Prohibitins and the Cytoplasmic Domain of CD86 Cooperate To Mediate CD86 Signaling in B Lymphocytes

This information is current as Christopher R. Lucas, Hector M. Cordero-Nieves, Robert S. of September 29, 2021. Erbe, Jaclyn W. McAlees, Sumeena Bhatia, Richard J. Hodes, Kerry S. Campbell and Virginia M. Sanders J Immunol 2013; 190:723-736; Prepublished online 12 December 2012;

doi: 10.4049/jimmunol.1201646 Downloaded from http://www.jimmunol.org/content/190/2/723

Supplementary http://www.jimmunol.org/content/suppl/2012/12/12/jimmunol.120164 Material 6.DC1 http://www.jimmunol.org/ References This article cites 67 articles, 35 of which you can access for free at: http://www.jimmunol.org/content/190/2/723.full#ref-list-1

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Prohibitins and the Cytoplasmic Domain of CD86 Cooperate To Mediate CD86 Signaling in B Lymphocytes

Christopher R. Lucas,*,† Hector M. Cordero-Nieves,*,† Robert S. Erbe,† Jaclyn W. McAlees,*,† Sumeena Bhatia,‡ Richard J. Hodes,‡ Kerry S. Campbell,x and Virginia M. Sanders†,{

CD86 engagement on a CD40L/IL-4–primed murine B cell activates signaling intermediates that promote NF-kB activation to increase Oct-2 and mature IgG1 mRNA and protein expression, as well as the rate of IgG1 transcription, without affecting class switch recombination. One of the most proximal signaling intermediates identified is phospholipase Cg2, a protein reported to bind tyrosine residues, which are absent in the cytoplasmic domain of CD86. Using a proteomics-based identification approach, we show that the tyrosine-containing transmembrane adaptor proteins prohibitin (Phb)1 and Phb2 bind to CD86. The basal expres- sion of Phb1/2 and association with CD86 was low in resting B cells, whereas the level of expression and association increased Downloaded from primarily after priming with CD40. The CD86-induced increase in Oct-2 and IgG1 was less when either Phb1/2 expression was reduced by short hairpin RNA or the cytoplasmic domain of CD86 was truncated or mutated at serine/threonine protein kinase C phosphorylation sites, which did not affect Phb1/2 binding to CD86. Using this approach, we also show that Phb1/2 and the CD86 cytoplasmic domain are required for the CD86-induced phosphorylation of IkBa, which we previously reported leads to NF-kB p50/p65 activation, whereas only Phb1/2 was required for the CD86-induced phosphorylation of phospholipase Cg2 and protein http://www.jimmunol.org/ kinase Ca/bII, which we have previously reported leads to NF-kB (p65) phosphorylation and subsequent nuclear translocation. Taken together, these findings suggest that Phb1/2 and the CD86 cytoplasmic domain cooperate to mediate CD86 signaling in a B cell through differential phosphorylation of distal signaling intermediates required to increase IgG1. The Journal of Immunology, 2013, 190: 723–736.

lso referred to as B7-2, CD86 is a 70-kDa transmem- might become phosphorylated owing to cellular activation stimuli, brane glycoprotein expressed primarily on APCs, in- suggesting that CD86 may signal directly. Studies have reported cluding macrophages, dendritic cells, and B cells (1, 2). that CD86 engagement induced a signal directly within the B cell

A by guest on September 29, 2021 CD86 is a well-known costimulatory molecule that ligates CD28 that increased IgG4 production in anti-CD40/IL-4–primed human and CTLA-4 expressed on a CD4+ T cell to increase or decrease, B cells (16), and the murine IgG4 homolog IgG1 production in respectively, T cell activation signals (3–6), and it is essential for CD40L/IL-4– (13, 17–20) or LPS-primed (21) murine B cells germinal center formation (7, 8). CD86 expression is low on in vitro, as well as in B cells from mice immunized with either resting B cells (1), but it increases in response to engagement of trinitrophenyl hapten-keyhole limpet hemocyanin (20) or influ- the BCR (1), CD40 (9), the IL-4R (10), the LPS receptor (11, 12), enza virus (22). It has also been reported that CD86 also signals to or the b2-adrenergic receptor (13, 14). CD86 contains a short regulate other Ig isotypes, including IgE (13, 16), and IgG2a (21), cytoplasmic domain that lacks tyrosine phosphorylation sites and an affect that may be controlled by the priming Ag or stimulus. was thought not to signal directly. However, the CD86 cytoplas- Collectively, these findings suggested that CD86 on a B cell plays mic domain contains three putative protein kinase C (PKC) serine/ a role in regulating the level of IgG1 produced. threonine phosphorylation sites. Additionally, a proposal by The initial functional results from these studies led to the search Lenschow et al. (15) reported that the CD86 cytoplasmic domain for signaling intermediates and transcription factors activated by CD86 engagement to mediate the increase in IgG1 production. CD86 engagement on the surface of a CD40L/IL-4–primed B cell *Integrated Biomedical Science Graduate Program, The Ohio State University, Co- was found to activate two cascades of signaling intermediates that lumbus, OH 43210; †Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, OH 43210; ‡Experimental Immu- ultimately allowed for NF-kB p50/p65 activation via phosphory- nology Branch, National Cancer Institute, National Institutes of Health, Bethesda, lation of IkBa and p65 phosphorylation, respectively (18). Inhi- x MD 20892; and Institute for Cancer Research, Fox Chase Cancer Center, Philadel- bition or loss of these signaling intermediates in a B cell phia, PA 19111; and {Institute for Behavioral Medicine Research, The Ohio State University, Columbus, OH 43210 eliminated the CD86-induced increase in Oct-2 expression (18, Received for publication June 14, 2012. Accepted for publication November 11, 19), Oct-2 binding to the 39-IgH enhancer (18, 19), the rate of 2012. mature IgG1 transcription (17), and the increase in IgG1 protein This work was supported by National Institutes of Health Grants R01-AI037326 and per cell (13), confirming their roles in mediating CD86 signals to R56-AI073663. affect the level of IgG1 produced. Importantly, CD86 engagement Address correspondence and reprint requests to Dr. Virginia M. Sanders, The Ohio on primed B cells failed to affect class switch recombination (13, State University, 319 Institute for Behavioral Medicine Research Building, 460 Medical 17–20), indicating that the increase in IgG1 was due to an effect Center Drive, Columbus, OH 43210. E-mail address: [email protected] on the amount of IgG1 produced per cell and not the number of The online version of this article contains supplemental material. cells that switched to IgG1. The increased level of signaling in- Abbreviations used in this article: Phb, prohibitin; PKC, protein kinase C; PLC, phos- pholipase C; qRT-PCR, quantitative real-time PCR; shRNA, short hairpin RNA; Syk, termediate activation and/or Oct-2 that was induced by CD86 spleen tyrosine kinase; WT, wild-type. engagement on primed B cells resulted in a 2- to 3-fold increase in www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201646 724 PROHIBITINS AND CYTOPLASMIC CD86 SIGNAL IN B CELLS

IgG1 as compared with primed B cells in the absence of CD86 spectrometry was performed on a Thermo Finnigan LTQ mass spectrom- engagement. Notably, clinical findings have shown that a 2- to 3- eter equipped with a nanospray source operated in positive ion mode. The fold increase in serum IgG correlates to a 3- to 9-fold increase in liquid chromatography system was an UltiMate 3000 system from Dionex (Sunnyvale, CA). Sequence information from the tandem mass spec- protection against Streptococcus pneumoniae and pertussis (23, trometry data were processed by converting the .raw files into a merged 24), suggesting that increases of this magnitude in the level of Ab file (.mgf) using an in-house program, RAW2MZXML_n_MGF_batch produced are potentially relevant clinically. (merge.pl, a Perl script). The resulting .mgf files were searched using In this study we sought to identify potential signaling intermediates Mascot Daemon by Matrix Science (Boston, MA) version 2.3.2. associated directly with CD86 and to verify its role in mediating Immunoblot analysis CD86 signaling. Such a directly associated protein must exist because Resting B cells (5–7 3 106) were primed as described above. CH12.LX one of the most proximal CD86-dependent signaling intermediates, B cells were transfected with scrambled negative control short hairpin phospholipase C (PLC)g2, functions classically by recruitment to RNA (shRNA) or Phb1/2-specific shRNA plasmids (1–5 mg/106 cells) Phb1 proteins containing tyrosine residues (25, 26), which are lacking (clones 1, 3, and 4) and Phb2 (clones 1 and 3) (SABiosciences) for 24 h followed by priming with CD40L/IL-4 for 16 h or with a FLAG-CD86 in the CD86 cytoplasmic domain. Using a proteomics approach, we expression plasmid (1 mg/106 cells) via nucleofection (Amaxa, program demonstrate a novel protein/protein interaction that occurred in O-003), followed by priming with CD40L/IL-4 for 16 h. In some experi- a CD40L/IL-4–primed B cell between CD86 and prohibitin (Phb) ments, cells were cultured under serum-free conditions for at least 30 min 1 and Phb2, which are transmembrane adaptor proteins that con- and engagement of CD86 with anti-CD86 (P03; eBioscience), species- and tain tyrosine residues. Furthermore, we report that both Phb1/2 isotype-matched control Ab (rat IgG2b; eBioscience), anti-FLAG (M2; Sigma-Aldrich), or species- and isotype-matched control Ab (mouse IgG1; and an intact CD86 cytoplasmic domain are required to mediate SouthernBiotech). Cell lysates were prepared as described previously (18). CD86 signaling that regulates the level of IgG1 produced by Nuclear-enriched protein lysates were prepared by washing 5 3 106 CH12. Downloaded from a B cell via differential activation of distal signaling intermediates. LX B cells with 13 PBS followed by the addition of 0.5% Nonidet P-40 lysis buffer containing 10 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 3 mM MgCl2. Nuclei were pelleted and resuspended in 13 lysis buffer (20 mM Materials and Methods Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, Animals 2.5 mM sodium pyrophosphate, 1 mM Na2VO4,1mg/ml leupeptin, 10 nM okadaic acid, and 10 nM tautomycin). Protein samples (5–12 mg) were run Female pathogen-free BALB/c mice were obtained from Taconic (Ger-

on a denaturing 10% polyacrylamide gel and transferred to Immobilon http://www.jimmunol.org/ mantown, NY) and were housed in the American Association Accreditation polyvinylidene difluoride membranes (Millipore). Membranes were blocked of Laboratory Animal Care–accredited Animal Research Facility at The with TBST as described previously (18) and incubated overnight with pri- Ohio State University (Columbus, OH). All mice were provided autoclaved mary Abs at 4˚C. Membranes were probed with HRP-labeled secondary Abs, food and deionized water ad libitum and used at 8–10 wk age. developed using a LumiGlo detection kit (Cell Signaling Technology), and Cell lines/transgenic B cells specific bands were visualized on Kodak BioMax MS film. Abs used were anti-CD86 (M-20), anti-CD86 (H-200), anti-GAPDH (FL-335), anti-PKCbII CH12.LX is a murine B cell lymphoma line previously described (27) and (C-18) (Santa Cruz Biotechnology), anti-Bap37 (Phb2; Poly6118) (Bio- was provided by Dr. G. Bishop (University of Iowa, Iowa City, IA). Wild- Legend), anti-Phb1, anti–phospho-PLCg2 (Y1217), anti-PLCg2, anti–phos- type (WT), transgenic B cells (line 7) that overexpress CD86 on a C57BL/6 pho-PKCa/bII (T638/641), anti-PKCa, anti–phospho-IkBa (S32; 14D4), background were previously described (28). Transgenic B cells that express a anti-IkBa (44D4), anti–phospho-p65 (S536; 93H1), anti-p65 (C22B4), anti-

CD86 cytoplasmic truncation (line I2) within a CD80/CD86 double-deficient Lamin A/C (4C11), anti-GAPDH (14C10), anti–b-actin (13E5) (Cell Sig- by guest on September 29, 2021 mouse on a C57BL/6 background were previously described (29). naling Technology), and anti–a-tubulin (DM1A) (Sigma-Aldrich).

B cell isolation/priming Immunoblot densitometric analysis Naive B cells were isolated using MACS anti-mouse CD43 beads (Miltenyi Specific protein bands detected via immunoblot analysis were quantified via Biotec, Auburn, CA) following the manufacturer’s directions. Cells were densitometry (ImageJ software, National Institutes of Health). The scanned primed with CD40L-expressing Sf9 cells or control Sf9 cells lacking image was inverted to measure the OD of a specific protein band. A CD40L expression at a B cell/Sf9 cell ratio of 10:1, 1 ng/ml IL-4 (eBio- measurement box was created around the broadest band on a given gel and science), 0.1 mg/ml anti-IgM, 1026 M terbutaline (Sigma-Aldrich), or 50 used to measure each band on the gel. The numerical value was recorded as mg/ml LPS (Sigma-Aldrich) for 16 h. Total mRNA and total protein were an OD. Background measurements of each band were recorded from the collected and analyzed via quantitative real-time PCR (qRT-PCR) and corresponding gel lanes and subtracted from the original ODs. The ODs of immunoblot, respectively. All reagents used were negative for the presence Phb1/2 total protein bands were normalized to GAPDH protein loading g of endotoxin, as determined by E-Toxate (Sigma-Aldrich), a Limulus ly- control band ODs. The ODs of phospho-PLC 2 (Y1217) protein bands g sate assay with a level of detection ,0.1 U/ml. were normalized to ODs of corresponding total PLC 2 protein bands. ODs from phospho-PKCa/bII (T638/641) protein bands were normalized to OD Immunoprecipitation values from both PKCa and PKCbII total protein bands. ODs obtained from phospho-IkBa (S32) and total IkBa protein bands were normalized Immunoprecipitations were performed using the ProFound mammalian to GAPDH total protein band ODs. The ODs recorded from phospho-p65 coimmunoprecipitation kit (Pierce). Briefly, 25 3 106 CH12.LX B cells (S536) protein bands were normalized to both total p65 protein band ODs were cultured and primed as described above, lysed with 0.5% Triton and ODs obtained from GAPDH protein bands. The ODs measured from X-100 buffer, immunoprecipitated with an anti-CD86 GL1 (eBioscience) nuclear phospho-p65 (S536) and nuclear p65 were normalized to both or species- and isotype-matched control Ab (rat IgG2a; eBioscience) fol- Lamin A and Lamin C protein band ODs. The data are expressed as mean lowing the manufacturers’ directions. The precipitates were analyzed via fold change 6 SD/SEM relative to resting, CD40L/IL-4 priming alone, or immunoblot for CD86, Phb2, and Phb1. Alternatively, 20 3 106 CH12.LX species- and isotype-matched control Ab conditions. 3 6 B cells and 50 10 WT B cells were activated and lysed as described Flow cytometry above, then immuoprecipitated with 50 mg either anti-FLAG Ab (M2; Sigma-Aldrich) or anti-CD86 Ab (GL1; eBioscience) overnight at 4˚C, The level of CD86 on GFP+ Phb1/2-shRNA+ CH12.LX B cells was de- followed by the addition of 50 ml protein G-agarose beads (Invitrogen). termined as previously described (13) using allophycocyanin-conjugated rat anti-mouse CD86 (GL1). Samples were analyzed using a FACSCanto II Mass spectrometry (BD Biosciences). Expression of FLAG-CD86 was measured via FITC- Mass spectrometry and protein identification were performed by the Mass conjugated anti-mouse FLAG (M2) (Sigma-Aldrich). The data were ana- Spectrometry and Proteomics Core Facility at The Ohio State University. lyzed using FlowJo software (Tree Star). The unique band present at ∼35 kDa in an anti-CD86 immunoprecipitate RNA-interfering Phb1 and Phb2 knockdown and qRT-PCR of CH12LX cells from a gel stained with SYPRO Ruby (Invitrogen) analysis was excised and digested with sequencing-grade trypsin from Promega (Madison, WI) using the MultiScreen Solvinert filter plates from Millipore CH12.LX B cells were transfected with scrambled negative control shRNA, (Bedford, MA). Capillary/liquid chromatography/nanospray tandem mass Phb1 (clones 1, 3, and 4)- or Phb2 (clones 1 and 3)-specific shRNA The Journal of Immunology 725

(SABiosciences) (1–5 mg/106 cells), or with a FLAG-CD86 expression performed according to the manufacturer’s instructions. Abs used to vector (1 mg/106 cells) via nucleofection (Amaxa, program O-003) detect phospho-PLCg2 (Y1217) and total PLCg2 in-cell protein levels were according to the manufacturer’s instructions, cultured for 24 h, followed anti–phospho-PLCg2 (Y1217) and anti-PLCg2 (Cell Signaling Technology). by CD40L/IL-4 priming for an additional 16 h. In some experiments, GFP+ The HRP-conjugate Ab was used at a 1:2000 dilution. cells were sorted (ICy-Reflection) and recultured in the presence of either anti-CD86 (PO3; eBioscience) or species- and isotype-matched control Ab Statistical analysis (rat IgG2b) (eBioscience) or CD28/Fc chimera (CD28/Ig) (R&D Systems) Data from multiple treatment groups were analyzed using a one-way ANOVA for 24 h. Total RNA was isolated via TRIzol (Invitrogen), cDNA was to determine whether an overall statistical change existed. Certain p values created via reverse transcription with random hexamer primers (Qiagen), were calculated using a Bonferroni post hoc analysis or a two-sided Stu- and qRT-PCR (SYBR Green; Roche) was conducted as previously described dent t test for comparison of two treatment groups. A p value #0.05 in- (17) for Phb1 mRNA levels, forward primer, 59-ATGGCTGCCAAAGTGT- dicated statistically significant results. TTGAGTC-39,reverseprimer,59-TCACTGGGGAAGCTGGAGAAGC-39; Phb2 mRNA levels, forward primer, 59-GTAGAAGCCAAGCAAGTGGC-39, reverse primer, 59-TTCAGCACAAGGTTGTCAGC-39;CD86mRNAlevels, Results forward primer, 59-CGAGCACTATTTGGGCACAGAG-39, reverse primer, CD86 associates with Phb1 and Phb2 in B lymphocytes 59-TTTCCAGAACACACACAACGGTC-39; cytoplasmic CD86 mRNA levels, forward primer, 59-GTCAATGAAGATTTCCTCCAAA-39, reverse CD86 engagement on a CD40L/IL-4–primed B cell is known to primer, 59-CAGGTTGATAGTCTCTCTGTCAG-39; Oct-2 mRNA levels, activate PLCg2 (19), a signaling intermediate that requires binding forward primer, 59-ATCAAGGCTGAAGACCCCAGTG-39, reverse primer, to proteins containing phospho-tyrosine residues for recruitment/ 59-TGGAGGAGTTGCTGTATGTCCC-39; germline IgG1 mRNA levels, for- ward primer, 59-CATCCTATCACGGGAGATTGGG-39, reverse primer, 59- activation and its ability to implement receptor function. We rea- ATCCTCGGGGCTCAGGTTTG-39; and mature IgG1 mRNA levels, forward soned that CD86, which does not contain tyrosine residues in the primer, 59-TATGGACTACTGGGGTCAAG-39, reverse primer, 59-CCT- cytoplasmic domain, must associate with a tyrosine-containing Downloaded from GGGCACAATTTTCTTGT-39 relative to actin mRNA levels, forward adaptor/scaffolding protein to mediate the activation of PLCg2. primer, 59-TACAGCTTCACCACCACAGC-39,reverseprimer,59-AAGG- Therefore, a proteomics-based approach was used to identify any AAGGCTGGAAAAGAGC-39 or GAPDH mRNA levels, forward primer, 59-ACCACAGTCCATGCCATCAC-39, reverse primer, 59-TCCACCACCC- protein that associated with CD86 in primed B cells. The murine TGTTGCTGTA-39. B cell line CH12.LX was cultured overnight in the presence of CD40L/IL-4 and then exposed to either an anti-CD86 or species- cDNA constructs and site-directed mutagenesis

and isoytpe-matched control Ab. CD86 was immunoprecipitated http://www.jimmunol.org/ Total RNA was isolated by TRIzol (Invitrogen) from CD40L (10 B cells/1 from cell lysates, and proteins were separated via SDS-PAGE and Sf9) and rIL-4 (1 ng/ml) (eBioscience)–primed murine splenocytes for 24 h stained with SYPRO Ruby (Fig. 1A). Three unique bands were and cDNA was generated using AccuScript RT (Stratagene). The murine ∼ CD86 cDNA was amplified by PCR using Easy-A high-fidelity PCR present at molecular masses of 50, 35, and 28 kDa in the anti- cloning (Stratagene) and the following primers: the forward primer con- CD86 Ab immunoprecipitate, but they were absent when the control taining an EcoRI site (59-GAATTCCGAGACGCAAGCTTATTTCAATG- Ab was used. The unknown ∼35-kDa protein was excised because GGACTGCATAT-39) and the reverse primer containing a BamHI site it was clearly isolated from other bands and identified by mass 9 9 (5 -TTTTGGATCCTCACTCTGCATTTGGTTTTGCTGAAGC-3 ). The spectrometry as the adaptor protein Phb2 (Fig. 1B). In all, 14 non- CD86 cDNA was then subcloned into the pCR2.1 TOPO TA vector. To generate a FLAG-CD86 expression construct, murine CD86 cDNA was redundant tryptic peptide fragments of murine Phb2 were identified. subsequently cloned into the pFLAG-CMV3 vector (Sigma-Aldrich), in- Although sequence analysis was not performed on the other two by guest on September 29, 2021 troducing an N-terminal FLAG epitope tag (DYKDDDK) before the start bands, we predict that the CD86-associated ∼28 kDa protein band of the mature polypeptide receptor. Mutant plasmids containing either is Phb1 because the literature suggests that Phb2 associates closely FLAG-CD86 cytoplasmic truncations at KKPD or 282D and/or single al- anine point mutations at positions S285, T291, and S303 were created by with the highly related Phb1 in a variety of intracellular locales using the QuikChange site-directed mutagenesis kit (Stratagene) according (30). The identity of the CD86-associated protein band at ∼50 kDa to the manufacturer’s instructions. The following primers were used to remains unclear, but it may be another adaptor/scaffolding protein generate specific mutant plasmids: KKPD, forward primer, 59-ATTGTA- similar to that reported for other B cell surface receptors, in- TGTCACAAGAAGTAGAATCAGCCTAGCAGGCCC-39, reverse primer, cluding CD40 (31–34) and the BCR (35–39) that are also devoid 59-GGGCCTGCTAGGCTGATTCTACTTCTTGTGACATACAAT-39;282D, forward, primer, 59-AACACAGCCTCTAAGTTATAGCGGGATAGTAACG- of tyrosine residues within the cytoplasmic domain, but that use CTGACAG-39,reverseprimer,59-CTGTCAGCGTTACTATCCCGCTATA- adaptor/scaffolding complexes to function. ACTTAGAGGCTGTGTT-39; S285A, forward primer, 59-GCCTCTAAGTT- We tested whether Phb1 and/or Phb2 could be specifically AGAGCGGGATGCTAACGCTGACAGAGAGACTATC-39, reverse primer, coimmunoprecipitated from B cells under primed versus resting 59-GGTTGATAGTCTCTCTGTCAGCGTTAGCATCCCGCTCTAACT- TAG-39; T291A, forward primer, 59-ATAGTAACGCTGACAGAGAGG- conditions. Parent CH12.LX cells, CH12.LX cells transfected with CTATCAACCTGAAGGAACT-39, reverse primer, 59-AGTTCCTTCAGG- FLAG-tagged CD86, or primary B cells were cultured overnight in TTGATAGCCTCTCTGTCAGCGTTACTAT-39; S303A, forward primer, the presence or absence of CD40L/IL-4 priming. Lysates were 59-AACTTGAACCCCAAATTGCTGCAGCAAAACCAAATGCAGA-39, prepared and immunoprecipitated with control and anti-CD86 Abs 9 reverse primer, 5 -TCTGCATTTGGTTTTGCTGCAGCAATTTGGGGT- and then analyzed for level of Phb1/2 and CD86 by immunoblot. As TCAAGTT-39. Integrity of all constructs was confirmed by sequencing at the Nucleic Acid Shared Resource Facility at The Ohio State University. shown in Fig. 1C, levels of Phb1 and Phb2 were low or unde- Surface expression of all constructs was confirmed by flow cytometry. tectable in CD86 immunoprecipitates from resting CH12.LX B cells, whereas coimmunoprecipitation of both proteins increased sub- In-cell ELISA stantially in primed B cells. This increase in Phb1/2 binding to In-cell ELISAs were performed to detect in-cell levels of phospho-PLCg2 CD86 may be due to a specific CD40L/IL-4–dependent recruit- (Y1217) and total PLCg2 protein using an in-cell colorimetric ELISA kit ment event or an increased level of CD86 present in the CD86 3 6 (Pierce). Briefly, 1 10 CH12.LX B cells were transfected under mock immunoprecipitates. Similar results were observed when CH12. conditions or with either scrambled negative control shRNA or Phb1/2- specific shRNA plasmids for 24 h followed by priming with CD40L/IL-4 LX B cells were transfected with a FLAG epitope–tagged CD86 or with a WT or cytoplasmic-deficient (KKPD) FLAG-CD86 expression construct and primary B cells were used (Fig. 1C). To determine plasmid followed by priming with CD40L/IL-4 for 16 h. Cells were cul- whether Phb1/2 associated with the cytoplasmic domain of CD86, tured under serum-free conditions for at least 30 min, followed by en- we tested CH12.LX B cells transfected with a cytoplasmic trun- gagement of either CD86 with anti-CD86 or FLAG-CD86 with anti-FLAG and appropriate controls (as described above) for 15–30 min. The cells were cated form of FLAG-CD86 (KKPD), as well as primary B cells then washed in 13 PBS. Cells (2 3 104) were plated on a 96-well culture from transgenic mice that express a truncated form of the CD86 plate and fixed with 8% formaldehyde. The remainder of the assay was cytoplasmic domain. Phb1 and Phb2 were found to associate with 726 PROHIBITINS AND CYTOPLASMIC CD86 SIGNAL IN B CELLS Downloaded from

FIGURE 1. CD86 associates with Phb1 and Phb2 in B lymphocytes. (A) CH.12LX B cells were primed with CD40L/IL-4 for 16 h followed by the addition of a species- and isotype-matched control Ab (anti-rat IgG2a ctrl Ab) or rat anti-mouse CD86 (GL1) (anti-CD86) for 15 min. Lysates were prepared, followed by immunoprecipitation (Pierce). Immunoprecipitates were separated via SDS-PAGE followed by total protein staining with SYPRO http://www.jimmunol.org/ Ruby. Unique bands in the CD86 immunoprecipitate are indicated by arrows at ∼50, ∼35, and ∼28 kDa. (B) The band marked with an arrow at ∼35 kDa was sequenced via liquid chromatography/tandem mass spectrometry. Tryptic peptide fragments were sequenced and matched with known tryptic peptides via a MASCOT search. Fourteen nonredundant tryptic peptides were identical to murine Phb2. (C) CH12.LX B cells alone, CH12.LX B cells expressing a FLAG-CD86 recombinant protein, or primary splenic B cells were primed as described above. Immunoprecipitates were immunoblotted for CD86, Phb2, and Phb1. Gels are representative of three independent experiments. (D) FLAG-CD86–transfected CH12.LX B cells with either full-length FLAG-CD86 or a truncated FLAG-CD86 (KKPD) or nontransfected B cells from WT or truncated CD86-expressing mice (Trunc CD86) were primed as described above, immunoprecipitated with either anti-FLAG or anti-CD86, and analyzed for Phb1/2 and CD86 protein levels via immunoblot. The gels shown are repre- sentative of three independent experiments (left panel) or one experiment (right panel). by guest on September 29, 2021 CD86 in the absence or presence of the cytoplasmic domain (Fig. had no effect on the level of Phb1/2 expression, whereas con- 1D), suggesting that a transmembrane-specific association oc- comitant engagement of the IL-4R with CD40 further increased curred between CD86 and Phb1/2. Taken together, these findings the level of Phb2, but not Phb1, mRNA and increased the level indicated that low levels of Phb1 and Phb2 were associated with of Phb1/2 protein as compared with CD40 engagement alone. CD86 in resting B cells, but that the levels increased after CD40L/ Treatment with LPS alone also increased levels of Phb1/2 mRNA IL-4 priming and were independent of most of the CD86 cyto- relative to resting B cells, although the increase was less than that plasmic domain. caused by CD40 engagement, and this LPS-induced increase in mRNA was further increased by the addition of IL-4. In contrast, The expression of Phb1 and Phb2 is regulated primarily by only a weak increase in Phb1/2 protein was detected in LPS- CD40 engagement exposed B cells in the absence or presence of IL-4. Other stimuli A number of studies have reported that Phb1 and Phb2 associate that were able to increase CD86, that is, IgM and b2-adrenergic with each other to form a large complex within the inner mito- receptor engagement on a B cell, were unable to upregulate Phb1/ chondrial membrane and that they function by stabilizing pro- 2 expression to any significant level in comparison with resting teins of the electron transport chain (40). If a similar stabilizer/ levels. Importantly, control Sf9 cells lacking CD40L expression molecular chaperone function existed for Phb1/2 to facilitate CD86 failed to induce Phb1/2 mRNA or protein levels (Fig. 2C). Taken stability, transport, and insertion into the plasma membrane, then together, these findings suggested that Phb1/2 protein expression Phb1/2 protein expression would be expected to increase con- may be regulated primarily by a CD40-dependent mechanism that comitantly with CD86 protein expression when the B cell was is independent from the mechanism involved in upregulating ex- primed with CD40L/IL-4, as well as with other B cell stimuli that pression of CD86. are known to increase CD86 protein expression. Primary resting B cells were cultured in the absence or presence of IL-4 overnight Phb1 and Phb2 play a role in mediating the effect of CD86 engagement on B cell function with either CD40L/anti-IgM to engage the BCR, the b2-adrenergic receptor agonist terbutaline, or LPS, all of which have been re- Although the data thus far showed that Phb1/2 associated with ported to increase CD86 expression on a B cell (1, 9–14) (see CD86, the functional significance of this association was unclear. Supplemental Fig. 1A). Total mRNA and protein were collected Recent studies have reported that Phb1 interacts with various sig- and analyzed for the level of Phb1/2 and CD86 expression by qRT- naling intermediates to modulate cellular processes, and thus the PCR and immunoblot, respectively. Engagement of CD40 alone possibility was supported that by associating with CD86 in primed promoted a robust increase in the level of expression for both B cells, Phb1 and Phb2 could be providing CD86 with a cytoplasmic Phb1 and Phb2 mRNA (Fig. 2A) and protein (Fig. 2B) as com- signaling platform to stimulate B cell responses. To test this pos- pared with resting B cells. IL-4R engagement on a resting B cell sibility directly, CH12.LX B cells were transfected with GFP- The Journal of Immunology 727

FIGURE 2. The expression of Phb1 and Phb2 is regulated primarily by CD40 engagement. Naive splenic B cells were isolated and cultured for 16 h in the absence or presence of CD40L, anti-IgM, terbutaline, or LPS in the ab- sence or presence of IL-4. (A) Total mRNA was collected and analyzed via qRT-PCR analysis of Phb2, Phb1, and GAPDH. Phb1/2 values were normal- ized to GAPDH and the data are repre- sented as a mean fold change in Phb1/2 mRNA from primed B cells relative to resting B cells and are expressed as the means 6 SEM of quadruplicate samples Downloaded from per condition from three independent experiments. (B) Total protein was col- lected and immunoblot analysis was used to measure total Phb2, Phb1, and GAPDH protein levels. Gels are repre- sentative of three independent experi- ments. Densitometry was performed http://www.jimmunol.org/ and measured Phb1/2 band intensity/ GAPDH band intensity and data are presented as the mean fold change in Phb1/2 protein from primed B cells relative to resting and expressed as the mean fold change 6 SEM from three independent experiments. (C)Naive splenic B cells were isolated and cul- tured for 16 h in the presence or absence of control Sf9 cells lacking expression by guest on September 29, 2021 of CD40L (ctrl Sf9 CD40L neg). mRNA and total protein were collected and analyzed for Phb1/2 expression levels relative to actin or GAPDH via qRT- PCR or immunoblot, respectively. The data are expressed as the mean fold change 6 SEM from either three (mRNA) independent experiments or fold change 6 SD from two to three (protein) inde- pendent experiments. Statistical dif- ferences are shown relative to resting. *p , 0.05.

containing shRNA expression vectors designed to suppress Phb1/2 17–20). CD86 engagement on primed GFP+ B cells resulted in com- expression. The effect of transfection on B cell CD86 expression parable levels of germline IgG1, Oct-2, and mature IgG1 mRNA rel- was measured before functional studies were performed. Flow ative to isotype control–treated B cells, whereas significant increases in cytometric analysis showed that CD86 surface expression following Oct-2 and mature IgG1 mRNA were observed owing to CD86 en- CD40L/IL-4 priming was comparable in the absence or presence gagement in GFP2 B cells (Fig. 3B). To ensure that the comparable of Phb1/2 knockdown (Supplemental Fig. 1B). qRT-PCR analysis levels of Oct-2 and mature IgG1 mRNA were not due to an off target of FACS-sorted GFP+ and GFP2 cells showed that both Phb1 and effect of the Phb1/2 shRNA plasmids, scrambled negative control Phb2 expression was reduced by at least 50% in shRNA-expressing shRNA plasmids were transfected into CH12.LX B cells as described. GFP+ cells (Fig. 3A), where reduction of Phb1 resulted in the loss CD86 engagement promoted significant increases in the level of Oct-2 of Phb2 and vice versa (Supplemental Fig. 2). and mature IgG1 mRNA produced (Fig. 3B), whereas germline IgG1 We next determined whether both Phb1 and Phb2 play roles in mRNA remained unchanged. Taken together, these findings indicated mediating the CD86-induced increases in Oct-2 and IgG1 without that Phb1 and Phb2 are both necessary for CD86 engagement on a change in class switch recombination, as reported previously (13, a primed B cell to enhance the level of B cell function. 728 PROHIBITINS AND CYTOPLASMIC CD86 SIGNAL IN B CELLS

FIGURE 3. Phb1 and Phb2 play a role in medi- ating the effect of CD86 engagement on B cell function. (A and B) CH12.LX B cells were trans- fected with either a GFP-containing Phb1, Phb2, or scrambled, negative control shRNA (neg ctrl shRNA) vector for 36 h to silence Phb1 and Phb2 gene expression. At 24 h after transfection, cells were primed with CD40L/IL-4 for 16 h. GFP cells con- taining the Phb1 or Phb2 shRNA vector were sep- arated by FACS then recultured with either an anti- CD86 Ab or a species- and isotype-matched control Ab (iso ctrl). On day 2 or day 6 after cell priming, Phb1/Phb2 mRNA (A) or Oct-2/germline IgG1 and mature IgG1 mRNA (B), respectively, were mea- sured via qRT-PCR analysis and normalized to ac- tin. GFP2 cells and scrambled negative control shRNA are depicted with gray bars and GFP+ cells containing Phb1/2 shRNA are depicted with black Downloaded from bars. Data are expressed as mean fold change 6 SEM relative to either no GFP (A) or isotype control of each group (B) and represent at least quadrupli- cate samples per condition from three independent experiments. Statistical differences are shown rela- tive to no GFP (A) or isotype control of each group B , ( ). *p 0.05. http://www.jimmunol.org/

The cytoplasmic domain of CD86 also plays a role in phosphorylation of the cytoplasmic domain of CD86 is impor- mediating the effect of CD86 engagement on B cell function tant for signaling function. The evidence thus far suggested that CD86 associated with Phb1/2 In addition to the use of CD86-transfected B cells, B cells were through transmembrane-specific interactions and that Phb1 and also used that had been isolated from mice transgenic for either WT by guest on September 29, 2021 Phb2 were essential to couple CD86 engagement to a downstream CD86 that overexpressed CD86 or a truncated form of CD86 that functional increase in IgG1. However, it remained unclear whether lacked the cytoplasmic domain. In B cells from the truncated form the cytoplasmic domain of CD86 played any role in mediating the of CD86 mice that were primed, Phb1/2 proteins remained asso- CD86-dependent increase in the B cell functional response. It was ciated with CD86 (Fig. 1D), but the level of mRNA expressed for proposed previously that the cytoplasmic domain of CD86 had the the cytoplasmic domain of CD86, Oct-2, and mature IgG1 was potential to be phosphorylated at one or more of several putative decreased in comparison with WT-expressing B cells, whereas PKC phosphorylation sites (15), which might provide a mecha- germline IgG1 was comparable (Supplemental Fig. 3). Thus, to nism for CD86 to recruit and/or activate downstream signaling our knowledge, these data collectively provide evidence that, in intermediates that are known to participate in the CD86-induced addition to Phb1/2, the cytoplasmic domain of CD86 is also re- increases in Oct-2 and IgG1. quired to mediate CD86 signaling to regulate B cell function, To test this possibility, a series of mutations/truncations were likely via phosphorylation of the cytoplasmic domain at serine introduced into the FLAG-CD86 cytoplasmic domain that included and/or threonine residues, as the present site-directed mutagenesis single alanine point mutations of the three putative PKC serine/ data show. threonine phosphorylation sites, as well as either partial or full truncation of the cytoplasmic domain as diagramed in Fig. 4A. Phb1 and Phb2 are necessary for the CD86-induced activation CH12.LX B cells were transfected with the FLAG-CD86 plas- of PLCg2 mids, and cells were primed with CD40L/IL-4 in culture for 16 h, Although findings thus far suggested that both Phb1/2 and the at which time either a species- and isotype-matched control Ab or CD86 cytoplasmic domain are required to mediate CD86 sig- anti-FLAG Ab (M2) was added. Cells were assessed, via qRT- naling to regulate a B cell functional response, the proximal PCR analysis, for levels of Oct-2/germline IgG1 mRNA on day molecular mechanism was unclear. One of the most proximal 2 or mature IgG1 mRNA on day 5 (Fig. 4B). The introduction CD86 signaling intermediates to date, PLCg2 (19), is classically of alanine point mutations at all PKC phosphorylation sites had recruited to phosphorylated tyrosine-containing protein/protein no effect on germline IgG1, but significantly reduced Oct-2 and complexes (25, 26). Additionally, it was reported that Phb1 binds mature IgG1 mRNA levels, except for Oct-2 induction by the T291A the protein tyrosine kinase spleen tyrosine kinase (Syk) upon and S285A mutants, as compared with WT controls. Partial and BCR engagement in murine B cells (41), indicating that Phb1 full truncation of the CD86 cytoplasmic domain resulted in sig- may be involved with the activation of protein tyrosine kinases nificant reductions of Oct-2 and mature IgG1 mRNA levels when specifically in B cells, and perhaps other membrane-associated compared with WT controls. Importantly, levels of either WT or signaling intermediates after receptor engagement. To address mutant FLAG-CD86 expressed on the B cell surface remained this, CH12.LX B cells were transfected with scrambled negative comparable (data not shown). These results emphasize that PKC control or Phb1/2-specific shRNA plasmids and either a WT or The Journal of Immunology 729

with both WT and KKPΔ FLAG-CD86 expression plasmids caused an increase in PLCg2 phosphorylation relative to a species- and isotype-matched control Ab (Fig. 5E). To extend our im- munoblot findings, the level of PLCg2 phosphorylation was measured via in-cell ELISA. Whereas mock- and scrambled nega- tive control shRNA–transfected CH12.LX B cells allowed for a CD86-induced increase in PLCg2 phosphorylation relative to priming alone, PLCg2 phosphorylation in the presence of Phb1/2 shRNA remained comparable to priming alone (Fig. 5F). Similar to our immunoblot findings, engagement of FLAG-CD86 on B cells transfected with both WT and KKPΔ FLAG-CD86 expres- sion plasmids caused an increase in PLCg2 phosphorylation rel- ative to a species- and isotype-matched control Abs (Fig. 5G). Taken together, these findings suggested that whereas Phb1 and Phb2 are critical for the CD86-induced activation of PLCg2, the CD86 cytoplasmic domain is not involved, indicating that Phb1/2 and the CD86 cytoplasmic domain must differentially activate signaling intermediates upon CD86 engagement. Downloaded from Phb1 and Phb2 are necessary for the CD86-induced activation of PKCa/bII Although our evidence suggested that Phb1 and Phb2 are necessary for the CD86-induced activation of PLCg2 independent of the CD86 cytoplasmic domain, whether Phb1 and Phb2 were essential

for the CD86-dependent downstream activity of PLCg2 remained http://www.jimmunol.org/ unknown. Because PLC classically allows for the activation of PKC via cleavage of phosphoinositides into inositol-3 phosphate and diacylglycerol, leading to an increase in Ca2+ (42), and be- cause our previous findings showed that CD86 engagement on a CD40L/IL-4–primed B cell promoted the activation of PLCg2 and the downstream activation of PKCa/bII (19), we considered PKCa/bII phosphorylation a viable measurement of PLCg2 ac- tivity. We predicted that Phb1/2 alone would be necessary to in-

FIGURE 4. The cytoplasmic domain of CD86 also plays a role in me- by guest on September 29, 2021 duce CD86-dependent PKCa/bII phosphorylation independent of diating the effect of CD86 engagement on B cell function. (A) A series of the CD86 cytoplasmic domain because Phb1/2 alone were suffi- single-alanine point mutations (S285A, T291A, S303A) or cytoplasmic cient to allow for CD86-induced PLCg2 activation. To address this truncations (KKPD and 282D) were introduced into a FLAG-CD86 ex- issue, CH12.LX B cells were either mock transfected or trans- pression plasmid. (B) Either WT FLAG-CD86 or mutant/truncated plas- mids were transfected into CH12.LX B cells before the cells were primed fected with scrambled negative control shRNA or with Phb1/2- with CD40L/IL-4. Cells were cultured for 16 h before an anti-FLAG specific shRNA plasmids followed by priming with CD40L/IL-4 Ab (M2) or species- and isotype-matched control Ab (mouse IgG1 iso ctrl) and the addition of a CD86 Ab during a 60-min time course. The was added to the culture. Twenty-four hours and 4 d later, respectively, the levels of PKCa/bII phosphorylation (pPKCa/bII) relative to total levels of germline IgG1/Oct-2 mRNA and mature IgG1 mRNA were de- PKCa and total PKCbII protein were measured via immunoblot. termined by qRT-PCR. Values were normalized to actin and data are Whereas CH12.LX B cells that were mock transfected and expressed as mean fold change 6 SEM relative to WT mouse IgG1 isotype scrambled negative control shRNA produced a time-dependent control and represent data of quadruplicate replicates per condition from CD86-induced PKCa/bII phosphorylation relative to priming three independent experiments. Statistical analysis was used to determine alone, the effect was prevented in the presence of Phb1/2-specific statistical significance relative to WT anti-FLAG. *p , 0.05. shRNA relative to priming alone (Fig. 6A). Engagement of FLAG- CD86 on B cells transfected with both WT and KKPΔ FLAG- CD86 expression plasmids caused an increase in PKCa/bII phos- cytoplasmic-deficient FLAG-CD86 expression plasmid (KKPΔ) phorylation relative to a species- and isotype-matched control followed by priming with CD40L/IL-4 and CD86 or FLAG- Ab (Fig. 6B). Collectively, these findings indicate that whereas CD86 engagement. Phb1/2 protein knockdown efficiency and Phb1 and Phb2 are necessary for CD86-dependent activation of the level of PLCg2 phosphorylation were measured by immuno- PKCa/bII, a downstream signaling intermediate promoted through blot. The presence of Phb1/2-specific shRNA resulted in modest PLCg2 activity, the CD86 cytoplasmic domain is not involved, reductions of Phb1/2 protein where reduction of Phb1 resulted providing further support that CD86 engagement allows for dif- in the loss of Phb2 (Fig. 5A) and vice versa. Importantly, levels ferential activation of distal signaling intermediates. of Phb1/2 among mock-transfected cells versus cells transfected with scrambled negative control shRNA were comparable (Fig. 5A). Phb1/2 and the CD86 cytoplasmic domain are each required Whereas mock- and scrambled negative control shRNA–trans- for the CD86-dependent activation of NF-kB fected CH12.LX B cells promoted a time-dependent CD86- Although our findings suggested that Phb1/2 alone is sufficient to induced phosphorylation of PLCg2 relative to primed cells alone promote CD86-dependent PLCg2andPKCa/bII activation, the (Fig. 5B), PLCg2 phosphorylation upon CD86 engagement in the molecular role of the CD86 cytoplasmic domain remained un- presence of Phb1/2 shRNA remained comparable to priming alone known. Previous reports demonstrated that engagement of CD86 (Figs. 5C, 5D). Engagement of FLAG-CD86 on B cells transfected on the surface of a CD40L/IL-4–primed B cell initiates two 730 PROHIBITINS AND CYTOPLASMIC CD86 SIGNAL IN B CELLS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021 FIGURE 5. Phb1 and Phb2 are necessary for the CD86-induced activation of PLCg2. (A) CH12.LX B cells were either mock transfected with scrambled negative control or Phb1/2-specific shRNA plasmids via nucleofection for 24 h followed by priming with CD40L/IL-4 for 16 h. Phb1/2 protein levels were measured via immunoblot relative to GAPDH. Densitometry was performed and the data are expressed as the mean percentage change 6 SEM relative to mock and are representative of three independent experiments. (B) CH12.LX B cells were either mock transfected or transfected with scrambled negative control shRNA (neg ctrl shRNA) via nucleofection for 24 h followed by priming with CD40L/IL-4 for 16 h. A CD86 Ab (anti-CD86) was administered for 5, 15, 30, and 60 min. Levels of phospho-PLCg2(pPLCg2) protein relative to total PLCg2weremeasured via immunoblot. (C and D) Phb1 or Phb2 shRNA plasmids were transfected into CH12.LX B cells followed by CD40L/IL-4 priming, and a CD86 Ab (anti-CD86) was added as described above. Levels of phospho-PLCg2 protein were measured relative to total PLCg2 via immunoblot. (E)WTand CD86 cytoplasmic-deficient (KKPΔ) FLAG-CD86 plasmids were transfected into CH12.LX B cells via nucleofection and primed with CD40L/IL-4 for 16 h. Either an anti-FLAG Ab or species- and isotype-matched control Ab (iso ctrl Ab) was added for 15 min. Levels of phospho-PLCg2 protein were measured relative to total PLCg2 via immunoblot. Representative gels (B–E) are shown from three independent experiments. Densitometry was per- formed and measured phospho-PLCg2 relative to PLCg2 band intensity and the data are presented as the mean fold change in phospho-PLCg2from primed B cells where CD86 was engaged relative to priming alone (B–D)orisotypecontrolAb(E) and expressed as the mean fold change 6 SEM from three independent experiments. Statistical differences are shown relative to mock (A), priming alone (B–D), or isotype control Ab (E). *p , 0.05. (F) CH12.LX B cells were either mock transfected or transfected with scrambled negative control shRNA (neg ctrl shRNA) via nucleofection for 24 h followed by priming with CD40L/IL-4 for 16 h. A CD86 Ab (anti-CD86) was administered for 30 min. Levels of phospho-PLCg2(pPLCg2) protein relative to total PLCg2 were measured via in-cell ELISA. (G) WT and CD86 cytoplasmic-deficient (KKPΔ) FLAG-CD86 plasmids were transfected into CH12.LX B cells via nucleofection and primed with CD40L/IL-4 for 16 h. Either an anti-FLAG Ab or species- and isotype-matched control Ab (iso ctrl Ab) was added for 15 min. Levels of phospho-PLCg2 protein were measured relative to total PLCg2 via in-cell ELISA. (F and G) Phospho-PLCg2 protein OD values were normalized to total phopho-PLCg2 protein OD values and the data are represented as a mean fold change in phospho-PLCg2 protein from CD86-engaged B cells relative to primed alone B cells of each group (F)orisotypecontrol(G) and are expressed as the mean 6 SD of quintuplicate samples per condition from at least two independent experiments. Statistical differences are shown relative to priming alone of each group (F) or isotype control (G). *p , 0.05. signaling cascades that converge to activate the p50/p65 subunits transfected or transfected with scrambled negative control or of NF-kB (18, 19). Whereas one cascade is essential for CD86- Phb1/2-specific shRNA plasmids and either WT-FLAG CD86 or dependent IkBa phosphorylation (pIkBa), the other promotes CD86 cytoplasmic domain-deficient (KKPΔ)expressionplas- the phosphorylation of p65 (p-p65) in a PKCa/bII-andPLCg2- mids followed by CD40L/IL-4 priming and engagement of either dependent manner (18, 19). To determine which CD86-induced CD86 or FLAG-CD86 during a 60- to 90-min time course. The signaling intermediate controlled NF-kB activation, that is, IkBa level of IkBa and p65 phosphorylation and the total levels of and p65 phosphorylation, CH12.LX B cells were either mock IkBa protein were determined via immunoblot. We observed The Journal of Immunology 731 Downloaded from

FIGURE 6. Phb1 and Phb2 are necessary for the CD86-induced activation of PKCa/bII.(A) CH12.LX B cells were either mock transfected or transfected with a scrambled negative control shRNA (neg ctrl shRNA) or with Phb1 or Phb2 shRNA plasmids into CH12.LX B cells via nucleofection for 24 h followed by priming with CD40L/IL-4 for 16 h. A CD86 Ab (anti-CD86) was administered for 5, 15, 30, and 60 min. Levels of phospho-PKCa/bII (pPKCa/bII) protein relative to total PKCa and PKCbII were measured via immunoblot. (B) WT and CD86 cytoplasmic-deficient (KKPΔ) FLAG-CD86 plasmids were transfected into CH12.LX B cells via nucleofection and primed with CD40L/IL-4 for 16 h. Either an anti-FLAG Ab or species- and isotype- http://www.jimmunol.org/ matched control Ab (iso ctrl Ab) was added for 30 min. Levels of phospho-PKCa/bII protein relative to total PKCa and PKCbII were measured via immunoblot. Representative gels are shown from three independent experiments. Densitometry was performed and measured phospho-PKCa/bII relative to PKCa and PKCbII band intensity and the data are presented as the mean fold change in phospho-PKCa/bII from primed B cells where CD86 was engaged relative to priming alone (A) or iso ctrl Ab (B) and expressed as the mean fold change 6 SEM from three independent experiments. Statistical differences are shown relative to mock priming alone (A) or isotype control Ab (B). *p , 0.05. a time-dependent increase in the level of phosphorylated IkBa NF-kB activation, it was likely that both Phb1/2 and the CD86 and a time-dependent decrease in the level of total IkBa protein cytoplasmic domain were required to promote CD86-dependent by guest on September 29, 2021 upon engagement of CD86 when both Phb1/2 and the CD86 p65 nuclear localization. To address this issue, CH12.LX B cells cytoplasmic domain were intact, whereas Phb1/2 depletion and were either mock transfected or transfected with scrambled neg- the lack of the CD86 cytoplasmic domain prevented a CD86- ative control shRNA and Phb1/2-specific shRNA plasmids to de- dependent increase in the level of IkBa phosphorylation and plete Phb1/2, and either WT-FLAG CD86 or CD86 cytoplasmic decrease in the level of total IkBa protein (Fig. 7). Furthermore, domain-deficient (KKPΔ) plasmids followed by CD40L/IL-4- the CD86-induced increase in p65 phosphorylation was pre- priming and either CD86 or FLAG-CD86 engagement for 90 min. vented when Phb1/2 was depleted (Fig. 7A) and when CD86 Nuclear enriched protein lysates were prepared and levels of p65 lacked the cytoplasmic domain (Fig. 7B) relative to either phosphorylation and total p65 protein levels relative to nuclear priming alone or isotype control Ab, respectively. However, we membrane proteins Lamin A/C were measured via immunoblot. speculated that the signaling intermediates Phb1 and Phb2, Whereas phosphorylated and total p65 nuclear localization in- which were essential for PLCg2andPKCa/bII activation, may creased upon CD86 engagement when both Phb1/2 and the CD86 also prevent CD86-induced p65 phosphorylation because previ- cytoplasmic were intact, the effect failed to occur upon CD86 ous findings showed that PKCa/bII-dependent p65 phosphory- engagement in the absence of the intermediates (Fig. 8). Fur- lationoccurreddistaltoIkBa phosphorylation (18). Taken thermore, successful cytoplasmic- and nuclear-enriched protein together, these findings indicated that both Phb1/2 and the CD86 lysate fractionation was demonstrated via the presence of a-tu- cytoplasmic domain are necessary for the CD86-dependent ac- bulin, b-actin, and GAPDH in cytoplasmic-enriched protein tivation of NF-kBviaIkBa phosphorylation/degradation, fol- lysates and the presence of Lamin A/C in nuclear-enriched protein lowed by p65 phosphorylation. lysates (Supplemental Fig. 4). Thus, our findings indicated that Phb1/2 and the CD86 cytoplasmic domain were necessary to Phb1/2 and the CD86 cytoplasmic domain are each required promote the CD86-dependent nuclear localization of p65 and, for the CD86-dependent nuclear localization of NF-kB (p65) therefore, extended the finding that both Phb1/2 and the CD86 Although our findings suggested that both Phb1/2 and the CD86 cytoplasmic domain are each required for the CD86-dependent cytoplasmic domain were required for the CD86-induced activation NF-kB activation. of NF-kB, what remained unclear was whether the intermediates Based on the findings of the present study and our previous were necessary for NF-kB (p65) nuclear entry promoted via CD86 reports, we propose the following CD86 signaling model (Fig. 9): engagement. Because previous findings from our laboratory showed Upon CD86 engagement, Phb1/2 and the CD86 cytoplasmic do- that CD86 engagement promoted an increase in the level of p65 main promote the phosphorylation of IkBa, allowing for the ac- phosphorylation present in the nucleus (18), and findings in the tivation of NF-kB p50/p65. Concurrently, Phb1/2 alone promote present study indicated that both Phb1/2 and the CD86 cyto- CD86-induced PLCg2 phosphorylation, allowing for subsequent plasmic domain were required to promote CD86-dependent PKCa/bII activation and phosphorylation of p65. NF-kB p50/p65 732 PROHIBITINS AND CYTOPLASMIC CD86 SIGNAL IN B CELLS Downloaded from http://www.jimmunol.org/

FIGURE 7. Phb1/2 and the CD86 cytoplasmic domain are each required for the CD86-dependent activation of NF-kB. (A) CH12.LX B cells were mock transfected, transfected with either scrambled negative control shRNA or with Phb1 or Phb2-specific shRNA plasmids via nucleofection for 24 h, followed by priming with CD40L/IL-4 for 16 h. The cells were then resuspended in serum-free conditions for at least 30 min. A CD86 Ab (anti-CD86) was added to cell cultures for 5, 15, 30, 60, and 90 min. Levels of phospho-IkBa (pIkBa), total IkBa, and phospho-p65 (p-p65) protein were measured by guest on September 29, 2021 relative to GAPDH or total p65 via immunoblot. (B) WT or CD86 cytoplasmic-deficient (KKPΔ) FLAG-CD86 plasmids were transfected into CH12.LX B cells via nucleofection and primed with CD40L/IL-4 for 16 h. An anti-FLAG Ab was added for 5, 15, 30, and 60 min relative to a species- and isotype-matched control Ab (iso ctrl Ab). Levels of phospho-IkBa,totalIkBa, and p-p65 protein were measured relative to GAPDH or total p65 via immunoblot. Representative gels are shown from three independent experiments. Densitometry was performed and measured pIkBa,totalIkBa,and p-p65 band intensity relative to GAPDH, and p-p65 band density relative to total p65 and the data are presented as the mean fold change in phospho- IkBa,totalIkBa, or p-p65 from primed B cells where CD86 was engaged relative to priming alone (A) or isotype control Ab (B) and expressed as the mean fold change 6 SEM from at least three independent experiments. Statistical differences are shown relative to priming alone (A) or isotype control Ab (B). *p , 0.05. then initiates Oct-2 transcription, which ultimately regulates 39- Phb2 and its closely related homolog Phb1 are evolutionarily IgH enhancer activity to elevate the rate of IgG1 transcription and conserved, multifunctional proteins that are expressed in most cell the level of IgG1 produced by a B cell. types and typically reside in a variety of cellular compartments, including the inner mitochondrial and plasma membranes (30). Discussion Both Phbs have previously been found to be associated with the In this study, we show for the first time, to our knowledge, that BCR (43) and they were recently found to colocalize with CD3 of expression of the tyrosine-containing adaptor/scaffolding proteins the TCR complex (44). Phb2 is also known as either Bap37 (BCR- Phb1 and Phb2 increases in a CD40L/IL-4–primed B cell to as- associated protein of 37 kDa) or REA (nuclear repressor of es- sociate with CD86, and that depletion of these proteins renders trogen receptor activity), whereas Phb1 is also referred to as CD86 unable to mediate an enhancement in the level of Oct-2 and Bap32 (BCR-associated protein of 32 kDa). Phbs are a family of IgG1 produced. To our knowledge, we also show for the first time proteins that possess various cellular and molecular functions (30, that Phb1/2 and the CD86 cytoplasmic domain are required for the 45, 46) that have been found to function independently of each CD86-induced phosphorylation of IkBa, which we previously re- other (30, 45), except within the context of the mitochondrial ported leads to NF-kB p50/p65 activation (18), whereas only Phb1 membrane, where they seem to function together (40, 47–49), and and Phb2 were required for the CD86-induced phosphorylation of when they associate with the BCR and CD3 of the TCR complex, PLCg2, which we previously reported leads to PKCa/bII activation where their function remains less well understood. Taken together and NF-kB (p65) phosphorylation (19). Thus, Phb1/2 association with findings that Phb1/2 residues can be phosphorylated, in- with CD86 and expression of an intact CD86 cytoplasmic domain cluding tyrosines (50–53), we considered Phb1 and Phb2 as viable are each necessary to mediate CD86 signaling function to enhance candidates to mediate CD86 signaling function. the level of the Oct-2/IgG1 response in a primed B cell through the Previous reports demonstrated that NF-kB (p65) phosphoryla- differential activation of distal signaling intermediates. tion is regulated via PKCa/bII, and that PKCa/bII activation is The Journal of Immunology 733 Downloaded from http://www.jimmunol.org/

FIGURE 8. Phb1/2 and the CD86 cytoplasmic domain are each required for the CD86-dependent nuclear localization of NF-kB (p65). (A) CH.12LX B by guest on September 29, 2021 cells were transfected either under mock conditions or with scrambled negative control shRNA or Phb1/2-specific shRNA plasmids via nucleofection for 24 h followed by CD40L/IL-4 priming for an additional 16 h. The cells were then resuspended in serum-free conditions for at least 30 min. A CD86 Ab (anti-CD86) was added to cell cultures for 90 min. Nuclear-enriched protein lysates were prepared and the level of p65 phosphorylation (p-p65) and total levels of p65 protein present in the nucleus were measured via immunoblot relative to Lamin A/C. (B) WT or CD86-cytoplasmic deficient (KKPΔ) FLAG-CD86 plasmids were transfected into CH12.LX B cells via nucleofection and primed with CD40L/IL-4 for 16 h. An anti-FLAG Ab was added for 90 min relative to a species- and isotype-matched control Ab (iso ctrl Ab). The level of p65 phosphorylation and total levels of p65 present in the nucleus were determined via immunoblot. Densitometry was performed and measured p-p65 and total p65 band intensity present in the nucleus relative to Lamin A/C and the data are presented as the mean fold change in p-p65 or total p65 from primed B cells where CD86 was engaged relative to priming alone (A) or isotype control Ab (B) and expressed as the mean fold change 6 SEM from three independent experiments. Statistical differences are shown relative to priming alone (A) or isotype control Ab (B). *p , 0.05.

PLCg2-dependent on CD86 engagement on a B cell (18, 19). Data IkBa phosphorylation, independently of the CD86 cytoplasmic in the present study showed that the CD86-induced PLCg2 domain. However, data in this study showed that both an intact phosphorylation failed to occur when Phb1/2 was reduced, but not CD86 cytoplasmic domain and Phb1/2 are required to activate when the CD86 cytoplasmic tail was deleted. Alternatively, CD86- IkBa upon CD86 engagement, suggesting that cross talk may induced IkBa phosphorylation failed to occur when either Phb1/2 exist between CD86-associated Phb1/2 and the cytoplasmic do- was reduced or the CD86 cytoplasmic domain was deleted, indi- main for IkBa activation. Data also showed that all alanine sub- cating that both were required for NF-kB activation. The present stitutions of the PKC phosphorylation sites within the CD86 evidence indicated that both Phb1/2 and the cytoplasmic domain cytoplasmic domain impaired CD86 function, with S303A causing of CD86 on a B cell, although not directly associated, appear to the most significant reduction of CD86-induced Oct-2 and mature each be necessary to elicit CD86-induced back signaling to en- IgG1 mRNA, suggesting that phosphorylation of the CD86 cyto- hance the level of Oct-2 and IgG1 responses. Previous reports plasmic domain may serve as a potential docking site for an un- showed in B cells that engagement of cell surface receptors that known common signaling intermediate that may also interact lack cytoplasmic tyrosine residues, including the BCR (54, 55) with Phb1/2 to regulate phosphorylation of IkBa. Although the and CD40 (56, 57), used adaptor protein complexes to signal in- mechanistic link between Phb1/2 and the CD86 cytoplasmic do- tracellularly to activate NF-kB. Because the CD86 cytoplasmic main is unclear at present, each appears to be required to promote domain is devoid of tyrosine residues, and because our evidence CD86-dependent phosphorylation of IkBa and the activation of suggested that a novel association exists between CD86 and Phb1/ NF-kB p50/p65. 2, which does expresses tyrosine residues, we initially thought is Previous reports showed that both Phb1 and Phb2 become was possible that Phb1/2 alone might mediate CD86-dependent tyrosine phosphorylated within their C-terminal domains upon 734 PROHIBITINS AND CYTOPLASMIC CD86 SIGNAL IN B CELLS

discrepancy could be due to a mixed population of Phb1/2 shRNA plasmids, as were used in the present study that targeted multiple regions of Phb1/2 transcripts, because Phb1 and Phb2 are highly homologous (40), thus promoting a higher likelihood of sup- pressing mRNA translation. To our knowledge, the present findings using RNA interference are the first to identify a role for Phb1 and Phb2 in mediating the CD86- induced enhancement of the levels of Oct-2 and IgG1 produced by a CD40L/IL-4–primed B cell. Phb1/2 shRNA-mediated depletion in the present study led to modest reductions in Phb1/2 protein levels, suggesting that only a modest amount of Phb1/2 protein in the cell is required to mediate signaling events induced by CD86, whereas a greater proportion is necessary to regulate mitochondrial function. This is supported by previous reports that showed Phb1/2 depletion resulted in the loss of mitochondrial membrane potential (50, 62), even though other reports have shown Phb1/2 depletion did not affect mitochondrial membrane potential (61, 63). Also, by using gene silencing, Phb1 was shown to have functional roles in cell

FIGURE 9. CD86 signaling model. Dotted arrows indicate proposed migration (64), regulation of angiogenic activity and cellular se- Downloaded from novel signaling events identified in the present study. CD86 engagement nescence (62), and cellular metabolic activity (65) upon engage- promotes the phosphorylation of IkBa via mechanisms regulated by ment of various cell surface receptors. Likewise, gene silencing of Phb1/2 and the CD86 cytoplasmic domain allowing for the activation of Phb2 indicated a functional role for Phb2 in muscle cell differ- NF-kB p50/p65. Concurrently, Phb1/2 alone promotes the CD86-induced entiation (66). Also, Phb1 and Phb2 have been reported to asso- PLCg2 phosphorylation, allowing for subsequent PKCa/bII activation and phosphorylation of p65. NF-kB p50/p65 is now poised to initiate ciate with the BCR (43) and, recently, CD3 of the TCR complex

Oct-2 transcription, which ultimately regulates 39-IgH enhancer activity (44), although the exact functional roles for the associations re- http://www.jimmunol.org/ to increase the rate of IgG1 transcription and the level of IgG1 produced main unclear. Furthermore, a direct protein/protein interaction has by a B cell. been shown to occur between Phb1 and Syk upon BCR engage- ment (41), whereas another report showed a decrease in MAPK signaling upon CD3 engagement when Phb1/2 function was engagement of cell surface receptors such as the TCR and CD28 blocked, indicating that Phb1/2 may serve functional roles in (50), whereas Phb1 alone was shown to undergo tyrosine phos- BCR/TCR signaling. Thus, the present findings using RNA in- phorylation upon insulin receptor engagement (51–53, 58). Re- terference provide further support that Phb1 and Phb2 participate cently, it was found that Phb1 and Phb2 are linked to the MAPK in mediating cell surface receptor–induced regulation of cellular signaling cascade upon CD3 engagement in T cells (44). There- functions. by guest on September 29, 2021 fore, it was possible that Phb1/2 may serve as critical interme- We originally hypothesized that the cytoplasmic domain of diates for the CD86-induced activation of PLCg2. We also CD86 associated directly with Phb1/2 to mediate CD86 signaling proposed that the CD86 cytoplasmic domain may not be in- function. However, our data showed that Phb1/2 associated with volved with the CD86 induction of PLCg2 owing to the lack CD86 even in the absence of the CD86 cytoplasmic domain, of tyrosine residues within this domain that would be necessary suggesting a transmembrane-specific interaction. The ability of for PLCg2 binding. Because prior reports revealed that PLCg2 CD86 to bind to transmembrane proteins is supported by a recent becomes recruited to tyrosine-containing proteins upon cell study in dendritic cells where it was shown that CD86 associated surface receptor engagement (25, 26), our findings indicate with an E3 ubiquitin ligase in the presence or absence of the CD86 a potential role for Phb1/2 alone in mediating CD86-induced cytoplasmic domain (67). It is also supported by previous reports PLCg2 phosphorylation, independent of the CD86 cytoplasmic showing that Phb1 and Phb2 exist as transmembrane proteins that domain, because Phb1 and Phb2 contain tyrosine residues, function to stabilize proteins in the electron transport chain and to whereas the cytoplasmic domain does not. Also, it was reported mediate functional changes induced by cell surface receptors (40, that Phb1 associates with Syk upon BCR engagement (41). 47–49, 62, 64–66). Taken together, these findings support the Because Syk has been reported to regulate the activation of proposal that CD86 associates with Phb1/2 via transmembrane- PLCg2 via the adaptor protein B cell linker protein upon BCR specific interactions in B cells. engagement (59), it is possible that Phb1 and Phb2 serve as a Findings from the present study may help us to understand the scaffolding complex to link CD86 engagement to PLCg2 activa- mechanism by which CD86 signals directly to a B cell to increase tion. Nonetheless, the modest interdependent reduction in Phb1/2 not only IgG1, but also IgE production, as shown previously (13, protein levels in the present study was sufficient to prevent CD86 16). Additionally, understanding this novel signaling mechanism induction of IgG1 and proximal signaling intermediates, suggesting mayhelpdirecttherationaldesignofCD86-targetedtherapeutic that Phb1 and Phb2 are each required to mediate CD86-induced strategies to increase the level of protective Ab in immunocom- signaling in B cells. promised patients. Conversely, CD86-targeted therapeutic strate- In this study, we used RNA interference to silence Phb1/2 be- gies might also be developed to decrease the level of harmful Abs cause both Phb1- and Phb2-deficient mice are embryonic lethal. produced in certain cases of autoimmunity or allergic asthma. Previous reports revealed that Phb1 and Phb2 protein expression levels were interdependent, with depletion of one causing the loss Acknowledgments of the other (60, 61), which is consistent with findings in the We thank Dr. Alan Smith and Matthew Gormley for critical experimental present study. Phb1/2 mRNA levels, however, were reported to be support and Todd Shawler for assistance with flow cytometry. We also comparable in the presence of non-gene–specific Phb1/2 siRNA thank Caroline Padro and Sara McMaken for critical evaluation of the (61), which is in contrast with findings of the present study. The manuscript. The Journal of Immunology 735

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