NFATc1 Induction in Peripheral T and B Lymphocytes Matthias Hock, Martin Vaeth, Ronald Rudolf, Amiya Kumar Patra, Duong Anh Thuy Pham, Khalid Muhammad, Tobias This information is current as Pusch, Tobias Bopp, Edgar Schmitt, Rene Rost, Friederike of October 2, 2021. Berberich-Siebelt, Dimitri Tyrsin, Sergei Chuvpilo, Andris Avots, Edgar Serfling and Stefan Klein-Hessling J Immunol 2013; 190:2345-2353; Prepublished online 30 January 2013; doi: 10.4049/jimmunol.1201591 Downloaded from http://www.jimmunol.org/content/190/5/2345

Supplementary http://www.jimmunol.org/content/suppl/2013/01/30/jimmunol.120159 http://www.jimmunol.org/ Material 1.DC1 References This article cites 37 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/190/5/2345.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

NFATc1 Induction in Peripheral T and B Lymphocytes

Matthias Hock,* Martin Vaeth,* Ronald Rudolf,* Amiya Kumar Patra,* Duong Anh Thuy Pham,* Khalid Muhammad,* Tobias Pusch,* Tobias Bopp,† Edgar Schmitt,† Rene Rost,*,1 Friederike Berberich-Siebelt,* Dimitri Tyrsin,*,2 Sergei Chuvpilo,*,3 Andris Avots,* Edgar Serfling,*,4 and Stefan Klein-Hessling*,4

NFAT transcription factors control the proliferation and survival of peripheral lymphocytes. We have reported previously that the short isoform NFATc1/aA whose generation is induced by immune stimulation supports the proliferation and inhibits the activation-induced cell death of peripheral T and B cells. We will show in this study that in novel bacterial artificial transgenic mice that express EGFP under the control of entire Nfatc1 locus the Nfatc1/Egfp transgene is expressed as early as in double-negative thymocytes and in nonstimulated peripheral T and B cells. Upon immune receptor stimulation, Nfatc1/Egfp expression is elevated in B, Th1, and Th2 cells, but only weakly in T regulatory, Th9, and Th17 cells in vitro whose generation is affected by TGFb. In naive lymphocytes, persistent immune receptor signals led to a 3–5 increase in NFATc1/aA RNA levels Downloaded from during primary and secondary stimulation, but a much stronger induction was observed at the level. Whereas anti-CD3+ CD28 stimulation of primary T cells induces both NFATc1/aA and their proliferation and survival, anti-IgM stimulation of B cells induces NFATc1/aA and proliferation, but activation-induced cell death after 3-d incubation in vitro. The anti-IgM–mediated activation-induced cell death induction of B cells in vitro is suppressed by anti-CD40–, LPS-, and CpG-mediated signals. In addition to inducing NF-kB factors, together with anti-IgM, these signals also support the generation of NFATc1/aA. According to these data and the architecture of its promoter region, the Nfatc1 resembles a primary response gene whose induction is http://www.jimmunol.org/ affected at the posttranscriptional level. The Journal of Immunology, 2013, 190: 2345–2353.

mmune receptors control the survival as well as the elimina- specific protein phosphatase (CN) contribute to the tion of peripheral lymphocytes. Signals that emerge upon inter- differentiation, elimination, and anergy induction of lymphocytes. I action of immune receptors with (presented) Ags activate To a large extent, this is mediated through , a family of tran- lymphocytes to proliferate and to differentiate into effector T and scription factors (TFs) that are final targets of the store-operated B cells, or into memory cells that mediate rapid immune responses Ca2+ entry and the subsequent activation of CN (3–5). The four genuine NFATc factors differ from all other eukaryotic

upon a secondary Ag exposure. Other immune receptor signals can by guest on October 2, 2021 lead to cell death by apoptosis or to anergy, a state of unrespon- TFs by an array of Ser/Pro-rich phosphorylation motifs within siveness of T and B cells that protects against hyperresponsiveness their regulatory region that are rapidly dephosphorylated by CN and rapid apoptosis (1, 2). The immune receptor-mediated rise in upon lymphocyte activation. This leads to the nuclear transloca- cytosolic Ca2+ and the activation of the Ca2+-dependent Ser/Thr- tion and activation of NFATc factors that, often in association with other TFs, orchestrate the induction of many in lymphocytes and numerous nonlymphoid cells (3–5). The genes encoding *Department of Molecular Pathology, Institute of Pathology, University of Wu¨rz- † ∼ burg, D-97080 Wu¨rzburg, Germany; and Institute of Immunology, University of murine and human NFATc1 consist of 11 exons and span 110- Mainz, D-55131 Mainz, Germany and 140-kb DNA, respectively. In lymphocytes, their transcription 1Current address: Institute of Molecular Biology and Biochemistry, Graz, Austria. is controlled by two promoters, P1 and P2, two polyA sites, pA1 2Current address: TheraMAB, Stolovy Pereulok 6, Moscow, Russia. and pA2, and alternative splicing events (6). In peripheral 3Current address: TheraMAB, Wu¨rzburg, Germany. resting lymphocytes, the transcription of the Nfatc1 gene appears 4E.S. and S.K.-H. share senior authorship of this publication. to be controlled by the constitutively active promoter P2 and the strong distal polyA site pA1. Stimulation of lymphocytes through Received for publication June 18, 2012. Accepted for publication December 21, 2012. their immune receptors results in a switch to P1 promoter and the This work was supported by the Deutsche Forschungsgemeinschaft, Wilhelm-Sander, use of proximal polyA site pA2. This leads to the predominant and Scheel foundations. synthesis of NFATc1/aA RNA encoding the inducible short Address correspondence and reprint requests to Dr. Edgar Serfling and Dr. Stefan NFATc1 isoform that lacks the C-terminal peptide of ∼250 aa Klein-Hessling, Josef-Schneider-Strasse 2, D-97080 Wu¨rzburg, Germany. E-mail ad- typical for most of the other NFATc . The C-terminal dresses: serfl[email protected] (E.S.) and stefan.klein-hessling@uni- wuerzburg.de (S.K.-H.) peptides of NFATc1/C and c2 harbor two SUMOylation motifs The online version of this article contains supplemental material. that, when sumoylated, direct NFATs to nuclear promyelocytic leukemia bodies and affect the transactivation capacity of NFAT Abbreviations used in this article: BAC, bacterial artificial chromosome; ChIP, chro- matin immune precipitation; CHX, cycloheximide; CN, calcineurin; CsA, cyclo- proteins (7, 8). sporin A; DC, dendritic cell; DKO, double-knockout; DN, double-negative; EGFP, The inducible P1 promoter consists of a stretch of 800 bp that enhanced GFP; ER, ; Ex., exon; GC, germinal center; iTreg, induc- ible regulatory T cell; LN, lymph node; MFI, mean fluorescence intensity; pAb, are highly conserved between mouse and human. Similar to the polyclonal Ab; TF, ; tg, transgenic; T+I, 12-O-tetradecanoyl shorter P2 promoter, the P1 promoter forms DNase I–hypersensi- phorbol-13-acetate and ionomycin; TPA, 12-O-tetradecanoyl phorbol-13-acetate; tive chromatin sites and contains a CpG island. P1 harbors multiple Treg, regulatory T cell. DNA motifs for the binding of inducible TFs, such as for CREB, Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 Fos, ATF, and NFAT itself, and a composite palindromic NFAT/ www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201591 2346 NFATc1 INDUCTION IN LYMPHOCYTES

NF-kB site to which both TFs can bind. However, when fused washed, and frozen in liquid nitrogen. In experiments of Fig. 5B, cells to a luciferase reporter gene, the P1 promoter did not show the were stimulated by anti-IgM for 0.5 h at 4˚C (pulsed), or for 24 h at 37˚C, typical induction behavior of the Nfatc1 gene in transfection washed with prewarmed PBS, and incubated for another 24–72 h with anti- CD40, CpG, or LPS. CD4+ T cells were isolated by negative selection assays. Whereas fusion with more upstream DNA did not affect using Miltenyi Biotec’s T cell isolation kit II (130-095-248) to a purity of the activity of P1 promoter, fusion with a 1-kb–long DNA segment .90%. They were stimulated by plate-bound anti-CD3+CD28 for 3 (to from intron 1 of the NFATC1 gene enhanced its overall activity 3- 5) d and restimulated by 10 ng/ml TPA and 0.5 mM ionomycin for 4 h. For + + to 5-fold, but did not change its mode of induction in T cells (6). Th differentiation, upon primary anti-CD3 CD28 stimulation, CD4 T cells were cultured for 3 d in IMDM containing 20 U/ml IL-2, 10 mg/ml These and further data suggest that, similar to other inducible anti–IL-4, and anti–IFN-g each without (Th cells) or with 10 ng/ml IL-12 genes, in addition to its promoters, the expression of NFATc1 gene (Th1). Or they were cultured in medium containing IL-2, 20 U/ml IL-4, is also controlled by remote enhancer elements. and anti–IFN-g without (Th2) or with 5 ng/ml TGFb (Th9), containing IL- 2, TGFb, anti–IFN-g and anti–IL-4, or 50 ng/ml IL-6, TGFb, anti–IFN-g, Often, the activity of enhancers controlling cell-type–restricted + genes is determined early in development. By the binding of key and anti–IL-4 (Th17). In some experiments (Fig. 6B), upon anti-CD3 CD28 treatment, T cells were also stimulated by the synthetic triacylated transcription factors, such as GATA4 or PU.1 (9, 10), chromatin lipoprotein Pam3CSK4 (2 mg/ml), a TLR 1/2 ligand, or CpG (1668; 1 nM), changes are induced that facilitate the binding of further factors, a TLR7 ligand, respectively. In proliferation assays, cells were stained including the inducible factors of NF-kB and AP-1 families, and with CFSE (Invitrogen; C34554) or PKH26 (Sigma-Aldrich; MINI26), a predisposed stage of transcriptional activity. To elucidate the ex- according to manufacturer’s protocols, and measured by flow cytometry. Using a conventional DEAE DNA transfection protocol, EL-4 thymoma pression of Nfatc1 gene in more detail, we established and investi- cells were transfected with luciferase reporter genes directed by the 2800- gated novel bacterial artificial chromosome (BAC) transgenic (tg) bp Nfatc1 P1 promoter or a 275-bp fragment of P1 (6) along with an mouse lines in which an enhanced GFP (EGFP) reporter gene is expression plasmid (Fig. 4E). Forty-eight hours posttransfection, luciferase expressed under the control of the entire Nfatc1 locus. We show that activity was measured from the cells that were either left untreated or Downloaded from as early as in double-negative (DN) thymocytes and in resting treated with TPA and ionomycin (T+I) for 24 h. Relative light units were corrected for the transfection efficacy due to total protein concentrations lymphocytes the Nfatc1/Egfp transgene is already expressed, (relative luciferase activity = 0.5/protein concentrations 3 absolute lucif- albeit at low level. Its expression is moderately enhanced upon erase activity). Normalized mean values of at least three independent immune receptor stimulation in B cells, Th1, and Th2 cells, but experiments are depicted in relative light units. weakly in Th9, Th17, and regulatory T cells (Tregs). At the RNA Flow cytometry level, we detected a moderate induction of NFATc1/aAinpe- http://www.jimmunol.org/ ripheral lymphocytes, and a much stronger induction in Western Cells were washed once in cold PBS containing 0.1% BSA (FACS buffer) before blocking with anti-FcgRII/FcgRIII (2.4G2; BD Pharmingen, San blot assays at the protein level. Although immune receptor signals Diego, CA). Stainings were performed on ice using FITC-conjugated CD4 are the primary stimulus to induce NFATc1/aA, other signals, such (GK1.5), CD8a (53-6.7), CD19 (1D3); PE-conjugated CD3ε (145-2C11), as those by anti-CD40, LPS, and CpG, which induce NF-kBin CD4 (RM4-5), CD19 (1D3), CD25 (PC61); allophycocyanin-conjugated B cells, elevate the generation of NFATc1/aA in combination with CD8a (53-6.7); biotin-conjugated CD4 (GK1.5) and CD90.2 (53-2.1), as immune receptor signals in peripheral lymphocytes. Along with well as secondary streptavidin-allophycocyanin or streptavidin-PE mAbs (all BD Pharmingen) in FACS buffer incubated for 20 min. Intracellular the architecture of its promoters (6, 11), these properties charac- Foxp3 (FJK-16s, FITC and PE conjugated) was performed according to terize the Nfatc1 gene as an early response gene whose induction manufacturer’s instruction (eBiosciences, San Diego, CA). After washing by guest on October 2, 2021 contributes to the survival of peripheral lymphocytes. with FACS buffer, cells were analyzed on a FACSCalibur (BD Pharmin- gen) using CellQuest (BD Pharmingen) and FlowJo software (Tree Star, Materials and Methods Ashland, OR). Mice RNA extraction and PCR assays C57BL/6 tg Nfatc1-Egfp reporter mice were generated upon insertion of RNA was isolated from washed and deep-frozen lymphocytes using Egfp cDNA into exon 3 of mouse Nfatc1 locus cloned in a BAC vector a standard TRIzol/isopropanol protocol. cDNA was synthesized using the spanning ∼210-kb Nfatc1 DNA (BAC RP23-361H16). As homologous iScript cDNA synthesis kit, according to the manufacturer’s (Bio-Rad) DNA sequences (Box A) a 1.2-kb DNA stretch from the 39 end of Nfatc1 instructions. Real-time PCR assays (Figs. 4–6) were performed using the intron 2 was amplified by PCR (forward, 59-TTGGCGCGCCCAGA- SYBR green master mix (Applied Biosystem) under the following con- GACCTGGAAATGCTTG-39; reverse, 59-TTGGATCCTGTTCTGGAAG- ditions: 1 cycle for 2 min at 50˚C and 10 min at 95˚C, and 40 cycles for 15 s AAAAGAGG-39) and cloned as AscI/BamHI fragment into pLD53.SC2 at 95˚C and 1 min at 60˚C with the following primers: Nfatc1 P1 promoter containing an Egfp gene with a SV40 polyA addition sequence (12). By (exon [Ex.] 1–Ex. 3), forward, 59- GGGAGCGGAGAAACTTTGC-39 and site-specific recombination, the chimeric Nfatc1/Egfp gene containing reverse, 59-CAGGGTCGAGGTGACACTAGG-39; Nfatc1 P2 promoter (Ex. ∼210 kb of chromosomal Nfatc1 locus was created in which the Egfp 2–Ex. 3), forward, 59-AGGACCCGGAGTTCGACTTC-39 and reverse, 59- indicator gene (+SV40 polyA signal) was fused in frame to codon 3 of GCAGGGTCGAGGTGACACTAG-39; Prdm1 (Blimp-1), forward, 59-TA- large exon 3 of the Nfatc1 gene (13). Thereby, due to the alternate pro- GACTTCACCGATGAGGGG-39 and reverse, 59-GTATGCTGCCAACAA- moter usage and alternate splicing of the Nfatc1 gene, the expression of CAGCA-39; Actb (b-actin), forward, 59-GACGGCCAGGTCATCACTAT- indicator gene is driven either by the inducible promoter P1 located in TG-39 and reverse, 59-AGGAAGGCTGGAAAAGAGCC-39. front of exon 1, or by promoter P2 before exon 2 (Fig. 1A) (6). Eight- to For mapping all major Nfatc1 transcripts (Fig. 7), the following primers 10-wk-old C57BL/6 wild-type mice were used in all experiments and were used (for Nfatc1 P1 and P2 promoters, see above): Nfatc1 isoform c1/ maintained under conventional conditions in accordance with institutional A (Ex. 8/9-UTR), forward, 59- ACCTGTGCAAGCCAAATTCC-39 and re- guidelines for animal welfare, which are approved and controlled by the verse, 59-AGAGTTACCATTGGCAGGAAG-39; Nfatc1 isoform c1/B (Ex. Regierung von Unterfranken, Wu¨rzburg. 10 [c1/B-specific region]-Ex. 11/10), forward, 59-acttcagccacccgcttc -39 and reverse, 59-GATCTCATTTACTGCGGCTG-39; Nfatc1 isoform c1/C Isolation, culture, and DNA transfection of lymphocytes (Ex. 10 [c1/C-specific region]-Ex. 11/10), forward, 59-GCAAGAGCC- Splenic B cells were isolated using Miltenyi Biotec’s B cell isolation kit TGAGGAATTGG-39 and reverse, 59-CAGCTCCGATGTGCTGAATT (130-090-862) to a purity of 95–98%. A total of 5 3 106 B cells was AG-39. cultured in 1 ml X-vivo 15 medium (BE04-418Q; Lonza) and stimulated In semiquantitative PCR assays (Fig. 1, Supplemental Fig. 1), RNA levels were normalized by b-actin PCR assays. by anti-IgM [10 mg/ml; F(ab9)2 fragment of goat anti-mouse IgM; Jackson ImmunoResearch Laboratories; 115-006-020], LPS (10 mg/ml; Sigma- Western blotting Aldrich; #L5293), anti-CD40 (5 mg/ml; R&D Systems; MAB440), CpG (0.5 or 1 mM; type B, ODN 1668, MWG), cyclosporin A (CsA; 100 ng/ml; Whole-protein extracts from B cells were prepared by lysis of frozen cells Sigma-Aldrich), forskolin (50 mM; AppliChem/Sigma-Aldrich), or 12- in 60–100 ml 50 mM Tris buffer (pH 7.5), containing 150 mM NaCl and O-tetradecanoyl phorbol-13-acetate (TPA; 10 ng/ml; Sigma-Aldrich) and 1% Triton X-100 on ice for 20 min, followed by centrifugation and mea- ionomycin (0.5 mM; Invitrogen), as indicated. The cells were harvested, surement of protein content. T cells were lysed by sonication in 100–300 ml The Journal of Immunology 2347

(depending on cell number and activation state) of 50 mM Tris buffer containing 150 mM NaCl, 1% Triton X-100, 1% Na-deoxycholate, 0.1% SDS, 1 mM EDTA, and freshly added protease inhibitors. Western blots were performed by fractionating protein extracts (50–100 mg protein per lane) on SDS-PAGE gels, followed by immunodetection of NFATc1 using the 7A6 mAb (BD Pharmingen) or the polyclonal Ab (pAb) IG-205 raised against the N-terminal half of NFATc1 (ImmunoGlobe, Himmelstadt). For detecting NFATc1/aA, a pAb (IG-457; ImmunoGlobe) raised against NFATc1’s a-peptide was used, for E2F1 detection a pAb (sc-193; Santa Cruz Biotechnology). Protein loading was controlled by Ponceau red staining of membrane. Signals were developed by chemiluminescence detection using Super Signal (Thermo Fisher Scientific). Chromatin immune precipitation assays Chromatin immune precipitation (ChIP) assays were performed, as de- scribed previously (14), using the pAbs IG-205 and IG-209 (Immuno- Globe, Himmelstadt) specific for NFATc1 and NFATc2, respectively. The following promoter-specific primers were used for detecting NFATc1 binding in semiquantitative RT-PCRs: Bcl2a1a/A1, forward, 59-GGGT- TTATAGCCGGATCGTTTTC-39 and reverse, 59-ACAGCGTCCTTCCT- TCTCGTCAC-39; Tnf/TNFa, forward, 59-TTTCAGTTCTCAGGGTCCT- ATAC-39 and reverse, 59-TGTGCAGACGGCCGCCTTTATAG-39; Nr4a1/

Nur77, forward, 59-GAGCTGTTGGCACCAGGAGC-39 and reverse, 59- Downloaded from CCTGGAATGTCTGCGCGCGTG-39; Il4/IL-4, forward, 59-AAAGGCC- GATTATGGTGTAATTTC-39 and reverse, 59-CAATAGCTCTGTGCCG- TCAGTG-39; Nfatc1 P1 promoter, forward, 59- GCAAGCAATCCAGT- TCGCTCAG-39 and reverse, 59-GGCGGGTGCCCTGAGAAAGC-39.

Results

NFATc1 induction in T lymphocytes http://www.jimmunol.org/ To study the expression of the murine Nfatc1 gene in lymphoid cells, we created BAC tg mouse lines that express an Egfp reporter gene under the control of the entire Nfatc1 locus. The scheme of the shuttle vector pLD53.SC2, which was used for the generation of the Nfatc1/Egfp BAC construct, and the organization of the FIGURE 1. Generation of Nfatc1/Egfp BAC tg mice and analysis of resulting chimeric Nfatc1/Egfp gene are shown in Fig. 1A. The Nfatc1-directed EGFP expression. (A) Structure of the shuttle vector Nfatc1/Egfp gene contains an Egfp cassette that was inserted be- pLD53.SC2 used for the construction of chimeric Nfatc1/Egfp genes hind amino acid codon 3 of Nfatc1 exon 3. Due to a 39 polyA cloned in a BAC vector for the generation of Nfatc1/Egfp tg mice. Box A by guest on October 2, 2021 addition motif, Nfatc1/Egfp expression leads to the synthesis of corresponds to a 1.2-kb DNA fragment from the 39 end of Nfatc1’s intron short NFATc1/EGFP proteins of ∼31 or 32 kDa, which contain 2, which is used for site-specific recombination. Below, a scheme of the either the N-terminal a peptide (see Supplemental Fig. 1C) or the murine Nfatc1 gene, as cloned in the BAC vector, is presented. The two b peptide of NFATc1 (15). promoters, P1 and P2, and the two polyA sites, pA1 and pA2, are indi- B When we investigated the expression of the chimeric Nfatc1/ cated. ( ) Detection of basal EGFP expression in unstimulated splenic T cells and thymocytes from Nfatc1/Egfp tg mice by flow cytometry. (C) Egfp gene, we observed similar RNA levels for the chimeric and 2 Sorted CD4+CD25 T cells and CD4+CD25+ natural Tregs from tg mice endogenous Nfatc1 genes (Supplemental Fig. 1A). Stimulation of were left unstimulated (w/o) or stimulated with plate-bound anti-CD3 or + + primary CD4 T cells with anti-CD3 CD28 for 24 h (lanes 2 and anti-CD3+CD28 for 16 and 48 h in the absence or presence of 100 ng/ml 3)or48h(lanes 4 and 5) led to an increase in P1-directed tran- CsA. EGFP expression was measured by flow cytometry. scripts from both the endogenous Nfatc1 gene (Ex. 1–3) and the chimeric Nfatc1/Egfp gene (Ex. 1-EGFP), which was suppressed by the CN inhibitor CsA (Supplemental Fig. 1A, lanes 3 and 5). In expression was detected for single-positive CD8+ thymocytes, contrast, no increase was detected in P2 transcripts (Ex. 2–3), and whereas single-positive CD4+ thymocytes and, in particular, double- CsA did not affect the generation of P2 transcripts (the suppres- positive CD4+8+ thymocytes showed a markedly weaker basal sion seen in Supplemental Fig. 1A, lane 5, for P2 [Ex. 2-EGFP] fluorescence. To our surprise, a strong basal Nfatc1/Egfp expres- transcripts from the Nfatc1/Egfp gene is atypical: see Supple- sion was observed in DN thymocytes (Fig. 1B, middle panel). This mental Fig. 1B). Similar results were obtained upon stimulation of is not due to pre-TCR formation because DN4 thymocytes bear- whole splenocytes by Con A (Supplemental Fig. 1B). These PCR ing a pre-TCR showed a weaker EGFP expression than pre-TCR– data were confirmed by Western blot assays. Stimulation of spleno- less DN3 thymocytes (Fig. 1B, right panel). We will show else- cytes with Con A or T+I for 24 h led to the appearance of NFATc1/ where that IL-7 exerts a stimulatory effect on NFATc1 expression aA or a chimeric EGFP protein consisting of EGFP and the N- in DN thymocytes (16). 2 terminal sequences of NFATc1 encoded by exon 1 (the so-called Stimulation of conventional CD4+CD25 T cells by anti-CD3 N-terminal a-peptide encoded by exon 1). CsA suppressed the or anti-CD3+CD28for16or48hledtoamarkedincreasein appearance of both NFATc1/aA and chimeric NFATc1a/EGFP Nfatc1/Egfp expression, whereas a slight increase was detected for protein (Supplemental Fig. 1C). CD4+CD25+ natural Treg (Fig. 1C). A similar induction of Nfatc1/ Flow cytometry of Nfatc1/Egfp expression in freshly prepared Egfp expression was also observed in activated CD4+ T cells in T cells showed that CD8+ T cells exhibit a somewhat higher basal a MLR assay. When CD4+ T cells from tg C57BL/6 mice were NFATc1 expression than CD4+ T cells (Fig. 1B, left panel). This incubated for 72 h with BALB/c splenocytes from which T cells was also observed for CD8+ and CD4+ T cells from peripheral were depleted, a 3- to 4-fold increase in Nfatc1/Egfp expression blood (Supplemental Fig. 1D). A strong constitutive Nfatc1/Egfp was detected in the population of activated (CD25+ or CD69+) 2348 NFATc1 INDUCTION IN LYMPHOCYTES

T cells (Supplemental Fig. 2A). Under the same conditions, a 2- to cultivation of conventional CD4+CD252 T cells in the presence of 3-fold increase in Nfatc1/Egfp expression was detected for CD8+ IL-2, but absence of TGFb, leads to an increase in Nfatc1/Egfp T cells (Supplemental Fig. 2B). induction upon anti-CD3 or anti-CD3+CD28 stimulation, addition of 5 ng/ml TGFb to the medium suppressed Nfatc1/Egfp expres- TGFb interferes with NFATc1 induction in T cells sion. Under these conditions, .70% of cells express Foxp3 and, When (naive) splenic T cells from BAC tg mice were polarized for therefore, correspond to iTreg (Fig. 2C). These observations on 72 h under conditions that support the generation of Th1, Th2, the poor Nfatc1 expression in Treg confirm our previous findings Th17, and Th9 cells or of inducible Tregs, we detected a strong on low NFATc1 level in Treg (7). Nfatc1/Egfp expression in unpolarized Th cells and in both Th1 and Th2 cells, and a relatively weak induction in Th17 and Th9 NFATc1 expression upon immunization in lymphocytes in vivo cells, and in inducible Treg (iTreg) (Fig. 2A). Because the in vitro The strong effect of TGFb on Nfatc1/Egfp induction in vitro does differentiation of the latter cells needs TGFb, we investigated not necessarily imply that upon immunization and differentiation the effect of TGFb on Nfatc1/Egfp expression. As expected, in- of Th cells in vivo the expression of Nfatc1 gene is markedly creasing amounts of TGFb (from 2.5 to 7.5 ng/ml) led to a re- affected. To investigate the effect of immunization on NFATc1 duction in Nfatc1/Egfp induction upon anti-CD3 treatment (Fig. expression in vivo, we challenged tg Nfatc1-Egfp reporter mice 2B). by injecting 20 mg OVA peptide (OVA) into the hind footpad of In iTreg, the suppression of Nfatc1/Egfp induction correlated mice. Ten days later, T cells from draining and nondraining well with the occurrence of Foxp3, which is known to be regulated poplietal lymph nodes (LNs) were prepared, sorted according to by TGFb-mediated activation of Smad factors (17). Whereas their GFP fluorescence intensity, and restimulated ex vivo by 40 mg OVA for 24 h in the presence of LN-resident CD11c+ dendritic Downloaded from cells (DCs; ratio T:DC 10:1). Whereas in three OVA immunization assays in cells from draining LNs a relative decrease of CD4+ T cell compartment (from 18.7 to 10.5%) was observed, their CD4+ T cells showed an increase of ∼20% in GFP fluorescence, and those cells with the brightest fluorescence an increase of 50% compared with those from nondraining LNs (Fig. 3A and data not http://www.jimmunol.org/ shown). Intracellular stainings of sorted CD4+ TcellsforT-bet (Th1 cells), RORgt (Th17), and Foxp3 (Treg) revealed the highest Foxp3 and RORgt levels in cells with no or low GFP fluorescence, whereas T-bet was expressed in T cells with intermediate GFP levels (Fig. 3A). We also investigated the Nfatc1/Egfp induction in splenic B cells upon immunization with the T cell–dependent Ag nitro- phenyl-keyhole limpet hemocyanin. As shown in Fig. 3B, 7 d by guest on October 2, 2021 upon immunization we observed a strong increase in GFP fluo- rescence in Fas+ or peanut lectin agglutinin+ germinal center (GC)–like B cells from immunized mice. And similar to IgM+ IgG3+ double-positive plasmablasts, which we observed previ- ously to express high GFP levels upon immunization with nitro- phenyl-Ficoll (14), we detected a high GFP fluorescence for IgM+ IgG1+ and IgM+IgG2b+ GC B cells (Fig. 3B). These findings support the view that NFATc1 plays an important role in Ig class switch. NFATc1 expression is correlated with the entry of lymphocytes into cell cycle

The majority of (naive) splenic lymphocytes are in the G0 phase of cell cycle, that is, they do not proliferate. They need ∼24 h to enter into the G1 phase of cell cycle, and within this time the Nfatc1 gene is activated upon anti-IgM stimulation in B cells (14) or upon anti-CD3+CD28 stimulation in T cells (18). Whereas stimulation for 48 h results in a moderate increase in NFATc1 levels, longer stimulation in vitro does not lead to an overall increase in NFATc1 FIGURE 2. TGFb suppresses NFATc1 induction during CD4+ T cell levels (Fig. 4A–C). Transcription factors whose expression and A differentiation. ( ) Elevated NFATc1/EGFP expression in Th, Th1, and activity are tightly connected with the G1 phase of cell cycle and + Th2 cells and poor expression in iTreg, Th9, and Th17 cells. CD4 T cells cell cycle regulation are the members of factor family (19). from Nfatc1/Egfp tg mice were differentiated for 3 d in vitro for the Stimulation of splenic B cells with anti-IgM (or LPS; data not generation of Th cells or iTreg, and their EGFP expression was analyzed shown) led to a marked increase in E2F1 levels within 24 h (Fig. by flow cytometry. (B) CD4+ T cells from tg mice were left unstimulated or 4D). When we cotransfected a luciferase reporter gene controlled stimulated by anti-CD3 Ab for 48 h in the absence or presence of TGFb. EGFP expression was measured by flow cytometry. (C) CD4+ T cells were by the Nfatc1 P1 promoter spanning 800 bp of Nfatc1’s 59 region cultivated for 48 h with IL-2 or IL-2 plus TGFb. A portion of cells was with a plasmid expressing E2F1 into EL-4 thymoma cells, we stimulated by anti-CD3 or anti-CD3+CD28 without or with CsA. The detected a 5 (to 10)-fold increase in the T+I-induced luciferase expression of EGFP or Foxp3 was measured by flow cytometry and is activity. No increase was detected for a luciferase gene directed by indicated as MFI and as percentage of Foxp3+ cells, respectively. 75 bp of immediate upstream region of the P1 promoter (Fig. 4E). The Journal of Immunology 2349

FIGURE 4. NFATc1 expression is correlated with the entry of T cells into cell cycle and E2F1 expression. (A–C) CD4+ T cells from Nfatc1/Egfp tg mice were stained with PKH-26 for measuring cell division. They were left unstimulated (0h) or stimulated by anti-CD3+CD28 for 48 and 72 h. EGFP expression and proliferation were measured by flow cytometry. (D) Downloaded from Western blot of whole-protein extracts from freshly isolated splenic B cells (lane 1) and B cells stimulated by anti-IgM for 24 h (lane 2)or48h(lane FIGURE 3. NFATc1/EGFP expression in Th cells and GC-like B cells 3). An E2F1-specific Ab was used for detection. NS, nonspecific band. (E) upon immunization. (A) Nfatc1/Egfp tg mice were immunized with OVA Transactivation of the Nfatc1 P1 promoter by E2F1. An expression vector (20 mg) emulsified in CFA in the hind footpad. Ten days later, draining and encoding E2F1 was cotransfected into EL-4 cells with luciferase genes nondraining LNs were prepared and CD4+ T cells were sorted on a FACS controlled either by the 2800-bp P1 promoter or a 275-bp segment of P1 ARIA II according to EGFP expression (P5 = EGFP-high, P6 = EGFP- http://www.jimmunol.org/ promoter. Twenty-four hours posttransfection, the cells were left unstim- medium, P7 = EGFP-low, P8 = EGFP-negative). Upon restimulation with ulated (2) or stimulated by T+I for 24 h. Mean values of three transfection OVA (40 mg) in the presence of LN-resident CD11c+ DCs for 24 h, the experiments are shown. (F) MCF-7 cells were transfected with a vector cells were intracellularly stained for the expression of T-bet, RORgt, and expressing inducible E2F1/ER protein. Transfected cells were selected Foxp3. Depicted are the percentages of T-bet–, RORgt-, and Foxp3- with puromycin for 3 d and either left untreated (lane 1)ortreatedby expressing cells among all CD4+ T cells from draining LN. Shown are tamoxifen (+) for 24 h (lane 2). Whole-cell protein was analyzed by im- data of one representative assay of three. (B) Strong Nfatc1-Egfp reporter munoblotting using the NFATc1 7A6 mAb (upper panel) or an Ab specific in GC-like B cells. Mice were immunized with NP– for E2F1 (lower panel). keyhole limpet hemocyanin emulsified in alumn, and 10 d later splenic B cells were analyzed by flow cytometry. One typical experiment from three by guest on October 2, 2021 assays is shown. whereas LPS or anti-CD40 stimulation exerted a poor stimulatory effect (14). In line with this observation, when splenic B cells Moreover, the inducible expression of E2F1 in MCF-7 cells, a pretreated by anti-IgM were stimulated by LPS, CpG, or anti- human breast cancer line (Fig. 4F), or in mouse embryonic CD40 for 24 h, NFATc1 levels decreased strongly, as shown in fibroblasts (data not shown) led to NFATc1 expression in these immunoblots using a a-peptide–specific Ab. However, after fur- cells, which otherwise express NFATc1 in minute amounts. The ther incubation for 72 h, NFATc1/aA reappeared and represented data suggest that E2F factors support the induction of NFATc1 in the most prominent NFATc1 protein (Fig. 5C). When B cells were B lymphocytes. stimulated for 2–4 d with LPS alone [which leads to a strong increase in Blimp-1 RNA (Supplemental Fig. 4A) that controls NFATc1 expression in splenic B cells plasma cell formation (20)] in immunoblots using the NFATc1- The expression of the Nfatc1/Egfp transgene does not distinguish specific mAb 7A6, a faster migrating band appeared (see asterisk between individual NFATc1 isoforms. Therefore, we analyzed at lane 15, Fig. 5C). However, this band could be detected by NFATc1 induction in primary lymphocytes cultured in vitro in neither the a-specific pAb (Fig. 5C, lower panel) nor the NFATc1- real-time PCR and Western blot assays. In PCR assays, primers specific pAb IG-205 (data not shown). But it was detected by an for the detection of P1- or P2-directed transcripts were used. Upon anti-mouse pAb coupled to peroxidase (see asterisk in Supple- stimulation of (naive) splenic B cells by anti-IgM for 24 and 48 h, mental Fig. 4B, lanes 7, 9, and 11), suggesting that this band does we observed a 3- to 4-fold increase in P1-directed transcripts not correspond to NFATc1, but rather to IgM protein, which is (Fig. 5A, left panel), whereas P2-directed RNAs decreased (right massively produced in plasmablasts. panel). LPS did not enhance, but suppress, both P1 and P2 activity (Fig. 5A), and a similar effect was detected for anti-CD40 treat- NFATc1 induction in T cells needs persistent receptor signals ment alone (data not shown). Adding the NF-kB inducers LPS or When freshly prepared (naive) CD4+ T cells were incubated with anti-CD40 (see Supplemental Fig. 3) to cells that were pretreated anti-CD3+CD28, we observed a 5-fold increase in P1 transcripts by anti-IgM for 24 h and washed afterward led to a marked de- and a weak, but reproducible decrease in P2-directed transcripts crease in P1-directed transcripts upon further incubation for 24 h upon incubation for 1 or 3 d (Fig. 6A). Removal of cells from the and, somewhat less, for 72 h. Ab-coated plates led to a rapid drop in P1 transcripts within 24 h. Similar effects of anti-IgM–, LPS-, or anti-CD40–mediated When T cells stimulated by anti-CD3+CD28 for 3 d and rested for stimulation of B cells were observed for the Nfatc1/Egfp expres- 2 d (without Abs) were stimulated by T+I for 4 h, a 10-fold in- sion (Fig. 5B). We showed previously in Western blots of whole- duction of P1 was observed (Fig. 6A). Incubation of CD4+ T cells cell protein extracts that anti-IgM stimulation of splenic B cells from Nfatc1/Egfp tg mice with anti-CD3+CD28 led to a marked for 24 h ex vivo leads to massive increase in NFATc1/aA level, increase of GFP fluorescence from ∼90 mean fluorescence in- 2350 NFATc1 INDUCTION IN LYMPHOCYTES

NFATc2 and c3 are dispensable for NFATc1/aA induction in Th1 effector cells Using primers for all prominent NFATc1 RNAs, we investigated also the appearance of individual NFATc1 RNAs in EL-4 thymoma cells, which express a constitutively active subunit Aa of CN (21), and of differentiated splenic T cells from wild-type and NFATc2+c3 double-knockout (DKO) mice upon secondary stimulation. T+I treatment of EL-4 cells for 4 h led to predominant induction of NFATc1/aA RNA and, therefore, P1 promoter activity (22), but not of longer transcripts spanning exon 10 and 11 sequences (Fig. 7A). The predominant NFATc1/aA induction correlated well with an increased binding of NFATc1 to the P1 promoter. This is shown in ChIP assays in which the binding of NFATc1 and c2 to the P1 promoter was compared with the binding to the Il4 and Tnf pro- moters, two well-known NFAT targets (23, 24), and the Bcl2a1a/A1 and Nr4a1/nur77 promoters, two putative NFAT targets (25, 26) (Fig. 7B). Whereas stimulation by T+I enhanced NFATc1 binding, CsA suppressed it, supporting our view on the autoregulation of P1-

directed NFATc1/aA induction (15). RNA from Th or Th1 cells Downloaded from restimulated by T+I for 4 h showed a similar 10-fold increase in P1- directed transcripts corresponding primarily to NFATc1/aARNA, and a similar strong P1 induction was observed for Th1 cells that were generated from splenic T cells of DKO mice (Fig. 7C). Contrary to a former report (27), this suggests that NFATc2 plays a minor role in NFATc1 induction. In Th and Th1 cells, we observed http://www.jimmunol.org/ high constitutive activity of P2 and a 1.5- to 3-fold transient increase in its activity upon stimulation for 30 min, which decreased after stimulation for 4 h (Fig. 7C). Whereas these transiently induced P2 transcripts were prominent in Th and Th1 wild-type cells, they were FIGURE 5. NFATc1 expression in splenic B cells. (A) Real-time PCR not observed in EL-4 cells and DKO Th1 cells. This correlates well assays for the detection of Nfatc1 P1- and P2-directed transcripts in splenic with the decrease in long exon 10 and 11 transcripts (compared with B cells. As indicated, untreated splenic B cells or B cells stimulated by shorter transcripts) in EL-4 and DKO Th1 cells and might be due to anti-IgM alone for 24 or 48 h, or stimulated for 24 h by anti-IgM, washed, the increased Ca2+/CN levels typical for those cells (21, 28). and then treated with anti-CD40 or LPS for additional 24 or 72 h. B cells by guest on October 2, 2021 were also stimulated for 48 or 96 h by LPS alone. (B) B cells from Nfatc1/ The N-terminal a-peptide confers a short half life time to Egfp tg mice were treated as in (A), and the EGFP fluorescence was NFATc1 proteins C measured by flow cytometry. ( ) Western blots of whole-protein extracts We were also interested to elucidate whether the individual NFATc1 from splenic B cells treated as in (A)(left panel). In lanes 5 and 6, anti- proteins differ in their stability. To investigate this in detail, we IgM–pretreated B cells were also treated with 1 mM CpG. In right panel, lanes 10–13, B cells were pulsed for 30 min with anti-IgM at 4˚C, washed, transfected expression vectors encoding individual NFATc1 pro- and treated, as indicated. Upper filters were incubated with NFATc1 7A6 teins into EL-4 cells that remained uninduced or were induced by mAb, and lower filters with the pAb specific for NFATc1’s N-terminal T+I for 2–8 h in the presence of cycloheximide (CHX) to inhibit a-peptide. The asterisk indicates an Ig-like band in protein from plasma- further protein synthesis. Immunoblots showed that NFATc1 pro- blasts. NS, Nonspecific band. teins bearing the N-terminal a-peptide, that is, NFATc1/aA and NFATc1/aC, were rapidly degraded, whereas NFATc1/b proteins spanning the N-terminal b-peptide appeared to be more stable, in tensity (MFI) to 184 and 240 MFI upon stimulation for 48 and particular upon T+I stimulation of EL-4 cells (Fig. 8A). To con- 96 h, respectively. And similar to the effect of TLR ligands on solidate this observation, we introduced DNA encoding the N- anti-IgM–induced B cells, adding CpG or Pam3CSK4 (a ligand of terminal a-andb-peptides into a vector expressing the estrogen + TLR2) to those CD4 T cells led to a reduction in Nfatc1/Egfp receptor (ER). The resulting vectors express chimeric proteins that expression after a further incubation for 24 or 72 h (Fig. 6B). In consist either of the N-terminal a-orb-peptide, a SV40 nuclear + immunoblots, whole-protein extracts from splenic CD4 T cells localization signal, and the ER. They were transfected into 293 + stimulated for 1 d by anti-CD3 CD28 revealed a strong increase human embryonic kidney cells that were left untreated or treated by in NFATc1 protein level, especially in NFATc1/aA (Fig. 6C). T+I for 3–12 h in the presence of CHX. The results of immunoblots Whereas the NFATc1 level remained almost constant upon per- show that the fusion of b-peptide to the ER led to a prolonged sistent stimulation of T cells for 3 d, a strong decline in NFATc1 appearance of chimeric b-peptide/ER protein, compared with the was observed when cells stimulated with Abs for 2 d were sub- ER protein (Fig. 8B). In contrast, fusion of b-peptide did not sequently incubated for 1 d without Abs (Fig. 6C). In particular, markedly prolong the short half life of ER protein. These data show the level of NFATc1/aA diminished, whereas that of the longer that N-terminal peptides of NFATc1 proteins contribute to the sta- NFATc1 isoforms remained almost constant. A similar pattern was bility of proteins that differ markedly in their half life time. found upon continuation of incubation without Abs for 3 d. Sec- ondary stimulation of such T cells with T+I for 4 h led both to an Discussion increase in short NFATc1/aA isoform and in a shift of the banding We show in this study that the Nfatc1 gene is expressed as early as pattern reflecting the dephosphorylation of all NFATc1 proteins in DN thymocytes, and in naive T and B lymphocytes of periph- (see lanes 5 and 6 in Fig. 6C). eral lymphoid organs. Unlike several lymphokine genes that be- The Journal of Immunology 2351

FIGURE 6. NFATc1 expression in peripheral T cells. (A) Real-time PCR assays for the detection of Nfatc1 P1- and P2-directed transcripts in pe- ripheral T cells. CD4+ T cells were left untreated (0) or treated by plate-bound anti-CD3+CD28 Abs for 1, 2, or 3 d. When indicated, the stimulus was removed and incubation was continued for 1–3 d in the ab- sence of Abs. At day 5, cells were restimulated by T+I for 4 h. Mean values of three assays are shown. (B) CD4+ T cells from Nfatc1/Egfp tg mice were left untreated (w/o) or treated for 48 or 96 h with anti- CD3+CD28 Ab (left panel), respectively. In the middle and right panels, T cells were treated by anti- CD3+CD28 Ab for 48 h, or by anti-CD3+CD28 Ab

for 24 h. The latter were washed and treated for an Downloaded from additional 24 or 72 h by the TLR ligands CpG or

Pam3CSK4. The EGFP fluorescence was measured by flow cytometry. (C) NFATc1 immunoblots of T cells treated as in (A). The left filter was incubated with the 7A6 mAb directed against all NFATc1 isoforms, and the right filter with a pAb recognizing the NFATc1 N-terminal a-peptide and, therefore, the http://www.jimmunol.org/ NFATc1/a isforms. NS, Nonspecific band.

come strongly induced upon activation in peripheral T cells (29, effector cells. One such event is the generation of effector Th1 by guest on October 2, 2021 30), in lymphoid progenitor cells and naive peripheral lymphocytes and Th2 cells from naive T cells. Upon secondary stimulation of the Nfatc1 gene appears not to be closed, but organized in an open T cells driven to Th1 effector cells, a 10-fold increase in Nfatc1 chromatin configuration. This view is supported by genome-wide transcripts was observed (Fig. 7), which correlates well with the mapping studies that showed that the DNase I–hypersensitive sites appearance of novel histone tail modifications at intron 10 of the of two Nfatc1 promoters (6) are already present in human CD34+ Nfatc1 locus, such as the appearance of H3K4me3 mark, an indi- bone marrow cells (31). This, however, does not exclude that the cator for active transcription (32), in Th1 and Th2 cells, but not chromatin architecture of Nfatc1 locus remains unchanged during in Th17 and Treg cells (11) in which the Nfatc1 gene is weakly the numerous differentiation events from lymphoid progenitor to expressed (Figs. 1, 2, Supplemental Fig. 3).

FIGURE 7. Nfatc1 RNA expression in EL-4 thy- moma cells and in peripheral CD4+ T cells upon secondary stimulation. (A) EL-4 cells were left un- treated or treated by T+I without or with CsA or forskolin (F), as indicated. RNA was isolated, and real-time PCR assays were performed with primers specific for Nfatc1 P1 (a/A,B,C)- and P2 (a/A,B,C)- directed transcripts, and of NFATc1/A (ab/A), NFATc1/B (ab/B), and NFATc1/C (ab/C) tran- scripts. (B) For ChIP assays, EL-4 cells and primary CD4+ T cells were treated with T+I, as indicated. The cross-linked chromatin was sheared and precipitated with pAbs raised against NFATc1 or c2. (C) CD4+ T cells from wild-type mice or mice double deficient (DKO) for NFATc2 and c3 were incubated for 5 d in vitro under conditions that favor the generation of Th or Th1 effector cells. They were restimulated by T+I, as indicated, and their RNA was analyzed in real-time PCR assays for Nfatc1 RNAs, as in (A). 2352 NFATc1 INDUCTION IN LYMPHOCYTES

T cells (Figs. 5, 6). Supported by persistent receptor signals, this autoregulatory network consisting of the CN-inhibitor Rcan1, the large subunit A of CN, NFATc1 (14), and, certainly, further mole- cules can keep constant high NFATc1/aA levels for days. How- ever, when interrupted, the generation of NFATc1/aARNAis stopped and NFATc1/aA protein disappears rapidly (Fig. 6C), which is facilitated by the short half life of NFATc1/aAin stimulated lymphocytes and other cells (Fig. 8). The striking differences in NFATc1 RNA and protein levels sug- gest that in primary lymphocytes NFATc1 expression is controlled at the posttranscriptional and posttranslational level. To our surprise, RNA Seq experiments revealed a high number of Nfatc1 transcripts FIGURE 8. Stability of NFATc1 proteins. (A) Expression vectors en- in nonstimulated, freshly prepared B and T cells, compared with coding the human NFATc1 isoforms NFATc1/aA, bA, aC, or bC(and fully activated cells (E. Serfling and V. Boisguerin, unpublished an N-terminal HA tag) were transfected into EL-4 T cells. Twenty-four observations). In those cells, almost no NFATc1 protein can be hours later, the cells were left unstimulated (unst.) or stimulated (st.) by detected in Western blots and histochemical stainings. Whereas T+I for 2–8 h in the presence of 10 mg/ml CHX. Whole-protein extracts upon primary stimulation for 1 d a moderate 3- to 5-fold increase in were immunoblotted using an Ab directed against the HA tag. Act., actin signal. (B) The 293 human embryonic kidney cells were transfected with NFATc1/aA RNA level and almost no change in all Nfatc1 tran- retroviral pEGZ vectors (7) encoding ER, or chimeric proteins consisting scripts are found, a conspicuous increase in protein levels is Downloaded from of an ATG initiation codon, the N-terminal NFATc1 a-orb-peptide, a detected in Western blots (Figs. 5, 6). Although a part of this dis- SV40 nuclear localization signal, and the ER (constructs a-ER and b-ER). crepancy could be due to technical caveats, other findings support Twenty-four hours later, the cells were left untreated (0) or treated with this assumption, as the distinct differences between NFATc1/aA 10 mg/ml CHX for 3–12 h. Whole cellular protein extracts were immu- RNA and protein induction in B cells upon double stimulation with noblotted using an Ab directed against ER protein. Act., Actin control anti-IgM and anti-CD40 or LPS (Fig. 5). Although in Western blots signal. a strong increase in NFATc1/aA was detected upon prolonged anti- CD40 or LPS stimulation of B cells pretreated with anti-IgM, no http://www.jimmunol.org/ Another differentiation event in which the Nfatc1 gene is increase, but a decrease, in level of P1-directed transcripts was ob- strongly expressed is the GC formation. In follicular Th cells, high served (compare Fig. 5C with Fig. 5A). It will be the task of further NFATc1 RNA levels were detected (33), and GC B cells from our experiments to elucidate by which posttranscriptional and posttrans- tg Nfatc1-Egfp reporter mice were found to express high levels of lational mechanisms the generation of NFATc1/aA is controlled. NFATc1/EGFP (14) (Fig. 3B). It remains to be shown whether the Another interesting observation that emerged from our study strong expression of Nfatc1 gene in (a portion) of GC B and is the collaboration between immune receptor and costimulatory T cells is accompanied by distinct changes in Nfatc1 chromatin signals, in particular CD40 and TLR signals, in NFATc1/aA in- structure. duction and B cell survival. The strong induction of NFATc1/aA by guest on October 2, 2021 Analyses of chromatin structure of CpG-containing promoters upon prolonged incubation of splenic B cells stimulated by anti- and CpG-less promoters indicated remarkable differences in their IgMand,subsequently,byanti-CD40,LPS,orCpG(Fig.5C) architecture and induction behavior. Primary response genes, such suggests a role for NF-kB factors in NFATc1/aA induction and as the FOS, JUN, EGR, ATF, and a few more TF genes, are usually a collaboration between NFATc1/aAandNF-kB factors in the associated with CpG islands. Before induction, they are associated survival (and differentiation) of splenic B cells. Although anti-IgM with RNA polymerase II and histone tail modifications that are stimulation alone is a strong inducer of NFATc1/aAinsplenic indicative for actively transcribed genes. Moreover, their induction B cells, these cells die without any further (co)stimulatory signals does not need the SWI/SNF nucleosomal remodelling complex upon incubation in vitro. The rescue of anti-IgM–stimulated splenic (34, 35). Whereas secondary response genes, for example, the IL6, B cells upon anti-CD40, LPS, or CpG stimulation suggests that NOS2, and IL12B genes in macrophages, are ∼1000-fold induced, NFATc1 and NF-kB signals cooperate in the survival (and dif- primary response genes show a moderate 5- to 10-fold induction ferentiation) of splenic B cells in adaptive immune responses. (36). Unlike the promoter of primary response genes, the chro- Whereas anti-IgM stimulation (and NFATc1 induction) alone leads matin opening of secondary response genes depends on the SWI/ to cell death, anti-CD40, LPS, or CpG signals all induce NF-kB SNF complex; their promoters are poor in CpG residues and factors (Supplemental Fig. 3), albeit through different receptors H3K4me3 marks and not bound by preactivated/poised by RNA and pathways (38). They enhance the generation of NFATc1/aA, polymerase II (36, 37). which, along with NF-kB factors, contributes to the survival and It remains to be shown whether the promoter induction in differentiation of B cells upon BCR stimulation. lymphocytes follows this scheme of promoter induction. The Collectively, our data indicate that, similar to induction of pri- open chromatin configuration and rapid, but moderate induction mary response genes, the induction of Nfatc1 gene is moderately of NFATc1/aA in effector Th cells classify the Nfatc1 gene as elevated at the transcriptional level in lymphoid cells. However, a primary response gene in these cells. The Nfatc1 promoter re- along with posttranscriptional and posttranslational events, it re- gion contains CpG islands and histone tail modifications for ac- sults in a strong increase in NFATc1/aA protein, which, together tive transcription (6, 11). The Nfatc1 P1 promoter is composed of with NF-kB factors, contributes to the survival of peripheral lym- numerous binding sites for inducible TFs, including multiple phocytes upon immune receptor and coreceptor stimulation. NFAT sites. Due to the in vitro binding and transactivation of the P1 promoter by NFATc1, we and others postulated an auto- Acknowledgments regulatory mechanism that maintains Nfatc1 transcription constant We are very much indebted to Doris Michel and Ilona Pietrowski for ex- (6, 15, 27). This view is supported by the binding of NFATc1 to P1 cellent technical assistance, to Dr. Heike Wagner (Central Animal Facility, in vivo (Fig. 7B) and the constant P1-directed NFATc1/aARNA Faculty of Medicine, University of Wu¨rzburg) and team for maintaining levels upon immune receptor stimulation for 1, 2, or 3 d in B and mice, and to Elena Wiese and Dr. Kurt Reifenberg (Mainz) for oocyte The Journal of Immunology 2353 injections. We thank Drs. Andreas Beilhack (Wu¨rzburg), Isabelle Bekered- isoforms with individual transcriptional properties are synthesized in jian-Ding (Bonn), Torsten Stiewe (Gießen), and Ju¨rgen Wienands (Go¨ttin- T lymphocytes. J. Immunol. 162: 7294–7301. 19. Rowland, B. D., and R. 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Supplementary Figures

FIGURE S1: Expression of chimeric Nfatc1/Egfp gene and endogenous Nfatc1 gene in T cells from tg mice. (A) Semi-quantitative RT-PCR assays for the detection of

Nfatc1 and Nfatc1/Egfp RNAs in CD4+ T cells. Purified splenic CD4+ T cells from tg mice were either left un-stimulated (lane 1) or stimulated by αCD3+CD28 for 24 h

(lane 2) or 48 h (lane 4) in the absence or presence of CsA (lanes 3 and 5).

Transcripts from the endogenous Nfatc1 gene resulted in the Ex1-3 (P1 promoter directed), Ex2-3 (P2) and Ex3-7 (P1+P2) PCR products, transcripts of chimeric

Nfatc1/Egfp gene in the Ex1-EGFP, Ex2-EGFP and EGFP products, respectively. (B)

PCR assays for the detection of Nfatc1 and Nfatc1/Egfp RNAs in Con A-stimulated

CD4+ T cells. T cells from tg mice were left un-stimulated (lane 1) or stimulated by

Con A for 24 h in the absence (lane 2) or presence of CsA (lane 3), or by T+I (lane

4). Transcripts from the endogenous Nfatc1 gene resulted in the Ex 1-3 (P1 promoter directed) or Ex 2-3 (P2) PCR products, transcripts of chimeric Nfatc1/Egfp gene in the Ex 1-EGFP and Ex 2-EGFP products. (C) Western blots for the detection of

NFATc1/EGFP expression in T cells from tg mice. Whole cell protein from un- stimulated cells (lanes 1+5) or cells which were stimulated for 24 h by Con A without

(lanes 2+6) or with CsA (lanes 3+7) or by T+I (lanes 4+8) were immunoblotted using

NFATc1-, EGFP- or β-actin-specific Abs. (D) Detection of EGFP expression in CD4+ and CD8+ T lymphocytes from peripheral blood of tg Nfatc1/Egfp mice by flow cytometry. Bars indicate mean values of three individual animals. FIGURE S2: Activation of Nfatc1/Egfp gene in CD4+ and CD8+ tg T cells in MLRs. (A)

Conventional CD4+ T cells from a tg C57/Bl6 Nfatc1/Egfp mouse were incubated with

T-cell depleted splenocytes from a BALB/c (allogeneic; red lines) mouse or C57/BL6

(syngeneic; filled curves) mouse for 72 h. CD4+/EGFP+ cells were analyzed for CD69 or CD25 expression by flow cytometry. In the right panels, the EGFP expression in

CD69low and CD25low cells from an allogeneic MLR is shown in grey, the EGFP expression of CD69high and CD25high cells in red. (B) MLR of CD8+ T cells from a tg

C57/BL6 Nfatc1/Egfp mouse were incubated with T-cell depleted splenocytes from a

BALB/c (allogeneic; red lines) mouse or C57/BL6 (syngeneic; filled curves) mouse for

72 h.

FIGURE S3: NF-κB induction in splenic B cells. EMSAs. (A) Effect of αIgM, αCD40,

LPS and CpG on NF-κB activation. Nuclear protein from 107 freshly prepared splenic

B cells (lane 1) or cells stimulated with αIgM (10 μg/ml) for 30 min (lane 2) or 120 min

(lane 3) at 40C, with αIgM for 30 min at 40C and, after washing, with αCD40 (5 μg/ml) for further 24 h (lane 4), with αIgM for 120 min at 40C and with αCD40 (5 μg/ml) for further 22 h at 370C (lane 5), with αIgM for 30 min at 40C and with LPS (10 μg/ml) for further 24 h (lane 6), with αIgM for 120 min at 40C and with LPS (10 μg/ml) for further

22 h (lane 7), with αIgM (lane 8), αCD40 (lane 9), LPS (lane 10) or CpG (1 μM; lane

11) for 24 h at 370C. (B) Supershift EMSAs. Splenic B cells were incubated either with 10 μg/ml αIgM for 24 h (lanes 1-6) or for 30 min with αIgM at 40C, washed, and followed by incubation with 5 μg/ml αCD40 for 24 h (lanes 7-12). As indicated, Abs against NFκB1/p50, RelA/p65, c-Rel, RelB or NFκB2/p52 were added to the assays.

- Shifted complexes are shown whereas the free probes are cut off. FIGURE S4: Detection of plasmablast formation in vitro. (A) Blimp-1 RNA levels in splenic B cells upon cultivation in vitro. Real-time PCR assays of splenic B cell left untreated or cultivated with 10 μg/ml LPS alone or in combination with 10 μg/ml αIgM as indicated. (B) Detection of immunoglobulins in plasmablasts generated by LPS or

CpG treatment for 3-4 d in vitro. Immunoblots. Splenic B cells either untreated or incubated with 10 μg/ml αIgM for 24 h in vitro. As indicated, cells were washed and incubated for further 24 h, 48 h or 72 h with LPS or αCD40. In lanes 9-11, splenic B cells were incubated for 96 h with LPS, αCD40 or CpG alone. For Ig detection, the membrane was incubated with a goat anti-mouse pAb coupled to peroxidase.

A αIgM [h] – 0.5 2 0.52 24 + 24 h

0 0 0 4 4 4 M D S D S D S G g C P C P I C P p α L α L α α L C

• p65, c-Rel, RelB

• p50/p50

1234567891011

B αIgM [h] 24 0.5 + 24 h αCD40

l l e B e B 0 5 l 2 0 5 l 2 R e R e Ab – 5 6 - 5 – 5 6 - 5 p p c R p p p c R p

• p65, c-Rel, RelB • p50/p50

12 3456 789101112

Suppl. Figure 3, Hock et al.