CARMA1 Controls Th2 Cell-Specific Cytokine Expression through Regulating JunB and GATA3 Transcription Factors

This information is current as Marzenna Blonska, Donghyun Joo, Patrick A. of September 27, 2021. Zweidler-McKay, Qingyu Zhao and Xin Lin J Immunol published online 27 February 2012 http://www.jimmunol.org/content/early/2012/02/26/jimmun ol.1102943 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 27, 2012, doi:10.4049/jimmunol.1102943 The Journal of Immunology

CARMA1 Controls Th2 Cell-Specific Cytokine Expression through Regulating JunB and GATA3 Transcription Factors

Marzenna Blonska,* Donghyun Joo,*,† Patrick A. Zweidler-McKay,‡ Qingyu Zhao,*,1 and Xin Lin*,†

The scaffold CARMA1 is required for the TCR-induced lymphocyte activation. In this study, we show that CARMA1 also plays an essential role in T cell differentiation. We have found that the adoptive transfer of bone marrow cells expressing constitutively active CARMA1 results in lung inflammation, eosinophilia, and elevated levels of IL-4, IL-5, and IL-10 in recipient mice. In contrast, CARMA1-deficient T cells are defective in TCR-induced expression of Th2 cytokines, suggesting that CARMA1 preferentially directs Th2 differentiation. The impaired cytokine production is due to reduced expression of JunB and GATA3 transcription factors. CARMA1 deficiency affects JunB stability resulting in its enhanced ubiquitination and degradation. In

contrast, TCR-dependent induction of GATA3 is suppressed at the transcriptional level. We also found that supplementation with Downloaded from IL-4 partially restored GATA3 expression in CARMA1-deficient CD4+ splenocytes and subsequently production of GATA3- dependent cytokines IL-5 and IL-13. Therefore, our work provides the mechanism by which CARMA1 regulates Th2 cell differ- entiation. The Journal of Immunology, 2012, 188: 000–000.

D4+ T cells, also known as Th cells, play a central role diseases, whereas overproduction of Th2 cytokines causes allergic

in immune protection by directing responses to various inflammation (1). http://www.jimmunol.org/ C infectious agents. Inappropriate regulation of the Th Previous studies have established the central and indispensible response results in chronic inflammation, autoimmune diseases, role of the NF-kB in TCR-induced prolifera- and/or allergies (1). Therefore, activation, differentiation, and ex- tion of CD4+ T cells (11). In resting cells, NF-kB is sequestered in pansion of Th cells are tightly regulated by specific transcription an inactive state by the cytoplasmic inhibitory IkB. Upon factors and cytokine network. Stimulation of the TCR leads to stimulation, the IkB kinase (IKK) complex, composed of IKKa a rapid activation of transcription factors, such as NF-kB, AP-1, and IKKb kinases and regulatory subunit NEMO, phosphorylates and NF-AT, that initiates IL-2 production and is required for cell IkB inhibitors, tagging them for ubiquitination and degradation. proliferation and expansion (2). Activated Th cells further dif- Released NF-kB is translocated to the nucleus and initiates tran- ferentiate into one of the four major effector populations, Th1, scription of its target (12). To date, several signaling mol- by guest on September 27, 2021 Th2, and Th17, and inducible regulatory T cells (1). Each Th ecules have been shown to transduce signals from TCR to the IKK lineage preferentially expresses distinct transcription factors and complex (13, 14). One of the critical molecules is CARMA1 (also cytokines. T-bet is upregulated in Th1 cells and controls IFN-g known as CARD11), a scaffold protein exclusively expressed in production (3); in contrast, GATA3, c-Maf, and JunB are ex- lymphoid and myeloid tissues (14, 15). Loss of CARMA1 impairs pressed at high levels in Th2 cells and induce IL-4, IL-5, and IL- AgR-induced IKK activation, NF-kB translocation, IL-2 pro- 13 (4–8). Th17 is a novel subset of effector cells that require duction, and proliferation of T cells (16–18). Ligation of TCR RORa and RORgt for its differentiation and produce IL-17 (9, and CD28 coreceptor results in activation of protein kinase Cu 10). Both, Th1 and Th17 have been associated with autoimmune (PKCu) (19) and phosphorylation of CARMA1 in the linker re- gion between the coiled-coil and PDZ domains (20, 21). This critical modification destabilizes an inhibitory conformation of *Department of Molecular and Cellular Oncology, University of Texas MD Anderson CARMA1 and uncovers the protein interaction sites (21). Acti- Cancer Center, Houston, TX 77030; †Cancer Biology Program, Graduate School of vated CARMA1 recruits its downstream signaling components, ‡ Biomedical Sciences, University of Texas, Houston, TX 77030; and Division of Bcl10, MALT1, and IKK, leading to activation of the IKK com- Pediatrics, University of Texas MD Anderson Cancer Center, Houston, TX 77030. plex and subsequently NF-kB (22, 23). Our previous study also 1Currentaddress:IntensiveCareUnit,Sun Yat-sen University Cancer Center, Guangzhou, People’s Republic of China demonstrates that the CARMA1–Bcl10 complex is indispensible Received for publication October 12, 2011. Accepted for publication January 22, for recruitment and assembly of the MAPK module that phos- 2012. phorylates JNK2 (24, 25). JNK2 is known to regulate the ex- This work was supported by National Institutes of Health Grants GM065899 and pression level of AP-1 family members c-Jun and JunB by targeting AI050848 (to X.L.). M.B. is a Special Fellow of the Leukemia and Lymphoma them for ubiquitination and degradation (26–28). CARMA1- Society. dependent JNK2 phosphorylation is required for stabilization and Address correspondence and reprint requests to Dr. Xin Lin, Department of Mo- accumulation of c-Jun in activated cells (24). lecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 108, Houston, TX 77030. E-mail address: xllin@ In this study, we demonstrate that stable expression of consti- mdanderson.org tutively active CARMA1 increases the basal levels of c-Jun and The online version of this article contains supplemental material. JunB in vitro, suggesting its role in cell proliferation. However, Abbreviations used in this article: BAL, bronchoalveolar lavage; ChIP, chromatin upregulation of CARMA1 in vivo results in Th2 cell-mediated immunoprecipitation; IKK, IkB kinase; ph, phospho; PKC, protein kinase C; PKCu, inflammation. Mice expressing constitutively active CARMA1 protein kinase C u; WT, wild-type. have elevated IL-4, IL-5, and IL-10 and spontaneously develop Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 pulmonary inflammation and eosinophilia. We found that CARMA1

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102943 2 CARMA1 REGULATES Th2 CYTOKINE EXPRESSION regulates JunB and GATA3 expression, which are crucial for the CD4+ T cell isolation and activation initiation and propagation of Th2 polarization. CARMA1-deficient + + CD4 T cells were isolated from mouse spleens and peripheral lymph CD4 T cells have severely impaired production of IL-4 because of nodes using magnetic beads (StemCell Technologies). The cells were the reduced JunB level and defective GATA3 induction. We also purified by negative selection with a panel of biotinylated Abs directed found that CARMA1-deficient CD4+ splenocytes could induce against CD8, CD11b, CD19, CD45R/B220, CD49b, TER119 (StemCell GATA3 in the presence of exogenous IL-4, and the supplementa- Technologies), and CD25 (eBioscence). After 3 h of resting, the cells were activated with plate-bound anti-CD3ε (2 mg/ml) and anti-CD28 (1 mg/ml) tion with IL-4 partially restored production of GATA3-dependent (both from BD Biosciences). For Th2 polarization, 10 mg/ml anti–IFN-g cytokines IL-5 and IL-13. (XMG1.2) and 10 ng/ml murine IL-4 (PeproTech) were added. Because CARMA1-deficient T cells are defective in IL-2 production, every cell Materials and Methods culture was supplemented with rIL-2 (30 U/ml). Reagents and plasmids ELISA 63/73 202 Abs specific for phospho (ph)–c-Jun (Ser ), c-Jun, ph-ERK1/2 (Thr / Cytokines were measured in cell culture supernatants and mouse plasma by 204 32/36 183 185 Tyr ), ph-IkBa (Ser ), ph-JNK (Thr /Tyr ), JNK2, and CARMA1 sandwich ELISA. IL-4, IL-5, IL-10, IL-13, and IFN-g Ready-SET-GO kits were purchased from Cell Signaling Technology. Abs specific for JunB, were purchased from eBioscience, and IL-2 OptEIA kit was obtained from JunD, GATA3, T-bet, c-Maf, NFAT, IkBa, , laminB, Ub (P4D1), and BD Biosciences. Results are presented as arithmetic mean of triplicate actin were obtained from Santa Cruz Biotechnology. Ab that recognizes cultures 6 SD or mean + SEM for each group of mice. ubiquitin linked at Lys48 (catalog number 05-1307) and Lys63 (catalog number 05-1308) was purchased from Millipore. JNK inhibitor (SP600125; Immunobloting and immunoprecipitation catalog number S5567) and IKKb-specific small-molecule inhibitor (TPCA-1; catalog number T1452) were obtained from Sigma-Aldrich. The cells were lysed in the buffer containing 50 mM HEPES (pH 7.4), 150 Downloaded from CARMA1 lentivirus expression vectors were constructed by inserting mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, CARMA1 or its mutant into the BamHI and EcoRI sites in pRV3 vector 1 mM PMSF, 1 mM DTT, and a protease inhibitor mixture (Roche that contains bicistronic IRES-GFP cassette downstream of the multiple Diagnostics). The cell lysates were subjected to SDS-PAGE and Western cloning sites. CARMA1del mutant was generated by overlapping PCR blot or immunoprecipitated with JunB Ab (sc-73; Santa Cruz Biotech- using wild-type (WT) CARMA1 cDNA and verified by sequencing. nology). The immunoprecipitates were washed with lysis buffer four times and eluted with 23 SDS loading buffer. After boiling (4 min), the samples Cell lines and transfection were fractionated on 10% SDS-PAGE and transferred to nitrocellulose membranes. Immunoblots were incubated with specific primary Abs, fol- http://www.jimmunol.org/ Jurkat T cells deficient in CARMA1 were described earlier (17). Jurkat lowed by HRP-conjugated secondary Abs, and were developed by the ECL T cells deficient in NEMO were provided by Dr. S.-C. Sun (University of method according to the manufacturer’s protocol (Pierce). Texas MD Anderson Cancer Center). Jurkat and primary T cells were cul- tured in RPMI 1640 medium supplemented with 10% FBS, penicillin, and Proliferation assay streptomycin. Human embryo kidney 293 and Phoenix cells were main- 3 3 4 tained in DMEM supplemented with 10% FBS and the antibiotics. Cells For [ H]thymidine incorporation assay, the cells (3 10 /well in a 96-well plate) were seeded in triplicate in normal growth medium. At the indicated were transfected using the calcium phosphate coprecipitation method. 3 Stable transfection of Jurkat and CARMA1-deficient cells with CAR- time points, cells were pulsed with 1 mCi [ H]thymidine for 6 h and MA1wt and CARMA1del was established by lentiviral infection. First, harvested onto glass fiber filters. The incorporated radioactivity was human embryo kidney 293T cells were transfected with expression vector measured by scintillation counting. Results are presented as the arithmetic (pRV3) together with packaging plasmids (pHep and pEnv) using the cal- mean of triplicate cultures plus SD. by guest on September 27, 2021 cium phosphate precipitation method. Jurkat cells were resuspended in viral EMSA supernatant in the presence of polybrene and incubated for 6 h. Next, cells were washed and cultured in the complete medium. The efficiency of viral Nuclear proteins were extracted from 3 to 4 3 106 cells as described before infection was determined by flow cytometry or Western blot analysis. (24). Nuclear extracts (4 mg) were incubated with a 32P-labeled, double- stranded, AP-1– or Oct-1–specific probe for 15 min at room temperature, Mice fractionated on a 5% polyacrylamide gel, and visualized by autoradio- CARMA1 null mice were described previously (16). Animals were graphy. AP-1 and Oct-1 probes were purchased from Promega. maintained under pathogen-free conditions in the institutional animal fa- Chromatin immunoprecipitation assay cility. All experiments were performed in compliance with the institutional guidelines and according to the protocol approved by the Institutional Chromatin immunoprecipitation (ChIP) was performed using a ChIP assay Animal Care and Use Committee of the University of Texas MD Anderson kit (Upstate Biotechnology). In brief, after cross-linking with 1% form- Cancer Center. aldehyde for 10 min at room temperature, cells were lysed in SDS buffer (50 mM Tris-Cl [pH 8.1], 10 mM EDTA, 1% SDS, and protease inhibitors). Bone marrow chimeric mice Lysates were sonicated using Bioruptor and diluted with a ChIP dilution Four days before bone marrow harvesting, the donor mice were injected buffer (20 mM Tris-Cl [pH 8.1], 1 mM EDTA, 150 mM NaCl, and 0.3% Triton X-100). Lysates were then precleared with protein G beads and with 5-fluorouracil (Acros Organics). Bone marrow cells were collected incubated with JunB (sc-73; Santa Cruz Biotechnology) or control rabbit from femurs and tibias and cultured in conditional media supplemented with murine stem cell factor, IL-3, and IL-6 for 16 h. Cells were infected by IgG at 4˚C overnight. Protein G-agarose was added to the lysates and spinoculation for two consequent days and injected to sublethally irradi- incubated for 2 h at 4˚C. Ab/protein/DNA complexes were eluted and ated recipient mice. The infection efficiency (15–20%) was determined by incubated at 65˚C overnight to reverse formaldehyde cross-linking, and flow cytometry. Peripheral blood samples were collected from tail veins DNA was recovered by a PCR purification kit (Qiagen). DNA fragments and analyzed by automatic hematological counter (Advia; Bayer). Leu- were amplified by PCR with specific primers. kocyte differential was verified by microscopic evaluation of blood smear Real-time PCR (May–Grunwald–Giemsa staining). At autopsy, organs were fixed with 10% formalin and embedded in paraffin. Sections were stained with H&E Total RNA was isolated using RNeasy kit (Qiagen) and reverse transcribed for histopathologic examination by light microscopy. using SuperScriptIII (Invitrogen). Quantitative PCR was performed in triplicates using Power SYBR Green PCR Master Mix (Applied Bio- Ova-induced allergic lung inflammation systems). The amounts of transcript were normalized to GAPDH. Melt Mice were immunized with two i.p. Ova/alum injections (100 mg; days curves were run to ensure amplification of a single product. 0 and 7) and intranasally challenged with 50 mg Ova in PBS (days 25–27). Luciferase assay Two days after the last challenge, bronchoalveolar lavage (BAL) was collected by passing 1 ml PBS (three times), and cells recovered from the Cells were transfected in triplicate with reporter plasmid encoding 53 BAL were counted using an automatic cell counter (Coulter). Ova-specific NF-kB-luc and pEF-Renilla-luc together with the expression vectors by IgE was measured in mouse serum using an ELISA kit from DS Pharma electroporation. Twenty hours later, cell lysates were prepared, and lucif- Biomedical. erase activities were measured with Dual-Luciferase assay kits (Promega). The Journal of Immunology 3

NF-kB activities were determined by normalization of NF-kB–dependent tive because of destabilization of an inhibitory conformation of Firefly luciferase to Renilla luciferase activity. CARMA1 (21). Jurkat cells were stably transfected with CAR- MA1del, WT (CARMA1wt), or empty vector as a control. We Results found that cells expressing a high amount of CARMA1wt or Expression of constitutively active CARMA1 increases the level expressing CARMA1del had elevated levels of c-Jun and JunB of Jun proteins and promotes T cell proliferation (Fig. 1D, upper panel), and c-Jun was phosphorylated at Ser73, Our previous study demonstrates that stimulation of the TCR in- which is known to induce its activity (29). Consistent with these duces rapid accumulation of c-Jun and JunB transcription fac- data, AP-1 binding activity was enhanced in the presence of tors, and this effect is completely diminished in CARMA1-deficient CARMA1del (Fig. 1D, lower panel). To test whether constitu- Jurkat T cells (Fig. 1A) (24). Moreover, prolonged stimulation tive activity of CARMA1 promotes cell proliferation, we used a results in persistent accumulation of Jun proteins (Supplemental [3H]thymidine incorporation assay (Fig. 1E). Indeed, the cells Fig. 1A). Because CARMA1 is directly phosphorylated by PKC expressing CARMA1del proliferated slightly faster than mock (20, 21), we also stimulated cells in the presence of PKC inhibitors cells. Taken together, these results demonstrate that constitutively and found that treatment with PKC inhibitors completely sup- active CARMA1 results in elevated levels of c-Jun and JunB and pressed TCR-induced c-Jun and JunB accumulation in Jurkat cells promotes cell proliferation in vitro. (Fig. 1B). In contrast, an IKK inhibitor that effectively blocks NF- kB activation had no effect on Jun protein level (Supplemental Fig. Upregulation of CARMA1 in vivo results in Th2 cell-associated 1B), indicating that NF-kB does not control signal-dependent c-Jun inflammation and JunB induction. Taken together, our results raised possibility Previous studies suggest that deregulation of NF-kB and/or AP-1 Downloaded from that prolonged CARMA1 activation might enhance T cell prolifer- signaling pathways leads to inappropriate immune responses ation through upregulation of c-Jun and JunB transcription factors. and contributes to the development of lymphoma (29–32). Be- To test this hypothesis, we generated constitutively active cause our studies demonstrate that CARMA1 controls NF-kB, CARMA1 by deletion of the linker region between the coiled-coil c-Jun, and JunB transcription factors in activated lymphocytes (17, and PDZ domains (CARMA1del) (Fig. 1C). This construct is ac- 20, 24), we decided to investigate biological consequences of http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Constitutively active CARMA1 increases the basal level of c-Jun and JunB and promotes T cell proliferation. (A) Jurkat and CARMA1- deficient Jurkat T cells (6 3 106 cells/sample) were stimulated with CD3 plus CD28 Abs (3 and 2 mg/ml, respectively) for different time points. Whole-cell lysates were subjected to SDS-PAGE and analyzed by Western blot using indicated Abs. (B) TCR-induced accumulation of c-Jun and JunB is PKC de- pendent. Jurkat T cells were pretreated with PKC inhibitors Ro31-822 and GF109203 (100 nM) for 30 min and then stimulated for indicated time points. The cell lysates were subjected to SDS-PAGE and examined by Western blots using indicated Abs. (C) A schematic diagram of CARMA1 constructs. WT CARMA1 (CARMA1wt) is composed of an N-terminal caspase recruitment domain (CARD), a coiled-coil domain (C-C), a PDZ domain, an Src homology (SH3) domain, and a guanylate kinase-like (GUK) domain in the C terminus. Constitutively active CARMA1 (CARMA1del) was generated by deletion of the linker region between the C-C and PDZ domains (436–660 residues). CARMA1-deficient Jurkat T cells were transfected in triplicate with empty vector, WT CARMA1 (CARMA1wt), or constitutive active mutant, CARMA1del, together with reporter plasmids encoding 53 NF-kB-luc and pEF-Renilla-luc. NF-kB activation was determined by normalization of NF-kB–dependent luciferase to Renilla luciferase activity. Expression levels of transfected proteins were determined in cells lysates using CARMA1 Abs. (D) CARMA1-dependent accumulation of c-Jun and JunB. Jurkat T cells were stably transfected with empty vector WT CARMA1 (CARMA1wt) or constitutive active CARMA1del. The cells were lysed and subjected to SDS-PAGE, followed by Western blot analysis using indicated Abs. Nuclear extracts from tested stable cell lines were analyzed by EMSA using 32P-labeled probes containing AP-1 or Oct-1 binding sites. (E) Proliferation assay. The cells (3 3 104/well in a 96-well plate) were seeded in triplicate in normal growth medium. At the indicated time points, cells were pulsed with 1 mCi [3H]thymidine for 6 h and harvested onto glass fiber filters. The incorporated radioactivity was measured by scintillation counting. Results are presented as mean of triplicate cultures 6 SD. 4 CARMA1 REGULATES Th2 CYTOKINE EXPRESSION

CARMA1 upregulation in vivo. We adoptively transferred bone CARMA1del mice (Fig. 2E, 2F). In contrast, control mice did not marrow cells transduced with CARMA1del, CARMA1wt, or develop any significant pulmonary inflammation. One mouse from mock control into sublethally irradiated mice (Supplemental Fig. CARMA1del group was sacrificed at week 15 because of severe 2A). Recipient mice were observed for 20 wk, focusing on their symptoms, such as short breath, hunched posture, itching, rush, weight (Supplemental Fig. 2B) and hematological parameters. and skin lesions (Supplemental Fig. 2E). These results suggest that RBC parameters and platelet count were comparable during the constitutive activity of CARMA1 might be sufficient to initiate Th whole observation period in all groups (Supplemental Fig. 2C). cell differentiation to IL-4– and IL-5–producing effectors and Although we did not find significant differences in total WBC Th2-like inflammation in vivo. Because Th cells require GATA3 count (Fig. 2A), we observed increased number of eosinophils in transcription factor for optimal Th2 differentiation and GATA3 peripheral blood from mice expressing CARMA1del (Fig. 2B, controls transcription of IL-5 (4), we examined whether CAR- 2C). Consistent with this finding, CARMA1del mice had elevated MA1del could induce GATA3 expression in T cells. Indeed, Jurkat levels of IL-5 (Fig. 2D), which is known to support growth, sur- cells transfected with CARMA1del expressed much higher levels vival, and effector function of eosinophils. We also detected of GATA3 than control cells (Supplemental Fig. 3A). significantly higher concentrations of IL-2, IL-4, and IL-10 (Fig. CARMA1 is required for expression of Th2 cytokines and 2D) but not IFN-g or IL-6 (Supplemental Fig. 2D) in all + plasma samples. Furthermore, tissue analysis at week 20 post- transcription factorsin CD4 T cells transplantation revealed lung inflammation (five of six mice) To examine whether CARMA1 is required for Th2 differentia- and enlarged peripheral lymph nodes (three of six mice) in tion, we isolated CD4+ T cells from the spleens of WT and Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Upregulation of CARMA1 in bone marrow chimeric mice results in Th2 cell-mediated inflammation. (A) WBC count (WBC) in peripheral blood of control mice (mock; n = 5) and mice expressing WT CARMA1 (CARwt; n = 6) or CARMA1del mutant (CARdel; n = 6) at week 20 post- transplantation. Blood samples were collected from tail veins and analyzed by an automatic hematological counter (Advia; Bayer). (B) Populations of WBC were analyzed by an automatic hematological counter, and leukocyte differentials were verified by microscopic evaluation of blood smear (May–Grun- wald–Giemsa staining). (C) Eosinophilia in CARMA1del mouse. Statistical analysis was performed using Prism 5 software (GraphPad), and p value was determined using Kruskal–Wallis test for three experimental groups. (D) Cytokine level in plasma was measured by ELISA. Results are presented as mean plus SEM for each group of mice, and p value was determined using Kruskal–Wallis test. (E) Comparison of lung inflammation in CARMA1wt and CARMA1del mouse. Lung tissues (week 20 posttransplantation) were fixed with 10% formalin and embedded in paraffin. Tissue sections were stained with H&E and examined by light microscopy (310 objective). The images were taken with an Olympus IX70 inverted FL microscope and edited with DP Controller software. (F) Enlarged inguinal lymph node in CARMA1del mouse. The Journal of Immunology 5

CARMA1-deficient (CARMA1KO) mice and stimulated the cells regulation occurs on the transcriptional level and is con- with plate-bound anti-CD3 and CD28. Every culture was supple- trolled by multiple transcription factors (2). Because the initial mented with rIL-2 because CARMA1-deficient cells are defective amount of IL-4 secreted from T cells is induced by JunB (6), we in production of this growth factor, resulting in reduced prolifer- assessed the recruitment of JunB to the IL-4 promoter in the ab- ation and cell survival. TCR-dependent production of cytokines sence of CARMA1. Indeed, we found that the binding of JunB to was measured in supernatants collected at 24-, 48-, and 72-h time this promoter was not observed in CARMA1-deficient cells during points. We found that CARMA1-deficient CD4+ splenocytes se- the first 16 h of TCR stimulation (Fig. 4A). creted significantly reduced levels of IL-4, IL-5, and IL-10, To determine how CARMA1 regulates JunB, we first measured whereas IFN-g production was slightly higher in comparison the JunB mRNA expression in WT and CARMA1-deficient cells with WT cells (Fig. 3A). Taken together, these results indicate that and found that CARMA1 deficiency did not significantly affect CARMA1 is required for the expression of Th2 cytokines. the transcription of JunB upon TCR ligation (Fig. 4B). Because It is well established that GATA3 directly controls IL-5 and previous studies suggest that JunB protein level is controlled together with JunB enhances IL-4 production (2, 4, 6, 8). JunB is by a ubiquitination-dependent mechanism (34), we examined the also implicated in IL-10 expression (33). Therefore, we examined level of JunB ubiquitination in the absence of CARMA1. We TCR-dependent induction of these transcription factors in the found that JunB was highly ubiquitinated in CARMA1-deficient absence of CARMA1. We used CD4+ T cells isolated from pe- cells (Fig. 4C, 4D), which correlated with the low expression of ripheral lymph nodes or spleens of WT and CARMA1-deficient JunB protein (Fig. 4C, 4D). We also confirmed that the majority of mice. The cells were stimulated with plate-bound anti-CD3/ detected Ub chains was linked through Lys48 (Fig. 4D, middle

CD28 plus IL-2 for 2 and 3 d, and whole-cell lysates were panel), but Lys63-linked chains were barely detectable (Fig. 4D, Downloaded from analyzed by immunoblotting (Fig. 3B, 3C). We found that lower panel). These results suggest that CARMA1 regulates JunB CARMA1-deficient cells did not upregulate both GATA3 and level through inhibiting an unknown ubiquitin E3 ligase that JunB, but Th1-specific factor T-bet was expressed at comparable induces Lys48-linked JunB polyubiquitination and proteosome- level in examined cells. Consistent with the results from mouse mediated degradation. Of note, we have found that c-Jun ubiq- primary T cells, Jurkat T cells were also defective in GATA3 and uitination also depends on CARMA1 (Supplemental Fig. 4), and it

JunB induction in the absence of CARMA1 (Fig. 3D). These data is highly probable that the same E3 ligase is involved in this http://www.jimmunol.org/ indicate that CARMA1 is required for induction of Th2 tran- process. scription factors, GATA3 and JunB, and controls IL-4, IL-5, and CARMA1 regulates GATA3 through a transcriptional IL-10 production. mechanism CARMA1 regulates JunB through a posttranslational To confirm that CARMA1 is required for GATA3 expression, we mechanism reconstituted CARMA1-deficient Jurkat T cells with wt CARMA1. IL-4 is a key Th2 cytokine that regulates humoral immunity, and Indeed, stable re-expression of CARMA1 efficiently rescued the excessive expression of IL4 may cause allergic inflammation. Il4 TCR-dependent induction of GATA3 (Fig. 5A). To further deter- by guest on September 27, 2021

FIGURE 3. Reduced expression of Th2 cytokines in CARMA1-deficient mice. (A) CARMA12/2 CD4+ cells have diminished production of Th2 cytokines. CD4+ splenocytes were stimulated with plate-bound anti-CD3ε (2 mg/ml) and anti-CD28 (1 mg/ml) in the presence of rIL-2 (30 U/ml). Pro- duction of IL-4, IL-5, IL-10, and IFN-g was measured in supernatants collected at 24-, 48-, and 72-h time points using ELISA. Results are presented as mean of triplicate cultures 6 SD. (B–D) Signal-dependent GATA3 and JunB expression is defective in CARMA1-deficient CD4+ T cells (B, C) and CARMA1-deficient Jurkat T cells (D). Cells were stimulated with CD3/CD28 Abs (2 and 1 mg/ml, respectively) for 48 and 72 h. Each culture of primary cells was supplemented with rIL-2 (30 U/ml). Whole-cell lysates were subjected to SDS-PAGE and analyzed by Western blot using indicated Abs. 6 CARMA1 REGULATES Th2 CYTOKINE EXPRESSION Downloaded from

FIGURE 4. CARMA1 regulates JunB through a posttranslational mechanism. (A) ChIP assay. CD4+ splenocytes were activated with plate-bound anti-

CD3/CD28 for 2 or 16 h. JunB–DNA complexes were precipitated with anti-JunB following with real-time PCR. Results are presented as means plus SD of http://www.jimmunol.org/ triplicates after normalization to input. (B) Jurkat and CARMA1-deficient Jurkat T cells were stimulated with CD3/CD28 Abs for indicated time points. Total RNA was reverse transcribed, and quantitative PCR was performed with SYBR Green PCR Master Mix (Applied Biosystems). The amounts of the JunB transcript were normalized to GAPDH. The graphs show mean plus SD of triplicates. (C and D) The cells were stimulated with CD3/28 (3 and 2 mg/ ml, respectively) for 24 h (C) or PMA plus ionomycin (P/I; 20 and 100 ng, respectively) for 2 h (D). Lysates from these cells were precipitated with JunB Abs. The immunocomplexes were subjected to Western blot analysis using ubiquitin or JunB Abs. mine the mechanism by which CARMA1 regulates GATA3 CARMA1-deficient mice are less susceptible for expression, we examined GATA3 mRNA level in CARMA1- T cell-mediated airway inflammation by guest on September 27, 2021 deficient T cells following TCR stimulation. We found that the To provide the functional significance of CARMA1-dependent GATA3 transcript was gradually augmented during first 18–36 h activation of JunB and GATA3 in vivo, we immunized WT or of stimulation in Jurkat cells but not in the absence of CARMA1 CARMA1-deficient mice with OVA and then rechallenged these (Fig. 5B). These results indicate that CARMA1 regulates GATA3 mice with Ova peptides intranasally. Consistent with a previous on the transcriptional level. study (36), CARMA1-deficient mice were less susceptible to the To determine whether TCR-induced NF-kB activation is re- allergic airway inflammation (Fig. 6A). We observed less in- quired for GATA3 expression, we used a Jurkat T cell line defi- flammatory cells in BAL fluid and a lower level of IL-5 in BAL cient in NEMO (also known as IKKg), a subunit of the IKK (Fig. 6B) and a lack of Ova-specific IgE in serum (Fig. 6C) in complex, which is completely defective in NF-kB activation. We found that signal-dependent induction of GATA3 was comparable CARMA1KO mice. On the basis of the results previously men- in NEMO-deficient Jurkat T cells and control cells re-expressing tioned, we propose a model of CARMA1-mediated regulation of wt NEMO (Fig. 5C). This result indicates that GATA3 expres- Th2 cytokines (Fig. 6D) in which initial IL-4 production is in- sion does not require the intact NF-kB activation. Therefore, duced by TCR in a CARMA1- and JunB-dependent manner. In CARMA1-dependent GATA3 expression is regulated through an turn, IL-4 enhances expression of GATA3 through the IL-4R/ NF-kB–independent mechanism. STAT6 signaling leading to optimal secretion of IL-4, IL-5, and It has been established that GATA3 expression is induced by IL-13. In contrast, CARMA1-dependent activation of NF-kBis both TCR signaling and the IL-4R/STAT6 signaling (35), which required for IL-2 production and expansion of effector cells. raises the possibility that stimulation of CARMA1-deficient cells in the presence of IL-4 may rescue signal-dependent GATA3 ex- Discussion pression. Indeed, we found that GATA3 expression could be CARMA1 is a scaffold molecule expressed in the hematopoietic partially rescued in the presence of exogenous IL-4 in CARMA1- cells. Its role in AgR-induced NF-kB activation is well established, deficient CD4+ splenocytes (Fig. 5D) and in CARMA1-deficient but its effect on other transcription factors has not been well Jurkat T cells (Supplemental Fig. 3B). Consistent with above re- studied. In this study, we show that stable expression of the con- sult, the supplementation with IL-4 could also partially restore stitutively active mutant of CARMA1 results in accumulation of the production of GATA3-dependent cytokines IL-5 and IL-13 two Jun family members, c-Jun and JunB, and activation of AP-1 in CARMA1-deficient cells (Fig. 5E). Importantly, we detected in the absence of exogenous stimulus. This activity may lead to the comparable level of IL-4R on the surface of WT and uncontrolled cell growth in vivo. However, we have not observed CARMA1KO lymphocytes (Supplemental Fig. 3C), suggesting enhanced proliferation of lymphocytes in mice adoptively trans- that attenuation of GATA3 expression in CARMA1-deficient cells ferred with the CARMA1-mutant transduced cells. Instead, re- is only partially attributable to the lack of CARMA1-dependent cipient mice displayed an enhanced Th2 phenotype. Our results IL-4 production. can be partially explained by the CARMA1-dependent induction The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. CARMA1 regulates GATA3 through a transcriptional mechanism. (A) Jurkat and CARMA1-deficient Jurkat T cells with or without re- constitution of CARMA1 were stimulated with CD3 plus CD28 Abs (3 and 2 mg/ml, respectively) for 48 h. Whole-cell lysates were subjected to Western blot analysis using indicated Abs. (B) Jurkat and CARMA1-deficient Jurkat T cells were stimulated with CD3/CD28 Abs for indicated time points. Total RNA was reverse transcribed, and quantitative PCR was performed with SYBR Green PCR Master Mix. The amounts of GATA3 transcript were normalized to GAPDH. The graphs show mean plus SD of triplicates. (C) NF-kB activation is not required for TCR-induced GATA3 expression. NEMO-deficient Jurkat T cells were stably transfected with empty vector (mock) or NEMO (NEMOwt). These cells were stimulated with PMA or PMA plus ionomycin. Whole-cell lysates were subjected to SDS-PAGE and analyzed by Western blot using indicated Abs. (D) CD4+ splenocytes were stimulated with plate-

bound anti-CD3e and anti-CD28 (2 and 1 mg/ml, respectively) in the presence of rIL-2 (30 U/ml), anti–IFN-g (10 mg/ml), and recombinant murine IL-4 (10 by guest on September 27, 2021 ng/ml) for 48 h. Cells were lysed and subjected to Western blot analysis using indicated Abs. (E) Supernatants from cells treated as in (D) were collected, and indicated cytokines were measured by ELISA. Results are shown as means plus SD of triplicate cultures. of JunB transcription factor, because a previous study indicates contrast, we have found that CARMA1 deficiency results in that JunB is selectively expressed in developing Th2 cells, and a decreased expression of JunB and Th2 cytokines, which is JunB transgenic mice have upregulated Th2 cytokines (6). In consistent with the finding that loss of JunB in T cells results in

FIGURE 6. Ova-induced lung in- flammation. (A) Inflammation around airways and blood vessels. Mice were immunized with two i.p. Ova/ alum injections (100 mg; days 0 and 7) and intranasally challenged with 50 mg Ova in PBS (days 25–27). The lungs were fixed in 10% formalin and processed into paraffin. Tissue sec- tions were stained with H&E and ex- amined by light microscopy (310 objective). The images were taken with an Olympus IX70 inverted FL microscope and edited with DP Con- troller software. (B) BAL was col- lected by passing 1 ml PBS (three times). Cells recovered from the BAL were counted using an automatic cell counter (Coulter). IL-5 concentration was measured by ELISA. (C) Serum levels of OVA-specific IgE were de- termined by ELISA. (D) A model of CARMA1-mediated cytokine pro- duction (description in the main text). 8 CARMA1 REGULATES Th2 CYTOKINE EXPRESSION a reduced production of IL-4 (37). Interestingly, JunB-deficient shown that persistent CARMA1 activity leads to Th2-like in- mice also exhibit an impaired allergen-induced airway inflam- flammation with elevated eosinophils and Th2 cytokines IL-4 and mation (37), similarly to CARMA1KO mice (this study and Refs. IL-5. To our knowledge, this is the first study that addresses bi- 36 and 38). ological consequences of prolonged CARMA1 activation in vivo To date, the molecular mechanism by which CARMA1 regulates in T cells. JunB expression has not been revealed. Our previous study dem- In summary, our study provides the molecular link between onstrates that CARMA1 is required for TCR-induced activation CARMA1 and Th2 cell differentiation. We demonstrate that of JNK2 (24), the key regulator of c-Jun and JunB stability (26– CARMA1 is a critical signaling molecule in the regulation of 28). In this study, we show that CARMA1 deficiency results JunB and GATA3 transcription factors and subsequent production in accelerated K48-linked JunB polyubiquitination, and the simi- of Th2 cytokines. Therefore, the selective role of CARMA1 in lar effect is observed for c-Jun. Therefore, we speculate that directing the immune response makes it a very attractive target for CARMA1 may suppress an E3 ligase that is responsible for in- immunosuppressive therapy. ducing Jun protein ubiquitination. Indeed, previous study suggests that JunB and c-Jun can be regulated by Itch, an E3 ligase, fol- Acknowledgments lowing TCR stimulation (34). However, this process requires We thank Dr. J. Penninger for CARMA1-knockout mice and Dr. Shao- initial phosphorylation of Itch by JNK1 (34). Because CARMA1 Cong Sun for NEMO-deficient Jurkat T cells. appears to suppress an E3 ligase activity even under unstimu- lated conditions, CARMA1-dependent regulation of JunB ubiq- Disclosures uitination is unlikely mediated through suppression of Itch. The authors have no financial conflicts of interest. Downloaded from Therefore, it remains to be determined which E3 ligase is reg- ulated by CARMA1 in T cells. The role of GATA3 transcription factor in regulation of IL-4, References IL-5, and IL-13 is well established (4, 8), but the mechanism 1. Zhu, J., H. Yamane, and W. E. Paul. 2010. Differentiation of effector CD4 T cell populations (*). Annu. Rev. Immunol. 28: 445–489. that regulates GATA3 expression is not fully defined. It has been 2. Glimcher, L. H., and K. M. Murphy. 2000. Lineage commitment in the immune demonstrated that can be induced by the system: the T helper lymphocyte grows up. Genes Dev. 14: 1693–1711. http://www.jimmunol.org/ TCR, IL-4R/STAT6, and IL-2R/STAT5A signaling pathways 3. Szabo, S. J., S. T. Kim, G. L. Costa, X. Zhang, C. G. Fathman, and L. H. Glimcher. 2000. A novel transcription factor, T-bet, directs Th1 lineage during Th cell differentiation (35, 39). Our current work reveals commitment. Cell 100: 655–669. that CARMA1 is required for TCR-dependent induction of 4. Pai, S. Y., M. L. Truitt, and I. C. Ho. 2004. GATA-3 deficiency abrogates the GATA3, and Jurkat T cells expressing constitutively active development and maintenance of T helper type 2 cells. Proc. Natl. Acad. Sci. USA 101: 1993–1998. CARMA1 have upregulated GATA3. This finding suggests that 5. Ho, I. C., M. R. Hodge, J. W. Rooney, and L. H. Glimcher. 1996. The proto- CARMA1 may transduce signals from the to the gata3 oncogene c- is responsible for tissue-specific expression of interleukin-4. gene-regulating transcriptional complex. Because CARMA1 is Cell 85: 973–983. 6. Li, B., C. Tournier, R. J. Davis, and R. A. Flavell. 1999. Regulation of IL-4 required for activation of the NF-kB transcription factor and a expression by the transcription factor JunB during T helper cell differentiation. previous study demonstrates that mice deficient in the p50 subunit EMBO J. 18: 420–432. by guest on September 27, 2021 of NF-kB have reduced GATA3 expression upon TCR stimulation 7. Kim, J. I., I. C. Ho, M. J. Grusby, and L. H. Glimcher. 1999. The transcription factor c-Maf controls the production of interleukin-4 but not other Th2 cyto- (40), it is conceivable that CARMA1-dependent NF-kB activation kines. Immunity 10: 745–751. contributes to the GATA3 expression. However, we have found 8. Zhu, J., B. Min, J. Hu-Li, C. J. Watson, A. Grinberg, Q. Wang, N. Killeen, that GATA3 induction is intact in NEMO-deficient cells, sug- J. F. Urban, Jr., L. Guo, and W. E. Paul. 2004. Conditional deletion of Gata3 shows its essential function in T(H)1-T(H)2 responses. Nat. Immunol. 5: 1157– gesting that TCR-induced NF-kB activation is not required for 1165. GATA3 expression. Instead, our data indicate that CARMA1- 9. Park, H., Z. Li, X. O. Yang, S. H. Chang, R. Nurieva, Y. H. Wang, Y. Wang, dependent IL-4 production may contribute to the induction of L. Hood, Z. Zhu, Q. Tian, and C. Dong. 2005. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat. Immunol. 6: GATA3, because exogenous IL-4 can partially rescue the defect of 1133–1141. GATA3 expression in CARMA1-deficient cells. Nevertheless, we 10. Martinez, G. J., R. I. Nurieva, X. O. Yang, and C. Dong. 2008. Regulation and are unable to rule out the possibility that CARMA1 links TCR to function of proinflammatory TH17 cells. Ann. N. Y. Acad. Sci. 1143: 188–211. 11. Zheng, Y., M. Vig, J. Lyons, L. Van Parijs, and A. A. Beg. 2003. Combined another transcription factor that controls gata3 gene. deficiency of p50 and cRel in CD4+ T cells reveals an essential requirement for A previous study demonstrates that CARMA1KO mice do not nuclear factor kB in regulating mature T cell survival and in vivo function. J. develop allergic airway inflammation upon immunization and Exp. Med. 197: 861–874. 12. Hayden, M. S., and S. Ghosh. 2008. Shared principles in NF-kB signaling. Cell aerosol challenge with chicken OVA (36). The authors provide 132: 344–362. in vivo evidence that CARMA1-deficient mice do not generate 13. Schulze-Luehrmann, J., and S. Ghosh. 2006. Antigen-receptor signaling to nu- Ag-specific Th2 effector cells (36). Similar results have been clear factor k B. Immunity 25: 701–715. 14. Blonska, M., and X. Lin. 2011. NF-kB signaling pathways regulated by obtained using PKCu-deficient mice (41), which is consistent with CARMA family of scaffold proteins. Cell Res. 21: 55–70. the role of PKCu as a direct activator of CARMA1 (20, 21). Al- 15. Bertin, J., L. Wang, Y. Guo, M. D. Jacobson, J. L. Poyet, S. M. Srinivasula, though the studies mentioned previously link CARMA1 to Th2 S. Merriam, P. S. DiStefano, and E. S. Alnemri. 2001. CARD11 and CARD14 are novel caspase recruitment domain (CARD)/membrane-associated guanylate differentiation, the work by Jun et al. (42) demonstrates that mice kinase (MAGUK) family members that interact with BCL10 and activate NF- expressing inactive CARMA1 mutant do not respond to Borde- kB. J. Biol. Chem. 276: 11877–11882. tella pertussis immunization, which is associated with Th1 re- 16. Hara, H., T. Wada, C. Bakal, I. Kozieradzki, S. Suzuki, N. Suzuki, M. Nghiem, E. K. Griffiths, C. Krawczyk, B. Bauer, et al. 2003. The MAGUK family protein sponse. In addition, Morinelo and Alegre report in their meeting CARD11 is essential for lymphocyte activation. Immunity 18: 763–775. abstract that CARMA1-deficient mice do not develop Th17-driven 17. Wang, D., Y. You, S. M. Case, L. M. McAllister-Lucas, L. Wang, experimental autoimmune encephalomyelitis (43). Therefore, P. S. DiStefano, G. Nun˜ez, J. Bertin, and X. Lin. 2002. A requirement for CARMA1 in TCR-induced NF-kB activation. Nat. Immunol. 3: 830–835. CARMA1 could be required for expansion of effector cells, and 18. Egawa, T., B. Albrecht, B. Favier, M. J. Sunshine, K. Mirchandani, W. O’Brien, the observed defects could be attributable to reduced T cell pro- M. Thome, and D. R. Littman. 2003. Requirement for CARMA1 in antigen liferation. In our study, we provide in vivo and in vitro evidence receptor-induced NF-kB activation and lymphocyte proliferation. Curr. Biol. 13: 1252–1258. that CARMA1 preferentially directs Th2 differentiation by regu- 19. Monks, C. R., H. Kupfer, I. Tamir, A. Barlow, and A. Kupfer. 1997. Selective lating JunB and GATA3 transcription factors. Moreover, we have modulation of protein kinase C-u during T-cell activation. Nature 385: 83–86. The Journal of Immunology 9

20. Matsumoto, R., D. Wang, M. Blonska, H. Li, M. Kobayashi, B. Pappu, Y. Chen, complex formation and Bcl-3 expression in classical Hodgkin, anaplastic large- D. Wang, and X. Lin. 2005. Phosphorylation of CARMA1 plays a critical role in cell, and other peripheral T-cell lymphomas. Blood 106: 4287–4293. T cell receptor-mediated NF-kB activation. Immunity 23: 575–585. 32. Karin, M. 2009. NF-kB as a critical link between inflammation and cancer. Cold 21. Sommer, K., B. Guo, J. L. Pomerantz, A. D. Bandaranayake, M. E. Moreno- Spring Harb. Perspect. Biol. 1: a000141. Garcı´a, Y. L. Ovechkina, and D. J. Rawlings. 2005. Phosphorylation of the 33. Wang, Z. Y., H. Sato, S. Kusam, S. Sehra, L. M. Toney, and A. L. Dent. 2005. CARMA1 linker controls NF-kB activation. Immunity 23: 561–574. Regulation of IL-10 gene expression in Th2 cells by Jun proteins. J. Immunol. 22. Hara, H., C. Bakal, T. Wada, D. Bouchard, R. Rottapel, T. Saito, and 174: 2098–2105. J. M. Penninger. 2004. The molecular adapter Carma1 controls entry of IkB 34. Gao, M., T. Labuda, Y. Xia, E. Gallagher, D. Fang, Y. C. Liu, and M. Karin. kinase into the central immune synapse. J. Exp. Med. 200: 1167–1177. 2004. Jun turnover is controlled through JNK-dependent phosphorylation of the 23. Wang, D., R. Matsumoto, Y. You, T. Che, X. Y. Lin, S. L. Gaffen, and X. Lin. E3 ligase Itch. Science 306: 271–275. 2004. CD3/CD28 costimulation-induced NF-kB activation is mediated by re- 35. Scheinman, E. J., and O. Avni. 2009. Transcriptional regulation of GATA3 in cruitment of protein kinase C-u, Bcl10, and IkB kinase b to the immunological T helper cells by the integrated activities of transcription factors downstream of synapse through CARMA1. Mol. Cell. Biol. 24: 164–171. the interleukin-4 receptor and T cell receptor. J. Biol. Chem. 284: 3037–3048. 24. Blonska, M., B. P. Pappu, R. Matsumoto, H. Li, B. Su, D. Wang, and X. Lin. 36. Medoff, B. D., B. Seed, R. Jackobek, J. Zora, Y. Yang, A. D. Luster, and 2007. The CARMA1-Bcl10 signaling complex selectively regulates JNK2 ki- R. Xavier. 2006. CARMA1 is critical for the development of allergic airway nase in the T cell receptor-signaling pathway. Immunity 26: 55–66. inflammation in a murine model of asthma. J. Immunol. 176: 7272–7277. 25. Blonska, M., and X. Lin. 2009. CARMA1-mediated NF-kB and JNK activation 37. Hartenstein, B., S. Teurich, J. Hess, J. Schenkel, M. Schorpp-Kistner, and in lymphocytes. Immunol. Rev. 228: 199–211. P. Angel. 2002. Th2 cell-specific cytokine expression and allergen-induced air- 26. Sabapathy, K., K. Hochedlinger, S. Y. Nam, A. Bauer, M. Karin, and way inflammation depend on JunB. EMBO J. 21: 6321–6329. E. F. Wagner. 2004. Distinct roles for JNK1 and JNK2 in regulating JNK activity 38. Ramadas, R. A., M. I. Roche, J. J. Moon, T. Ludwig, R. J. Xavier, and and c-Jun–dependent cell proliferation. Mol. Cell 15: 713–725. B. D. Medoff. 2011. CARMA1 is necessary for optimal T cell responses in 27. Fuchs, S. Y., B. Xie, V. Adler, V. A. Fried, R. J. Davis, and Z. Ronai. 1997. c-Jun a murine model of allergic asthma. J. Immunol. 187: 6197–6207. NH2-terminal kinases target the ubiquitination of their associated transcription 39. Zhu, J., J. Cote-Sierra, L. Guo, and W. E. Paul. 2003. Stat5 activation plays factors. J. Biol. Chem. 272: 32163–32168. a critical role in Th2 differentiation. Immunity 19: 739–748. 28. Fuchs, S. Y., L. Dolan, R. J. Davis, and Z. Ronai. 1996. Phosphorylation- 40. Das, J., C. H. Chen, L. Yang, L. Cohn, P. Ray, and A. Ray. 2001. A critical role dependent targeting of c-Jun ubiquitination by Jun N-kinase. Oncogene 13: for NF-kB in GATA3 expression and TH2 differentiation in allergic airway in- Downloaded from 1531–1535. flammation. Nat. Immunol. 2: 45–50. 29. Eferl, R., and E. F. Wagner. 2003. AP-1: a double-edged sword in tumorigenesis. 41. Marsland, B. J., T. J. Soos, G. Spa¨th, D. R. Littman, and M. Kopf. 2004. Protein Nat. Rev. Cancer 3: 859–868. kinase Cu is critical for the development of in vivo T helper (Th)2 cell but not 30. Mathas, S., M. Hinz, I. Anagnostopoulos, D. Krappmann, A. Lietz, F. Jundt, Th1 cell responses. J. Exp. Med. 200: 181–189. K. Bommert, F. Mechta-Grigoriou, H. Stein, B. Do¨rken, and C. Scheidereit. 42. Jun, J. E., L. E. Wilson, C. G. Vinuesa, S. Lesage, M. Blery, L. A. Miosge, 2002. Aberrantly expressed c-Jun and JunB are a hallmark of Hodgkin lym- M. C. Cook, E. M. Kucharska, H. Hara, J. M. Penninger, et al. 2003. Identifying phoma cells, stimulate proliferation and synergize with NF-kB. EMBO J. 21: the MAGUK protein Carma-1 as a central regulator of humoral immune responses

4104–4113. and atopy by genome-wide mouse mutagenesis. Immunity 18: 751–762. http://www.jimmunol.org/ 31. Mathas, S., K. Jo¨hrens, S. Joos, A. Lietz, F. Hummel, M. Janz, F. Jundt, 43. Molinero, L., and M. -L. Alegre. 2011. TCR-CARMA1-NF-kB controls Th17 I. Anagnostopoulos, K. Bommert, P. Lichter, et al. 2005. Elevated NF-kB p50 differentiation. J. Immunol. 186: 152.4 (Abstr.). by guest on September 27, 2021