IFN Regulatory Factor 4 Regulates the Expression of a Subset of Th2 Cytokines Ayele-Nati N. Ahyi, Hua-Chen Chang, Alexander L. Dent, Stephen L. Nutt and Mark H. Kaplan This information is current as of October 1, 2021. J Immunol 2009; 183:1598-1606; Prepublished online 10 July 2009; doi: 10.4049/jimmunol.0803302 http://www.jimmunol.org/content/183/3/1598 Downloaded from

References This article cites 45 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/183/3/1598.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on October 1, 2021

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IFN Regulatory Factor 4 Regulates the Expression of a Subset of Th2 Cytokines1

Ayele-Nati N. Ahyi,*† Hua-Chen Chang,* Alexander L. Dent,† Stephen L. Nutt,‡ and Mark H. Kaplan2*†

Th2 cells can be subdivided into subpopulations depending on the level of a cytokine and the subsets of cytokines they produce. We have recently identified the ETS family PU.1 as regulating heterogeneity in Th2 popula- tions. To define additional factors that might contribute to Th2 heterogeneity, we examined the PU.1 interacting IFN-regulatory factor (IRF)4. When Th2 cells are separated based on levels of IL-10 secretion, IRF4 expression segregates into the subset of Th2 cells expressing high levels of IL-10. Infection of total Th2 cells, and IL-10 nonsecreting cells, with retrovirus-expressing IRF4, resulted in increased IL-4 and IL-10 expression, no change in IL-5 or IL-13 production and decreased Il9 transcription. Transfection of an IRF4-specific small interfering RNA into Th2 cells decreases IL-10 produc- Downloaded from tion. IRF4 directly binds the Il10 as evidenced by chromatin immunoprecipitation assay, and regulates Il10 control elements in a reporter assay. IRF4 interacts with PU.1, and in PU.1-deficient T cells there was an increase in IRF4 binding to the Il10 gene, and in the ability of IRF4 to induce IL-10 production compared with wild-type cells and Il10 promoter activity in a reporter assay. Further heterogeneity of IRF4 expression was observed in Th2 cells analyzed for expression of multiple Th2 cytokines. Thus, IRF4 promotes the expression of a subset of Th2 cytokines and contributes to Th2 heterogeneity. The Journal of Immunology, 2009, 183: 1598–1606. http://www.jimmunol.org/

D4ϩ Th cells play a critical role in modulating both in- IFN-regulatory factor (IRF)3-4 was first identified as a protein nate and adaptive immune responses. Cytokines direct recruited by the transcription factor PU.1 to an Ig ␬ 3Ј enhancer in C the differentiation of precursor CD4ϩ Th cells into com- a cooperative manner (9–12) and contributes to the differentiation mitted Th phenotypes, Th1, Th17, and Th2 cells, which are defined of Th2, Th17, and some functions of Treg cells (13–15). IRF4 also by their function and the array of secreted cytokines (1–3). Th2 has distinct effects on cytokine production in naive vs memory T cells mediate humoral immune responses by secreting IL-4, IL-5, cell populations (16). In Th2 cells, IRF4 binds to a site adjacent to IL-9, IL-13, and other cytokines including the anti-inflammatory a NFAT binding element and cooperates with NFATc1, NFATc2,

cytokine IL-10. The differentiation of Th2 cells in vivo upon in- and c- in the transcription of IL-4 (17, 18). These interactions by guest on October 1, 2021 fection or in vitro generates a heterogenous population of Th2 cells are likely important as the activation of T cells triggers a rapid that express and secrete various combinations of Th2-type cyto- induction of IRF4 (19, 20) and IRF4-deficient T cells secrete de- kines at different levels (4–6). Although Th2 heterogeneity is well creased levels of Th2 cytokines (13). Ectopic IRF4 has been shown documented, the origins are still unclear. Guo et al. (7) reported to increase the level of IL-10 secreted by Jurkat cells, but the that the Il4 gene in IL-4-producing and nonproducing cells has mechanism by which IRF4 controls the expression of Th2-cyto- differential levels of CpG methylation and histone acetylation. kines in differentiated Th2 cells has not been examined. In this These modifications translate in an open DNA conformation and report, we determine that IRF4 is a regulator of a subset of Th2 gene activation in IL-4-producing cells. We reported that PU.1 cytokines, and contributes to Th2 heterogeneity. plays a critical role in establishing these phenotypes by preventing GATA-3 binding to the IL-4 locus resulting in an IL-4 low Th2 Materials and Methods population (8). Knock-down of PU.1 expression resulted in in- Mice creased homogeneity of Th2 cells (8). In addition to PU.1, it is Wild-type (WT) C57BL/6 and BALB/c mice (Harlan Bioscience) were likely that additional factors are involved in the establishment of used for Th1 and Th2 differentiation. Conditional mutant Sfpi1 mice on the Th2 subpopulations. C57BL/6 background were previously described (21) and were mated to lck-Cre expressing mice (noted as Sfpi1lckϪ/Ϫ). Mice were maintained in pathogen-free conditions and all studies were approved by the Indiana University School of Medicine Animal Care and Use Committee. *Department of Pediatrics, Herman B. Wells Center for Pediatric Research, †Depart- ment of Microbiology and Immunology, and the Walther Cancer Institute, Indiana University School of Medicine, Indianapolis, IN 46202; and ‡The Walter and Eliza Th1 and Th2 cell differentiation Hall Institute of Medical Research, Victoria, Australia CD4ϩ T cells were purified from spleen and lymph nodes by positive Received for publication October 2, 2008. Accepted for publication May 31, 2009. selection using magnetic beads (Miltenyi Biotec) with purity Ͼ97% by ␮ The costs of publication of this article were defrayed in part by the payment of page FACS analysis. Cells were activated with 2 g/ml plate-bound anti-CD3 charges. This article must therefore be hereby marked advertisement in accordance (145–2C11) plus 1 ␮g/ml anti-CD28, in addition to 10 ng/ml IL-4 and 10 with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by U.S. Public Health Service Award AI057459 (to 3 Abbreviations used in this paper: IRF, IFN-regulatory factor; WT, wild type; ChIP, M.H.K.) from the National Institutes of Health. chromatin immunoprecipitation assay; siRNA, small interfering RNA; qPCR, quan- 2 Address correspondence and reprint requests to Dr. Mark H. Kaplan, Department of titative PCR; DAPA, DNA affinity purification assay; GATA-3, GATA binding Pediatrics, and Microbiology and Immunology, Wells Center for Pediatric Research, protein-3. Indiana University School of Medicine, 702 Barnhill Drive, RI 2600, Indianapolis, IN 46202. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803302 The Journal of Immunology 1599

␮g/ml anti-IFN-␥ (R4/6A2 or XMG) for Th2 differentiation, or 10 ng/ml were labeled using the mouse IL-10 Secretion Assay (Miltenyi Biotec) IL-12 plus 10 ␮g/ml anti-IL-4 (11B11) for Th1 differentiation. After 3 days before sorting with FACSVantage SE or FACSAria from Becton Dick- of incubation, cells were expanded for a total of 5 or 6 days. Differentiated inson. The cells were rested for 1 or 2 days before restimulation, anal- cells were restimulated with 2 ␮g/ml anti-CD3 at a concentration of 106 ysis, or transduction. cells/ml for real-time PCR and ELISA as previously described (8, 22). Statistics were performed using a t test with SPSS software (v. 16.0; SPSS). IL-10 high and low Th2 cocultures ϫ 6 Immunoprecipitation and Western blot analysis IL-10 high and low Th2 (1 10 ) cells were sorted as described above and cocultured with either 0.5 ϫ 106 splenic CD11cϩ cells purified by positive Total cell extracts were prepared by lysing Th2 cells with lysis buffer (10% selection using magnetic beads (Miltenyi Biotech) or 1 ϫ 106 total spleno- glycerol,1% Igepal, 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA cytes from BALB/c mice. Cells supplemented with RPMI 1640 medium (pH 8)) for 15 min on ice before centrifugation at 14,000 rpm for 15 min were activated with 5 ␮g/ml anti-CD3 and 4 ␮g/ml LPS. dendritic cell at 4°C. Nuclear and cytoplasmic were prepared from differentiated coculture was harvested after 3 days for IFN-␥ and IL-6 ELISA and total Th2 cells using Nuclear and Cytoplasmic Extraction Reagents from Pierce splenocyte coculture was harvested after 5 days for IgG1 ELISA. Biotechnology. Protein extracts (25 ␮g) from naive T, Th1, Th2, and Phoe- nix cells were separated on 4–12% SDS-PAGE and transferred onto Ny- Chromatin immunoprecipitation assay (ChIP) tran membranes (Schleicher and Schuell Bioscience). Immunoprecipitation CD4 T cells cultured under Th2 conditions for 5 days were fixed with was performed as previously described (8). Nuclear cell lysates (1 mg Th2 formaldehyde to cross-link protein-DNA complexes and 5 ϫ 106 cells extract) were incubated with control Ab, anti-IRF4, anti-PU.1, or anti- were used per ChIP reaction. The ChIP assay was performed essentially as GATA-3 conjugated with protein G beads (Santa Cruz Biotechnology) described (25) except that cells were resuspended in nuclear lysis buffer (50 overnight at 4°C before precipitation with protein G beads. Immunocom- nM Tris (pH 8.0), 10 mM EDTA (pH 8.0), 1% SDS, and protease inhibitor) plexes were separated by SDS-PAGE and immunoblots were probed with at 4°C for 15 min before ultrasonication and the addition of one LiCl wash the precipitating Abs. with the LiCl buffer (0.25 M LiCl, 1% Igepal, 1% sodium deoxycholate,1 Downloaded from DNA affinity precipitation assay mM EDTA (pH 8.0), and 10 mM Tris (pH 8.0)), and two Tris-EDTA buffer washes (1 mM EDTA and 10 mM Tris (pH 8.0)). Real-time PCR was done Th2 nuclear lysate (250 ␮g) was incubated with double stranded biotinyl- with 2 ␮l (1.7 ϫ 105 cells) of immunoprecipitated DNA for 30 cycles. ated oligonucleotides as described (23). The sequences for Il-10 promoter DNA was then analyzed using quantitative PCR (qPCR). To calculate per- oligonucleotide are biotin-TGAGGTCTGAAGAAAATCAGCCCTCTCG cent input, ChIP results for the specific Ab were determined using a stan- GG and the reverse complement. For the competition assay, the competitor dard curve of input DNA from the same cells. Control IgG ChIP results are oligonucleotide was incubated with the nuclear protein for 15 min at room subtracted from specific Ab ChIP results and results are shown as the mean Ϯ temperature before the addition of the biotinylated Il-10 promoter oligo- specific binding of replicates SD. Primers for Il10 and Il4 ChIP have http://www.jimmunol.org/ nucleotide. The IRF4 consensus binding site was deleted in the IRF4 mu- been previously described (8, 26). Primers for the Il9 promoter were CAG tant competitor oligonucleotide and the sequence is TGAGGTCTAT- TCT ACC AGC ATC TTC CAG TCT AGC and GTG GGC ACT GGG CAGCCC TCTCGGG and the reverse complement. The sequence for the TAT CAG TTT GAT GTC. ␭B IRF4 competitor and GATA-3 oligonucleotide were previously de- scribed (8, 24). Protein-DNA complexes were separated by SDS-PAGE Reporter assay and immunoblots were probed with anti-IRF4. EL4 cells were cultured for 3 days before transfection of 106 cells by Retroviral vectors and transduction Amaxa nucleofection or by electroporation with expression plasmid, re- porter vector containing the regulatory sequence and ␤-galactosidase in The retroviral vector MIEG-hCD4 was previously described (8). The cod- DMEM. Cells were immediately transferred to six-well plates. After 24 h,

ing region for human IRF4 cDNA was amplified by PCR and cloned into the cells were harvested, washed with PBS, and cultured in the presence or by guest on October 1, 2021 MIEG-hCD4. The Phoenix GP packaging cells line was transiently trans- absence of 0.2 ␮g/ml ionomycin and 20 ng/ml PMA for 24 h. Harvested fected with 15 ␮g of purified plasmid by calcium phosphate precipitation. cells were lysed with reporter lysis buffer (Promega) and the luciferase CD4ϩ T cells cultured under Th1 or Th2 conditions for 2 days were trans- activity was measured for each sample and divided by the protein concen- duced by centrifugation at 1,800 rpm, 20°C for 2 h with 1.5 ml of retroviral tration and the ␤-galactosidase activity of the sample. The Il4 and Il10 supernatant containing 8 ␮g/ml polybrene, 100 U/ml human IL-2, and reporters were described previously (26). The NFAT reporter is a trimer of cytokines and Abs for either Th1 or Th2 differentiation. Transduced Th2 a distal Il2 promoter-binding site and was generously provided by Gerald were stained with anti-human CD4-PE from BD Biosciences and purified Crabtree (Stanford University, Stanford, CA). by sorting before restimulation for real-time PCR or ELISA. Results Nucleofection of siRNA IRF4 expression defines IL-10 low and IL-10 high phenotype WT CD4ϩ T cells were cultured under Th2 condition for 5 days as de- scribed above before Amaxa nucleofection with scrambled small interfer- Having established a role for PU.1 in regulating Th2 heterogene- ing RNA (siRNA) or IRF4 specific siRNA, a pool of two different siRNA ity, we wanted to determine whether other factors required for Th2 constructs (siRNA2 and siRNA3) described by Bru¨stle et al. (14). After differentiation also contribute to the expression of subsets of Th2 4 h, treated cells were restimulated with 5 ␮g/ml, anti-CD3, 1 ␮g/ml anti- cytokines. We separated Th2 cultures into IL-4 high and low, and ␮ CD28, 10 ng/ml IL-4, and 10 g/ml XMG for 40 h. Cells were then har- IL-10 high and low populations and examined the expression of vested and either analyzed by RT-PCR or restimulated with 2 ␮g/ml anti- CD3 for ELISA. Th2-associated transcription factors in each population. As previ- ously described, PU.1 segregated into the IL-4 low population (8). Flow cytometry IL-10 high cells expressed 15-fold more Il10 mRNA and secreted Cells were restimulated with 4 ␮g/ml ␣-CD3 for 3 h before 3-h treatment 50-fold more IL-10 than IL-10 low cells (Fig. 1, A–C). Il4 expres- with 3 ␮M monensin. Intracellular cytokine staining was performed using sion was 2-fold higher in IL-10-high than in IL-10-low cells. The fluorochrome-conjugated Abs (BD Pharmingen) and ␣-IL-4 PECy7 (eBio- IL-10-high and low phenotypes were stable as there was still a ␣ science). IRF4 staining was performed using -IRF4 (Santa Cruz Biotech- significant difference in IL-10 production from these cells after an nology) and the secondary donkey anti-goat Ab (Jackson ImmunoResearch Laboratories) conjugated with Cy5 (Cyanine 5). Stained cells were ana- additional week in culture (Fig. 1B). In contrast to the enrichment lyzed with an LSRII instrument. for IL-4 and IL-10 production, the level of Il9 mRNA is 9-fold higher in IL-10 low than in IL-10 high cells (Fig. 1D). To deter- Isolation of IL-10 high and low populations mine whether IL-10 high and low cells had different functional CD4ϩ T cells were differentiated into Th2 cells for 7 to 10 days as characteristics in culture, we cultured CD11cϩ splenocytes or total described above. The day before sorting, the differentiated cells were splenocytes in the absence or presence of either IL-10 high or low harvested counted and plated at 107 cells/ml in RPMI 1640 for 6 h. After this resting period, the cells were restimulated for 6 h with 10 Th2 cells. We observed that IL-10 low cells stimulated increased ng/ml PMA and 1 ␮g/ml ionomycin. Cells were harvested and washed IL-6 and IFN-␥ production from LPS-stimulated dendritic cells, with MACS buffer. The IL-10 secreting and nonsecreting populations while IL-10-high cells has less of an effect on IL-6 production and 1600 IRF4 AND Th2 HETEROGENEITY Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. IRF4 expression segregates between IL-10 high and IL-10 low Th2 cells. A, CD4 T cells were cultured under Th2 conditions for 7–10 days and separated into IL-10 high and IL-10 low populations using cytokine capture and sorting by flow cytometry. The purity of the IL-10 low and IL-10 high cells was determined by flow cytometry. B, IL-10 low and IL-10 high cells were stimulated for 1 day directly after or 6 days after sorting. Supernatants were tested for IL-10 concentration using ELISA. C, Cells cultured and sorted as in A were stimulated with anti-CD3 for 4 h and RNA was isolated for qPCR analysis of Il4 and Il10 mRNA, or stimulated for 1 day and supernatants were tested for IL-4 concentration using ELISA. D, Cells cultured and sorted as in A were stimulated with anti-CD3 for 4 h and RNA was isolated for qPCR analysis of Il9 mRNA. E, Splenic CD11cϩ cells were cultured alone or with IL-10 low or IL-10 high Th2 cells and stimulated for 3 days with LPS and anti-CD3. Supernatants were tested for IL-6 and IFN-␥ using ELISA. F, Splenocytes were cultured alone or with IL-10 low or IL-10 high Th2 cells and stimulated for 5 days with LPS and anti-CD3. Supernatants were tested for IgG1 using ELISA. G, RNA was isolated from sorted IL-10 low and IL-10 high cells and analyzed using qPCR for Irf1, Irf2, and Irf4. H, IRF4 protein level was analyzed in naive T cells and Th2 cells treated with monensin for the last3hofa6-hstimulation with anti-CD3 before intracellular staining for IL-10 and IRF4. IRF4 expression is shown in cells gated for high or low expression of IL-10. Data in all panels are representative of at least three experiments.

did not affect IFN-␥ production (Fig. 1E). Conversely, IL-10-high high and low cells for expression of a number of factors associated cells, but not IL-10-low cells were able to induce class switching with regulating the Th2 phenotype, including Sfpi1, Maf, Bcl6, to IgG1 in LPS-stimulated splenocyte cultures (Fig. 1F). These Zfpm1, Zbtb32, Runx1, and Irf4. We observed that Irf4 expression data suggest that IL-10 high and low populations in Th2 cultures was enriched 5-fold in the IL-10 high population (Fig. 1G). To represent distinct states that have the potential for separate biolog- confirm that IRF4 protein was also differentially expressed be- ical functions in vivo. tween IL-10 high and low cells, we performed intracellular stain- To determine whether there were transcription factors that were ing for IL-10 and IRF4. There was an 8-fold difference in expres- associated with differences in IL-10 expression, we screened IL-10 sion of IRF4 in cells gated for IL-10 high or IL-10 low expression The Journal of Immunology 1601

(Fig. 1H). Because IRF4 expression is induced upon anti-CD3 stimulation of naive CD4ϩ T cells, we compared the expression of IRF4 in naive cells activated with anti-CD3 for 6 h with the ex- pression in IL-10 low and high cells. Although IRF4 was induced following activation, IRF4 expression was still higher in IL-10 low cells, suggesting that IL-10 low cells are not activated but undifferentiated cells (Fig. 1H). To further show specificity for the expression of Irf4, we also examined the expression of re- lated factors Irf1, Irf2, and Irf8. Irf1 and Irf2 were not differ- entially expressed between IL-10 high and low cells (Fig. 1G). Irf8 mRNA was barely detectable in either subset in the absence of stimulation with anti-CD3 and expression was not different following activation of IL-10 high or low cells (data not shown). These studies demonstrate a specific correlation of IRF4 expression with IL-10 production. IRF4 function in Th cells IRF4 was reported to be important for Th2 development because IRF4-deficient T cells secrete decreased levels of Th2 cytokines while IFN-␥ levels increase (13). However, the role of IRF4 in Downloaded from differentiated Th2 cells has not been clearly defined. The require- ment for IRF4 in Th2 development suggested that ectopic expres- sion of IRF4 into Th2 cells during differentiation would increase the level of secreted Th2 cytokines. To test this, we generated an IRF4-expressing bicistronic retroviral vector (Fig. 2A) and trans- duced differentiating Th2 cells on the second day of a 5-day culture http://www.jimmunol.org/ period. Cells sorted for hCD4 expression were restimulated with anti-CD3 and evaluated for cytokine production using ELISA. Ec- topic expression of IRF4 in Th2 cells increased production of IL-10 and IL-4 by 8-fold and 4-fold, respectively, with no signif- icant effect on IL-5 and IL-13 (Fig. 2B). Because transduction of IRF4 increases specific cytokines in differentiating Th2 cells, we next tested whether it would alter the phenotype of IL-10 low cells isolated from differentiated Th2 pop- by guest on October 1, 2021 ulations. To assess the change in IL-10 low phenotype, we sorted IL-10-low cells from cells cultured under Th2 conditions for 10 days as described in Materials and Methods. IL-10 low cells were transduced with IRF4 RV and cultured for 2 days before selection and stimulation with anti-CD3 to assess the level of cytokine pro- duction. Transduction of IRF4 in IL-10 low cells enhanced the production of IL-10 and IL-4 by 6-fold and 4-fold, respectively, with no significant effect on IL-5, but a decrease in Il9 mRNA (Fig. 2C). These results demonstrate that IRF4 specifically increases IL-4 and IL-10 production from Th2 cells but does not induce other Th2 cytokines. To further show that IRF4 is required for IL-10 expression in Th2 cultures, we differentiated Th2 cultures in vitro and trans- fected cells with IRF4-specific or scrambled siRNA. Because Irf4 is expressed at high levels in effector Th2 cells, the IRF4- FIGURE 2. IRF4 transduction increases production of specific Th2 cy- specific siRNA resulted in only a partial reduction of Irf4 ex- tokines. A, Schematic of control and bicistronic retroviral vectors contain- pression (Fig. 2D). However, there was a commensurate decrease in ing cDNAs for human IRF4 (hIRF4) and human CD4 (hCD4, selectable IL-10 production from cells transfected with IRF4-specific siRNA marker). B, BALB/c CD4 T cells were transduced with control (MIEG) or (Fig. 2D). IRF4 containing retroviruses on the second day of a 5-day culture under Th1 cells also secrete IL-10, though at levels much lower than Th2 conditions. HCD4-positive cells were sorted and stimulated with anti- those produced by Th2 cells and with fewer IL-10ϩ cells in the CD3. Supernatants were analyzed for concentration of the indicated cyto- Th1 population than Th2 cultures, though most IL-10ϩ cells are kines using ELISA. C, CD4 T cells cultured under Th2 conditions for 7 also IFN-␥ϩ (Fig. 3A and data not shown) (27, 28). As IRF4 is also days and sorted for low IL-10 secretion were transduced with control or IRF4-expressing retrovirus and cultured for an additional 3 days. HCD4- expressed in Th1 cells (Fig. 3B), we wanted to determine whether positive cells were then sorted and stimulated with anti-CD3. Supernatants IRF4 expression also segregated with IL-10 expression in Th1 cul- were analyzed for concentration of the indicated cytokines using ELISA. tures. Intracellular staining for IL-10 and IRF4 in Th1 cells was Il9 expression was determined using qPCR. D, CD4ϩ T cells cultured similar to the pattern in Th2 cells where higher IRF4 was observed under Th2 condition for 5 days were transfected with scrambled siRNA or in IL-10 high cells (Fig. 3C). To further demonstrate that IRF4 IRF4 siRNA. After 40 h, cells were either stimulated with anti-CD3 for 2 h functioned in Th1 cells to increase IL-10 production, we trans- and RNA was isolated for qPCR analysis of Irf4 mRNA or stimulated for duced differentiating Th1 cell with control or IRF4-expressing ret- 1 day and supernatants were tested for IL-10 concentration using ELISA. 1602 IRF4 AND Th2 HETEROGENEITY

IRF4 was previously shown to transactivate a reporter gene with the Il4 promoter (17), which we also observed in this study (Fig. 4D). To determine whether IRF4 could transactivate gene expres- sion from the Il10 regulatory elements, we used a luciferase re- porter containing either the Il10 promoter region or the Il10 CNS3 region, both of which contain IRF4 binding sites (Fig. 4A) (26). Upon transfection of EL4 cells with either of the Il10 reporters and IRF4-expressing or control pCEP4 vectors, the transactivation was measured by assessing luciferase activity. Cotransfection of IRF4 induced increased luciferase activity from promoter and CNS3 reporters (Fig. 4D). Stimulation of cells with PMA plus iono- mycin increased basal reporter activity and IRF4 cotransfection was able to further increase reporter activity (Fig. 4D). As a negative control, IRF4 did not activate an NFAT reporter plas- mid (Fig. 4D). Thus, IRF4 binds and directly transactivates Il10 regulatory elements. Functional interactions of PU.1 and IRF4 PU.1 was demonstrated to interact with GATA binding pro- tein-3 (GATA-3) using recombinant protein binding assays (29) Downloaded from and in Th2 cell coimmunoprecipitates (8). PU.1 is also known to interact with IRF4 in B cells (10, 12, 24, 30, 31). To inves- tigate the interaction of PU.1 and IRF4 in Th2 cells, we used anti-PU.1, anti-IRF4, or anti-GATA-3 to immunoprecipitate complexes from Th2 nuclear lysate. PU.1, IRF4, and a small http://www.jimmunol.org/ FIGURE 3. IRF4 expression segregates between IL-10 high and amount of GATA-3 were immunoprecipitated from Th2 cells IL-10 low Th1 cells. A, CD4 T cells were cultured under Th1 or Th2 with anti-IRF4 and anti-PU.1 (Fig. 5A). Immunoprecipitation conditions for 5 days before stimulation with anti-CD3. Supernatants with anti-GATA-3 confirmed the interaction between PU.1 and were analyzed for IL-10 concentration using ELISA. B, IRF4 protein GATA-3, though little IRF4 was precipitated with this complex level was assessed by immunoblot in control (MIEG) and IRF4-trans- (Fig. 5A). Thus, although PU.1 interacts with IRF4 and fected phoenix cells, as well as unstimulated and 6-h stimulated naive GATA-3, these data suggest that PU.1-IRF4 and PU.1-GATA-3 T cells, Th1, and Th2 cells. C, IRF4 protein level was analyzed in Th1 are largely separate complexes, as little IRF4 was associated cells treated with monensin for the last3hofa6-hstimulation with with GATA-3. Because IRF4 expression segregates in IL-10 anti-CD3 before intracellular staining for IL-10 and IRF4. IRF4 expres- high cells, these interactions would only be meaningful if PU.1 sion is shown in cells gated for high or low expression of IL-10. D, by guest on October 1, 2021 Differentiating Th1 cells were transduced with control (MIEG) or IRF- were also expressed in the same cells. To test this, we examined 4-expressing retrovirus as in Fig. 2B. Data in all panels are represen- expression of Sfpi1, encoding PU.1, in IL-10 high and low cells tative of two to three experiments. and observed that Sfpi1 mRNA was enriched in the IL-10-high population in a pattern similar to Irf4 expression (Fig. 5B). Because IL-10 high cells are comprised of both IL-4 high and low cells, this does not contradict previous data showing a seg- roviruses as done with Th2 cells in Fig. 2B and observed that IRF4 regation of PU.1 expression in IL-4 high and low populations. increased IL-10 production from Th1 cells (Fig. 3D). To determine whether the association of IRF4 and PU.1 had Together, these data demonstrate that IRF4 contributes to IL-10 functional consequences, we used mice carrying a conditional al- production in Th subsets. lele of the Sfpi1 gene, crossed to lck-Cre transgenic mice (denoted as Sfpi1lckϪ/Ϫ). We then compared the function of IRF4 in WT and IRF4 binding to Il10 and Il4 Sfpi1lckϪ/Ϫ Th2 cells. ChIP demonstrated that IRF4 binding to the To determine whether IRF4 is directly regulating Il10 and Il4, Il10 promoter is greater in Sfpi1lckϪ/Ϫ Th2 cells than in C57BL/6 we tested the direct binding of IRF4 to the Il10 locus (Fig. 4A) Th2 cells (Fig. 5C). A similar trend of IRF4 binding was observed using DAPA and chromatin immunoprecipitation. We first used in Il4 promoter (data not shown). The level of Il4 DNase hyper- a biotinylated oligonucleotide corresponding to an IRF4 con- sensitivity site VA (32, 33) in the IRF4 precipitates was also sensus binding site in the Il10 promoter. Strepavidin-agarose greater in Sfpi1lckϪ/Ϫ Th2 cells than in WT Th2 cells though bind- was able to precipitate IRF4 in the presence but not the absence ing to Il10 CNS3 was only modestly affected by PU.1-deficiency. of oligonucleotide using a DNA affinity purification assay Th2 cultures from Sfpi1lckϪ/Ϫ mice produced slightly more IL-10 (DAPA) protocol (Fig. 4B). To demonstrate specificity for this than WT cultures, supporting observations in our previous report interaction, IRF4 was competed with an oligonucleotide from (8). Concomitant with increased IRF4 binding to the Il10 locus, the Ig␭ enhancer that contains an IRF4 binding site, but not transduced IRF4 induced more IL-10 in Sfpi1lckϪ/Ϫ Th2 cells with the Il10 promoter oligonucleotide with the IRF4 binding than in WT Th2 cells. We did observe that IRF4 transduction site deleted or with a GATA-3 consensus oligonucleotide (Fig. had a less robust increase in IL-10 production in C57BL/6 back- 4B). To test this interaction in vivo we used chromatin immu- ground cells, compared with BALB/c Th2 cells (Fig. 5D vs Fig. noprecipitation and observed the Il10 promoter, the Il10 CNS 2B) although IRF4 expression as assessed by flow cytometry element (26), and as controls, the Il4 promoter and enhancer was similar between BALB/c and C57BL/6 cells, and between were enriched in IRF4 immunoprecipitates compared with con- C57BL/6 WT or Sfpi1lckϪ/Ϫ cells (data not shown). To further trol Ab precipitates (Fig. 4C). In contrast, IRF4 had minimal demonstrate the ability of PU.1 to interfere with gene activation binding to the Il9 promoter (Fig. 4C). by IRF4 we repeated the reporter assay in Fig. 4D with the The Journal of Immunology 1603

addition of a condition where PU.1- and IRF4-expressing vec- tors were cotransfected. Results demonstrate that PU.1 also in- terferes with the IRF4-dependent induction of the Il4 and Il10 promoter reporter vectors (Fig. 5E). Overall, these results sug- gest that PU.1 interactions limit the ability of IRF4 to transac- tivate Th2 cytokines.

IRF4 expression profile in subpopulations of Th2 cells The analysis in Fig. 1 is based on two-state separation of cells into IL-10 high and low cells. However, IL-10 high and low cells can be further divided based on coexpression of other cytokines. Be- cause IRF4 induced production of IL-4 and IL-10, we examined the expression of IRF4 in populations of IL-4- and IL-10-positive cells by intracellular staining. IRF4 expression was highest in Th2 cells that were double-positive for IL-4 and IL-10, and lowest in cells that did not secrete either cytokine (Fig. 6). Interestingly, expression was intermediate but similar in IL-4- and IL-10-single positive cells. Results are shown for BALB/c cells and similar patterns are observed for C57BL/6 cells. Thus, while IRF4 pro- Downloaded from motes IL-4 and IL-10 production, other factors also contribute to the decision of a cell to make one or both cytokines.

Discussion IRF4 plays an important role in the development of Th2 and Th17 cells (14). In Irf4Ϫ/Ϫ mice, development of Th2 cells is decreased, suggesting that it plays a role in the differentiation process (14). http://www.jimmunol.org/ However, a role in differentiation does not preclude involvement in the regulation of specific cytokines in differentiated Th2 cells. In this report, we demonstrate that IRF4 contributes to the heteroge- neity of Th2 populations by increasing production of IL-4 and IL-10 and decreasing expression of Il9, while having no effects on IL-5 or IL-13. IRF4 expression segregates in Th2 cells between IL-10 high and low cells, directly binds to and transactivates the

Il10 gene, and ectopic expression of IRF4 can increase IL-10 pro- by guest on October 1, 2021 duction from IL-10-low cells. Thus, IRF4 is an instructive factor in establishing Th2 heterogeneity. IL-10 is a regulatory cytokine produced by a number of cells, including Th2 cells and Il10 regulation in each cell type may be distinct. IL-10 plays a critical role in controlling inflammation in vivo by selectively suppressing the expression of proinflammatory cytokines. In this report, we show that IL-10 high and low cells have differing effects on cocultured cells in vitro, and it is likely that functions differ in vivo as well. In various cell types, the mo- lecular mechanism regulating the expression of IL-10 involves binding of IRF1 and STAT3 to the promoter region of the Il10 gene locus (27, 34), and the regulation of the level of IL-10 mRNA FIGURE 4. IRF4 binds to the IL-10 promoter and transactivates report- by Sp1 and Sp3 (35). In Th2 cells, GATA-3 remodels the Il10 ers with Il10 regulatory regions. A, Representation of the mouse IL-10 gene with the arrow indicating the translational start and black boxes represent- locus (34), and Jun family proteins bind the CNS3 region to induce ing exons. The regions used for promoter and CNS3 reporters and ChIP are Il10 transcription (26). Moreover, IL-10 production requires re- indicated. B, For DAPA analysis, Th2 nuclear extract was incubated with peated stimulation to be completely imprinted within the Th2 pop- (ϩ) or without (Ϫ) a biotinylated oligonucleotide corresponding to an ulation (36). In this report, we also show that IRF4 contributes to IRF4 consensus binding site in the Il10 promoter, before precipitation with Il10 expression in Th2 and Th1 cells. The IL-12-dependent pro- streptavidin-agarose and IRF4 immunoblot. Competitors (fold excess in- duction of IL-10 in Th1 cells has been documented in human and dicated) to demonstrate specificity of the Il10p oligonucleotide-IRF4 in- mouse cells (27, 37, 38). Clearly, additional factors contribute to teraction included oligonucleotides containing an IRF4 binding site from the Ig␭ gene, the Il10 promoter oligonucleotide containing a deletion of IRF4 binding site, and an oligonucleotide containing a GATA-3 consensus site. Competitors were incubated with Th2 extract for 15 min before in- mean specific binding of replicates Ϯ SD and are representative of three cubation with the biotinylated oligonucleotide. Direct immunoblot of Th2 experiments. D, Analysis of luciferase assays from EL4 cells transfected extract is indicated as Th2. C, ChIP assay of IRF4 binding to Il10, Il4, and with reporter vectors containing Il4 promoter, Il10 promoter, and CNS3 Il9 promoter regions; Il10 CNS3; and Il4Va. Quantification of IRF4 bind- regulatory regions, or an NFAT reporter and either empty vector or an ing was performed using qPCR and is shown as percent input. To calculate IRF4-expressing vector. Relative luciferase (normalized to EL4 cells trans- percent input, ChIP results for the specific Ab were determined using a fected with empty vector) are shown in cells that were cultured in the standard curve of input DNA from the same cells. Control IgG ChIP results presence (stimulated) or absence (unstimulated) of PMA plus ionomycin. .p Ͻ 0.05 ,ء .are subtracted from specific Ab ChIP results and results are shown as the Results are representative of two to three experiments 1604 IRF4 AND Th2 HETEROGENEITY Downloaded from

FIGURE 5. PU.1 interacts with and limits the activity of IRF4. A, Nuclear protein (1 mg) from Th2 cells was incubated overnight with Abs to PU.1, IRF4, or GATA-3 (IP) or control Ab (Ab). Immunocomplexes were precipitated with protein G beads. Immunoblot (IB) was performed for PU.1, IRF4, and GATA-3 in each of the precipitates. B, RNA was isolated from IL-10 low and IL-10 high cells sorted as in Fig. 1A and analyzed using qPCR for Sfpi1 expression. C, ChIP assay for IRF4 binding to Il10 promoter and CNS3 regions, and Il4Va regions was performed as described in Fig. 4B with chromatin from WT and SfpilckϪ/Ϫ CD4 T cells cultured under Th2 conditions. IRF4 ChIP was performed as in Fig. 4C. Results are shown with control IgG ChIP Ϫ Ϫ subtracted and indicate mean Ϯ SD of replicate samples that are representative of three experiments. D,WTandSfpilck / CD4 T cells were cultured under http://www.jimmunol.org/ Th2 conditions and transduced with control or IRF4-expressing retroviruses as described in Fig. 3B. IL-10 levels were determined by ELISA. E, Analysis of luciferase assays from EL4 cells transfected with reporter vectors containing Il10 promoter or Il4 promoter, and either empty vector, IRF4-expressing vector, or IRF4- and PU.1-expressing vectors. Relative luciferase (normalized to EL4 cells transfected with empty vector) are shown in cells that were .p Ͻ 0.05 ,ء .cultured in the presence of PMA plus ionomycin. Results are representative of two to three experiments

the difference in IL-10 production by Th1 and Th2 cells, including that GATA-3 is not expressed in Th1 cells, c-jun and JunB bind to

the Il10 CNS3 in Th2 but not Th1 cells (26), and Ets-1 is a neg- by guest on October 1, 2021 ative regulator of IL-10 production in Th1 cells (39). We demon- strate that in Th2 cells, IRF4 binds directly to the Il10 locus and is able to transactivate Il10 regulatory element reporter plasmids. Our results parallel the recent description of a role for IRF4 in Treg cells where Il10 was one of the prominently regulated (15). The fact that IRF4 binding sites exist in the Il10 promoter and CNS3 regions, and that it binds to the same regulatory element as Jun containing complexes suggests that these factors may cooper- ate. We did not observe strong interactions of IRF4 with GATA-3, suggesting that while these factors both contribute to Il10 expres- sion, direct interactions are not required. There are interactions of IRF4 with PU.1, which we previously demonstrated decreased IL-10 production (8). In the absence of PU.1, IRF4 had greater potential to bind Il10, transactivate the Il10 promoter, and increase IL-10 production, suggesting that, at least in part, PU.1 is able to modulate Il10 through the ability to inter- fere with IRF4 activity. It was surprising that we did not see in- creased IL-10 in Sfpi1lckϪ/Ϫ control-transduced cells (Fig. 5D), and this may be a result of the culture conditions required for retroviral transduction. The effects of PU.1 deficiency resulting in increased Th2 cytokine production are most dramatic when TCR stimulation is limiting (H. C. Chang, S. L. Nutt, and M. H. Kaplan, submitted for publication). However, transduction of cells with limiting TCR stimulation was not efficient, and as a result of using FIGURE 6. IRF4 heterogeneity in Th2 cells. CD4 T cells were cultured optimal stimulation conditions for the retroviral transduction, we under Th2 conditions and IRF4 protein level was analyzed in cells treated with monensin for the last3hofa6-hstimulation with anti-CD3 before did not observe altered IL-10 production in the absence of IRF4 intracellular staining for IL-4, IL-10, and IRF4. The mean fluorescence transduction. The role of PU.1 must also be placed in the context intensity (MFI) of IRF4 staining is shown in the table for each of the gated of interactions with multiple transcription factors (Fig. 5A) and populations. with the heterogeneity of the Th2 population where PU.1 is The Journal of Immunology 1605 differentially expressed in subpopulations of cells. As previously cells, which binds IRF4 and decreases binding to target genes in- noted, PU.1 is expressed highly in IL-4 low cells, and in this report cluding Il10. Future work will examine how these factors interact is also expressed in IL-10-high cells. This might suggest that PU.1 to generate the population phenotype and what signals determine is most highly expressed in an IL-4 low/IL-10 high population. the expression of each factor within individual cells. However, it has thus far been difficult to sort cells stained for two cytokines preventing a more thorough analysis of this issue. As Acknowledgments PU.1 expression is only present in a subpopulation of Th2, the We thank Dr. Q Yu for help with primer design. effects of deficiency on IRF4 function might be obscured in a bulk Th2 population, unless IRF4 is overexpressed (Fig. 5D). More- Disclosures over, as ectopic PU.1 expression decreased IL-5 and IL-13 pro- The authors have no financial conflict of interest. duction, as well as IL-4 and IL-10, it is clear that interactions with IRF4 only account for a portion of the observed function of PU.1 References in Th2 cells. 1. Murphy, E., K. Shibuya, N. Hosken, P. Openshaw, V. Maino, K. Davis, The reciprocal regulation of Il10 and Il9 in Th2 cells is striking K. Murphy, and A. O’Garra. 1996. Reversibility of T helper 1 and 2 populations is lost after long-term stimulation. J. Exp. Med. 183: 901–913. and distinct from the IL-9 and IL-10-producing cells present in 2. Weaver, C. T., L. E. Harrington, P. R. Mangan, M. Gavrieli, and K. M. Murphy. cultures primed with TGF␤ and IL-4 (40, 41). Although transduc- 2006. Th17: an effector CD4 lineage with regulatory T cell ties. Immunity tion of IRF4 in Th2 cells decreases Il9 expression, we observed 24: 677–688. 3. Murphy, K. M., W. Ouyang, J. D. Farrar, J. Yang, S. Ranganath, H. Asnagli, only minimal IRF4 binding to the Il9 promoter in a ChIP assay. M. Afkarian, and T. L. Murphy. 2000. Signaling and transcription in T helper This suggested that the effects of IRF4 could be indirect, through development. Annu. Rev. Immunol. 18: 451–494. 4. Bucy, R. P., L. Karr, G. Huang, J. Li, D. Carter, K. Honjo, J. A. Lemons, Downloaded from the induction of IL-10. Indeed, neutralizing IL-10 in IL-10-high K. M. Murphy, and C. T. Weaver. 1995. Single cell analysis of cytokine gene cells, that express low levels of Il9, modestly increased Il9 mRNA coexpression during CD4ϩ T-cell phenotype development. Proc. Natl. Acad. Sci. (data not shown). However, it is not clear which IL-10 activated USA 92: 7565–7569. 5. Jankovic, D., M. C. Kullberg, N. Noben-Trauth, P. Caspar, W. E. Paul, and pathways might be responsible for this regulation. It is also not A. Sher. 2000. Single cell analysis reveals that IL-4 /Stat6 signaling is not clear why a cell would be specialized to express only one of these required for the in vivo or in vitro development of CD4ϩ with a Th2 cytokines. IL-9 is a pleiotropic cytokine involved in the pathologic cytokine profile. J. Immunol. 164: 3047–3055. 6. Karulin, A. Y., M. D. Hesse, M. Tary-Lehmann, and P. V. Lehmann. 2000. http://www.jimmunol.org/ and physiologic evolution of asthma by recruiting eosinophils and Single-cytokine-producing CD4 memory cells predominate in type 1 and type 2 lymphocytes to the lung, inducing mucus hypersecretion, mast immunity. J. Immunol. 164: 1862–1872. 7. Guo, L., J. Hu-Li, J. Zhu, C. J. Watson, M. J. Difilippantonio, C. Pannetier, and cells hyperplasia in concert with IL-4, IL-5, and IL-13 (42), while W. E. Paul. 2002. In TH2 cells the Il4 gene has a series of accessibility states IL-10 is a suppressive cytokine that may modulate many of these associated with distinctive probabilities of IL-4 production. Proc. Natl. Acad. Sci. processes. It is possible that secretion of IL-9 by Th2 cells would USA 99: 10623–10628. 8. Chang, H.-C., S. Zhang, V. T. Thieu, R. B. Slee, H. A. Bruns, R. N. Laribee, only be effective if target cells did not receive a conflicting signal M. J. Klemsz, and M. H. Kaplan. 2005. PU. 1 expression delineates heterogeneity generated by IL-10. In this manner, Th2 heterogeneity may reflect in primary Th2 cells. Immunity 22: 693–703. functional specialization of cell types within the inflammatory 9. Eisenbeis, C. F., H. Singh, and U. Storb. 1995. Pip, a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator. Genes Dev. 9: microenvironment. 1377–1387. by guest on October 1, 2021 Increasing evidence suggests that the establishment of Th2 het- 10. Escalante, C. R., L. Shen, M. C. Escalante, A. L. Brass, T. A. Edwards, H. Singh, and A. K. Aggarwal. 2002. Crystallization and characterization of PU.1/IRF-4/ erogeneity is not stochastic, but rather instructive, based on the DNA ternary complex. J. Struct. Biol. 139: 55–59. expression of specific factors. As such, a growing list of transcrip- 11. Pongubala, J. M., S. Nagulapalli, M. J Klemsz, S. R. McKercher, R. A. Maki, and tion factors has specific effects on individual Th2 cytokines. IRF4 M. L. Atchison. 1992. PU. 1 recruits a second nuclear factor to a site important for immunoglobulin ␬3Ј enhancer activity. Mol. Cell Biol. 12: 368–378. is induced following T cell activation, and expression is further 12. Yee, A. A., P. Yin, D. P. Siderovski, T. W. Mak, D. W. Litchfield, and increased following Th2 differentiation. Importantly, the level of C. H. Arrowsmith. 1998. Cooperative interaction between the DNA-binding do- IRF4 in IL-10 low cells is increased compared with that in recently mains of PU.1 and IRF4. J. Mol. Biol. 279: 1075. 13. Lohoff, M., H.-W. Mittru¨cker, S. Prechtl, S. Bischof, F. Sommer, S. Kock, activated T cells. As we have shown, IRF4 activates IL-10 and D. A. Ferrick, G. S. Duncan, A. Gessner, and T. W. Mak. 2002. Dysregulated T IL-4 production, while decreasing IL-9 and having little effect on helper cell differentiation in the absence of interferon regulatory factor 4. Proc. Natl. Acad. Sci. USA 99: 11808–11812. IL-5 or IL-13. C-maf regulates IL-4 production but is not required 14. Brustle, A., S. Heink, M. Huber, C. Rosenplanter, C. Stadelmann, P. Yu, for production of other Th2 cytokines (43). Similarly, Pias1 in- E. Arpaia, T. W. Mak, T. Kamradt, and M. Lohoff. 2007. The development of creases IL-13 production without affecting IL-4 or IL-5 expression inflammatory TH-17 cells requires interferon-regulatory factor 4. Nat. Immunol. 8: 958–966. (44). We have shown that PU.1 decreases expression of many Th2 15. Zheng, Y., A. Chaudhry, A. Kas, P. deRoos, J. M. Kim, T. T. Chu, L. Corcoran, cytokines, but increases expression of CCL22, a chemokine asso- P. Treuting, U. Klein, and A. Y. Rudensky. 2009. Regulatory T-cell suppressor ciated with Th2 inflammation (8). BOB.1/OBF.1 regulates PU.1 program co-opts transcription factor IRF4 to control TH2 responses. Nature 458: 351–356. expression in Th2 cells and also affects the potential for Th2 cy- 16. Honma, K., D. Kimura, N. Tominaga, M. Miyakoda, T. Matsuyama, and K. Yui. tokine production (45). Moreover, the expression levels of each of 2008. Interferon regulatory factor 4 differentially regulates the production of Th2 cytokines in naive vs. effector/memory CD4ϩ T cells. Proc. Natl. Acad. Sci. USA these factors, and other factors that contribute to Th2 cytokine 105: 15890–15895. production exist in gradients that correlate with cytokine produc- 17. Hu, C.-M., S. Y. Jang, J. C. Fanzo, and A. B. Pernis. 2002. Modulation of T cell ing phenotypes (Fig. 6). The similar level of expression of IRF4 in cytokine production by interferon regulatory factor-4 (IRF-4). J. Biol. Chem. 277: 49238–49246. both IL-4 and IL-10 single-positive cells supports the idea that 18. Rengarajan, J., K. A. Mowen, K. D. McBride, E. D. Smith, H. Singh, and other positive- or negative-acting factors overlay on the IRF4 gra- L. H. Glimcher. 2002. Interferon regulatory factor 4 (IRF4) Interacts with dient to generate the specific patterns of cytokine secretion. The NFATc2 to modulate interleukin 4 . J. Exp. Med. 195: 1003–1012. mosaic of transcription factor gradients ultimately results in the 19. Matsuyama, T., A. Grossman, H. W. Mittrucker, D. P. Siderovski, F. Kiefer, heterogeneity observed in cytokine production from individual T. Kawakami, C. D. Richardson, T. Taniguchi, S. K. Yoshinaga, and T. W. Mak. cells. 1995. Molecular cloning of LSIRF, a lymphoid-specific member of the interferon regulatory factor family that binds the interferon-stimulated response element In this report, we have identified IRF4 as a regulator of Th2 (ISRE). Nucleic Acid Res. 23: 2127–2136. heterogeneity by enhancing or decreasing the production of spe- 20. Yamagata, T., J. Nishida, S. Tanaka, R. Sakai, K. Mitani, M. Yoshida, T. Taniguchi, Y. Yazaki, and H. Hirai. 1996. A novel interferon regulatory factor cific cytokines. IRF4 function, like GATA-3 as described in our family transcription factor, ICSAT/Pip/LSIRF, that negatively regulates the ac- previous report (8), is limited by the expression of PU.1 in Th2 tivity of interferon- regulated genes. Mol. Cell Biol. 16: 1283–1294. 1606 IRF4 AND Th2 HETEROGENEITY

21. Dakic, A., D. Metcalf, L. Di Rago, S. Mifsud, L. Wu, and S. L. Nutt. 2005. PU.1 34. Shoemaker, J., M. Saraiva, and A. O’Garra. 2006. GATA-3 directly remodels the regulates the commitment of adult hematopoietic progenitors and restricts gran- IL-10 locus independently of IL-4 in CD4ϩ T cells. J. Immunol. 176: 3470–3479. ulopoiesis. J. Exp. Med. 201: 1487–1502. 35. Brightbill, H. D., S. E. Plevy, R. L. Modlin, and S. T. Smale. 2000. A prominent 22. Mathur, A. N., H.-C. Chang, D. G. Zisoulis, G. L. Stritesky, Q. Yu, J. T. role for Sp1 during lipopolysaccharide-mediated induction of the IL-10 promoter O’Malley, R. Kapur, D. E. Levy, G. S. Kansas, and M. H. Kaplan. 2007. Stat3 in macrophages. J. Immunol. 164: 1940–1951. and Stat4 direct development of IL-17-secreting Th cells. J. Immunol. 178: 36. Lohning, M., A. Richter, T. Stamm, J. Hu-Li, M. Assenmacher, W. E. Paul, and 4901–4907. A. Radbruch. 2003. Establishment of memory for IL-10 expression in developing 23. Chang, H.-C., S. Zhang, I. Oldham, L. Naeger, T. Hoey, and M. H. Kaplan. 2003. T helper 2 cells requires repetitive IL-4 costimulation and does not impair pro- STAT4 requires the N-terminal domain for efficient phosphorylation. J. Biol. liferation. Proc. Natl. Acad. Sci. USA 100: 12307–12312. Chem. 278: 32471–32477. 37. Gerosa, F., C. Paganin, D. Peritt, F. Paiola, M. T. Scupoli, M. Aste-Amezaga, 24. Escalante, C. R., A. L. Brass, J. M. Pongubala, E. Shatova, L. Shen, H. Singh, and I. Frank, and G. Trinchieri. 1996. Interleukin-12 primes human CD4 and CD8 T A. K. Aggarwal. 2002. Crystal structure of PU.1/IRF-4/DNA ternary complex. cell clones for high production of both interferon-␥ and interleukin-10. J. Exp. Mol. Cell 10: 1097–1105. Med. 183: 2559–2569. 25. O’Sullivan, A., H.-C. Chang, Q. Yu, and M. H. Kaplan. 2004. STAT4 is required 38. Mo, C., W. Chearwae, J. T. O’Malley, S. M. Adams, S. Kanakasabai, for interleukin-12-induced chromatin remodeling of the CD25 locus. J. Biol. C. C. Walline, G. L. Stritesky, S. R. Good, N. B. Perumal, M. H. Kaplan, and Chem. 279: 7339–7345. J. J. Bright. 2008. Stat4 isoforms differentially regulate inflammation and demy- 26. Chang, H.-D., C. Helbig, L. Tykocinski, S. Kreher, J. Koeck, U. Niesner, and elination in experimental allergic encephalomyelitis. J. Immunol. 181: A. Radbruch. 2007. Expression of IL-10 in Th memory lymphocytes is condi- 5681–5690. tional on IL-12 or IL-4, unless the IL-10 gene is imprinted by GATA-3. Eur. 39. Grenningloh, R., B. Y. Kang, and I. C. Ho. 2005. Ets-1, a functional cofactor of J. Immunol. 37: 807–817. T-bet, is essential for Th1 inflammatory responses. J. Exp. Med. 201: 615–626. 27. Yao, Y., W. Li, M. H. Kaplan, and C. H. Chang. 2005. Interleukin (IL)-4 inhibits 40. Veldhoen, M., C. Uyttenhove, J. van Snick, H. Helmby, A. Westendorf, J. Buer, IL-10 to promote IL-12 production by dendritic cells. J. Exp. Med. 201: B. Martin, C. Wilhelm, and B. Stockinger. 2008. Transforming growth factor-␤ 1899–1903. ‘reprograms’ the differentiation of T helper 2 cells and promotes an interleukin 28. Wang, Z.-Y., H. Sato, S. Kusam, S. Sehra, L. M. Toney, and A. L. Dent. 2005. 9-producing subset. Nat. Immunol. 9: 1341–1346. Regulation of IL-10 gene expression in Th2 cells by Jun proteins. J. Immunol. 41. Dardalhon, V., A. Awasthi, H. Kwon, G. Galileos, W. Gao, R. A. Sobel, 174: 2098–2105. M. Mitsdoerffer, T. B. Strom, W. Elyaman, I. C. Ho, et al. 2008. IL-4 inhibits 29. Rekhtman, N., F. Radparvar, T. Evans, and A. I. Skoultchi. 1999. Direct inter- TGF-␤-induced Foxp3ϩ T cells and, together with TGF-␤, generates IL-9ϩ IL- Downloaded from action of hematopoietic transcription factors PU. 1 and GATA-1: functional an- 10ϩ Foxp3Ϫ effector T cells. Nat. Immunol. 9: 1347–1355. tagonism in erythroid cells. Genes Dev. 13: 1398–1411. 42. Temann, U.-A., P. Ray, and R. A. Flavell. 2002. Pulmonary overexpression of 30. McKercher, S. R., C. R. Lombardo, A. Bobkov, X. Jia, and N. Assa-Munt. 2003. IL-9 induces Th2 cytokine expression, leading to immune pathology. J. Clin. Identification of a PU.1-IRF4 protein interaction surface predicted by chemical Invest. 109: 29–39. exchange line broadening. Proc. Natl. Acad. Sci. USA 100: 511–516. 43. Kim, J. I., I. C. Ho, M. J. Grusby, and L. H. Glimcher. 1999. The transcription 31. Gross, P., A. A. Yee, C. H. Arrowsmith, and R. B. Macgregor. 1998. Quantitative factor c-Maf controls the production of interleukin-4 but not other Th2 cytokines. hydroxyl radical footprinting reveals cooperative interactions between DNA- Immunity 10: 745–751.

binding subdomains of PU.1 and IRF4. Biochemistry 37: 9802–9811. 44. Zhao, X., B. Zheng, Y. Huang, D. Yang, S. Katzman, C. Chang, D. Fowell, and http://www.jimmunol.org/ 32. Baguet, A., and M. Bix. 2004. Chromatin landscape dynamics of the Il4-Il13 W. P. Zeng. 2007. Interaction between GATA-3 and the transcriptional coregu- locus during T helper 1 and 2 development. Proc. Natl. Acad. Sci. USA 101: lator pias1 is important for the regulation of Th2 immune responses. J. Immunol. 11410–11415. 179: 8297–8304. 33. Avni, O., D. Lee, F. Macian, S. J. Szabo, L. H. Glimcher, and A. Rao. 2002. Th 45. Brunner, C., A. Sindrilaru, I. Girkontaite, K-D. Fischer, C. Sunderko¨tter, and cell differentiation is accompanied by dynamic changes in histone acetylation of T. Wirth. 2007. BOB. 1/OBF. 1 controls the balance of Th1 and Th2 immune cytokine genes. Nat. Immunol. 3: 643–651. responses. EMBO J. 26: 3191–3202. by guest on October 1, 2021