al. 1997. In characterizing tmTNF knock-in Foxp3-GFP reporter mice, A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from we observed fewer CD4+ Foxp3+ T cells in the SPLN and MLN, cells. Nature 385: 729–733. http://www.jimmunol.org/ 16. Grell, M. 1995-1996. 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Expanding TRAF function: TRAF3 secondary lymphoid organ organization (75). Moreover, B cells as a tri-faced immune regulator. Nat. Rev. Immunol. 11: 457–468. are the main producers of TNF in the SPLN, whereas T cells 20. Ho¨fer, T., O. Krichevsky, and G. Altan-Bonnet. 2012. Competition for IL-2 by guest on October 3, 2021 generate the majority of TNF in the LNs (75). Our results in between Regulatory and Effector T Cells to Chisel Immune Responses. Front. + Immunol. 3: 268. tmTNF mice suggest that sTNF plays a role in maintaining CD4 21. Wang, X., Y. Zhang, X. O. Yang, R. I. Nurieva, S. H. Chang, S. S. Ojeda, Tregs in the SPLN and MLNs. 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