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T Cells+Producing CD4− IL-17Homeostasis by Limiting the Number of

Protein-1 Contributes to Intestinal Mucosa Induced Maturation−B Lymphocyte

Meffre, Stephan Targan and Gislâine A. MartinsWillen, Michael Couse, Joao S. Silva, Deepti Dhall, Eric Soofia Salehi, Rashmi Bankoti, Luciana Benevides, Jessica

http://www.jimmunol.org/content/189/12/5682doi: 10.4049/jimmunol.1201966November 2012;

2012; 189:5682-5693; Prepublished online 16J Immunol 

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The Journal of Immunology

B Lymphocyte–Induced Maturation Protein-1 Contributes toIntestinal Mucosa Homeostasis by Limiting the Number ofIL-17–Producing CD4+ T Cells

Soofia Salehi,* Rashmi Bankoti,* Luciana Benevides,*,† Jessica Willen,* Michael Couse,*

Joao S. Silva,† Deepti Dhall,‡ Eric Meffre,x Stephan Targan,*,{ and Gislaine A. Martins*,{

The transcription factor B lymphocyte–induced maturation protein-1 (Blimp-1) plays important roles in embryonic development

and immunity. Blimp-1 is required for the differentiation of plasma cells, and mice with T cell–specific deletion of Blimp-1 (Blimp-

1CKO mice) develop a fatal inflammatory response in the colon. Previous work demonstrated that lack of Blimp-1 in CD4+ and

CD8+ T cells leads to intrinsic functional defects, but little is known about the functional role of Blimp-1 in regulating differen-

tiation of Th cells in vivo and their contribution to the chronic intestinal inflammation observed in the Blimp1CKO mice. In this

study, we show that Blimp-1 is required to restrain the production of the inflammatory cytokine IL-17 by Th cells in vivo. Blimp-

1CKO mice have greater numbers of IL-17–producing TCRb+CD4+cells in lymphoid organs and in the intestinal mucosa. The

increase in IL-17–producing cells was not restored to normal levels in wild-type and Blimp-1CKO–mixed bone marrow chimeric

mice, suggesting an intrinsic role for Blimp-1 in constraining the production of IL-17 in vivo. The observation that Blimp-1–

deficient CD4+ T cells are more prone to differentiate into IL-17+/IFN-g+ cells and cause severe colitis when transferred to Rag1-

deficient mice provides further evidence that Blimp-1 represses IL-17 production. Analysis of Blimp-1 expression at the single cell

level during Th differentiation reveals that Blimp-1 expression is induced in Th1 and Th2 but repressed by TGF-b in Th17 cells.

Collectively, the results described here establish a new role for Blimp-1 in regulating IL-17 production in vivo. The Journal of

Immunology, 2012, 189: 5682–5693.

Interleukin-17 is an inflammatory cytokine produced by Th17cells (1), a Th subset that develops independently (2, 3) ofthe transcription factors T-bet and GATA-3, which are re-

quired for the development of Th1 and Th2 cells, respectively (4).Th17 cells are associated with protection against bacterial andfungal infections encountered at mucosal surfaces (5) but also withseveral autoinflammatory disorders, including rheumatoid arthritis,psoriasis, multiple sclerosis, corticosteroid-resistant asthma, andinflammatory bowel disease (6, 7).Differentiation of Th17 cells is induced by the cytokines TGF-b,

IL-6, and IL-1b (8–10), and it is dependent on the expression of

the transcription factors RORgt and RORa (11, 12), which inducetranscription of the Il17a gene (11). ROR-gt acts in cooperationwith RORa and other transcription factors, including STAT3, IFNregulatory factor-4, BATF, and Runx1, to induce full commitmentof precursors to the Th17 subset (13–15). Activation of RORgtalso promotes expression of the receptor for IL-23 (11), which isthought to maintain the expansion and pathogenesis of matureTh17 cells (16).Although Th17 cells are clearly a distinct Th subset, recent data

demonstrate that similar to other Th subpopulations, Th17 cells areconsiderably plastic and can acquire features and carry on effectorfunctions characteristics of other Th subsets (17, 18). Th17 cells canexpress the regulatory T (Treg) cell specifying factor Foxp3 undercertain conditions, and in the context of infectious and autoimmuneinflammation, Th17 cells can also produce IFN-g and IL-17 si-multaneously (17, 19). IL-17/IFN-g double-positive (IL-17+/IFN-g+)T cells are found in elevated numbers in inflamed tissues of bothhumans and mice (20–22), and similar populations are observed inTh17 cells differentiated in vitro from human and murine naiveCD4+ T cells (21–23). Recent studies indicate that IL-23 signalingis important for the differentiation of IFN-g+/IL-17+ CD4+ T cellsboth in vivo and in vitro (23, 24), but the transcriptional mechanismsunderlying the differentiation and/or conversion of these cells fromnon-IFN-g–producing Th17 cells are poorly understood.B lymphocyte–induced maturation protein-1 (Blimp-1; also

called PRDI-BF1 in humans) is a transcription factor (encoded bythe PRDM-1 gene) that plays crucial roles in regulating B andT lymphocyte function. Blimp-1 is expressed in both Treg andconventional T cells (25–27). Previous studies show that inFoxp3+ Treg cells Blimp-1 interacts with IFN regulatory factor-4to induce IL-10 production (26). However, little is known aboutBlimp-1’s role in non Treg cell function and how lack of Blimp-1

*F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute,Cedars–Sinai Medical Center, Los Angeles, CA 90048; †Department of Immunologyand Biochemistry, School of Medicine of Ribeirao Preto, Sao Paulo University,Ribeirao Preto, Sao Paulo 14049, Brazil; ‡Department of Pathology, Cedars–SinaiMedical Center, Los Angeles, CA 90048; xDepartment of Immunobiology, YaleUniversity, New Haven, CT 06511; and {Research Division of Immunology, Depart-ment of Biomedical Sciences, Cedars–Sinai Medical Center, Los Angeles, CA 90048

Received for publication July 18, 2012. Accepted for publication October 13, 2012.

This work was supported by National Institutes of Health‑National Institute of Al-lergy and Infectious Diseases Grant AI083948-01 (to G.A.M.), F. Widjaja FoundationInflammatory Bowel and Immunobiology Institute funds, and a predoctoral scholar-ship from Fundacao de Amparo a Pesquisa e ao Ensino do Estado de Sao Paulo(Brazil) (FAPESP#2008/04606-2) (to L.B.).

Address correspondence and reprint requests to Dr. Gislaine A. Martins, Cedars-SinaiMedical Center, Davis Research Building, Room 4092, 110 North George BurnsRoad, Los Angeles, CA 90048. E-mail address: martinsg@csmc.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: BFA, brefeldin A; Blimp-1, B lymphocyte–inducedmaturation protein-1; CSMC, Cedars–Sinai Medical Center; ICC, intracellular cyto-kine staining; LI, large intestine; LP, lamina propria; mLN, mesenteric lymph node;qRT-PCR, quantitative real-time PCR; SP, spleen; Treg, regulatory T; TSS, transcrip-tion start site; YFP, yellow fluorescent protein.

Copyright� 2012 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/12/$16.00

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contributes to the development of chronic mucosal inflammation.Despite their documented deficiency in producing IL-10, Blimp-1–deficient Treg cells can suppress T cell–mediated colitis (25,28) suggesting that defects in other T cell subsets may underlie thechronic intestinal inflammation developed in the Blimp-1CKOmice. The studies described in this paper address this possibilityand identify a new, Treg-independent role for Blimp-1 in con-trolling intestinal mucosal homeostasis by limiting the numbers ofIL-17–producing Th cells in vivo.

Materials and MethodsMice

C57BL/6 Prdm1flox/floxCD4-Cre+ (Blimp-1 CKO) and Prdm1+/+ CD4-Cre(control) mice were generated as described previously (25, 29). Previouslydescribed (30) C57BL/6 Prdm1flox/floxCD19-Cre+ mice were obtained fromDr. M. McHeyzer-Williams (Department of Immunology and MicrobialScience, Scripps Research Institute, La Jolla, CA). Mice bearing a bacte-rial artificial chromosome transgene encoding yellow fluorescent protein(YFP) under the control of Blimp-1 regulatory elements (Blimp-1 YFPreporter mice) (31) were also described previously (32). In these mice,YFP expression closely recapitulates Blimp-1 mRNA expression (32).Foxp3-IRES-GFP knockin (33) (Foxp3 reporter) mice were obtained fromV. Kuchroo (Brigham and Women’s Hospital, Boston, MA) and crossed toC57BL/6 Prdm1flox/floxCD4-Cre+ mice to generate Ctrl and Blimp-1CKOFoxp3 reporter mice. All mice were maintained in a specific pathogen-freeanimal facility at the Cedars-Sinai Medical Center (CSMC) and handled inaccordance with the institutional guidelines.

Large intestines lamina propria lymphocyte isolation

Large intestines (LI) (cecum, colon, and rectum) were removed, openedlongitudinally, cleaned and cut into strips 1 cm in length. Tissues werewashed in ice-cold PBS and subjected to enzymatic digestion as describedpreviously (34). Lamina propria (LP) mononuclear cells were purified ona 45/72% Percoll gradient by centrifugation for 20 min at 25˚C and 600 3g with no brake.

Abs and flow cytometry

Abs used for cell surface staining were Alexa 700–conjugated TCR-b, PacificBlue–conjugated anti-CD4, PE-conjugated anti-CD25, allophycocyanin-conjugated anti-CD44 (all from BioLegend), PE-conjugated anti–IL-17A, Alexa 647–conjugated anti–IL-17F, PE-conjugated anti–IL-4, andallophycocyanin-conjugated IFN-g (eBioscience). Cell surface stainingwas performed as described previously (25). Samples were analyzed ona LSRII analyzer (BD Biosciences, San Jose, CA).

Determination of cytokine production

Total cell suspensions obtained from the LI-LP, spleen (SP), and mes-enteric lymph nodes (mLN) were stimulated with plate-bound anti-CD3(5 mg/ml) plus anti-CD28 (2.5 mg/ml) for 24 h with addition of brefeldinA (BFA; 1 mg/ml) for the last 6 h. The production of IL-17 and IFN-gwas determined by ELISA on supernatants collected at 18 h (before BFAaddition) using eBioscience ELISA kits, following the manufacturer’sinstructions. Where indicated, IL-17, IL-4, and IFN-g were also detectedby intracellular staining, as previously described (29). Cells were ana-lyzed on a LSRII analyzer. Cytokine staining was evaluated in live CD4+

TCRb+-gated lymphocytes.

Quantitative real-time PCR

Total mRNA was isolated using RNAeasy kits (Qiagen) according to themanufacturer’s instructions. Reverse transcription was performed on equalamounts of RNA (as determined by Nanodrop measurements) for eachsample using Superscript III (Invitrogen). SYBR Green incorporationquantitative real-time PCR (qRT-PCR) was performed using a FastStartSYBR Green Master mix (Roche) in the Realplex2 Mastercycler ep gra-dient S (Eppendorf). Primers used are described on Supplemental Table I.

Mixed bone marrow chimera

Recipient mice (CD45.1+ wild-type) were irradiated (10 Gy) for depletionof hematopoietic cells. Donor Ctrl (CD45.1+) or Blimp-1CKO (CD45.2+)Lin2 bone marrow cells, enriched for hematopoietic progenitors by magneticbead depletion (StemCell Technologies), were injected i.v. and chimeraswere analyzed 11 wk later.

T cell transfer model of colitis

Induction of colitis by transfer of naive CD4+ T cells was performed aspreviously described (24, 35), with the following modifications: C57BL/6Rag12/2 mice were injected i.p. (4 3 105 cells in 200 ml PBS) with naiveCD4+CD252CD45RBHigh or CD4+CD252CD44low sorted from SP andLN from 4- to 6-wk-old Ctrl or Blimp-1CKO mice or CD4+CD252

CD44lowFoxp32 cells sorted from Ctrl or Blimp-1CKO-Foxp3 reportermice. Mice were weighed weekly and inspected for clinical signs of dis-ease (including weight loss, hunched appearance, pilo-erection of fur coat,and loose stool). Mice presenting clinically severe disease were sacrificedaccording to the CSMC Animal Care and Use Committee guidelines. At 7–12 wk posttransfer, recipient mice were sacrificed and colons were re-moved, cleaned in ice-cold PBS, and pieces of ∼0.5 cm in length wereobtained from the proximal, middle, and distal portion of the colon andfixed immediately in 10% formalin. Fixed tissue was later embedded inparaffin, and 3-mm sections were cut and stained with H&E. Samples werecoded and scored by a pathologist in a blinded fashion as described pre-viously (35).

T cell isolation and in vitro Th differentiation

Naive CD4+ (CD252CD44low) or effector (CD252CD44High) T cells weresorted from SP and lymph nodes cell suspensions, using a FACSAria IIIfluorescent cell sorter (BD Biosciences, San Jose, CA). Purity of sortedcells preparations were .98%. For in vitro Th differentiation, sorted naiveCD4+ T cells were stimulated in Iscove’s DMEM (Cellgro, Manassas, VA)supplemented with 10% FBS (Omega Scientific, Tarzana, CA) and penicillin/streptomycin (Cellgro) with plate-bound anti-CD3 (5 mg/ml), anti-CD28(2.5 mg/ml) (both from BioXCell, West Lebanon, NH), and rHuIL-2 (25 U/ml; Roche) (neutral conditions). Experimental Th1 conditions also in-cluded rMuIL12 (5 ng/ml) plus anti–IL-4 (10 mg/ml) in addition to IL-2.Th2 conditions included rMuIL-4 (10 ng/ml) and anti–IFN-g (10 ng/ml).Th17 conditions excluded IL-2 and included IL-1b (20 ng/ml), IL23 (50ng/ml), IL-6 (10 ng/ml), TGF-b (5 ng/ml) (Promega), anti–IL-4, and anti–IFN-g. With the exception of TGF-b, all recombinant cytokines wereobtained from eBioscience. Neutralizing Abs anti–IFN-g and anti–IL-4were obtained from BioXCell. Cells were split every 3 d, and fresh me-dium (with the appropriate recombinant cytokines) added each time cellswere split. For experiments in which TGF-b was used to repress Blimp-1expression, recombinant TGF-b was only added at the beginning of thecultures and not replaced upon splitting of the cells. Restimulation wasperformed with PMA (50 ng/ml) and ionomycin (500 ng/ml) (both fromSigma-Aldrich) for 4 h; BFAwas added in the last 2 h or with plate-boundanti-CD3 for 6 h (BFA added in the last 2 h).

Western blots

Whole-cell lysates (30 ng/sample) were subjected to SDS-PAGE transferredto Invitrolon polyvinylidene difluoride membranes (Invitrogen) and immuno-blotted with monoclonal anti-mouse Blimp-1 (3H2E8) or monoclonal anti-mouse b-actin (Sigma-Aldrich).

Chromatin immunoprecipitation

Chromatin immunoprecipitation assays were performed as described pre-viously (29). Briefly, naive CD4+ T cells were sort-purified from cellsuspensions of lymph nodes and SPs from wild type mice and stimulatedwith plate bound anti-CD3, plus anti-CD28 under Th2 polarizing con-ditions. After seven to eight days cells were restimulated with PMA andionomycin for 4 h before crosslinking by fixation with 1.1% paraformal-dehyde for 10 min at room temperature. Sonicated chromatin from 4 to5 3 107 cells was immunoprecipitated with 25 ml of either rabbit anti–Blimp-1 polyclonal Ab (clone 267, recognizing the C terminal of Blimp-1)(36) or preimmune serum as a control. qRT-PCR using SYBR Green in-corporation was performed in DNA recovered from IP and input samples(see Supplemental Table II for primers sequences). Fold enrichment foreach sample was calculated by dividing the percentage of input valuesobtained with anti-Blimp by the values obtained with control Ab, followedby normalization to the values obtained from the input chromatin. Analysisof sequence homology and identification of putative Blimp-1 consensussites were performed using Evolutionary Conserved Regions browser(http://ecrbrowser.dcode.org) and rVista 2.0 software. All genomic sequenceswere obtained from Ensembl.

Retroviral gene transduction

Recombinant retroviruses encoding Blimp-1 (in the MigR1 vector, con-taining an internal ribosomal entry site–GFP cassette) were produced fromtransfected Plat-E packaging cells (37) and were used to transduce naive

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CD4+ T cells stimulated under Th17-polarizing conditions or CD32

B220+ cells sorted from the SPs of Prdm1Flox/FloxCD19-CRE+/2 andPrdm1Flox/ FloxCD19 cre2/2 mice and stimulated for 24 h with 2 mg/mlLPS (Sigma-Aldrich). Transduction was performed by mixing the B cells(24 h after stimulation) or the T cells (48–56 h after stimulation) withsupernatant-containing virus and Polybrene (8 mg/ml; Sigma-Aldrich)and then centrifuged at room temperature for 90 min (10,000 3 g). Af-ter transduction B cells were replated in regular media and T cells werereplated under Th17-polarizing conditions, B cells were restimulated withLPS (10 mg/ml) 2 d after transduction and analyzed 36 h after LPSrestimulation. T cells were analyzed 3 and 4 d after transduction and uponrestimulation with PMA and ionomycin.

Statistics

Student t test with two-tailed distribution of equal variances was used tocalculate p values, using the JMP software (SAS Institute, NC).

ResultsLack of Blimp-1 is associated with accumulation of CD4+

T cells in the colon and increased production of IL-17 in vivo

To identify the cellular mechanisms leading to the inflammatoryresponse observed in the intestinal mucosa of the Blimp-1CKOmice, we first investigated the presence of T cells in the in-flammatory infiltrate observed in 8- to 16-wk-old mice with signsof colitis development. We found that the absolute numbers ofmononuclear cells recovered from the LI (cecum, colon, andrectum)-LP were significantly higher in the Blimp-1CKO mice(Fig. 1A). Both the percentages and absolute numbers of TCRb+

CD4+ were elevated in the LI-LP of Blimp-1CKO mice; CD4+

T cells percentages were on average 2-fold higher in the Blimp-1CKO mice (Fig. 1B), whereas the absolute numbers were 3- to4-fold higher in the Blimp-1CKO mice (Fig. 1B). Thus, Blimp-1–deficient CD4+ T cells accumulate in the colonic LP.To characterize the CD4+ T cells that accumulated in the in-

testinal mucosa of Blimp-1CKO mice, we next investigated theproduction of the cytokines IFN-g and IL-17A by these cells,because these two cytokines have been shown to play effectorsroles in intestinal inflammation. LI-LP were isolated from Ctrland Blimp-1CKO mice and stimulated with plate-bound anti-CD3 plus anti-CD28 for 24 h, and IFN-g and IL-17A produc-tion were measured in the supernatants (Fig. 1C). The amount ofIFN-g detected in the supernatant of these cultures was similar inCtrl and Blimp-1CKO mice (Fig. 1C, right), but LI-LP fromBlimp-1CKO mice produced significantly more IL-17A thancells from Ctrl mice (Fig. 1C, left).To determine whether the increased production of IL-17A and

IFN-g was restricted to the LI mucosa, we measured productionof these cytokines in SP and mLN of Blimp-1CKO mice afterin vitro TCR stimulation. Intracellular cytokine staining (ICC)and FACS analysis showed increased percentages (Fig. 1D, 1E)and absolute numbers (Fig. 1F) of IL-17A–producing cells inboth SP and mLN of Blimp-1CKO mice. In addition, we alsoobserved an increase in the percentage of IL-17F+ cells in themLN of Blimp-1CKO mice (Fig. 1D). In both Ctrl and Blimp-1CKO mice, ∼50% of the IL-17A+ cells were also IL-17F+, butCKO mice had 2- to 3-fold more IL-17–producing cells (Fig.1D). Different from the observed for IL-17 production, thepercentages of IFN-g–producing cells were elevated in themLNs but not in the SP of Blimp-1CKO mice (Fig. 1D, 1E), andno significant differences were observed in the absolute numbersof IFN-g–producing cells in Ctrl and Blimp-1CKO mice (Fig.1F). Secretion of IL-17A was also significantly increased in theSP and mLNs of the CKO mice (Fig. 1G). Thus, lack of Blimp-1is associated with increased production of IL-17 and, to a lesserextent, IFN-g in vivo.

Blimp-1–deficient effector/memory CD4+ T cells haveincreased expression of genes associated with the Th17differentiation program

Characterization of the IL-17–producing cells present in the Blimp-1CKO mice revealed that they were all Ag-experienced cells,which expressed high levels of CD44 (data not shown). Sort-purified CD44highCD252CD4+ cells from pooled mLN and SPfrom Blimp-1CKO had higher frequency of IL-17 producers andsecreted significantly more IL-17A protein than Ctrl cells uponin vitro restimulation (Fig. 2A, 2B). They also expressed signifi-cantly higher amounts of Il17a, Rorc, and Il23r but not Batf mRNAthan Ctrl cells when stimulated in vitro (Fig. 2C). Thus, the ex-pression of Th17 signature genes is upregulated in Blimp-1–defi-cient Ag-experienced CD4+ T cells, suggesting that Blimp-1 isrequired to control the differentiation and/or accumulation of Th17cells in vivo.

Regulation of IL-17 production by Blimp-1 is intrinsic to CD4+

effector T cells

Increased numbers of IL-17–producing CD4+ T cells in Blimp-1CKO mice could result from intrinsic effects of Blimp-1 inregulating the differentiation/accumulation of Th17-cells in vivoor could result from the previously described impaired Treg cellfunction in these mice (25, 26), because defective Treg cellresponses can facilitate priming and development of IL-17– andIFN-g–producing cells (38–40). Alternatively, the increased numbersof IL-17–producing cells could be a secondary effect of the on-going inflammation in the colon of these mice (25). To distinguishthese possibilities, we next analyzed the expression of IL-17 inCD4+ T cells from chimeric mice generated with a mixture ofwild-type and Blimp-1CKO bone marrow cells. To delay thedevelopment of severe inflammation in the chimeric mice, wedesigned our experiments such that the hematopoietic compart-ment in the reconstituted mice would contain significantly moreCtrl than Blimp-1–deficient cells. Thus, in all sites analyzed, themajority of TCRb+CD4+ cells were Blimp-1 sufficient (Fig. 3A).In both compartments, Blimp-1–sufficient and Blimp-1–defi-

cient, the frequency of Foxp3+ Treg cells was similar (Fig. 3C). Inthe Blimp-1–deficient compartment; however, the frequency ofIL-17A–producing (but not of IFN-g–producing) cells was sig-nificantly increased in comparison with the wild-type compart-ment in all sites evaluated, including the LI-LP (Fig. 3). Thus,Blimp-1–deficient CD4+ T cells are intrinsically more prone todifferentiate into IL-17–producing cells.

Blimp-1–deficient CD4+ T cells differentiate into IL-17+/IFN-g+ cells and cause severe colitis in Rag1-deficient mice

To determine whether the absence of Blimp-1 can lead to thepreferential differentiation of IL-17– and/or IFN-g–producing cellsin vivo, we transferred CD4+ naive (CD44low/CD252) Ctrl orBlimp-1CKO cells into Rag1-deficient (Rag12/2) mice and moni-tored colitis development in the recipient mice. Rag12/2 mice re-ceiving Ctrl cells developed colitis and wasting disease as indicatedby histological analysis and total body weight loss. Rag12/2 micereceiving Blimp-1–deficient cells showed signs of disease earlierthan mice receiving Blimp-1–sufficient cells (Fig. 4A) and alsodeveloped more severe colitis, with more prominent inflammationin the colon (Fig. 4B) and significantly increased colitis scores(average score = 36 0.9) than in those receiving Ctrl cells (averagescore = 1 6 0.7) (Fig. 4C).Evaluation of cytokine production upon ex vivo TCR stimulation

of cells from mLN and LI-LP revealed that while the percentagesof IFN-g or IL-17–single producer cells were only marginallyincreased in the Blimp-1CKO mice, the percentage of IFN-g and

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FIGURE 1. Accumulation of CD4+TCRb+ cells in the colon and increased amounts of IFN-g– and IL-17–producing cells in Blimp-1 CKO mice. (A)

Total numbers of mononuclear cells in the LI (cecum, colon, and rectum) LP in 8- to 16-wk-old Prdm1+/+CD4-CRE+/2 (Ctrl) and Prdm1F/FCD4-CRE+/2

(CKO) mice. Each symbol represents one animal (d, Ctrl; :, CKO), and bars represent average for each group. Results shown are representative of five

independent experiments. (B) Percentages of TCRb+CD4+ T cells (FACS plots) and total numbers of TCRb+CD4+ T cells (chart) in LI-LP from Ctrl and

Blimp-1CKO mice. (C) Production of and IL-17A (left) and IFN-g (right) measured by ELISA on supernatants of LI-LP cells stimulated with plate-bound

anti-CD3 plus anti-CD28 for 24 h. (D) ICC for IL-17A and IFN-g (D, left plots) or IL-17A and IL-17F (D, right plots) and FACS analysis of CD4+TCRb+

cells in mLN (top row) and SP (SP, bottom row) from Ctrl and CKO mice. SP and mLN total cell suspensions were stimulated as in C) before analysis (BFA

was added in the last 6 h). Data shown are from gated TCRb+CD4+ cells. (E and F) combined results of the percentage (E) or absolute numbers (F) of IL-

17A or IFN-g+CD4+ T cells in SP or mLN in five to six independent experiments for a total of six to eight mice per group. (G) Production of IL-17A

(ELISA) by cells from SP or mLN after 18 h stimulation with plate-bound anti-CD3 plus anti-CD28. Data shown are the average and SEM from three

different experiments with three to seven mice per group.

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IL-17 double producer cells were significantly increased in themLN of mice transferred with Blimp-1-deficient cells (Fig. 4D,4E). However, the absolute numbers of IL-17+, IFN-g+, and IL-

17+/IFN-g+ cells were all significantly increased in the LI-LP ofmice injected with Blimp-1–deficient cells in comparison withmice injected with Ctrl cells (Fig. 4F). To determine whether the

FIGURE 2. Increased expression of il17a and

Th17-signature genes in Blimp-1–deficient effector/

memory cells. CD4+ Ag-experienced (CD44High)

T cells were sorted from pooled SP and mLN from

Ctrl or Blimp-1 CKO mice and stimulated in vitro

with plate-bound anti-CD3 plus anti-CD28 and IL-

17 production was measured by ICC staining and

FACS analysis at 6 h (A) or in the supernatants by

ELISA at 48 h (B). Il17a, Il23r, RORC, and BATF

steady-state mRNA levels (C) were measured by

qRT-PCR in Ctrl and Blimp-1CKO stimulated as in

(A). Results shown in (A)–(C) are representative of

two to three independent experiments with three to

five mice per group. Bars show average, and error

bars represent SEM. Results in (C) are presented

relative to 18S expression.

FIGURE 3. Increased production of IL-17 by Blimp-1–deficient CD4+ T cells in mixed-bone marrow chimeric mice. Wild-type CD45.1-irradiated mice

received a total of ∼43 106 HSC-enriched mixed BM cells from Ctrl (CD451.1) and Blimp-1CKO (CD451.2) mice. Eleven weeks after bone marrow cells

transfer, mLN, SP, and LI-LP cells were isolated from chimeric mice, stimulated as described in Fig. 1D and IL-17 (A) production was analyzed in TCRb+

CD4+ cells in the CD45.22 (Ctrl) or CD45.2+ (Blimp1CKO) gates. (B) Frequency of IFN-g–producing TCRb+CD4+ cells from the mLN and SP from

chimeric mice [stimulation and analysis done as in (A)]. (C) Frequency of Foxp3-expressing cells in the Ctrl and Blimp-1CKO compartments in the SP and

mLN from chimeric mice [gated as shown in (A)]. Plots (A)–(C) (bottom) show compiled results from four to nine different chimeric mice.

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FIGURE 4. Blimp-1 CKO CD4+ T cells differentiate into IL-17 and IFN-g double producer cells in vivo and cause severe colitis in Rag12/2 mice.

Rag12/2 mice were injected i.p. with 43 105 naive (CD44lowCD252) CD4+ T cells sorted from Ctrl or Blimp-1CKO mice and monitored for symptoms of

colitis development and wasting disease, including body weight loss daily (A). Six to 7 wk after T cell transfer, recipient mice were euthanized and colons

were collected and processed for H&E staining [(B), 320 magnification] and histological analysis. Slides were analyzed blindly. Colitis score (C) was

graded semiquantitatively from 0 to 4, as previously described (35) each symbol represent one animal (the circles and triangles represent Ctrl and CKO,

respectively). (D) ICC staining and FACS analysis of cells from mLN from recipient mice 7 wk after adoptive T cell transfer of Ctrl (d) or Blimp1CKO

(:) cells. mLN cells were stimulated in vitro for 24 h with plate-bound anti-CD3 plus aCD28 before staining. FACS plots show cells in the TCRb+CD4+

gate. (E) Combined results (each symbol represent one animal, and bars represent average of five to six different experiments) of the percentage of IL-17A+,

IFN-g+, or IL-17A+/IFN-g+ CD4+ T cells in the mLN (left panel), or LI-LP (right panel) for a total of six to seven mice per group. (F) Absolute numbers of

IL-17A+, IFN-g+, or IL-17A+/ IFN-g+ CD4+ T cells in the LI-LP based on the percentages shown in (E). (G) Percentages of TCRb+ CD4+Foxp3+ cells in

the mLN (left) or LI-LP (right) of Rag2/2 mice injected with Ctrl or Blimp-1CKO 4 3 105 naive [CD44lowCD252GFP2 (Foxp32)] CD4+ T cells and

analyzed six to seven weeks later. Data are representative of five [(A)–(C)] or three (E, F) different experiments, with a total of four to seven mice per group.

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differences we observed between Ctrl and Blimp-1CKO cells-mediated colitis was due to differential induction Foxp3+ Tregcells from the injected naive cells, we repeated these experimentsusing naive CD4+ T cells isolated from Ctrl or Blimp-1CKO micethat had been previously bred with Foxp3 reporter mice. Thisstrategy allowed us to exclude Foxp3+ cells from the pool of naivecells adoptively transferred to the Rag12/2 mice and subsequentlymonitor the development of Foxp3-expressing cells in the recip-ient mice. Rag12/2 mice injected with CD4+ naive (CD44low

CD252GFP2 [Foxp32] cells) from Blimp-1CKO mice developedmore severe colitis than mice injected with Blimp-1–sufficientcells (data not shown), similarly to that described above, indi-cating that the increased severity of colitis caused by Blimp-1–deficient cells was not due to differential contamination of thetransferred cells with Foxp3+ Treg cells. Moreover, analysis ofGFP expression in cells from the recipient mice showed thatFoxp3+ cells developed in greater numbers from Blimp-1–defi-cient than from Ctrl cells (Fig. 4G), indicating that increased in-flammation in mice injected with Blimp-1–deficient was not dueto impaired induction of Foxp3+ cells.

Blimp-1 expression is selectively regulated during Thdifferentiation

The results described above indicated that expression of Blimp-1suppresses the differentiation of Th17 cells in vivo and that in theabsence of Blimp-1 more cells tend to differentiate into IL-17+

or IL-17+/IFN-g+ cells. We reasoned that if Blimp-1 antagonizesTh17 differentiation, its expression should be downregulated incells differentiated under Th17 conditions, as opposed to cells thatusually do not produce IL-17, such as Th1 and Th2 cells. Previousstudies have shown that Blimp-1 is expressed in CD4+ T cellsdifferentiating into either Th1 or Th2 conditions, but whereasone group showed increased expression of Blimp-1 in Th2 cells(41), another showed similar expression in Th1 and Th2 cells (28).Neither of these studies investigated Blimp-1 expression in Th17cells. To clarify whether Blimp-1 expression is, in fact, differen-tially regulated during Th differentiation, we used cells frompreviously described Blimp-1-YFP reporter mice (29, 32) to an-alyze Blimp-1 expression at the single cell level during Th dif-ferentiation in vitro.Sorted naive (CD44lowCD252) CD4+T cells from Blimp-1-YFP

reporter mice were stimulated under neutral, Th1, Th2, or Th17conditions (Supplemental Fig. 1) and Blimp-1 mRNA expression(as reported by YFP) was analyzed at different time points. Asexpected from previous reports (41, 42), stimulation of naiveCD4+ T with plate-bound anti-CD3 plus anti-CD28 induced ex-pression of Blimp-1 in a small percentage of cells; addition ofIL-2 to these cultures (neutral conditions) increased expression,especially at later time points (Fig. 5A). Cells cultured under Th1or Th2 conditions began to express Blimp-1 sooner than cellscultured in neutral conditions, but Th1 cells expressed signifi-cantly more than Th2 cells at earlier time points (Fig. 5A).However, at day 7.5, .70% of Th1 and Th2 cells expressed highlevels of Blimp-1 (Fig. 5A-B). In contrast to cells stimulated underneutral, Th1 or Th2 conditions, cells cultured under Th17 con-ditions did not upregulate Blimp-1 significantly throughout theexperiment, and ,5% of the cells expressed Blimp-1 at day 7.5poststimulation (Fig. 5B). Analysis of Blimp-1 steady-statemRNA (Fig. 5C) and protein levels (Fig. 5D) in these cells con-firmed the differential expression of Blimp-1 during Th differen-tiation. Thus, Blimp-1 is expressed in Th1 and Th2 cells but not inTh17 cells.We next investigated the regulation of Blimp-1 expression by

different Th-polarizing cytokines. Previous studies (28, 41, 42)

have reported contradictory results on the induction of Blimp-1 byTh1- and Th2-inducing cytokines. We found that IL-2, IL-4, IL-6,and IL-12 were each capable of enhancing expression of Blimp-1by anti-CD3 plus anti-CD28 stimulation (which alone inducedlittle expression of Blimp-1) (Fig. 6A), but at different magni-tudes, with IL-12 being the best inducer, followed by IL-2 andthen IL-4. Among Th17-inducing cytokines, IL-6 was the bestinducer of Blimp-1 expression, although in comparison with IL-2,IL-4, and IL-12, IL-6 was a poor inducer. Neither IL-1b nor IL-23induced Blimp-1 expression; in fact, these cytokines partiallyinhibited Blimp-1 expression induced by IL6 (Fig. 6A). Becausethe expression of Blimp-1 in cells stimulated with a combinationof IL-6, IL-23, and IL-1b was higher than in cells stimulatedunder Th17 conditions (Fig. 6A) (which included TGF-b in ad-dition to IL-6, IL-23, and IL-1b),we suspected that TGF-b couldhave an inhibitory effect on Blimp-1 expression. We thereforecompared Blimp-1 expression induced by the combination of TCR(and costimulation) and IL-2, IL-12, or IL-4 in the absenceor presence of TGF-b. We found that TGF-b led to significantinhibition of Blimp-1 expression in these cultures (Fig. 6A).Evaluation of Blimp-1 steady-state mRNA levels confirmed theseresults (Fig. 6B).We next sought to determine whether TGF-b could inhibit

expression of Blimp-1 induced by the combination of IL-2 andIL-12. TGF-b inhibited Blimp-1 expression induced by TCR andcostimulation in combination with IL-2 and IL-12 in a dose-dependent manner (Fig. 6C). Even at concentrations lower thanthe required amounts used to induce Th17 differentiation in vitro,TGF-b led to significant inhibition Blimp-1 expression in cellscultured simultaneously with IL-12 and IL-2. These results werealso confirmed by mRNA expression measured by qRT-PCR (Fig.6D). Together, these results establish TGFb as a potent suppressorof TCR and cytokine-induced Blimp-1 expression during Th17differentiation.

Blimp-1 binds to the Il17a gene in vivo but is not sufficient torepress Th17 differentiation

The observation that lack of Blimp-1 is associated with increasedexpression of Il17a mRNA and protein as well as several otherTh17 genes (Fig. 2) and that Blimp-1 expression is selectivelydownregulated during Th17 differentiation (Fig. 5) suggested thatBlimp-1 could function as a repressor of the Th17 program.Blimp-1 has potent transcriptional repression capabilities and di-rectly regulates the transcription of several cytokine genes (26, 29,41). Thus, we first sought to determine whether Blimp-1 responseelements could be identified in the Il17a gene. We identified manyputative Blimp-1 binding sites at the Il17a promoter region as wellas in regions downstream of the transcription start site (data notshown). We then tested whether Blimp-1 could bind to three of thesites we identified. Using wild-type Th2-polarized cells, we foundsignificant enrichment for Blimp-1 at least one of these sites(located at ∼3.3 kb upstream of the transcription start site [TSS]).We also detected some enrichment for Blimp-1 binding on a sitelocated 7.2 kb upstream of the TSS, but in this case, there was nosignificant difference in comparison with nonspecific bindingon a control gene (Fig. 7A). The third site investigated is locatedfurther downstream (∼44.9 kb from the TSS) showed no enrich-ment for Blimp-1 binding (Fig. 7A). Thus, in Th2 cells, Blimp-1can bind to at least one site in the Il17a gene.To determine whether Blimp-1 could suppress Il17a activity in

Th17 cells, we used retrovirus transduction to enforce Blimp-1expression in cells stimulated under Th17-polarizing conditionsand measured IL-17 production. Expression of Blimp-1 (as indi-cated by a GFP reporter) and confirmed by qRTPCR (data not

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shown) did not result in significant decrease of IL-17A or IL-17Fproduction (Fig. 7B, 7C). However, transduction with the sameretroviral construct was able to partially restore plasma cell forma-tion in Blimp-1–deficient B cells stimulated in vitro (SupplementalFig. 2), indicating that functional Blimp-1 protein is expressed fromthis construct. Therefore, although Blimp-1 is bound to the Il17agene in Th2 cells, it is not sufficient to repress Il17a transcription incells stimulated under Th17 conditions.

DiscussionThe results of this study provide evidence for a nonredundant rolefor the transcriptional regulator Blimp-1 in constraining thenumbers of IL-17–producing Th cells in vivo. We showed thatspontaneous development of chronic intestinal inflammation inmice with T cell-specific deletion of Blimp-1 is associated withaccumulation of IL-17–producing Th cells in vivo. Furthermore,Blimp-1–deficient naive CD4+ T cells preferentially differentiatedinto IL-17A and IFN-g double-producing cells and caused severecolitis when transferred to Rag12/2 mice. Consistent with a rolefor Blimp-1 in keeping the numbers of Th17-producing cells un-der control, we demonstrated that Blimp-1 was selectively ex-pressed in Th1 and Th2 but not in Th17 cells.Our observation that IL-17A–producing cells selectively accu-

mulate in the Blimp-1–deficient CD4+ T cell compartment of Ctrland Blimp-1CKO mixed-bone marrow chimeric mice (Fig. 3)indicate a Treg-independent, effector T cell-intrinsic role forBlimp-1 in repressing the production of IL-17A in vivo. This is

further supported by our findings that Blimp1-deficient naiveCD4+ T cells generated more IL-17A–producing cells than controlcells and caused severe colitis upon adoptive transfer to Rag12/2

mice. These results cannot be attributed to a defective Treg re-sponse in the absence of Blimp-1, because Blimp-1–deficientCD4+ T cells led to the generation of Foxp3+ Treg cells in greaternumbers than control cells. Although IL-10 production is defec-tive in a small subset of Foxp3+ Blimp-1–deficient Tregs (25, 28,41), IL-10–deficient Treg cells can block adoptive T cell transfer–induced colitis (43, 44) and similar findings have been reported forBlimp-1–deficient Tregs (28).The mechanisms underlying repression of IL-17 production/

Th17 differentiation by Blimp-1 remains to be fully elucidated.Our observation that Blimp-1–deficient CD4+ T cells had in-creased amounts of Il17a mRNA, suggested that Blimp-1 couldregulate IL-17A production at the transcriptional level. In addi-tion, Blimp-1–deficient CD4+ effector T cells had increasedamounts of Il23r and RORC mRNA, suggesting that Blimp-1might directly repress these genes. Consistent with this possibil-ity Blimp-1 consensus binding sites can be found in conserved,putatively regulatory regions in both loci (data not shown). Al-ternatively, because expression of Il23r and Il17a are both posi-tively regulated by Rorc (encoding RORgt), Blimp-1 might alsoregulate Il23r and Il17a indirectly by acting as a repressor of Rorc.Our observation that Blimp-1 can bind to at least one site on the

Il17a promoter in Th2 cells suggests that Blimp-1 might functionto directly repress the Il17a gene during Th2 differentiation. In

FIGURE 5. Blimp-1 is expressed in Th1 and Th2 but not Th17 cells. (A) Blimp-1 expression as reported by YFP in naive CD4+ T cells sorted from YFP

transgenic Blimp-1 reporter mice and stimulated under neutral (with IL2), Th1,Th2 or Th17 conditions for 3.5 (top row) or 7.5 (bottom row) days. FACS

Plots shown are from the gated live, CD4+ cells in the well. (B) Average percentages (and SD) of YFP-positive cells shown in (A) for five independent

experiments for a total of seven different mice. (C) Steady-state Blimp-1 mRNA at day 7.5 in cells from experiment shown in (A). (D) Immunoblotting of

total cell lysates obtained from cells stimulated as in (A) for 5.5 d (Ntrl = Neutral), using Abs to Blimp-1 or b-actin.

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fact, Il17a site -3.2, which shown significant enrichment forBlimp-1 binding is located ∼2 kb downstream of conserved noncoding sequence 2 (CNS2, also called CNS5), an important reg-ulatory region in the il17a promoter (45, 46). Consistent with theidea that Blimp-1 could repress Il17a in Th2 cells, deficiency ofthe histone lysine methyltransferase G9a, which can function asa cofactor for Blimp-1–mediated repression (47), leads to in-creased production of IL-17A in Th2-polarized cells (48). How-ever, forced expression of Blimp-1 in cells differentiating underTh17 conditions did not result in a significant decrease of IL-17A

or IL-17F production (Fig. 7B, 7C). Expression of Prdm1 mRNAin transduced Th17 cells that expressed Blimp-1 was similar tothat of developing Th2 cells (data not shown). Nevertheless, it ispossible that repression of Il17a in Th17 cells requires higheramounts of Blimp-1 protein than achieved in our experiments.Alternatively, repression of the Il17a by Blimp-1 might requirecorepressors that are selectively expressed in Th2 (and potentiallyTh1) but not in Th17 cells. Such a mechanism could play a role inpreserving Il17a activity in Th17 cells that are exposed to con-ditions where Blimp-1 expression can be induced.

FIGURE 6. Blimp-1 expression is induced by Th1- and Th2-inducing cytokines but repressed by TGF-b in Th17-developing cells. (A) Naive CD4+

T cells from Blimp-1 reporter mice were stimulated with plate-bound anti-CD3 plus anti-CD28 alone or in the presence of the indicted cytokines and YFP

(Blimp-1) expression was analyzed by FACS at day 5. (B) qRT-PCR analysis of Blimp-1 mRNA expression in the same cells from experiment shown in (A).

(C) Dose-dependent inhibition of Blimp-1 expression by TGF-b in Blimp-1 reporter naive CD4+ T cells stimulated as indicated in the presence of different

amounts of TGF-b for 5 d and then analyzed by FACS. (D) qRT-PCR analysis of Blimp-1 mRNA expression in the same cells from experiment shown in

(C). Data shown are from one experiment with pooled cells from three mice; similar results were obtained in two independent experiments.

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Our results presented here show that TGF-b signaling is a potentrepressor of Blimp-1 expression and might function to repressBlimp-1 expression during Th17 differentiation. Interestingly,a recent study using in vitro-generated Th17 cells to induce colitisupon transfer into Rag12/2 mice demonstrate that the extinctionof IL-17 production in Th17 cells that become IFN-g producersin the presence of IL-12 and IL-23 is contingent upon limitedamounts or complete absence of TGF-b (23). The presence ofTGF-b resulted in maintenance of IL-17 production in thesecells (23). Using experimental autoimmune encephalomyelitis asa model of inflammation, Ghoreschi et al. (49) made similarobservations, showing that Th17 cells generated in the presence ofIL23 and absence of TGF-b retained the capacity to make IL-17while simultaneously turning on IFN-g production (49). On thebasis of these observations, our results showing that Blimp-1–deficient T cells generate more IL-17+/IFN-g+ cells under inflam-matory conditions suggest a model whereby induction of Blimp-1expression could be required to repress IL-17 production in Th17cells converting into IFN-g–producing cells. According to thismodel, under highly inflammatory conditions where IL-12 isabundant and TGF-b scarce, IL-12 would lead to induction of

Blimp-1 expression in Th17 cells, in addition to inducing ex-pression of T-bet and IFN-g. Blimp-1 expression under theseconditions would result in the repression of both IL-17 andIL23R, thus restraining the formation and/or maintenance of theIL-17+/IFN-g+ cells. In the absence of Blimp-1, IL-12 would leadto IFN-g production, but would fail to repress IL-17 and favor thegeneration/maintenance of the IL-17+/IFN-g+ cells.One caveat of this model is the assumption that IL12 could

induce Blimp-1 and IFN-g simultaneously, which is at odds withour previous observation that in CD4+ T cells stimulated underneutral conditions, Blimp-1 binds to and suppresses transcriptionof both Tbx21 and Ifng (41). It is possible, however, that the Ifnggene regions bound by Blimp-1 are not equally accessible inT cells stimulated under neutral conditions and in cells respondingto IL-12 and transitioning into Th1 cells (or “ex Th17 cells”)developing during mucosal inflammation. In addition to differ-ential accessibility of Blimp-1 binding regions at the Ifng locus,these different Th subpopulations may also diverge in the ex-pression of coactivators and corepressors, which could interactwith Blimp-1 to counteract or favor repression of Ifng. The samearguments can be made to explain the fact that Th1 cells express

FIGURE 7. Blimp-1 binds to the murine Il17a gene in Th2 cells, but it is not sufficient to repress IL-17 production in Th17 cells. (A) Chromatin

immunoprecipitation analysis of wild-type Th2-polarized cells restimulated with PMA and ionomycin, followed by immunoprecipitation of chromatin with

anti–Blimp-1 or control Ab and quantitative PCR analysis of binding of Blimp-1 at different regions upstream (23.3,27.2 kb) or downstream (+44.9 kb) of

the transcription start site of the murine Il17a gene or a negative control region in the SNAIL3 gene; results were normalized to those of the input chromatin,

followed by the ratio of results obtained with anti–Blimp-1 Ab and control Ab (nonspecific background). Data are representative of three to four inde-

pendent experiments (mean and SEM). (B and C) Production of IL-17 upon enforced expression of Blimp-1 in Th17 cells. Naive CD4+ T cells were

stimulated under Th17 conditions and transduced with retrovirus expressing GFP only (Ctrl RV) or GFP and Blimp-1 as a bicistronic message (Blimp-1

RV); cells were restimulated 3 d (B) or 4 d (C) after transduction and analyzed for IL-17A (top plots) or IL-17F (bottom plots) production. Plots shown are

from CD4+ (B) or CD4+GFP+ (C) cells. Chart in (C) shows the average and SEM of three independent experiments.

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high levels of Blimp-1 while making high amounts of IFN-g.Evaluation of Blimp-1 binding sites occupancy in cells stimulatedunder neutral conditions side by side with cell transitioning fromTh17 to Th1 will be required to clarify this.In line with our observation that IL-17–producing cells accu-

mulate in the Blimp-1CKO mice and that Blimp-1–deficient T cellsfail to turn off the Il17 gene under inflammatory conditions, recentgenome-wide association studies reveal strong association betweenthe gene encoding Blimp-1 (PRDM1) and inflammatory boweldisease (50, 51). One of the forms of inflammatory bowel disease,Crohn’s disease, is also associated with the accumulation ofIL-17+/IFN-g+ cells in the intestinal lesions (21, 22). Although thefunctional implications of the PRDM1 polymorphisms associatedwith IBD remain unknown, one intriguing possibility is that thesepolymorphisms cause decreased expression or partial loss of func-tion of Blimp-1 resulting in the accumulation of IL-17+/IFN-g+

cells in the intestinal lesions of CD patients.Plasticity of the CD4+ T cells is now a recognized trait of im-

mune responses, especially under conditions associated with in-flammatory disorders. Understanding the mechanisms regulatingTh cell plasticity in these conditions will be essential in consid-ering new therapeutic approaches to treat these diseases. The in-volvement of Blimp-1 in counteracting the differentiation of thehighly inflammatory IL-17+/IFN-g+ producing cells in vivo as wellas its potential role in repressing Il17a expression in Th1 and Th2cells that we report in this paper might represent initial steps to-ward the understanding the mechanisms regulating effector T cellresponse under chronic inflammatory conditions.

AcknowledgmentsWe thank Dr. Kathryn Calame for invaluable support of these studies and

Dr. Jonathan Kaye for helpful discussions and for critical reading of the

manuscript. We also thank Brian de la Torre and Asha Kadavallore for

helping with mice irradiation and retrovirus supernatant production, res-

pectively, and the CSMC Flow Cytometry core, especially Gillian Hultin,

for sorting. We also thank Carol Landers and Richard Deem for technical

help and Loren Karp for editorial assistance.

DisclosuresThe authors have no conflicting financial interests.

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