Inhibition of Terminal Differentiation of B Cells Mediated by CD27 and CD40 Involves Signaling through JNK

This information is current as Shuchismita Satpathy, Gautam N. Shenoy, Sheetal Kaw, of September 28, 2021. Tushar Vaidya, Vineeta Bal, Satyajit Rath and Anna George J Immunol published online 25 October 2010 http://www.jimmunol.org/content/early/2010/10/25/jimmun ol.0903229 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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 25, 2010, doi:10.4049/jimmunol.0903229 The Journal of Immunology

Inhibition of Terminal Differentiation of B Cells Mediated by CD27 and CD40 Involves Signaling through JNK

Shuchismita Satpathy,*,1,2 Gautam N. Shenoy,*,1 Sheetal Kaw,*,1 Tushar Vaidya,† Vineeta Bal,*,3 Satyajit Rath,*,3 and Anna George*,3

B cells responding to cognate Ag in vivo undergo clonal expansion that is followed by differentiation into Ab-secreting plasma cells or into quiescent restimulable memory. Both these events occur in the germinal center and require that cells exit from proliferation, but the signals that lead to one or the other of these mutually exclusive differentiation pathways have not been definitively char- acterized. Previous experiments have shown that signals transduced through the TNFRs CD27 and CD40 at the time of stimulation in vitro or in vivo can influence this cell fate decision by inhibiting terminal differentiation and promoting memory. In this study, we show that the PIQED domain of the cytoplasmic tail of murine CD27 and the adapter molecule TNFR-associated

factor 2 are involved in this effect. Using pharmacological inhibitors of signaling intermediates, we identify JNK as being necessary Downloaded from and sufficient for the observed inhibition of terminal differentiation. While JNK is involved downstream of CD40, inhibition of the MEK pathway can also partially restore generation, indicating that both signaling intermediates may be involved. We also show that inhibition of induction of IFN regulatory factor 4 and B induced maturation 1 are downstream events common to both receptors. The Journal of Immunology, 2010, 185: 000–000.

-dependent B cell responses in peripheral lymphoid organs successfully for help and for Ag presented on follicular http://www.jimmunol.org/ are initiated at the border of T and B cell zones, leading to dendritic cells (6, 7). Whereas the GC environment is conducive to T the establishment of primary foci where clonal expansion rapid and sustained B cell proliferation, some cells manage to occurs and isotype switching may be initiated (1). Although many exit cell cycle and to differentiate either into relatively long-lived proliferating cells die, some undergo terminal differentiation to plasma cells or into memory cells, and the signaling events that form relatively short-lived plasma cells (2) and a few others lead to this exit remain unclear (8–10). migrate into the B cell follicles and establish germinal centers Plasma cells downregulate most surface molecules that are (GCs). Within the GC, B cells proliferate extensively, upregulate synonymous with B cell identity but upregulate CD138, CXCR4,

activation-induced deaminase (AID), and undergo isotype switch- and CD44 [molecules that facilitate migration to and retention in by guest on September 28, 2021 ing and somatic hypermutation (3–5). Although most mutations the bone marrow (11–13)] and the protein synthesis machinery lead to BCRs with unchanged or lower affinity, a few high-affinity required for high-level Ab secretion (14). Memory cells, however, variants are also generated, and they get selected as they compete retain high-level expression of B220, BCR, CD19, CD86, and MHC class II and can recirculate to peripheral lymphoid organs. A large number of transcription factors, including B cell leukemia/ † *National Institute of Immunology, New Delhi 110067; and Center for Cellular and lymphoma-6 (BCL-6), B lymphocyte induced maturation protein Molecular Biology, Hyderabad 500007, India 1 (BLIMP-1), paired box gene 5 (PAX5), X-box binding protein 1 1S.S., G.N.S., and S.K. contributed equally to this work. (XBP-1), microphthalmia-associated transcription factor (Mitf), 2Current address: Department of Microbiology and Immunology, University of Louisville Health Sciences Center, Louisville, KY. and IFN regulatory factor 4 (IRF4) have been implicated in commitment to the plasma cell lineage (15). However, the precise 3V.B., S.R., and A.G. contributed equally to this work. signaling pathways and transcriptional events that determine cell Received for publication October 2, 2009. Accepted for publication September 21, 2010. fate decision, especially for memory generation in the GC, remain This work was supported in part by grants from the Department of Science and uncharacterized (16). Technology and the Department of Biotechnology, Government of India (to A.G., We have shown previously that immunization of mice under S.R., V.B., and T.V.). The National Institute of Immunology is supported by the cover of anti-CD27 Ab skewed the commitment of activated cells to Department of Biotechnology, Government of India. The Center for Cellular and Molecular Biology is supported by the Council for Scientific and Industrial Research, the memory pool and inhibited plasma cell generation without Government of India. affecting B cell activation, proliferation, or survival (17, 18). CD40 Address correspondence and reprint requests to Anna George, National Institute of ligation also enhanced commitment of responding B cells to the Immunology, Aruna Asaf Ali Road, New Delhi 110067, India. E-mail address: anna@ nii.res.in memory pool; however, it affected multiple events including pro- liferation, survival, and isotype switching after B cell activation. The online version of this article contains supplemental material. In this study, we have attempted to characterize the signaling Abbreviations used in this paper: D, deletion; AID, activation-induced deaminase; Bach2, BTB and CNC homology1, basic leucine zipper transcription factor 2; BCL- events downstream of CD27 in B cells and to determine whether 6, B cell leukemia/lymphoma-6; BLIMP-1, B lymphocyte induced maturation protein inhibition of terminal differentiation by CD27 and CD40 involves 1; EGFP, enhanced GFP; FL, full-length; GC, germinal center; IRF4, IFN regulatory factor 4; MFI, mean fluorescence intensity; Mitf, microphthalmia-associated tran- similar pathways. We report that of the two putative TNFR- scription factor; PAX5, paired box gene 5; PI, propidium iodide; pJNK, phosphory- associated factor (TRAF) binding domains present in the cyto- lated JNK; TRAF, TNFR-associated factor; WCL, whole-cell lysates; XBP-1, X-box plasmic tail of CD27 (19, 20), the region PIQED and specifically binding protein 1. residues P and D are required for inhibition of plasma cell gen- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 eration after CD27 ligation. We also report that JNK is an essential

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903229 2 SIGNALING THROUGH JNK INHIBITS PLASMA CELL GENERATION signaling intermediate downstream of CD27 and that the tran- Cloning of murine CD27 and its C-terminal deletion mutants scription factors IRF4 and BLIMP-1, but not BTB and CNC ho- Splenocytes were stimulated with 4 mg/ml Con A (Sigma) for 48 h, CD27 mology 1, basic leucine zipper transcription factor2 (Bach2), are induction confirmed by flow cytometry, RNA extracted (TRIzol; Sigma), modulated by CD27 ligation. Yet inhibition of plasma cell gen- and 2 mg total RNA used to synthesize cDNA with oligo-deoxythymine eration by CD40 ligation involves JNK as well as MEK; however, using First Strand cDNA synthesis (Invitrogen, Carlsbad, CA) ac- cording to the manufacturer’s recommendations. The 752-bp CD27 was they appear to lead to modulation of the same transcription factors then PCR-amplified from total cDNA using gene-specific primers, with as are modulated after CD27 ligation. Our data indicate that both EcoR1 and BamH1 sites incorporated into the forward and reverse primers, TNFRs influence B cell differentiation via similar signaling respectively. The amplicon was resolved on an agarose gel, eluted, and pathways and that CD27 may prove valuable in the further un- purified using QIAquick Gel Extraction kit (Qiagen, Valencia, CA), and blunt-end ligated into an SrfI-digested pPCR-Script vector (PCR-Script derstanding of lineage commitment after B cell activation. Amp cloning kit; Stratagene) by overnight incubation. Competent cells (DH5a, Escherichia coli) were transformed with the ligation mixture and plated on Luria–Bertani agar (Difco Laboratories, Detroit, MI; BD Bio- Materials and Methods sciences) plates containing 100 mg/ml ampicillin (Sigma). Single colonies Mice were screened by plasmid DNA extraction (Qiagen columns), insert checked by digestion with EcoR1 and BamH1, and confirmed by se- C57BL/6 and BALB/c mice used in the study were obtained from The quencing (University of Delhi, South campus facility, New Delhi, India). Jackson Laboratory (Bar Harbor, ME) and were bred and maintained in the Myc-tagged FL CD27 and its mutants were PCR-amplified from pPCR- Small Animal Facility of the National Institute of Immunology (New Delhi, Script Amp containing CD27 DNA using appropriate forward and reverse India). Mice were used for experiments at 8–12 wk of age. Approval from primers containing Xho1 and BamH1 sites, respectively. The amplicons

the institutional animal ethics committee was obtained for all experimental were cloned into pGEM-T Easy, the clones obtained were sequenced Downloaded from procedures involving animals. (LabIndia Systems, New Delhi, India), and the constructs subcloned into the Xho1-BamH1 sites of the LNCX2-IRES-EGFP vector. Reagents The following reagents were used: LPS, Triton X-100, sodium orthova- B cells nadate, PMSF, KCl, EDTA (Sigma, St. Louis, MO), Tris base (Amresco, Single-cell suspensions of cells from spleen were obtained by mechanical Solon, OH), Blotto (Santa Cruz Biotechnology, Santa Cruz, CA), leupeptin disruption, and erythrocytes in the splenocyte population were lysed by (Boehringer Ingelheim India, Mumbai, India), aprotinin (Merck India, treatment of the cell pellet with Gey’s solution. B cells were enriched by http://www.jimmunol.org/ Mumbai, India), azide-free anti-myc (clone 9E10; Roche Applied Scien- treatment of the cell suspension with biotinylated anti-IgD (Southern ces, Mannheim, Germany), anti-CD40 (clone 1C10; eBioscience, San Biotechnology, Birmingham, AL) followed by streptavidin microbeads Diego, CA), anti-CD8 (clone 53-6.7; BD Biosciences, San Jose, CA), (Miltenyi Biotec, Auburn, CA) or with anti-B220 magnetic beads (Mil- PE-streptavidin, fluorescein-streptavidin, FITC/PE donkey anti-rabbit IgG tenyi Biotec) and separated on MACS columns (Miltenyi Biotec) (Jackson ImmunoResearch Laboratories, Westgrove, PA), propidium io- according to the manufacturer’s instructions. Purified cells were routinely dide (PI; Sigma), CFSE (Molecular Probes, Eugene, OR), fluorescein/ $90% pure by flow cytometric analysis. biotin/PE-Cy5 anti-B220, PE anti-CD138, CD44, CD27, CD138, BLIMP-1, Cells were stimulated in 96-well flat-bottom plates or in 24- or 6-well Bach2, IRF4 (BD Biosciences or eBioscience), and anti-phospho SAPK/ plates (BD Falcon, Franklin Lakes, NJ) with 10 mg/ml LPS in Click’s JNK (Thr183/Tyr185; Cell Signaling Technology, Danvers, MA). ECL re- medium (Irvine Scientific, Santa Ana, CA) or RPMI 1640 (Biological agent and x-ray films were from Amersham Pharmacia Biotech, Little

Industries, San Diego, CA) supplemented with 10% FBS, 2 mM L-gluta- by guest on September 28, 2021 Chalfont, U.K. mine, 0.1 mM 2-ME, and penicillin (100 U/ml) and streptomycin (100 Inhibitors used in culture were obtained from Calbiochem or Sigma and mg/ml) (Life Technologies, Carlsbad, CA) in the presence or absence of m included JNK-1, JNK-II, JNK-3 (30–3 M), rottlerin, AG1478, bisindo- the indicated amounts of anti-CD40. Wherever necessary, viable cells were m lylmaleimide I (10–0.1 M), LY294002, AG490, SB203580, genistein isolated on a Ficoll-Hypaque gradient (Cedarlane, Hornby, Ontario, Can- m m (30–0.3 M), N-acetylcysteine (30–0.3 mM), olomoucine (100–0.1 M), ada) before further analysis. To assess proliferation, cells were labeled m and PD98059 and caffeine (10–1 M). with 10 mM CFSE (Molecular Probes) before stimulation, and 72–96 h later, CFSE dilution was estimated after excluding dead cells with 1 mg/ml Primers PI. For B cell transfection, cells were activated with 10 mg/ml LPS for 48 h, For cloning full-length (FL) CD27: forward 59-CGGAAATTCATGGCA- washed, and seeded at a density of 5 3 105/ml/well of OptiMEM in 6-well TGGCCACCTCCCTA-39, reverse 59-CGGGATCCAGGGTAGAAAGCA- GGCTCGG-39. For generating myc-tagged FL CD27: forward 59-GG- plates and transfected with plasmid DNA using TransIT-LT or TransIT- GGGGCTCGAGCGGATGGAGCAAAAACTCATCTCTGAAGAAGATC- Express (Mirus Bio, Madison, WI) transfection reagent, according to the 9 9 manufacturer’s recommendations. OptiMEM was replaced with complete TGATGGCATGGCCACCTCCCTA-3 , reverse 5 -GGGGGGGATCCTCA- m AGGGTAGAAAGCAGGCTCGG-39; CD27 deletion (D)10: reverse 59- medium containing LPS 4 h later, and 16 h after transfection, 10 g/ml anti-myc Ab was added to ligate CD27 on transfected cells. Cells were GGGGGGGATCCTCAGTCCTCCTGGATAGGGATAG-39;CD27D16: re- analyzed by flow cytometry after a further 48 h. verse 59-GGGGGGGATCCTCAAGCACTGCCCTCCTCTTCC-39;CD27D23: For retroviral transduction of primary B cells, the retroviral packaging reverse 59-GGGGGGGATCCTCAGGGGCAGCTGTAAGGACAAG-39 (all cell lines Plat-E (Cell Biolabs, Cambridge, U.K.) or RetroPack PT67 from Bio Basic). Point mutants in the PIQED domain of the CD27 tail were m generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, (Clontech) were transfected with 4 g plasmid DNA using FuGENE 6 Cedar Creek, TX) as per the manufacturer’s instructions. Primers used for mu- (Roche) at a DNA/reagent ratio of 3:2 in OptiMEM in 6-well plates, and tagenesis were as follows: forward 59-GAGGGCAGTGCTATCGCTATCCAG- medium replaced 12 h later as described earlier. The efficiency of trans- fection was checked 24 h later and was routinely .60%. Culture medium GAGGACTAC-39,reverse59-GTAGTCCTCCTGGATAGCGATAGCACTGC- was replaced at this time, the plates transferred to 32 C for virus pro- CCTC-39 for proline to alanine; forward 59-GCAGTGCTATCCCTATCGCG- GAGGACTACCGGAAACC-39,reverse59-GGTTTCCGGTAGTCCTCCGC- duction, and the supernatant harvested 24 h later and filtered to remove cell 9 9 debris. LPS-activated B cells were spin-infected with virus supernatant in GATAGGGATAGCACTGC-3 for glutamine to alanine; forward 5 -GCTATC- m CCTATCCAGGAGGCCTACCGGAAACCCGAGCC-39,reverse59-GGCTC- the presence of 8 g/ml Sequabrene (Sigma) and anti-myc added to ligate GGGTTTCCGGTAGGCCTCCTGGATAGGGATAGC-39 for aspartic acid to CD27 on transfected cells. alanine (Bioserve Biotech, Beltsville, MD). Mutations were confirmed by se- quencing with forward 59-AGCTCGGACTGCATCCGGATC-39. Immunoprecipitation Vectors The B cell line A20 was spin-infected with virus expressing FL CD27 or various deletion mutants in the presence of 12 mg/ml Sequabrene, and G418 pPCR-Script Amp (Stratagene) and pGEM-T Easy (Promega, Madison, WI) (1 mg/ml; Sigma) was added 24 h later. Two weeks into selection, more were used for cloning, and pLNCX2-IRES-enhanced GFP (EGFP) was than 95% of cells expressed GFP as determined by flow cytometry. The used for eukaryotic expression. The latter was generated by insertion of the stable transductants were either left unligated or ligated with 10 mg/ml IRES-EGFP cassette with flanking Xho1 and Not1 sites from pIRES-EGFP anti-myc for 15 or 30 min. Cells were washed twice with cold PBS after (Clontech, Mountainview, CA) into pLNCX2 (Clontech). which they were lysed in TKM buffer (1% Triton X-100, 50 mM Tris [pH The Journal of Immunology 3

7.4], 25 mM KCl, 1 mM EDTA) supplemented with protease inhibitors vector (19) (Supplemental Fig. 2). The D-10 mutant contains both (10 mg/ml leupeptin, 10 mg/ml aprotinin, 1 mM sodium orthovanadate, 1 domains, D-16 lacks the PIQED domain, and D-23 lacks both mM PMSF) for 30 min on ice. Lysates were immunoprecipitated with anti- PIQED and EEEG domains. The myc tag enabled ligation of the myc Ab on Protein G beads (Amersham Pharmacia) and resolved by SDS-PAGE and transferred to nitrocellulose membrane (mdi; Membrane mutant CD27 molecule with an anti-myc Ab, leaving endogenous Technologies, Ambala Cantonment, India). Membranes were blocked with CD27 molecules untouched. All transfected cells expressed GFP 5% Blotto and probed with Abs specific for TRAFs 2, 3, and 5 (Santa Cruz and myc (Fig. 2A and microscopy data not shown). The constructs Biotechnology). Blots were stripped (2% SDS, 12.5 mM Tris [pH 6.0], were then transfected into primary B cells stimulated for 48 h with 385 mM 2-ME) and probed with anti-myc Ab (BD Biosciences) and bands visualized by ECL. HRP-linked secondary Abs (anti-rabbit IgG and anti- LPS, and 12 h later, anti-myc Ab was added to ligate the trans- mouse IgG) were purchased from Santa Cruz Biotechnology and Cell fected CD27 molecules. Viable cells were isolated on Ficoll- Signaling Technology, respectively. Hypaque (Cedarlane) gradients 48 h later and stained for B220 and CD138. Plasma cell frequencies were then assessed on gated Flow cytometry GFP+ cells. As shown in Fig. 2B, and as expected from Fig. 1B, Cells were incubated with appropriate staining reagents in buffer containing ligation of transfected FL CD27 led to near-complete abrogation 0.1% sodium azide (Sigma) and 1% FBS for 45 min on ice. Samples were of plasma cell generation. No inhibition was seen after ligation of run on a BD-LSR or FACS Aria (BD Biosciences) flow cytometer. For the D-23 mutant, which lacks both domains, or with the D-16 determination of cell cycle progression, cells in cold PBS were permeabilized with ice-cold 70% ethanol and treated with 5 mg/ml PI in PBS. For in- molecule, which lacks only the PIQED domain, indicating that tracellular staining, cells were stained for surface markers and per- this region and not the EEEG domain is involved in signaling. Par- meabilized with 100 ml Cytoperm/Cytofix buffer (BD Biosciences) on ice tial inhibition was seen after ligation of the D-10 molecule, which for 15 min. Abs directed against intracellular markers were then added, contains both domains. These data indicate that the PIQED do- Downloaded from diluted in Permwash buffer (BD Biosciences). Data were analyzed with FlowJo software (Tree Star, San Carlos, CA). main in the cytoplasmic tail of CD27 is likely involved in the recruitment of factors responsible for signaling events that lead to Statistical analysis inhibition of terminal differentiation. To confirm this, we gener- Data were analyzed by Student t test. ated point mutants in the PIQED domain by mutating residues P, Q, or D to A. The resultant constructs were cloned into a retro-

viral vector as before and transduced into LPS-stimulated B cells. http://www.jimmunol.org/ Results Ligation of the Q/A mutant led to abrogation of plasma cell gen- CD27 signaling in primary B cells inhibits plasma cell eration, as with the FL molecule (Fig. 3). However, ligation of ei- generation ther the P/A or the D/A mutant had no effect, indicating that these We have previously shown that ligation of CD27 on activated B two residues are critical for recruitment of adapters to the cytosolic cells with an Ab inhibits plasma cell generation in vitro and in tail of CD27. vivo (17, 18). To elucidate the mechanisms underlying this in- hibition, we cloned myc-tagged FL CD27 into a bicistronic EGFP- TRAF2 is recruited to the CD27 tail expressing retroviral vector for transfection or retroviral trans-

To identify specific TRAFs recruited to the PIQED domain of the by guest on September 28, 2021 duction of primary B cells (see Materials and Methods). The GFP CD27 tail, we transduced the B cell line A20 with the FL CD27 tag was included to enable detection of transfected cells by flow construct and the D-10, the Q/A, and the D/A mutants, and stable cytometry, and the myc tag was included to enable specific liga- lines expressing GFP were generated by selection in G418. Cells tion of the transfected molecule with an anti-myc Ab. Expression were stimulated and immunoprecipitated with anti-myc Ab and of myc and CD27 on GFP+ transfectants was confirmed, and re- blots probed with Abs specific for various TRAFs. We found (Fig. lative levels of CD27 on the transfected cells relative to endoge- 4A) that TRAF2 was recruited to the FL molecule as well as to the nous levels were found to be high (Fig. 1A,1B and microscopy Q/A mutant. Recruitment to the D-10 mutant was partial, and there data not shown). The construct was then transfected into primary was no recruitment to the D/A mutant. B cells stimulated for 48 h with LPS, and 12 h later, anti-myc Ab was added to ligate transfected CD27 molecules. As shown in Fig. 1C, ligation of transfected FL CD27 led to near-complete abro- CD27 signals through JNK in primary B cells gation of plasma cell generation, as read out by surface expression To identify the signaling molecules involved downstream of CD27 of CD138 and of intracellular BLIMP-1. When FL CD27 was ligation, we transfected FL CD27 into primary B cells and ligated transfected into cells and left unligated, no effect on plasma cell myc in the presence or absence of a range of pharmacological generation was seen, indicating that overexpression of CD27 had inhibitors. We reasoned that pharmacological inhibition of a relevant no deleterious effects (Supplemental Fig. 1). The transfection data signaling pathway downstream of CD27 would reverse the effect of reproduce what we have reported earlier with anti-CD27 Ab and CD27 ligation and lead to equivalent proportions of CD138+ plasma are therefore consistent with the physiological possibility in- cells in the GFP2 and GFP+ populations. After testing a wide range dicated in our earlier data of a role for CD27 in skewing cell fate of inhibitor concentrations and identifying a range that had no ef- determination away from terminal differentiation and toward the fect on LPS-mediated cell proliferation or differentiation (data memory lineage (17, 18). not shown), we found that addition of JNK-II inhibitor led to a dose-dependent reversal of the effect of CD27 ligation (Fig. 4B). The PIQED region of the CD27 tail is involved in inhibition of Other inhibitors tested (rottlerin, AG1478, bisindolylmaleimide-I, terminal differentiation LY294002, AG490, SB203580, genistein, N-acetyl cysteine, olo- Signal transduction downstream of CD27 ligation requires the re- moucine, and PD98059) had no effect and failed to restore LPS- cruitment of TRAF molecules. The cytoplasmic domain of CD27 induced plasma cell generation at concentrations that did not oth- contains two putative TRAF-binding domains: a PIQED domain, erwise affect B cell activation, proliferation, or differentiation which could recruit the activating TRAFs 2, 3, and 5, and an EEEG (Supplemental Fig. 3). Ligation of FL CD27 on retrovirally trans- domain, which could recruit TRAFs 1 and 2 (19, 20). To elucidate duced B cells also led to inhibition of induction of IRF4, and the which of these regions was involved in the signaling in B cells, induction was restored when ligation was done in the presence of we next cloned three deletion mutants into the EGFP-expressing JNK inhibitor (Fig. 4C). 4 SIGNALING THROUGH JNK INHIBITS PLASMA CELL GENERATION

Inhibition of plasma cell generation by CD40 ligation involves JNK, MEK, and IRF4 but not Bach2 CD40 is the other TNFR expressed on B cells that is known to inhibit terminal differentiation and to enhance memory generation (17, 18, 21). We tested the effect of CD40 ligation on primary B cells by addition of stimulatory anti-CD40 Ab to LPS-stim- ulated cultures. As seen in Fig. 5, addition of anti-CD40 leads to inhibition of terminal differentiation in a dose-dependent fashion. At the higher concentrations, however, the Ab also in- duced prolific expansion of B cells (18) (data not shown), and so we used 4 mg/ml for all experiments. We then tested pharmaco- logical inhibitors of signaling intermediates for their ability to reverse the CD40-mediated inhibition, and we found that three different inhibitors of JNK were able to partially restore plasma cell generation (Fig. 6A). As expected, and as seen with CD27 ligation, abrogation of plasma cell generation was accompanied by reduction in levels of intracellular BLIMP-1 (Fig. 7). Pro- liferation, as assessed by CFSE dilution, was similar in the pres- ence or absence of inhibitor (Supplemental Fig. 4), and similar Downloaded from proportions of live cells were seen in the two groups (45.8 versus 45.2%), indicating that the concentrations of inhibitor used did not inhibit proliferation or induce . Various other inhibitors were tested, and apart from the MEK inhibitor PD98059, they had minimal effects on rescue of terminal differentiation in the pres- ence of anti-CD40 (Supplemental Fig. 5A). To show more directly http://www.jimmunol.org/ that JNK is involved downstream of the two receptors, we looked for phosphorylation of the enzyme after ligation of CD27 or CD40. As shown in Fig. 6B, JNK is phosphorylated after treat- ment of primary B cells with anti-CD40 as well as after treatment of A20 cells stably expressing FL CD27 with anti-myc, and this phosphorylation is inhibited in the presence JNK-1. We also confirmed that the inhibitor did not inhibit the phosphorylation of other MAPKs (Supplemental Fig. 4B,4C). by guest on September 28, 2021 As seen with CD27 (Fig. 4C), ligation of CD40 also led to lower levels of IRF4 (Fig. 7A,7B), and addition of JNK-II inhibitor to such cultures partially rescued plasma cell generation as read out by CD138, BLIMP-1, and IRF4 induction. Together, the data in- dicate that CD27 and CD40 can both signal through JNK to suppress induction of IRF4 and BLIMP-1 and thereby to suppress plasma cell generation. We also looked at intracellular levels of Bach2, a molecule that has been reported to be involved in plas- ma cell generation (15, 22). We found that Bach2 was downreg- ulated equivalently by 24 h after LPS stimulation in the presence or absence of added anti-CD40 Ab and stayed downregulated at 48, 72, and 96 h. (Fig. 7C and data not shown). Hence, it is un- likely to be involved in negatively influencing terminal differen- tiation after CD40 ligation. It is also unlikely to be involved downstream of CD27 as the ligation of myc in the transfection and transduction experiments occurs 60 h after LPS stimulation. To- gether, the data indicate that CD27 and CD40 can both signal through JNK to suppress induction of IRF4 and BLIMP-1 and thereby suppress plasma cell generation. Discussion Plasma cell differentiation is initiated after activation of B cells by FIGURE 1. Expression of cloned myc-tagged, EGFP-expressing FL CD27. A, Coexpression of CD27 and myc with GFP on PT67 cells or LPS- specific Ags or by LPS. The precise mechanisms leading to plasma activated B cells respectively transfected with myc-CD27-EGFP vector (CD27) or empty vector (vector). Representative of two independent experiments. B, CD27 levels on ex vivo B cells (shaded histogram) and on after transduction (hatched line, from the GFP+ gate in C). The dotted line B cells stimulated with 10 mg/ml LPS for 48 h. C, Gating for GFP+ and represents endogenous levels of CD27 at 96 h (from the GFP+ gate in C). GFP2 cells 48 h after retroviral transduction of FL CD27. D, Relative E, Abrogation of plasma cell generation by myc ligation. Primary B cells expression of endogenous CD27 on ex vivo B cells (shaded histogram) and were transfected with reagent alone, empty vector, or FL CD27 (as la- on B cells 48 h after stimulation with LPS (unbroken line) both gated as in beled), treated with anti-myc Ab and stained for surface CD138 and in- B, and of retrovirally transduced CD27 96 h into LPS stimulation, but 48 h tracellular BLIMP-1. One experiment representative of three is shown. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 3. The residues P and D in the CD27 tail are required for its ability to abrogate plasma cell generation. Proportion of B220low by guest on September 28, 2021 CD138high cells in LPS-activated B cell cultures transduced with virus expressing CD27 carrying point mutations (P/A, Q/A, or D/A) in the PIQED domain, or with the D-10 mutant, followed by myc ligation. Analysis is on live cells gated for GFP. Total CD138+ cells are 49.3 (control), 47.7 (P/A), 24 (Q/A), 48.2 (D/A), and 37.3% (D-10).

cell commitment remain unclear, but some transcription factors, especially BLIMP-1, IRF4, and XBP-1, appear to be critical for plasma cell formation and maintenance (23–26). BLIMP-1 has been considered a “master switch” for plasma cell generation (23, 27); however, recent data indicate that it is necessary but not sufficient for the induction (25, 28). IRF4 regulates isotype switching and terminal differentiation by controlling the expres- sion of AID and BLIMP-1, respectively. Thus, restoration of AID in IRF42/2 B cells rescues isotype switching, and restoration of Blimp-1 rescues plasma cell generation (28). XBP-1 expands ele- ments of the secretory pathway and enhances the unfolded pro- tein response (29, 30). Vav may be upstream of some of these factors, as mature marginal zone B cells from Vav-null mice show induction of IRF4 but no induction of BLIMP-1 or XBP-1 and fail to undergo terminal differentiation (31). On the other hand, the transcription factors BCL-6 and PAX5, which can repress XBP-1 FIGURE 2. Inability of CD27 lacking the C-terminal PIQED motif to and BLIMP-1, respectively, are downregulated during plasma cell abrogate plasma cell generation. A, Coexpression of GFP with CD27 and differentiation (32–34). BCL-6 is required for GC development myc after transfection of PT67 cells or LPS-activated B cells, respectively, and may be one of the primary molecules that influence differ- with the cloned deletion mutants. Representative of two independent entiation (35–37). There is a considerable amount of cross-talk experiments. B, CD138 and BLIMP-1 in gated GFP+ cells after trans- between these factors. Thus, upregulation of BLIMP-1 represses fection of B cells with empty vector or with the various CD27 constructs PAX5, a gene product that maintains B cell identity (38, 39) and (labeled) followed by myc ligation. No scatter gate has been used for the also represses BCL-6 (26). analysis. Representative of three independent experiments. Most of these studies have relied on the use of cell lines or on B cells from mice deficient for various factors, and they have 6 SIGNALING THROUGH JNK INHIBITS PLASMA CELL GENERATION Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Inhibition of plasma cell generation by CD27 is mediated through recruitment of TRAF2, activation of JNK, and inhibition of IRF4. A, Immunoprecipitation of A20 cells expressing either FL CD27, the D-10 mutant, or point mutations in the PIQED domain after ligation of the overexpressed molecule. Immunoprecipitation with anti-myc Ab was fol- lowed by a Western blot for TRAF2. Blots were stripped and reprobed for FIGURE 5. Ligation of CD40 inhibits plasma cell generation. CD138 myc. Whole-cell lysates (WCL) served as controls. B, CD138 expression on staining on B cells stimulated with LPS in the presence or absence of ti- gated GFP+ cells after ligation of transfected CD27 on LPS-stimulated B trating doses of anti-CD40 Ab or isotype-matched anti-CD8 Ab. Repre- cells in the presence or absence of JNK-II inhibitor. C, Induction of IRF4 sentative of two independent experiments. after ligation of transduced CD27 in the presence or absence of JNK in- hibitor. B cells transduced with FL CD27 were treated with 10 mg/ml anti- myc in the presence or absence of 3 mMor10mM JNK-II inhibitor, and 48 h stimulable memory cell. Previous studies have indicated a role for later, viable cells isolated on a Ficoll-Hypaque gradient were stained for Ag affinity in controlling this process (40, 41) and also a role for surface B220, fixed, permeabilized, and stained for intracellular IRF4. Left the TNFRs CD27 and CD40 in this cell fate determination (17, 18, panel, Shaded histogram, vector control; thin line, cells transduced with FL 21). The experiments reported here followed from these earlier CD27 and treated with anti-myc; thick line, cells transduced with FL CD27 data, which showed that ligation of CD27 or CD40 with an Ab m and treated with anti-myc in the presence of 3 M JNK-II inhibitor; dotted could specifically inhibit terminal differentiation in vitro (17). line, cells transduced with FL CD27 and treated with anti-myc in the Physiological relevance for the finding came from the observation presence of 10 mM JNK-II inhibitor. Right panel, Fold change in mean fluorescence intensity (MFI) of IRF4 in GFP+ cells from three independent that a single dose of anti-CD27, injected at the time of immuni- experiments when transduced CD27 was left unligated, ligated, or was li- zation, led to higher memory cell generation in mice by three gated in the presence of 10 mM JNK inhibitor. Rescue with 3 mM inhibitor independent readouts: limiting dilution assays, adoptive transfer did not achieve statistical significance over multiple experiments. experiments for secondary responses, and the tracking of Ag- specific B cells in vivo over time. As expected, treatment of mice with anti-CD40 Ab also enhanced memory cell frequencies. Sig- provided a wealth of information on the complex regulatory in- nificantly, we also showed that secondary responses were higher to teractions between the transcription factors involved. However, the T-independent Ag 4-hydroxy-3-nitrophenylacetyl–Ficoll when little is known about the cell surface molecules and the signaling immunization was done under cover of anti-CD27 treatment (18). intermediates that are involved in initiating this cascade of tran- Thus, CD27, at levels expressed on unmanipulated mouse B cells scriptional events in normal B cells. Further, little is known about can trigger signal modulations leading to altered cell-fate com- signaling events that determine whether a cell exiting from cycle mitment. CD40-mediated signals achieve a plethora of effects in in the GC will undergo terminal differentiation or become a re- B cells, whereas CD27-mediated signals appear to be selectively The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 7. IRF4 and BLIMP-1, but not Bach2, are modulated after CD40 ligation. A, Induction of CD138 on, and of BLIMP-1 and IRF4 in, cells treated with LPS alone or with LPS plus anti-CD40 in the presence or absence of JNK-II inhibitor. B, Bar diagrammatic representation of similar

data from five experiments. C, Bach2 expression in untreated B cells by guest on September 28, 2021 (dotted line) and in B cells treated with LPS in the absence (solid line) or presence (dashed line) of anti-CD40. Isotype control: shaded histogram. Representative of three experiments. pp = 0.0003; ppp = 0.0006; pppp = 0.00001. NS, not significant.

In pursuit of that aim, we have used a transfection-driven system to express CD27 mutants to understand the signaling intermediates involved in the plasma cell-memory B cell decision alteration effect of CD27 ligation. Identification of the tail regions of CD27 that are likely to be involved in these effects, most likely TRAF re- cruitment, required transfection/transduction of CD27 tail mutants into primary B cells and the ability to ligate selectively the mutant molecules while leaving the endogenous molecule untouched. FIGURE 6. JNK is involved in abrogation of plasma cell generation by Both molecules signal through the recruitment of TRAFs to their CD40 ligation. A, CD138 expression on cells stimulated with LPS with or without 4 mg/ml anti-CD40 in the absence or presence of titrating doses of cytoplasmic tail. In B lineage cells, CD40 has been shown to recruit JNK-1, JNK-II, or JNK-3 inhibitors (as labeled). Cells stimulated with LPS TRAFs 1, 2, 3, 4, 5, and 6 (42), but little is known about TRAFs alone are included as a control. Representative of three independent recruited by CD27 in primary B cells. Two putative TRAF binding experiments. B, JNK is phosphorylated after ligation of CD40 and CD27. domains, PIQED and EEEG, have been identified in the cyto- Primary B cells or A20 cells expressing FL CD27 were treated with 10 mM plasmic tail of CD27 (19, 20), and they have been shown, in JNK-1 inhibitor (for 2 h or 1 h, respectively), stimulated with anti-CD40 other cell lineages, to recruit TRAFs 2, 3, and 5. In this study, we for 15 min or with anti-myc for 30 min, respectively, in the continued pres- cloned and expressed FL CD27 and three deletion mutants that ence of inhibitor, washed, fixed with 2% p-formaldehyde, and stained either retained both PIQED and EEEG domains, lacked both for intracellular phosphorylated JNK (pJNK). Histogram overlays for pJNK domains, or lacked only the PIQED domain, and used them to expression after CD40 (left panel) or CD27 (right panel) ligation: shaded transfect or virally transduce primary B cells in culture. We found histogram, conjugate control; thin line, unstimulated cells; thick line, stim- D ulated cells; dashed line, cells stimulated in the presence of JNK inhibitor. that ligation of the overexpressed FL molecule or the -16 mol- ecule, which lacks the PIQED domain, or the D-23 molecule, restricted to the alteration of the plasma cell-memory B cell de- which lacks both domains, abrogates plasma cell generation, as cision. Thus, characterization of CD27-mediated effects will pro- read out by CD138 and BLIMP-1 staining. However, ligation of vide a tool for understanding the signals mediated through TNFR the D-10 molecule, which contains the PIQED domain, continues family members, such as CD40, for the induction of B cell memory. to inhibit plasma cell generation, albeit not completely (Fig. 2). 8 SIGNALING THROUGH JNK INHIBITS PLASMA CELL GENERATION

Immunoprecipitation experiments identified TRAF2 as being re- because JNK signaling is uniquely involved in this effect, our data cruited to the CD27 tail after receptor ligation (Fig. 4). Some level indicate that intervention strategies involving this molecule may of constitutive association of TRAF2 with CD27 was seen, and be possible for the rational design of vaccines. this is in keeping with earlier reports indicating that TRAF2 is constitutively associated with many TNFRs including CD40, Disclosures CD30, and CD27 (19, 43, 44). The authors have no financial conflicts of interest. A previous study in which CD27 was overexpressed in 293 cells identified a 13 aa region in the cytoplasmic tail containing both References EEEG and PIQED domains as necessary for recruitment of TRAF2 k 1. Jacob, J., R. Kassir, and G. Kelsoe. 1991. In situ studies of the primary immune and activation of NF- B after CD27 ligation. However, a construct response to (4-hydroxy-3-nitrophenyl)acetyl. I. 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