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The Journal of Immunology, 2006, 176: 6727-6735.
Copyright © 2006 by The American Association of Immunologists

CD86 Stimulation on a B Cell Activates the Phosphatidylinositol 3-Kinase/Akt and Phospholipase C{gamma}2/Protein Kinase C{alpha}beta Signaling Pathways1

Nicholas W. Kin and Virginia M. Sanders2

Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, OH 43210


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Stimulation of CD86 on a CD40L/IL-4-activated murine B cell increases the rate of mature IgG1 transcription by increasing the level of NF-{kappa}B activation, as well as Oct-2 expression and binding to the 3'-IgH enhancer. The signal transduction pathway activated by CD86 proximal to NF-{kappa}B activation is unknown. In this study, we show that CD86 stimulation on an activated B cell increases the activity of PI3K and the phosphorylation of phosphoinositide-dependent kinase 1, Akt, and I{kappa}B kinase {alpha}beta. In addition, CD86 stimulation induces an increase in the phosphorylation of phospholipase C{gamma}2 and protein kinase C {alpha}beta. CD86-mediated activation of these two signaling pathways leads to increased Oct-2 expression, increased gene activity mediated by NF-{kappa}B and 3'-IgH enhancer increased activity. These results identify a previously unknown signaling pathway induced by CD86 to regulate the level of B cell gene expression and activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Also known as B7-2, CD86 is a costimulatory molecule belonging to the Ig superfamily (1). It is a 70-kDa glycoprotein that was cloned in 1993 as a counterreceptor for CD28 and CTLA-4 (2, 3), both of which are expressed on a T cell. CD86 is primarily expressed on APCs, including B cells, dendritic cells, and macrophages (2, 3). Resting naive B cells express CD86 at low levels (2), but up-regulate it following stimulation of the BCR (2), MHC class II (4), CD40 receptor (5), IL-4R (6), LPS receptor (7, 8), and the beta2-adrenergic receptor (9, 10). CD86 on a B cell interacts with CD28 on a T cell, increasing T cell expression of CD40L and secretion of IL-4, which interact with CD40 and the IL-4R, respectively, on a B cell to induce class switch recombination to IgG1 (11, 12). The interaction of APC-bound CD86 with T cell-bound CD28 is required for optimal T cell activation (13) and for generation of an optimal IgG1 response (14). Thus, CD86 was classified as a costimulatory molecule required for CD28 stimulation, but was thought to possess no signaling potential itself due to a lack of identifiable docking sites within the cytoplasmic tail.

However, CD86 possesses three putative PKC phosphorylation sites in its cytoplasmic tail (15), suggesting that it may be able to transduce a signal directly within the APC. Recent data show that stimulation of CD86 on a B cell increases the rate of IgG1 production (9, 16, 17), as well as the level of antiapoptotic factors (18), CD80 expression (19), NF-{kappa}B activation (20), and Oct-2 expression and binding to the 3'-IgH enhancer (20). NF-{kappa}B, a family of transcription factors, consists of five major subunits, NF-{kappa}B1 (p105/p50), NF-{kappa}B2 (p100/p52), Rel A (p65), Rel B, and c-Rel, that form various homodimeric and heterodimeric complexes (reviewed in Ref. 21). NF-{kappa}B remains sequestered in the cytoplasm of a resting cell in a complex with I{kappa}B proteins. Upon activation of the classical NF-{kappa}B pathway, I{kappa}B proteins are phosphorylated, polyubiquitinated, and degraded within the proteosome, causing the release of NF-{kappa}B dimers that translocate to the nucleus to regulate gene activity. Recent reports indicate that CD86 stimulation on a B cell activates the classical NF-{kappa}B pathway, leading to a protein kinase C (PKC)3-independent phosphorylation and degradation of I{kappa}B{alpha}, and subsequent nuclear localization of p50/p65 (20). In addition, CD86 is reported to increase the phosphorylation of p65 in a PKC-dependent manner (20).

However, the signaling components activated proximal to NF-{kappa}B activation are unknown. Because NF-{kappa}B is involved in numerous biological responses, therapeutic interventions aimed at either suppressing or enhancing Ab production via CD86 signaling would need to target the specific intermediates activated by CD86 upstream of NF-{kappa}B activation. In this study, we report the CD86 signal transduction pathway in a B cell that is proximal to the activation of NF-{kappa}B and the regulation of gene activity by these intermediates. We show for the first time that CD86 increases the activity of PI3K, as well as the phosphorylation state of phosphoinositide-dependent kinase 1 (PDK-1), Akt, I{kappa}B kinase (IKK) {alpha}beta, phospholipase C (PLC) {gamma}2, and PKC{alpha}beta, to increase Oct-2 expressions gene activity mediated by NF-{kappa}B, and 3'-IgH enhancer activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines

CH12.LX is a murine B cell lymphoma line that has been described previously (22) and was provided by Dr. G. Bishop (University of Iowa, Iowa City, Iowa). A20 ({gamma}2b-hs1–4) is a B lymphoma cell line that has been stably transfected with a {gamma}2b reporter gene under the control of the 3'-IgH enhancer, as described previously (23), and was provided by Dr. L. Eckhardt (Hunter College, New York, NY).

Animals

Female BALB/c (H-2d) and PI3K-deficient (PI3K–/–) mice were purchased from Taconic Farms. CD86-deficient (CD86–/–) mice (H-2d) were provided by Dr. A. Sharpe (Brigham and Woman’s Hospital, Boston, MA) and were bred and housed within the pathogen-free facility at Taconic Farms. C3H/HeJ and TLR4-deficient mice (TLR4–/–) were purchased from The Jackson Laboratory. Upon arrival at The Ohio State University, all mice were housed in microisolator cages within a laminar flow barrier and provided autoclaved food and water ad labium. Mice were used at 7–8 wk of age and all experiments complied with the Animal Welfare Act and the National Institutes of Health guidelines for the care and use of animals in biomedical research.

Resting B cell isolation and activation

Murine spleens were collected, splenocytes were isolated, and RBC were lysed using 0.4% ammonium chloride. The splenocytes were negatively sorted with rat anti-mouse CD43 magnetic beads (Miltenyi Biotec) following the manufacturer’s directions (Miltenyi Biotec) using the AutoMacs machine (Miltenyi Biotec). Resting B cells (CD43–) were cultured at 5 x 105 cells/ml in either a 96-, 24-, or 6-well plate in a final volume of 0.2, 2.0, and 5.0 ml of culture medium, consisting of RPMI 1640 medium (CellGro), 10% FBS (Atlas Biologicals), 20 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 50 µM 2-ME, in a humidified atmosphere at 37°C with 5% CO2. Resting B cells were activated in the presence of CD40L-expressing Sf9 cells (CD40L), prepared as described previously (16), at a B cell to Sf9 cell ratio of 10:1, and IL-4 (1 ng/ml (eBioscience). After 16 h, either a CD28/Ig fusion protein (R&D Systems), a rat anti-mouse CD86 Ab (clone PO3 (eBioscience)), a species- and isotype-matched control Ab (rat IgG2b, {kappa}, clone A95-1; BD Pharmingen), or a recombinant human IgG1 Fc (R&D Systems) was added at a final concentration of 1 µg/ml. Pharmacologic inhibitors were added 30 min before stimulation of CD86, including the PI3K inhibitors LY-294002 (5 µM; Biomol Research Laboratories) and wortmannin (50 nM; BIOMOL), the PKC inhibitors calphostin C (0.5 nM; BIOMOL) and GF-109203X (5 nM; Biomol), the IKK inhibitors SC-514 (100 µM (Calbiochem) the IKK inhibitor peptide (50 µg/ml (Calbiochem)) and the IKK control peptide (50 {upsilon}g/ml (Calbiochem)), and the PDK-1 inhibitor OSU-03012 (5 µM (obtained from Dr. C.-Shih Chen, Ohio State University, Columbus, OH)). All reagents used were negative for the presence of endotoxin, as determined by Etoxate (Sigma-Aldrich), a Limulus lysate assay with a level of detection <0.1 U/ml.

Western blot

Resting B cells (5–10 x 106 cells) were activated as described above. Cells were collected and lysed with 250 µl of 1x lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, 10 nM okadaic acid, and 10 nM tautomycin). Protein samples (5–20 µg) were run on a denaturing 7.5% polyacrylamide gel and transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore). Membranes were blocked with TBST (140 mM NaCl, 25 mM Tris-HCl (pH 7.5), 3 mM KCl, and 0.05% Tween 20) plus 5% dried milk for 1 h at room temperature, probed with primary Abs diluted in TBST plus 5% dried milk for 2 h at room temperature or overnight at 4°C. Membranes were probed with HRP-labeled secondary Abs diluted in TBST plus 1% dried milk at room temperature for 1 h. HRP-labeled Abs were detected using the LumiGlo Detection kit (Cell Signaling) and specific bands were visualized on Kodak Biomax MS film using an intensifying screen enabled film cassette. Abs used were goat anti-human actin C-11 (Santa Cruz Biotechnology), rabbit anti-mouse I{kappa}B-{alpha} Ab, rabbit anti-mouse phospho-I{kappa}B-{alpha} (Ser32), rabbit anti-human phospho-PLC{gamma}2 (Tyr1217), rabbit anti-human PLC{gamma}2, rabbit anti-human phospho-PKC{alpha}beta (Thr638/641), rabbit anti-human phospho-IKK{alpha}beta, rabbit anti-mouse Akt, rabbit anti-mouse phospho-Akt (Thr308), rabbit anti-mouse phospho-Akt (Ser473), and rabbit anti-human phospho-PDK1 (Ser241) (Cell Signaling). All anti-human Abs cross-react with mouse.

PI3K ELISA

Resting naive B cells were activated as described above and total cellular protein was isolated 10–30 min after addition of CD28/Ig. Five microliters of a rabbit anti-mouse PI3K Ab (Upstate Biotechnology) was added and samples were rocked for 1 h at 4°C. Sixty microliters of a 50% protein A-agarose beads in PBS was added and samples were rocked for 1 h at 4°C. Samples were collected and washed three times with buffer A (137 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM CaCl2, 1 mM MgCl2, and 0.1 mM sodium orthovanadate) plus 1% Nonidet P-40, three times with (0.1 M Tris-HCl (pH 7.4), 5 mM LiCl, and 0.1 mM sodium orthovanadate), and two times with TNE (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 0.1 mM sodium orthovanadate). The immunoprecipitated proteins were used to analyze the PI3K activity using the PI3K ELISA (Echelon BioSciences). The level of PI3K activity in each treatment group was quantitated using a standard curve of known PI3K activity.

Quantitative real-time PCR

Quantitative real-time PCR was performed as described previously (16). Briefly, a common master mix (LightCycler-FastStartDNA SYBR Green I (Roche), 2 mM MgCl2, 0.5 µM gene-specific primer) was used and the concentration of gene-specific cDNA was quantified using a standard curve diluted 1/10 for a concentration range of 1 ng/ml to 1 fg/ml. A melting curve was generated after each real-time reaction and samples were run on a 1.0% agarose gel to ensure that only one gene-specific PCR product was generated. Real-time PCR was preformed using the Roto-gene 2000 real-time Cycler (Phenix Research Products). The following primers were used. beta-Actin 5'-TACAGCTTCACCACCACAGC-3' and 5'- AAGGAAGGCTGGAAAAGAGC-3' (annealing temperature 60°C, 206-bp product); Oct-2 5'-ATCAAGGCTGAAGACCCCAGTG-3' and 5'- TGGAGGAGTTGCTGTATGTCCC-3' (annealing temperature 60°C, 128-bp product); mature IgG1 transcript 5'-TATGGACTACTGGGGTCAAG-3' and 5'-CCTGGGCACAATTTTCTTGT-3' (annealing temp 63°C, 205-bp product).

ELISA

A20 cells were collected on day 5 after activation and lysed with a 0.5% Nonidet P-40 lysis buffer and samples were frozen at –80°C until analysis. Briefly, Costar 96-well flexi plates (Fisher Scientific) were coated with goat anti-mouse IgG (2 µg/ml) (BD Biosciences), followed by blocking with 20% FBS solution in PBS plus 0.02% azide. Twenty microliters of each sample was incubated on the plate, a standard curve for IgG2b was prepared using known quantities of recombinant IgG2b protein in a range of 1 µg/ml to 1 ng/ml. A secondary Ab, goat anti-mouse IgG2b (BD Biosciences), that was linked to alkaline phosphatase was used for detection. p-Nitrophenyl phosphate (Sigma-Aldrich) was added and color development was determined on a Spectramax Plus microplate reader (Molecular Devices) at a wavelength of 405 nm.

Chromatin immunoprecipitation (ChIP)

ChIP analysis was conducted as described previously with minor modification (20). Briefly, B cells (10 x 106 cells) were activated as described above and collected on day 3, fixed for 20 min on ice with one-tenth the volume of 11% formaldehyde solution (in 0.1 M NaCl, 1 mM EDTA, 0.5 mM EGTA and 50 mM HEPES (pH 8.0)), and cross-linking was stopped by the addition of glycine at a final concentration of 0.125 M for 5 min. Cells were rinsed with cold PBS and resuspended in 10 ml of lysis buffer (50 mM HEPES-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Nonidet P-40, 0.25% Triton X-100 and the following protease inhibitors: 1 ng/ml leupeptin, and 5 ng/ml aprotinin) and were gently rocked for 10 min at 4°C. Nuclei were pelleted, resuspended, and gently rocked for 10 min at room temperature in buffer 2 (0.2 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl (pH 8.0) and protease inhibitors). The nuclei were pelleted again and resuspended in 6 ml of sonication buffer (1 mM EDTA, 0.5 mM EGTA and 10 mM Tris-HCl (pH 8.0) and protease inhibitors). The suspension was sonicated 10 times for 30 s, with a 1-min cooling period on ice in-between. Debris was removed from samples and 250 µl were adjusted to 1% Triton X-100, 0.1% sodium deoxycholate and protease inhibitors in a final volume of 500 µl of TE buffer (10 mM Tris (pH 8) and 1 mM EDTA), and precleared with protein A/protein G agarose beads that had been blocked with sonicated salmon sperm DNA and 10 mg/ml BSA for 3 h with gentle rocking at 4°C. The beads were removed and chromatin samples were incubated at 4°C with various Abs overnight. Immunocomplexes were precipitated for 3 h by the addition of blocked protein A/protein G agarose beads. The precipitates were washed seven times for 5 min each with 1 ml of radioimmunoprecipitation assay buffer (50 nM HEPES at pH 7.6, 1 mM EDTA, 0.7% sodium deoxycholate, 1% Nonidet P-40, 0.5 M LiCl, and protease inhibitors) and resuspended in 100 µl of TE buffer. The samples were adjusted to 0.5% SDS, 100 µg/ml RNase A, and 200 µg/ml proteinase K and incubated at 55°C for 3 h, followed by an overnight incubation at 65°C to reverse the formaldehyde cross-links. The DNA was purified by phenol-chloroform extraction, precipitated in the presence of 20 µg of glycogen and resuspended in 100 µl of TE buffer.

PCR was done with 2 µl of the immunoprecipitated DNA for 30 cycles (45 s at 95°C, 45 s at 56°C, and 2 min at 72°C, completed by 10 min in 72°C) with various primers. As a control, the PCR was done directly on input DNA purified from chromatin before immunoprecipitation. PCR products were resolved on 1.5% agarose gels and visualized with ethidium bromide. The Abs used were anti-p50 and anti-p65, and anti-CREB as a control Ab (Santa Cruz Biotechnology). The following primers were used, hs4, 5'-AGAACAGGAACCACAGAGCAGAGG-3' and 5'-GGTCATTGAAACTCATCCATAGCC-3' (225-bp product).

Transfections

The NF-{kappa}B-sensitive wt and mut luciferase reporter plasmids were provided by Dr. D. Guttridge (Ohio State University, Columbus, OH) and the 3'-IgH enhancer plasmids were provided by Dr. L. Eckhardt (Hunter College, New York, NY) and were described in detail previously (24). Transfections were performed using program K-03 of the nucleofector device (Amaxa) following manufacturers directions. Briefly, 5 x 106 CH12.LX cells were combined with 5 µg of experimental plasmid DNA, 1 µg of a control pRL-TK Renilla plasmid (Promega), and 100 µl of transfection solution (Amaxa). Program K-03 was used for the electroporation and cells were cultured as described above until analysis.

Transfection isolation and reporter gene assay

Cells were isolated at various time points (24–48 h) after transfection using 1x lysis buffer from the Dual-Luciferase Reporter Assay System (Promega). The luciferase assays were performed following the manufacturer’s directions. Briefly, cells were collected, washed two times with PBS and lysed with 1x passive lysis buffer (Promega). Twenty microliters of cell lysate were combined with 100 µl of Luciferase Assay Substrate (Promega). Firefly luciferase activity was measured on a luminominar. One-hundred microliters of the Stop and Glo Reagent (Promega) were added and Renilla luciferase activity was measured. The amount of firefly luciferase activity is normalized to the amount of Renilla luciferase activity as a transfection control.

Statistics

Data with three or more groups were analyzed by a one-way ANOVA followed by post hoc analysis, while data with two groups were analyzed by a two-tailed paired t test to determine whether an overall statistically significant change existed. Statistically significant results were determined by a p value of <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD86 activates IKK{alpha}beta

CD86 activates the classical NF-{kappa}B pathway through increased I{kappa}B{alpha} phosphorylation and degradation, and subsequent nuclear localization of p50 and p65 (20). However, the signaling intermediates activated by CD86 proximal to NF-{kappa}B activation are unknown. A hallmark of the classical NF-{kappa}B pathway is the phosphorylation and degradation of I{kappa}B{alpha}. The IKK complex, which consists of two catalytic (IKK{alpha} and IKKbeta) and one regulatory (IKK{gamma}) subunits, is responsible for I{kappa}B{alpha} phosphorylation and requires its own phosphorylation for activation (reviewed in Ref. 21). To determine whether CD86 stimulation activates the IKK complex, B cells were activated with CD40L/IL-4 for 16 h before addition of CD28/Ig and total protein was isolated and analyzed using Western blot analysis. CD86 maximally increased the phosphorylation of IKK{alpha}beta within 5 min by ~2.5-fold above CD40L/IL-4 alone, and remained 2-fold above baseline throughout 30 min (Fig. 1a). In contrast, CD86-deficient B cells failed to change the phosphorylation state of IKK{alpha}beta (Fig. 1b). Thus, these data show CD86 increases the level of IKK{alpha}beta phosphorylation, suggesting that it is the signaling intermediate proximal to, and responsible for, the increase in I{kappa}B{alpha} phosphorylation.


Figure 1
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  		FIGURE 1. CD86 signals through IKK{alpha}beta. Western blot analysis of phospho-IKK{alpha}beta and total actin in (a) Wt and (b) CD86-deficient B cells activated with CD40L/IL-4 for 16 h before addition of CD28/Ig from one representative experiment of three. Band density was determined using densitometry and the data represent the mean fold increase in phospho-IKK{alpha}beta normalized to actin ± SEM from three independent experiments. c, Western blot analysis of phospho-I{kappa}B{alpha} and total I{kappa}B{alpha} in B cells activated with CD40L/IL-4 for 16 h and then pretreated with an IKK inhibitor peptide for 30 min before addition of CD28/Ig. Band density was determined using densitometry and the data represent the mean fold increase in phospho-I{kappa}B{alpha} normalized to I{kappa}B{alpha} ± SEM from three independent experiments. d, Real-time PCR analysis of Oct-2 mRNA and total actin in B cells activated with CD40L/IL-4 for 16 h and then pretreated with either an IKK inhibitor peptide, IKK control peptide, or the IKK inhibitor SC-514 for 30 min before addition of CD28/Ig. Data represent the mean fold increase in Oct-2 mRNA normalized to actin ± SEM from three independent experiments. *, A p value of <0.05 in comparison to the CD40L/IL-4 alone group.

 
One downstream effect of the CD86-induced activation of the classical NF-{kappa}B pathway appears to be an increase in the expression of the B cell-specific transcription factor Oct-2 (20). Oct-2 expression is dependent on activation of the classical NF-{kappa}B pathway (25). Because the promoter of Oct-2 is not fully characterized, it remains unknown as to whether NF-{kappa}B increases Oct-2 expression through regulation of promoter activity directly, or indirectly, through the up-regulation of another factor that regulates Oct-2 promoter activity directly. To determine whether the IKK complex mediates the CD86-induced increase in I{kappa}B{alpha} phosphorylation and is required for the increase in Oct-2 mRNA, B cells were cultured as described above, and either an IKK inhibitor peptide or a pharmacological IKK inhibitor (SC-514) was added 30 min before addition of CD28/Ig. Total protein or mRNA was isolated and analyzed using Western blot analysis or quantitative real-time PCR. Both inhibitors prevented the CD86-induced increase in the level of I{kappa}B{alpha} phosphorylation (Fig. 1c), degradation (Fig. 1c), and increase in Oct-2 mRNA (Fig. 1d). In contrast, addition of a control peptide that is unable to inhibit IKK activity was unable to block the increase in Oct-2 mRNA. Taken together, these multiple lines of evidence suggest that CD86 stimulation leads to increased IKK{alpha}beta phosphorylation, which appears to be required for the phosphorylation/degradation of I{kappa}B{alpha} and the increase in Oct-2 mRNA expression.

CD86 activates PDK-1 and Akt

Multiple signaling intermediates are known to play a role in the phosphorylation and activation of the IKK complex, including Akt (26) and NF-{kappa}B-inducing kinase (27). CD86 stimulation activates the classical NF-{kappa}B pathway, as indicated by the activation and nuclear localization of p50/p65 (20), suggesting that Akt as opposed to the NF-{kappa}B-inducing kinase is involved. The activation of Akt involves two key phosphorylation sites, serine 473 and threonine 308. To determine whether CD86 activates Akt through phosphorylation of either serine 473 or threonine 308, B cells were cultured as described above and total protein was analyzed using Western blot analysis. CD86 increases the level of phospho-Akt (Thr308) throughout a 30-min period to ~2.5-fold above CD40L/IL-4 alone (Fig. 2a), an effect not seen when CD86-deficient B cells were used (Fig. 2b). In contrast, the level of phospho-Akt (Ser473) was unchanged following stimulation of CD86 on an activated B cells (data not shown). To determine whether the CD86-induced increase in phospho-Akt (Thr308) is PI3K dependent, B cells were pretreated with wortmannin or LY294002, PI3K inhibitors, and total protein was isolated and analyzed for phospho-Akt (Thr308). Both PI3K inhibitors were able to completely inhibit the CD86-induced increase in phospho-Akt (Thr308) (Fig. 2c), suggesting that the increase in the level of phospho-Akt (Thr308) was PI3K dependent. Taken together, these data show that CD86 stimulation on an activated B cell increases the level of Akt activation by targeting an increase in the phosphorylation of threonine 308 in a PI3K-dependent manner.


Figure 2
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  		FIGURE 2. CD86 signals through PDK-1 and Akt. Western blot analysis for the level of phospho-Akt (Thr308) and total Akt in (a) wild-type (Wt), (b) CD86-deficient, and (c) LY294002 pretreated B cells were activated with CD40L/IL-4 for 16 h before addition of CD28/Ig. The level of phospho-PDK-1 and PDK-1 in (d) Wt and (e) CD86-deficient B cells was also measured. Western blot analysis of (f) phospho-Akt (Thr308) and total Akt in Wt B cells activated as described above and also pretreated with a PDK-1 inhibitor (OSU-03012) for 30 min before addition of CD28/Ig. Band density was determined using densitometry and the data represent the mean-fold increase in phospho-Akt normalized to total Akt or phospho-PDK1 normalized to total PDK1 ± SEM from three independent experiments. *, A p value of <0.05 in comparison to the CD40L/IL-4 alone group.

 
PDK-1, a serine/threonine kinase, is known to phosphorylate Akt at threonine 308 (28). To determine whether CD86 stimulation activates PDK-1, B cells were cultured as described above and total protein was isolated and analyzed using Western blot analysis. The level of PDK-1 phosphorylation was increased by 5 min following stimulation of CD86 and remained elevated through 30 min (Fig. 2d), an effect not seen when CD86-deficient B cells were used (Fig. 2e). To confirm that PDK-1 was playing a role in the CD86 signaling pathway, B cells were pretreated with OSU-03012, a PDK-1 inhibitor, and the level of phospho-Akt (Thr308) was analyzed by Western blot analysis. PDK-1 inhibition prevented the CD86-induced increase in phospho-Akt (Thr308) (Fig. 2f). Furthermore, PDK-1 inhibition also prevented the CD86-induced increase in phosphorylated IKK{alpha}beta and I{kappa}B{alpha} (data not shown). Taken together, these multiple lines of evidence show that CD86 stimulation activates both PDK-1 and Akt, and suggests that the PDK-1/Akt pathway is upstream of NF-{kappa}B activation.

CD86 activates PI3K

PDK-1 and Akt, through their pleckstrin homology domains, are recruited to the plasma membrane to interact with PIP3. Because PI3K facilitates the conversion of PIP2 to PIP3 and is required for Akt activation (reviewed in Ref. 29), we sought to determine whether CD86 stimulation activates PI3K. B cells were cultured as described above, protein lysates were obtained, PI3K was immunoprecipitated with an anti-PI3K Ab, and its activity was measured in vitro. CD86 stimulation induced an increase in PI3K activity, an effect not seen when CD86-deficient B cells were used (Fig. 3a). Because the PI3K/Akt pathway appears to be required for the CD86 activation of NF-{kappa}B, and because Oct-2 expression is NF-{kappa}B dependent (20, 25), we sought to determine whether the CD86-induced increase in Oct-2 mRNA expression was also PI3K dependent. Wild-type or PI3K-deficient B cells were cultured as described above, and either wortmannin or LY294002 was added for 30 min before addition of CD28/Ig. Total RNA was isolated and analyzed for the level of Oct-2 and actin mRNA using real-time PCR analysis. CD86 increases Oct-2 mRNA expression ~2.0-fold above CD40L/IL-4 alone (Fig. 3b), as has been shown previously (20). The use of both PI3K inhibitors (Fig. 3b) and PI3K-deficient B cells (Fig. 3c) blocked the CD86-induced increase in the level of Oct-2 mRNA. Taken together, these multiple lines of evidence show that CD86 stimulation on an activated B cell appears to increase the level of Oct-2 mRNA expression in a PI3K-dependent manner, and indirectly links CD86 activation of the PI3K/Akt pathway to the NF-{kappa}B-dependent expression of Oct-2 mRNA.


Figure 3
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  		FIGURE 3. CD86 signals through PI3K. a, PI3K ELISA analysis of the PI3K activity in BALB/c and CD86-deficient B cells activated with CD40L/IL-4 for 16 h before addition of CD28/Ig. Data represent the mean fold increase in PI3K activity ± SEM from three independent experiments. Real-time PCR analysis of Oct-2 and actin mRNA levels in (b) BALB/c and (c) PI3K-deficient B cells activated as stated above in the absence or presence of either PI3K inhibitor, LY294002 or wortmannin. Data represent the mean fold increase in Oct-2 mRNA normalized to total actin mRNA ± SEM from three independent experiments. *, A p value of <0.05 in comparison to the CD40L/IL-4 alone group.

 
CD86 activates PLC{gamma}2 and PKC{alpha}beta

Previous data from our laboratory suggested that CD86 stimulation on a CD40L/IL-4-activated B cell activated two signaling pathways to increase the level of Oct-2 expression, one that was PKC independent and another that was PKC dependent. Although the use of pharmacological inhibitors provided indirect evidence to indicate a role for PKC (20), we sought to determine whether CD86 stimulation on an activated B cell increased the level of PKC phosphorylation and activation. B cells were activated, as described above and total protein was isolated and analyzed using Western blot analysis. CD86 stimulation increased the level of PKC{alpha}beta phosphorylation ~2.0-fold above CD40L/IL-4 alone to a maximal level by 15 min after stimulation, and returned to baseline level by 30 min (Fig. 4a). This effect was lost when CD86-deficient B cells were used (Fig. 4b). Thus, CD86 stimulation on an activated B cell increases the level of PKC{alpha}beta phosphorylation and activation.


Figure 4
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  		FIGURE 4. CD86 signals through PKC{alpha}beta and PLC{gamma}2. Western blot analysis for the level of phospho-PKC{alpha}beta and total actin in (a) Wt and (b) CD86-deficient B cells activated with CD40L/IL-4 for 16 h before addition of CD28/Ig. The level of phospho-PLC{gamma}2 and total PLC{gamma}2 was also measured in (c) Wt and (d) CD86-deficient B cells. The data represent the mean fold increase in the band density of phospho-PKC{alpha}beta normalized to total actin and phospho-PLC{gamma}2 normalized to total PLC{gamma}2 ± SEM from three independent experiments. *, A p value of <0.05 in comparison to the CD40L/IL-4 alone group.

 
The activation of classical PKC isoforms are dependent on the activation of PLC, which cleaves phosphoinositides into inositol-3-phosphate and diacylglycerol, leading to increased intracellular Ca2+ and PKC activation (30). PLC{gamma}2 is the predominant form of PLC in a B cell and requires phosphorylation for full activation (31). To determine whether CD86 stimulation increases the level of phosphorylation and activation of PLC{gamma}2, B cells were cultured as described above and total protein was isolated and analyzed using Western blot analysis. CD86 increases phospho-PLC{gamma}2 ~2.0-fold above CD40L/IL-4 alone by 5 and 15 min, and returns to baseline by 30 min (Fig. 4c). This effect was lost when CD86-deficient B cells were used (Fig. 4d). Thus, CD86 stimulation on an activated B cell increases the level of PLC{gamma}2 phosphorylation and activation.

CD86 stimulation increases NF-{kappa}B- and 3'-IgH enhancer-dependent gene activity

Because data from our laboratory showed that CD86 stimulation on an activated B cell increased nuclear localization of p50/p65 dimers (20), we sought to determine whether CD86 induced a subsequent increase in gene activity that was mediated by NF-{kappa}B. A transient transfection system was used in which a B lymphoma cell line, CH12.LX, was transfected with an NF-{kappa}B-sensitive luciferase reporter plasmid, controlled by {kappa}B-binding sites within the promoter region. CH12.LX cells were cultured as described for normal B cells. As shown in Fig. 5a, a baseline level of NF-{kappa}B gene activity was induced by CD40L and IL-4 alone, as has been reported previously (32), and addition of a CD28/Ig increased luciferase gene activity ~2.0-fold above CD40L/IL-4 alone. In contrast, luciferase activity was undetectable when a plasmid containing mutated NF-{kappa}B-binding sites was used, indicating that NF-{kappa}B regulates the transcription of the luciferase reporter gene specifically. Thus, CD86 stimulation on an activated B cell increases the level of NF-{kappa}B-mediated gene activity within the B cell.


Figure 5
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  		FIGURE 5. CD86 stimulation increases NF-{kappa}B- and 3'-IgH enhancer-mediated gene activity. CH12.LX B cells, cotransfected with plasmid constructs of either an (a) NF-{kappa}B-binding element ({blacksquare}), (a) a mutated NF-{kappa}B-binding element ({cjs2113}), or the (b) hs4 region of the 3'-IgH enhancer, all containing the firefly luciferase gene, and a control plasmid containing the Renilla luciferase gene (pRL-TK). The cells were activated with CD40L/IL-4 for 16 h before addition of either a control Ig (–) or CD28/Ig. Data represent the mean fold increase in firefly luciferase activity normalized to Renilla luciferase activity ± SEM from three independent experiments. c, A20 cells, stably transfected with a plasmid construct containing the entire 3'-IgH enhancer followed by a {gamma}2b reporter gene, were activated with CD40L/IL-4 for 16 h before addition of CD28/Ig. Cells were lysed on day 5 following initial activation and the lysate was analyzed for the level of {gamma}2b by ELISA. Data represent the mean fold increase in {gamma}2b ± SEM from three independent experiments. d, Resting naive B cells were activated with CD40L/IL-4 for 16 h before addition of CD28/Ig. Three days after activation, cells were fixed, nuclei isolated, and the DNA sheared into 200- to 500-bp fragments by sonication. p50 or p65 bound to the DNA were immunoprecipitated using either an anti-p50 or anti-p65 Ab. The amount of p50 and p65 bound to {kappa}B sites contained within the hs4 region were analyzed using semiquantitative PCR. The amount of p50 or p65-specific PCR product for each sample was determined by subtracting out the nonspecific PCR product from samples that received no Ab during the immunoprecipitation and normalized to the amount of in-put DNA. Data represent the mean fold increase in p50 or p65 binding ± SEM from three independent experiments with a representative gel inlay. *, A p value of <0.05 in comparison to the CD40L/IL-4 alone group.

 
Previous data from our laboratory also showed that CD86 increases Oct-2 expression and binding to the hs1,2 and hs4 region of the 3'-IgH enhancer (20). Of the four hs regions that comprise the 3'-IgH enhancer, hs1,2 and hs4 regions appear to exert the most enhancer activity in transient transfection studies (24), although a synergistic effect is evident when all four hs regions are activated (33). The hs4 region contains both {kappa}B and octamer binding sites that play a role in regulating its activity (34), and the 3'-IgH enhancer is known to regulate the level of mature IgG1 transcript (35). CD86 stimulation on an activated B cell is reported to up-regulate the rate of mature IgG1 transcription per B cell, which is associated with an increase in Oct-2 binding to the hs1,2 and hs4 regions of the 3'-IgH enhancer (16, 20), suggesting that CD86 is able to regulate 3'-IgH enhancer activity. Therefore, we sought to determine directly if CD86 stimulation increases 3'-IgH enhancer activity of the hs4 region. A transient transfection system was used in which CH12.LX cells were transfected with a plasmid containing the hs4 region followed by a luciferase reporter gene. In addition, to determine whether CD86 increases the activity of the entire 3'-IgH enhancer region, another transfection system was used in which a B lymphoma cell line, A20, was stably transfected with the entire 3'-IgH enhancer region followed by a {gamma}2b reporter gene, as described previously (23). A20/CH12.LX cells were activated and CD86 was stimulated as described for normal B cells. When compared with the level induced by CD40L/IL-4 alone, CD86 increased the level of hs4-mediated gene activity by ~2.0-fold (Fig. 5b) and increased the entire 3'-IgH enhancer-mediated gene activity by ~2.5-fold (Fig. 5c). To determine whether NF-{kappa}B was playing a role in the CD86-induced regulation of 3'-IgH enhancer activity, ChIP was performed. When the level of p50 or p65 bound to {kappa}B sites located within the hs4 region were analyzed, CD86 stimulation increased the level of bound p50 and p65 as compared with the CD40L/IL-4 alone group (Fig. 5d). In contrast, a control Ab showed no difference with addition of CD28/Ig (data not shown). Taken together, these data show that CD86 stimulation increases the level of gene activity that is regulated by NF-{kappa}B and/or the 3'-IgH enhancer. The increase in p50/p65 binding to the hs4 region suggests that NF-{kappa}B plays a role in the regulation of 3'-IgH enhancer transcriptional activity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous data show that CD86 stimulation on a CD40L/IL-4-activated B cell increases the level of NF-{kappa}B activation, Oct-2 expression and binding to the 3'-IgH enhancer, and mature IgG1 mRNA and protein (20). The present data show that CD86 stimulation on a CD40L/IL-4-activated B cell activates the PI3K/Akt and PLC{gamma}2/PKC{alpha}beta signaling pathways, increases the level of gene activity mediated by NF-{kappa}B and the 3'-IgH enhancer, and increases p50 and p65 binding to the hs4 region (see model in Fig. 6). The reason for the activation of two signaling pathways to mediate the CD86-induced effects on gene activity is becoming clearer. Previous data from our laboratory suggested that one pathway activated a PKC-independent phosphorylation of I{kappa}B{alpha}, while the other activated a PKC-dependent phosphorylation of p65, with both pathways converging to increase Oct-2 and mature IgG1 mRNA expression (20). Alternatively, it is possible that the PKC{alpha}beta pathway is needed to phosphorylate Oct-2. A lack of Oct-2 phosphorylation, using cells transfected with a mutated form of the Oct-2 protein, was reported to result in decreased gene activity, suggesting that the phosphorylated form of Oct-2 may be necessary for binding to DNA (36). Therefore, PKC{alpha}beta might also phosphorylate Oct-2 following CD86 stimulation to increase binding to the 3'-IgH enhancer. However, to make this determination, an Ab that recognizes the phosphorylated form of Oct-2 is needed, but is unavailable at present. The present findings suggest that the PI3K/Akt pathway is needed to activate the IKK complex to phosphorylate I{kappa}B{alpha} allowing for NF-{kappa}B activation, while the PLC{gamma}2/PKC{alpha}beta pathway is needed to enhance p65 activity by mediating its phosphorylation. Thus, PKC-independent and PKC-dependent pathways are activated by CD86 stimulation on a CD40L/IL-4-activated B cell, and both pathways appear to play an important role in mediating the CD86-induced increase in B cell activity.


Figure 6
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  		FIGURE 6. Proposed CD86 signaling pathway in an activated B cell. Activation of the CD86 signaling pathway is dependent on prior B cell activation by CD40L/IL-4, which activates signaling intermediates responsible for CSR to IgG1 (11 12 13 ). Stimulation of CD86 increases the activity of PI3K and the phosphorylation of PDK-1, Akt (Thr308), and IKK{alpha}beta. The activation of IKK{alpha}beta leads to the phosphorylation of I{kappa}B{alpha}. Phosphorylated I{kappa}B{alpha} is rapidly degraded, allowing for the nuclear localization of NF-{kappa}B, specifically p50 and p65 (22 ). Additionally, stimulation of CD86 increases the phosphorylation of PLC{gamma}2 and PKC{alpha}beta, leading to the phosphorylation of p65. NF-{kappa}B-dependent Oct-2 gene activity is increased, leading to an increase in the level of Oct-2 mRNA and protein. Finally, NF{kappa}B- and 3'-IgH enhancer-mediated gene activity is increased via increased NF-{kappa}B and Oct-2 binding to their consensus sequences (22 ), leading to an increase in the rate of mature IgG1 transcription and protein production (18 ).

 
The present findings indicate that CD86 stimulation by CD28/Ig induces an increase in the level of the activated signaling intermediates PI3K, Akt, PLC{gamma}2, PKC{alpha}beta, and NF-{kappa}B. However, because CD28/Ig binds both CD86 and CD80 (37), it is possible that the present results are due to the stimulation of either one or both of these molecules. We think that this possibility is unlikely for a number of reasons. First, our data show that CD28/Ig induces the same signaling pathway within a B cell as was shown previously in response to a highly selective stimulatory anti-CD86 Ab (16, 20). Second, the kinetics of CD80 and CD86 expression differ dramatically. Detectable CD86 surface expression occurs by 6 h and maximally by 24–48 h (8), in contrast to CD80 that is detectable by 24 h and maximally by 48–72 h (38). This difference in expression kinetics makes it unlikely that CD80 is expressed to any detectable level 16 h after B cells are activated with CD40L/IL-4, which is the time that CD28/Ig is added in our model system. In contrast, CD86 is expressed maximally at this time point of CD28/Ig addition. And finally, the signaling pathway induced by CD28/Ig and/or an anti-CD86 Ab is absent when CD86-deficient B cells are used (9, 16, 20). Collectively, these findings indicate that the signaling intermediates found to be induced in an activated B cell exposed to CD28/Ig result from CD86 stimulation specifically.

The specific target genes activated by NF-{kappa}B in a B cell after CD86 stimulation remain unknown. Our finding of increased p50 and p65 binding to the hs4 region of the 3'-IgH enhancer suggests that the hs4 region is one target of the CD86-induced NF-{kappa}B activation. The hs4 region, which is known to be regulated directly by NF-{kappa}B and octamer binding proteins (34, 36), may be the specific target of the CD86 signaling pathway required for an increase in IgG1 on a per cell basis. A second potential target of the CD86-induced increase in NF-{kappa}B activity is the Oct-2 gene, which published data show, indirectly, is regulated by NF-{kappa}B (25). Furthermore, the present findings that an IKK inhibitor blocked the CD86-induced activation of NF-{kappa}B and the increase in Oct-2 expression, further suggests, indirectly, that NF-{kappa}B regulates Oct-2 expression. What remains to be determined is whether or not NF-{kappa}B binds to putative {kappa}B-binding site within the Oct-2 promoter, but such an experiment requires a characterization of the Oct-2 promoter sequence, which has not been reported to date. Thus, these findings suggest that the Oct-2 promoter and the hs4 region of the 3'-IgH enhancer are likely targets for the NF-{kappa}B activated by CD86 stimulation.

A third potential target of the CD86 activated NF-{kappa}B is the CD80 gene. CD80 expression is regulated by {kappa}B-binding sites located within its promoter region (39), and CD86 stimulation on a B cell is known to increase the level of CD80 mRNA and surface expression (19). Until now, the signaling pathway activated by CD86 to increase CD80, as well as the functional relevance of such regulation, is unknown. We propose that CD86 may have a dual function during an IgG1 response in that it may enhance the level of IgG1 by increasing NF-{kappa}B activity, as well as Oct-2 expression and binding to the 3'-IgH enhancer, but suppress the level of IgG1 indirectly by increasing CD80 expression to dampen the response. Although the ability of CD80 to signal is unknown, support for this proposal includes the early and late kinetics for CD28/CD86 (8, 40) and CTLA-4/CD80 (38, 41) expression, respectively, on T and B cells, making CD86 and CD80 ideal candidates to strengthen and dampen the B cell response, as has been documented for CD28 and CTLA-4 (reviewed in Ref. 42). In dendritic cells, CD28-Ig enhanced activation of NF-{kappa}B, p38 MAPK, and IL-6, while CTLA-4-Ig activated the immunosuppressive pathway of tryptophan catabolism (43). The ability of CD28-Ig to induce a positive signal and CTLA-4-Ig to induce a negative signal has not been tested directly in B cells. Suvas et al. (18) showed in B cells that an anti-CD86 Ab induced antiapoptotic genes, while an anti-CD80 Ab induced proapoptotic genes, suggesting CD28-Ig and CTLA-4-Ig could potentially activate both positive and/or negative signals within the B cell. Although the present data in B cells are the first to show that stimulation with CD28-Ig enhances IgG1 production in a CD86-dependent manner, the ability of CTLA-4-Ig to affect B cell function remains unknown. Collectively, these findings suggest that B cells and T cells express costimulatory molecules during different phases of an immune response that possibly mediate opposing effector functions, with CD28/CD86 involved in immune enhancement and CTLA-4/CD80 involved in immune suppression.

Previous findings in CD40L/IL-4-activated B cells indicated that CD86 stimulation increased specifically the level of Oct-2 expression and binding to the 3'-IgH enhancer, which was associated with an increase in the rate of IgG1 produced per B cell, without affecting class switch recombination (CSR) (16, 20). However, whether a direct link existed between CD86 and 3'-IgH enhancer activity was unknown. The present results establish that a direct link exists, and may explain why a CD86-induced effect on CSR does not occur. First, CD40 preferentially activates the p50/RelB and p50/p65 dimers (11, 44), while CD86 specifically activates p50/p65 dimers alone (20). And second, RelB appears to play a role in regulating germline IgG1 production (11, 44), while p50 and p65 appear to play a role in regulating the level of IgG1 protein produced (45). These findings lend support to the proposal that the CD86-induced effect on the rate of IgG1 production is dissociated from the CD40-induced effect on CSR, but not from the CD40-induced effect on 3'-IgH enhancer activity.

A previous finding showed that CD86 induced an increase in the level of expression of the antiapoptotic proteins Bcl-xL and Bcl-w, and a decrease in the level of expression of the proapoptotic factor caspase 8 (18), in LPS-activated B cells. Therefore, another possibility is that CD86 stimulation increases the level of IgG1 by increasing the level of B cell survival signals. The PI3K/Akt pathway is known to phosphorylate the Bcl-2 family protein Bad to prevent it from inactivating prosurvival factors, such as Bcl-xL (46), as well as to phosphorylate and inactivate the proapoptotic factors caspase 9 (47) and members of the Forkhead family (48). Our finding that CD86 activates Akt provides a mechanism by which the anti-apoptotic effects may occur in LPS-activated B cells. However, it is not yet clear whether the effect on cell survival is associated with the rate of IgG1 produced in CD40L/IL-4-activated B cells, because cell survival was unaffected by CD86 stimulation in our model system (16, 20).

The finding that CD86 appears to selectively induce the phosphorylation of Akt at thr308 as opposed to ser473 is interesting and novel. Akt activation and phosphorylation at thr308 is thought to be dependent on PI(3,4,5)P3 (49), which our data would support based on the observed increase in total PI3K activity. The increase in PI3K activity measured in vitro would suggest an increased amount of PI(3,4,5)P3 generation within the cell, allowing for more PDK-1 and Akt to associate with PI(3,4,5)P3 and become activated. SHIP, a serine/threonine phosphatase, converts PI(3,4,5)P3 to PI(3,4)P2 and negatively regulates the activation of Akt (reviewed in Ref. 29). Therefore, our finding that Akt phosphorylation at Thr308 is increased following CD86 stimulation would suggest that SHIP is not playing a role in the enhancement of Akt activation, although a CD86-induced suppression of SHIP activity has not been eliminated.

Prior B cell activation appears to be necessary for CD86 to function as a B cell regulatory molecule, suggesting two possibilities. First, CD86 is an inactive signaling molecule that requires induction of the expression and/or binding of an adapter protein, as has been shown for the TCR and BCR complexes (50). The presence of 3 putative PKC phosphorylation sites on the short cytoplasmic tail of CD86 (15) suggests that a clustering of CD86 on the B cell surface may activate PKC to phosphorylate these sites to provide signaling capacity to the otherwise inert molecule. Our results support this possibility because PKC activation is clearly increased when CD86 is stimulated on a CD40L/IL-4-activated B cell. Alternatively, CD86 may possess innate signaling potential, but because the level of CD86 is low on a resting B cell, any signaling intermediates generated may be below the level of detection. In previous studies designed to determine whether CD86 stimulation affects B cell functional activity, it was found that prior activation of the B cell with LPS (18), the BCR (9, 19), or CD40L/IL-4 (16, 17, 20) was needed for CD86 stimulation to exert an effect on the level of Ab produced. Importantly, these activating stimuli increased the level of CD86 expressed on a B cell, suggesting that B cell activation may induce a critical level of CD86 that is needed for a signal to be detectable. Alternatively, when two ligands were used that induce an increase in the level of CD86 expression without activating the B cell, namely IL-4 (6) and a beta2-adrenergic receptor agonist (10), stimulation of CD86 alone was unable to generate a detectable level of signaling intermediates, suggesting that CD86 may not possess innate signaling potential, regardless of the level of CD86 expressed on the B cell surface. Thus, the apparent inability of CD86 alone to signal on a resting B cell is not simply due to the low level of CD86 expression, but may be associated with the lack of a competency signal or factor afforded by B cell activation.

One important B cell activation receptor is the BCR. Cross-linking of the BCR on the surface of the B cell leads to activation of numerous signaling intermediates (reviewed in Ref. 51), including PLC{gamma}2 and PI3K. Because the present findings suggest that CD86 activates PLC{gamma}2, future studies will determine whether CD86 also activates two downstream effectors of PLC{gamma}2, namely intracellular Ca2+ and phospho-Erk. The present data would suggest that intracellular Ca2+ may be increased, as suggested by the activation of PLC{gamma}2 and PKC{alpha}beta. Erk activation has not been shown to regulate either Oct-2 expression, 3'-IgH enhancer activity, or IgG1 production, therefore, we think it is unlikely to be involved as a CD86 signaling intermediate. Because CD86 and BCR stimulation both generate some common signaling intermediates, future studies will determine whether CD86 either stimulates the BCR indirectly or uses the same adapter proteins to activate the PI3K/Akt and PLC{gamma}2/PKC{alpha}beta signaling pathways. It is also possible that CD86 will gain access to the latter adapter proteins only after B cell activation occurs. Experiments to address these questions would involve a detailed analysis of the cytoplasmic tail of CD86 for potential signaling and/or protein binding motifs.

The ability of CD40 and IL-4R stimulation to regulate the amount of IgG1 produced per B cell in the absence or presence of CD86 stimulation raises the possibility that B cells might be activated in vivo to produce a T cell-dependent Ab in an Ag nonspecific manner. The current study uses a model system where naive B cells are stimulated with CD40L and IL-4 16 h before addition of CD28-Ig to stimulate CD86. This model system was developed to allow for characterization of the CD86 signaling pathway, and was not meant to suggest that Ag-independent B cell activation occurs in vivo. The original model system used by Kasprowicz et al. (9) stimulated the BCR, waited 24 h, and then stimulated concurrently CD40, IL-4R, and CD86. The latter sequence of stimuli was designed to mimic the in vivo situation where the BCR would endocytose the Ag, process it, and present the peptide in association with MHC class II to a previously activated T cell that now expresses CD28 and CD40L, as well as secretes IL-4. In that model system, stimulation of the BCR was required for CD86 to become competent to signal, potentially because the BCR up-regulates CD86 expression (2). With BCR stimulation, it would have been extremely difficult to dissect the CD86 signaling pathway when stimulated at the same time as CD40 and the IL-4R. Consequently, a new model system was developed in which CD40 and IL-4R were stimulated in the absence of BCR stimulation and anti-CD86 was added 16 h later. Because CD86 appeared competent to signal and able to increase IgG1 production in this new model system of B cell activation (16), it was chosen to dissect the CD86 signaling pathway. However, now that the CD86 signaling pathway has been characterized, it will be important to bring back the BCR signal into the model system.

CD86 stimulation on an activated B cell increases the level of IgG1 2- to 2.5-fold higher than that induced by CD40L/IL-4 alone (16, 20). The biological significance of such a modest change in Ab level remains to be determined. Data in humans show that people immunized with pertussis toxoid produced IgG Abs, but that some individuals produced a 2- to 3-fold higher level of serum total IgG, which was associated with a 3- to 9-fold increase in the level of protection (52). It would be interesting to determine the level of CD86 on the B cells from the latter individuals to determine whether an association exists between the level of CD86 expressed and the level of total serum IgG produced. Although a change in the level of CD86 might affect the level of IL-4, and the subsequent level of IgG1 switching that occurs, our data would suggest that such a change might also affect the level of signals generated in a B cell to increase the rate of IgG1 produced on a per cell basis. Thus, the ability of CD86 stimulation to increase the level of IgG1 by 2-fold may be both biologically significant and clinically relevant.


   Acknowledgments
 
We gratefully acknowledge Dr. Joe Podojil (Northwestern University, Chicago, IL) for his early involvement in these studies, as well as Drs. Laurel Eckhardt (Hunter College of City University of New York, New York), Denis Guttridge (The Ohio State University, Columbus, OH), Gail Bishop (University of Iowa, Iowa City, Iowa), Ching-Shih Chen (The Ohio State University, Columbus, OH), and Arlene Sharpe (Brigham and Woman’s Hospital, Boston, MA) for generously sharing valuable reagents with us. We also thank Robert Erbe (The Ohio State University, Columbus, OH) for his assistance with Western blot analyses and Dr. William Lafuse (The Ohio State University, Columbus, OH) for helpful discussions regarding the research.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by research funds from the National Institutes of Health (NIH) Grants AI37326 and AI47420. N.W.K. is a recipient of a Training Grant Award from NIH Grant T32 AI55411. This research is part of the dissertation research conducted by N.W.K. who is a predoctoral student in the Integrated Biomedical Graduate Program, The Ohio State University (Columbus, OH). Back

2 Address correspondence and reprint requests to Dr. Virginia M. Sanders, Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, 2194 Graves Hall, 333 West 10th Avenue, Columbus, OH 43210. E-mail address: sanders.302{at}osu.edu Back

3 Abbreviations used in this paper: PKC, protein kinase C; IKK, I{kappa}B kinase; PLC, phospholipase C; ChIP, chromatin immunoprecipitation; PDK-1, phosphoinositide-dependent kinase 1. Back

Received for publication January 5, 2006. Accepted for publication March 15, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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