The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 1516 -1523
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kin, N. W.
Right arrow Articles by Sanders, V. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kin, N. W.
Right arrow Articles by Sanders, V. M.

CD86 Regulates IgG1 Production via a CD19-Dependent Mechanism1

Nicholas W. Kin2 and Virginia M. Sanders3

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
 
CD86 signals directly in a B cell to activate PI3K and increase the rate of IgG1 production, without affecting germline transcription. However, the mechanism by which CD86 activates PI3K in a B cell and the relevance of CD86 stimulation in vivo remains unknown. We show that the addition of CD28/Ig to CD40 ligand/IL-4-activated wild-type, but not CD86- or CD19-deficient, B cells increased the level of phosphorylation for Lyn and CD19, as well as the amount of Lyn, Vav, and PI3K that immunoprecipitated with CD19. Adoptive transfer of CD86-deficient B cells and wild-type CD4+ T cells into RAG2-deficient mice and immunization with trinitrophenylated keyhole limpet hemocyanin resulted in an IL-4 and germline IgG1 response equivalent to control mice, but a decrease in serum IgG1. Thus, our findings suggest that CD86 plays a key role in regulating the level of IgG1 produced in vitro and in vivo, and that Lyn and CD19 may be the signaling intermediates activated by CD86 proximal to PI3K.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD86 was cloned in 1993 as a counterreceptor for both CD28 and CTLA-4 that are expressed on T cells (1, 2, 3). CD86 is expressed primarily on APCs, such as B cells, macrophages, and dendritic cells (DC)4 (2, 3), and functions as an important costimulatory molecule. Resting B cells express CD86 at low levels (2), but rapidly up-regulate CD86 following activation of the BCR (2), CD40 (4), IL-4R (5), LPS receptor (6, 7), and beta2-adrenergic receptor (8, 9). CD86 on a B cell interacts with CD28 on a T cell to activate a signaling pathway in the T cell that increases cell activation and expression of CD40 ligand (CD40L) and secretion of IL-4 (10), which are essential molecules that interact with CD40 and the IL-4R on a B cell, respectively, to induce class switch recombination to IgG1 (11, 12). CD86- and CD28-deficient mice produce less IgG1 following immunization with a T cell-dependent Ag when compared with wild-type (WT) mice (13, 14), likely due to the loss of the CD86/CD28 interaction, which is required for costimulation of the T cell to increase CD40L and IL-4 expression during the generation of an optimal T cell-dependent Ab response.

However, an alternative explanation for the results using CD86- and CD28-deficient mice would be that CD86 signaling to the B cell was lost, and that this signal was required to regulate the level of IgG1 produced directly. Recent studies revealed that stimulation of CD86 with either an anti-CD86 Ab or CD28/Ig on an activated murine B cell increases the rate of mature IgG1 transcription, as determined by nuclear run-on analysis (15), the level of murine IgG1 (8, 16), and human IgG4 protein (17), anti-apoptotic factors (18), CD80 expression (19), NF-{kappa}B (p50/p65) activation (15), Oct-2 expression, and binding to the 3'-IgH enhancer, as determined by chromatin immunoprecipitation assay (15) and 3'-IgH enhancer activity, as shown using transiently and stably transfected B cell lines (20). Recently, the signaling pathway activated by CD86 proximal to NF-{kappa}B activation in a B cell was identified to involve PI3K/PDK1/Akt and PLC{gamma}2/PKC{alpha}beta (20). CD86 signaling is also activated in DC exposed to CD28/Ig or CTLA-4/Ig to stimulate CD86. In these cells, CD86 stimulation increases the level of p38 MAPK activation, the production of IL-6, IFN-{alpha}, and IDO (21, 22), but only in DC-expressing CD19 (21), suggesting that CD19 may be necessary for CD86 to function. However, the question remains as to the mechanism by which CD86 stimulation activates PI3K, particularly because CD86 fails to express tyrosine residues in the cytoplasmic domain that are essential for PI3K activation.

CD19 is a coreceptor primarily expressed on the B cell and plays an important role in B cell development, as well as BCR- and CD21-induced signal transduction (23). CD19-deficient mice show abnormal B-1 cell development and impairment of T cell-dependent Ab responses in vivo (24, 25). CD19 functions as a signaling partner for several B cell surface receptors, including the CD21/CD81/Leu13 complex (26, 27), the BCR (28), CD180 (RP105) (29), and MHC class II (30). The cytoplasmic tail of CD19 contains nine tyrosine residues, that when phosphorylated, allow for the interaction with the SH2 domain-containing proteins, including Lyn, Vav, Grb2, and p85{alpha} PI3K (reviewed in Ref. 23). Lyn is one protein tyrosine kinase (PTK) that phosphorylates CD19 (31) and is reported to play both a positive and negative role in regulating B cell activation (32). Thus, CD19 and Lyn work together as important signaling intermediates to mediate the activation of PI3K in B cells following BCR and CD21 stimulation and, therefore, may also serve as potential signaling intermediates to mediate PI3K activation following CD86 stimulation.

In the current study, we show that the addition of CD28/Ig to CD40L/IL-4-activated B cells on WT, but not CD86- or CD19-deficient, B cells increased the level of phosphorylation for Lyn and CD19, as well as the amount of Lyn, Vav, and PI3K proteins that immunoprecipitated with CD19. Serum IgG1 levels were decreased in mice receiving CD86-deficient B cells when compared with mice receiving WT B cells. The decrease in serum IgG1 was associated with a decrease in the level of B cell-associated Oct-2 mRNA and protein, but a normal level of germline IgG1 mRNA. Thus, our findings suggest that CD86 plays a key role in regulating the level of IgG1 produced in vitro and in vivo, independently of class switch recombination.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Female BALB/c and CD19-deficient (CD19–/–) mice were purchased from Taconic Farms. CD86-deficient (CD86–/–) mice were provided by Dr. A. Sharpe (Brigham and Woman’s Hospital, Boston, MA). Mice were bred and housed within the pathogen-free facility at Taconic Farms. C3H/HeJ and TLR 4-deficient mice (TLR4–/–) were purchased from The Jackson Laboratory.

Resting B cell isolation and activation

Resting B cell isolation and activation was performed as described previously (20). In brief, resting B cells were isolated using the AutoMacs machine (Miltenyi Biotec) following the manufacturer’s instructions and were activated with CD40L-expressing Sf9 cells 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) or a recombinant human IgG1 Fc (R&D Systems) was added at a final concentration of 1 µg/ml. Pharmacologic inhibitors SU6656 and PP2 (Calbiochem) were added 30 min before the stimulation of CD86. 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.

In vivo cell transfer and immunization

Seven days before immunization, 5 x 106 CD4+ T cells and either 20 x 106 WT B cells or CD86-deficient B cells were adoptively transferred into RAG2-deficient animals in a volume of 100 µl PBS i.v. in the lateral tail vein. One week following the adoptive transfer of cells, mice were administered 100 µg TNP-keyhole limpet hemocyanin (KLH) in alum i.p., and serum samples were collected 7 and 14 days later. The level of serum IgG1 was determined in various dilutions of serum samples using ELISA.

Western blot

Western blot analysis was performed as described previously (20). In brief, following activation, B cells were lysed, protein samples (5–20 µg) were resolved by electrophoresis on 10% polyacrylamide gels, transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore), probed with either anti-CD19, anti-phospho-CD19, anti-Vav, anti-Lyn, anti-phospho-src family (Cell Signaling Technology), and anti-p85 PI3K (Upstate Biotechnology), detected with HRP-labeled secondary Abs, and developed with the LumiGlo Detection Kit (Cell Signaling Technology).

Immunoprecipitation

Immunoprecipitations were performed using the Profound Mammalian Coimmunoprecipitation Kit (Pierce). In brief, B cells were activated as described above, protein isolated, immunoprecipitated with either an anti-Lyn or anti-CD19 (Cell Signaling Technology) Ab following the manufacturer’s direction. The precipitates were analyzed using Western blot analysis as described above.

Flow cytometry

The number of B220+ and CD4+ cells was determined by FACS analysis as described previously (16). In brief, total splenocytes were collected on day 6 following immunization and stained with PE-conjugated rat anti-mouse B220, FITC-conjugated rat anti-mouse CD4, and allophycocyanin-conjugated rat anti-mouse IL-4 (BD Biosciences). For intracellular FACS analysis, total splenocytes on day 6 were stimulated for 4 h with PMA (50 ng/ml; Sigma-Aldrich) and ionomycin (500 ng/ml; Sigma-Aldrich) in the presence of brefeldin A (1 µg/ml; BD Biosciences). Cells were then washed, fixed, and permeabilized using the Cytofix/Cytoperm (BD Biosciences) kit according to the manufacturer’s directions and stained with a rat anti-mouse IL-4 (clone BVD6–24G2) (eBioscience). All samples were analyzed using a FACSAria flow cytometer (BD Biosciences). The data were analyzed using FlowJo software (Tree Star).

PI3K ELISA

The PI3K ELISA was performed as described previously (20). In brief, B cells were activated as described above, and protein was isolated and immunoprecipitated with a rabbit anti-mouse PI3K Ab (Upstate Biotechnology) for 1 h at 4°C. Protein A/G-agarose beads in PBS were added for 1 h at 4°C, and samples were collected and washed. The immunoprecipitated proteins were used to analyze the PI3K activity using the PI3K ELISA (Echelon BioSciences) following the manufacturer’s directions.

Quantitative real-time PCR

Quantitative real-time PCR was performed as described previously (20). The following primers were used: beta-actin 5'-TACAGCTTCACCACC ACAGC-3' and 5'-AAGGAAGGCTGGAAAAGAGC-3' (annealing temperature, 60°C, 206-bp product); Oct-2 5'-ATCAAGGCTGAAGAC CCCAGTG-3' and 5'-TGGAGGAGTTGCTGTATGTCCC-3' (annealing temperature, 60°C, 128-bp product); mature IgG1 transcript 5'-TATG GACTACTGGGGTCAAG-3' and 5'-CCTGGGCACAATTTTCTTGT-3' (annealing temperature, 63°C, 205-bp product).

IgG1 ELISA

For in vitro IgG1 determination, B cell culture supernatants were collected on days 4–7, and for in vivo IgG1 determination, serum samples were collected and frozen immediately at –80°C until analysis. Costar 96-well flexiplates (Fisher Scientific) were coated with goat anti-mouse IgG (2 µg/ml; BD Biosciences), and a standard curve for IgG1 was prepared using known quantities of recombinant IgG1 protein in a range of 1 µg/ml to 1 ng/ml. A secondary Ab, goat anti-mouse IgG1-alkaline phosphatase (BD Biosciences) 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.

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-deficient B cells produce less IgG1 and Oct-2 in vivo

Although the in vitro data to date show that direct stimulation of CD86 on a B cell regulates the amount of IgG1 produced, without affecting class switch recombination, animal models are required to prove in vivo relevance. A previous study from our laboratory used scid mice reconstituted with resting B cells alone that were activated by the i.p. injection of both an anti-CD40 Ab and rIL-4 with or without an anti-CD86 Ab. Serum levels of IgG1 on day 14 following activation were ~3-fold higher in the presence of an anti-CD86 Ab as compared with anti-CD40/IL-4 alone (15), suggesting that CD86 stimulation on some CD86-expressing cells affected the level of IgG1 produced. However, testing whether CD86 signals directly in a B cell when T cells are present to provide B cell help during a T cell-dependent IgG1 response in vivo proved more difficult, because any attempt to stimulate CD86 would interfere with T cell costimulation. Therefore, we established an adoptive transfer model system in which only the B cell would lack CD86, while all other APCs, namely DC and macrophages, would express CD86 normally and allow for optimal T cell activation to occur. CD4+ T cells and resting B cells from WT and/or CD86-deficient mice were adoptively transferred to Rag2-deficient mice, resulting in mice that expressed WT-T/WT-B or WT-T/CD86-deficient B cells. To determine whether equal numbers of T and B cells were localized in the spleens of these mice 6 days following immunization with TNP-KLH, flow cytometry was performed and the percentage of B220+ and CD4+ cells was found to be equivalent between the mice receiving WT-T/WT-B (Fig. 1A) or WT-T/CD86-deficient B cells (Fig. 1B). To determine whether a similar level of T cell activation occurred in the two groups of mice, the level of IL-4 mRNA produced by splenocytes was measured using real-time PCR, and was found to be equivalent (Fig. 1C). Likewise, flow cytometry was performed on single CD4+ T cells stained for intracellular IL-4 expression, and the data showed that the number of cells expressing IL-4 and the amount expressed per cell were equivalent (Fig. 1D). Because the induction of germline IgG1 mRNA is dependent on the presence of IL-4 (11, 12), and was not dependent on CD86 stimulation (33), the level of germline IgG1 mRNA was measured in splenocytes using real-time PCR, and was also found to be equivalent (Fig. 1C). Thus, all findings indicated that the level of T cell activation was equivalent in both adoptive transfer groups.


Figure 1
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 1. CD86-deficient B cells produce less IgG1 and Oct-2 in vivo. WT CD4+ T cells and WT B cells (WT) or WT CD4+ T cells and CD86-deficient B cells (CD86–/–) were adoptively transferred to RAG2-deficient mice. One week later, all mice received one i.p. injection of 100 µg TNP-KLH in alum. The percentage of total splenic B cells (B220+) and CD4+ T cells (CD4+) in the WT (A) and CD86–/– (B) reconstituted mice was determined by FACS analysis. C, The level of Oct-2, IL-4, and germline IgG1 mRNA on day 4 and mature IgG1 mRNA on day 6 was determined in total splenocytes from mice receiving WT B cells ({square}) or CD86-deficient B cells ({blacksquare}) using real-time PCR. D, The expression level of IL-4 in the CD4+ T cells from WT (24.6%) and CD86–/– (26.1%) was determined by intracellular FACS analysis. E, Serum was collected on day 14 following immunization and the level of IgG1 was determined by ELISA. F, Protein was isolated from total splenocytes on day 6 following immunization and the level of Oct-2 and actin was determined by Western blot analysis. *, p < 0.05 compared with the CD86–/– group.

 
When the reconstituted Rag2-deficient mice were immunized with the T cell-dependent Ag, TNP-KLH, mice receiving CD86-deficient B cells produced ~2-fold less of both serum IgG1 (Fig. 1E) and splenocyte-mature IgG1 mRNA (Fig. 1C), but equivalent amounts of germline IgG1 when compared with mice that received WT B cells. Similarly, total splenocyte Oct-2 mRNA (Fig. 1C) and protein (Fig. 1F), which were reported to increase in CD86-stimulated B cells (15), were also ~2-fold less in the mice receiving CD86-deficient B cells as compared with WT B cells. Taken together, these findings in vivo suggest that CD86 stimulation on a B cell during a T cell-dependent Ab response is needed to activate an intracellular signaling pathway in the B cell to up-regulate the level of IgG1 above the level induced by B cell activation alone, but independently of class switch recombination.

CD86 signaling in a B cell is CD19 dependent

The signaling pathway activated by CD86 proximal to NF-{kappa}B activation was recently identified to include PI3K/PDK1/Akt and PLC{gamma}2/PKC{alpha}beta (20). CD86, however, does not contain a tyrosine residue in the cytoplasmic domain, which is critical for the binding and activation of PI3K. The finding that CD86 signaling in a DC was limited to only those DC that expressed CD19 (21) suggested to us that CD86 might require CD19 to activate PI3K. Therefore, to determine whether CD19 was required for CD86 stimulation to increase PI3K activity, WT, CD86-, and CD19-deficient B cells were stimulated with CD40L/IL-4 for 16 h before the addition of CD28/Ig. As shown in Fig. 2A, exposure of CD28/Ig to WT B cells increased PI3K activity ~3-fold above that induced by CD40L/IL-4 alone, while CD86- and CD19-deficient B cells produced equivalent baseline levels of PI3K activity, but were unable to regulate PI3K activity in response to CD28/Ig. Previous data using a PI3K inhibitor showed that stimulation of CD86 on an activated B cell required PI3K to increase the level of Oct-2 mRNA (20). Consequently, WT, CD86-, and CD19-deficient B cells were activated as described above, and the level of Oct-2 mRNA was measured on day 2. CD86- and CD19-deficient B cells exposed to CD28/Ig failed to up-regulate Oct-2 mRNA, while WT B cells exposed to CD28/Ig increased the level of Oct-2 mRNA ~2-fold (Fig. 2B). The level of IgG1 mRNA (Fig. 2C) and protein (Fig. 2D) induced by the addition of CD28/Ig on WT, but not CD86- and CD19-deficient B cells, was ~2-fold higher when compared with the level induced by CD40L/IL-4 alone. To rule out the possibility that CD19-deficient B cells failed to up-regulate CD86 upon CD40/IL-4 stimulation, FACS analysis was performed, and the level of CD86 surface expression was equivalent between WT and CD19-deficient B cells (data not shown). In addition, the CD40L/IL-4-induced levels of PI3K activity, Oct-2, and IgG1 appeared to be equivalent between the WT, CD86-, and CD19-deficient B cells. We concluded that the inability of CD28/Ig to regulate these signaling pathways was due to a lack of CD86 signaling and not an inherent defect in the CD86- or CD19-deficient B cells. When TLR4-deficient B cells were activated with CD40L/IL-4, the level of Oct-2 and mature IgG1 mRNA (Fig. 3B) and protein (Fig. 3A) induced by addition of CD28/Ig was similar to that seen in WT cells (Fig. 2, D and E). Thus, collectively, these data show that CD86 stimulation on an activated B cell requires the presence of CD19 to increase the level of PI3K activity, Oct-2 expression, and IgG1 produced, suggesting that CD19 is a potential link between CD86 stimulation and the activation of PI3K in a B cell.


Figure 2
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 2. CD19 expression is required for CD86 signaling. A–D, WT ({square}), CD86-deficient (Figure 2), and CD19-deficient ({blacksquare}) B cells were activated in vitro with CD40L/IL-4 for 16 h before the addition of CD28/Ig or isotype control Ab. A, The level of PI3K activity was measured using an in vitro ELISA-based kinase assay. Data represent the mean (ng of PIP3) ± SEM from three independent experiments. The level of Oct-2 mRNA on day 2 (B) and mature IgG1 mRNA on day 5 (C) was determined using real-time PCR analysis and normalization to total actin. Data represent the mean ± SEM from three independent experiments. D, Cell supernatants were collected on day 7 and analyzed for total IgG1 by ELISA. Data represent the mean (ng/ml IgG1) ± SEM from three independent experiments. *, p < 0.05 compared with the CD40L/IL-4 alone group.

 

Figure 3
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 3. CD86 stimulation on TLR4-deficient B cells produces similar results to WT B cells. TLR4-deficient B cells were activated with CD40L/IL-4 in the absence or presence of CD28/Ig and supernatants were collected on day 7. The level of total IgG1 was determined using ELISA. The data represent the mean (ng/ml IgG1) ± SEM from three independent experiments. B, The level of Oct-2 mRNA on day 2 and mature IgG1 mRNA on day 5 was determined from WT B cells activated with CD40L/IL-4 in the absence ({square}) or presence ({blacksquare}) of CD28/Ig using real-time PCR analysis. The data represent mean ± SEM from three independent experiments. *, p < 0.05 compared with the CD40L/IL-4 alone group.

 
CD19 is activated by CD86 stimulation

Because CD86 failed to signal in a CD19-deficient B cell, the question remained as to the mechanism by which CD86 activated CD19. CD19 contains nine tyrosine residues in the cytoplasmic domain that are phosphorylated to promote CD19 interaction with a number of SH2 domain-containing proteins, including Lyn, Vav, Grb2, and p85{alpha} PI3K (reviewed in Ref. 23). To determine whether CD86 stimulation increased tyrosine phosphorylation of CD19, Western blot analysis was performed on whole cell lysates from B cells activated as described above. The level of tyrosine phophorylation of CD19 (Tyr513), which is the critical residue for CD19 activation of PI3K (34), was increased at 5 and 15 min following addition of CD28/Ig to WT B cells (Fig. 4A), but not when added to CD86-deficient B cells (Fig. 4B). When TLR4-deficient B cells were used, the data showed that the level of phosphorylated CD19 was increased to a similar level as seen using WT B cells (Fig. 4C). Because tyrosine phosphorylation of the CD19 cytoplasmic domain promotes the binding of SH2 domain-containing proteins, such as PI3K, it was possible that CD86 activated CD19 to bind and activate PI3K. B cells were activated as described above, and total protein was isolated and immunoprecipitated with an anti-CD19 Ab, after which time the precipitates were analyzed by Western blot analysis. The amount of Lyn, Vav, and p85{alpha} PI3K that immunoprecipitated with CD19 increased following the addition of CD28/Ig (Fig. 4D). The blots were also probed for the presence of CD86, but failed to show an interaction with CD19 (data not shown), suggesting that CD86 activates CD19 through a mechanism other than direct interaction. Thus, these results suggest that CD86 stimulation on a B cell activates CD19 to potentially mediate the activation of PI3K.


Figure 4
View larger version (50K):
[in this window]
[in a new window]

  		FIGURE 4. CD86 stimulation increases the activation of CD19. A–C, The level of phospho-CD19 and total actin was analyzed using Western blot analysis from WT (A), CD86-deficient (B), and TLR4-deficient B cells (C) activated with CD40L/IL-4 for 16 h before the addition of CD28/Ig. Band density was determined using densitometry and the data represent the mean fold increase in phospho-CD19 normalized to actin ± SEM from three independent experiments. D, Protein samples from resting, CD40L/IL-4-activated, and CD40L/IL-4/CD28/Ig-activated B cells were immunoprecipitated (IP) with an anti-CD19 Ab, and the precipitates were analyzed for the presence of CD19, phospho-CD19, Vav, p85{alpha} PI3K, and Lyn by Western blot analysis. One representative gel from three independent experiments is shown.

 
CD86 stimulation activates the PTK Lyn

Lyn is a src-family PTK reported to be capable of phosphorylating CD19 (31). However, Lyn kinase must first be activated through tyrosine phosphorylation. Y397, which is located in the kinase domain of Lyn, positively regulates activity, while Y508, which is located in the regulatory domain of Lyn, negatively regulates Lyn activity (reviewed in Ref. 32). To determine whether CD86 stimulation requires a PTK to positively regulate the phosphorylation of CD19, we designed three experiments. First, B cells were activated as described above, and two different src-family kinase inhibitors, SU6656 and PP2, were added 30 min before addition of CD28/Ig. Pretreatment with either one of the src-family kinase inhibitors prevented the CD86-induced increase in phospho-CD19 (Fig. 5A). Second, to further confirm that a PTK was involved in the CD86-induced signaling pathway, B cells were activated as described above in the presence or absence of a src-family kinase inhibitor. In the presence of the src-family kinase inhibitor, CD28/Ig failed to increase the level of Oct-2 mRNA (Fig. 5B) above that induced by CD40L/IL-4 alone. And third, because the PTK Lyn was immunoprecipitated with CD19 (Fig. 4C), it was possible that CD86 stimulation regulated the phosphorylation of Lyn. To test this possibility, B cells were activated as described above, and total protein was isolated and immunoprecipitated with an anti-Lyn Ab. The level of phospho-Lyn (Y397) that positively regulates Lyn activity was increased at 5 min following CD86 stimulation on WT B cells (Fig. 5C). Thus, these results suggest that Lyn is the PTK that is activated by CD86 stimulation to increase the phosphorylation of CD19.


Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 5. Lyn kinase plays a role in CD86 signaling. A, Western blot analysis of phospho-CD19 and total actin in B cells activated with CD40L/IL-4 for 16 h and then pretreated with an src-family kinase inhibitor, SU6656 (100 nM), for 30 min before the addition of CD28/Ig. Band densities were determined using densitometry, and the data represent the mean fold increase in phospho-CD19 normalized to total actin ± SEM from three independent experiments. One representative gel from three independent experiments is shown. B, Real-time PCR analysis of Oct-2 and actin mRNA in B cells was activated as described above. Data represent the mean fold increase in Oct-2 mRNA normalized to actin ± SEM from three independent experiments. C, Protein samples from resting, CD40L/IL-4-activated, and CD40L/IL-4/CD28/Ig-activated B cells were immunoprecipitated (IP) with an anti-Lyn Ab, and the precipitates were analyzed for the presence of phospho-Lyn and total Lyn by Western blot analysis. One representative gel from three independent experiments is shown. *, p < 0.05 compared with the CD40L/IL-4 alone group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The goal of the present study was to identify the mechanism by which CD86 activated PI3K, and to confirm the relevance of CD86 stimulation on a B cell responding to a T cell-dependent Ag in vivo. Using an adoptive transfer model, the present data show that the loss of CD86 expression on a B cell alone prevents the generation of an optimal IgG1 response, even in the presence of a normal IL-4 and germline IgG1 response. We also show, for the first time, that CD86 stimulation on a CD40L/IL-4-activated WT, but not CD86- or CD19-deficient, B cell in vitro increases the phosphorylation of Lyn and CD19, as well as the amount of Lyn, Vav, and p85{alpha} PI3K that immunoprecipitated with CD19. Thus, our findings suggest that CD86 on a B cell plays a key role in regulating the level of IgG1 produced in vitro and in vivo, independently of class switch recombination, and that Lyn and CD19 may serve as the signaling intermediates activated by CD86 proximal to PI3K.

Initially, CD86 was thought to be a costimulatory molecule that was unable to signal itself. However, a number of studies to date in both B cells and DC have suggested that CD86 is able to signal directly. For example, stimulation of CD86 with CD28/Ig on either a B cell or DC increased NF-{kappa}B-mediated gene transcription, as was shown using transient transfection systems (20, 22), suggesting that CD86 on either a B cell or dendritic cell activate at least one common signaling intermediate. This finding also suggests that the signaling intermediates activated proximal to NF-{kappa}B may also be the same. A recent study in DC suggested that only a subset of DC that expressed CD19 responded to CD86 stimulation (21), suggesting that CD19 may be required for CD86 signaling to occur in DC. The present findings using WT and CD19-deficient B cells showed that CD86 stimulation on an activated WT B cell increased the phosphorylation and activation of CD19, whereas the activation of CD19-deficient B cells did not, strengthening our hypothesis that CD19 expression is required for CD86 to function in a B cell. Therefore, because CD86 signaling has been shown in two different cell types to involve CD19 and NF-{kappa}B, it is becoming more likely that CD86 is able to signal directly and can no longer be considered simply a costimulatory molecule for CD28 on a T cell.

Yet, because CD19-deficient B cells have been reported to respond differently to activation stimuli, it was possible that the inability of CD19-deficient B cells to respond to CD28/Ig may have been due to an inherent defect of CD40 signaling in these cells, as opposed to a defect in CD19-dependent CD86 signaling. This possibility was particularly relevant because CD40 stimulation is reported to be associated with CD19 activation (35). For example, the ability of CD19-deficient B cells to proliferate upon CD40 stimulation was reported to be less than that in WT B cells (36), while another report showed that the proliferation remained unchanged (25). One possible explanation for these contrasting results is that the ligands used to stimulate CD40 differed, i.e., anti-CD40 Ab decreased proliferation in CD19-deficient B cells when compared with WT B cells (36), while recombinant CD40L induced no change (25). If these two CD40-stimulating reagents activate different signaling intermediates, as has been reported previously (37), then it is possible that each signaling intermediate affected proliferation differently. Our findings using recombinant CD40L expressed on the surface of Sf9 cells would support the finding of Rickert et al. (25) and expand it to the IgG1 response because WT and CD19-deficient B cells activated with CD40L/IL-4 produced comparable levels of IgG1 (Fig. 2D). This latter finding was also true for CD40/IL-4-activated B cells that made IgE (38). Another possible explanation is that proliferation results may be misleading when interpreting Ab results. For example, proliferation in the B cell was measured at 72 h, while regulation of the role of mature IgG1 transcription was measured on day 5, at a time when any change in proliferation that may have occurred at 72 h may not be relevant to the IgG1 response that occurs days later. Taken together, the loss of CD86 signaling in a CD19-deficient B cell appears to be due to a lack of CD19, and not to an inherent defect in the CD19-deficient B cell to make Ab after CD40 stimulation.

Previous data showed that CD86 stimulation increases PI3K activity (20), although the mechanism by which this occurs remained unknown because CD86 lacks expression of a tyrosine residue in the cytoplasmic domain that is required for PI3K activation. If previous reports are correct that CD19 stimulation activates PI3K (39, 40), and if our present finding is correct that CD86 stimulation activates CD19, then CD19 may be the link between CD86 stimulation and PI3K activation. However, what remains unclear is how CD86 mediates the activation of CD19. One possibility may be that CD86 activates CD19 through a direct physical interaction. For example, CD21/CD81 are reported to activate CD19 via a direct physical interaction using their extracellular and transmembrane domains (26, 41). In contrast, MHC class II (30) and CD180 (RP105) (29) are reported to activate CD19 without a detectable physical interaction. Our unpublished findings using coimmunoprecipitation suggest that CD86 and CD19 may not physically interact with each other. Although this finding does not preclude the possibility that CD86 and CD19 formed a weak interaction that was undetectable using our immunoprecipitation protocol, it does suggest that some mechanism would need to exist other than direct physical interaction. We propose, based on the present data, that this mechanism involves the phosphorylation of Lyn, phospho-Lyn binding to CD19, and subsequent phosphorylation of the CD19 cytoplasmic domain. However, the mechanism by which CD86 might activate Lyn directly remains unknown, but may be similar to the mechanism used by other B cell-associated receptors that also activate CD19. For example, crosslinking of the BCR (42) or CD21/CD19/CD81 (43) is known to recruit these receptor complexes into lipid raft domains where Lyn is selectively enriched (44). If CD86 uses a similar mechanism, then it also would need to be recruited into a lipid raft domain following either CD40L/IL-4 and/or CD86 stimulation, providing a localized region in which CD86 would cluster with Lyn and/or CD19. In support of this hypothesis are the findings that crosslinking of either CD86 or CD40 on mature DC recruited CD86 to lipid raft domains (45). If the mechanism by which CD86 signals in a DC is similar to a B cell, which may involve recruitment of CD86 into lipid raft domains and clustering with signaling intermediates to allow for CD19 activation, then this clustering may be the mechanism by which CD86 activates Lyn, CD19, and PI3K in a B cell.

Previous studies used an anti-CD86 Ab to stimulate CD86 on the B cell surface. However, we recently switched to using CD28/Ig to more closely mimic the endogenous ligand found on T cells, i.e., CD28. Because studies in DC using CD28/Ig suggested a role for both CD80 and CD86 signaling in some of the functional responses measured (22), and because CD28/Ig can bind to both CD80 and CD86 (46), the possibility existed that the loss of CD86 in a CD86-deficient B cell prevented CD80/CD86 heterodimerization and, therefore, might explain the loss of CD28/Ig-induced signaling when CD86-deficient B cells were used. We think that this possibility is unlikely because fluorescence resonance electron transfer technology showed that CD80 exists primarily as a homodimer on the B cell surface, while CD86 exists primarily as a monomer (47). Likewise, the chemical properties of the potential dimer interfaces of CD80 and CD86 were shown to be very different. CD80 expresses a hydrophobic interface that would promote dimerization, while CD86 expresses a hydrophilic interface that would make it less prone to dimerization (48). Therefore, it is unlikely that CD80 and CD86 interact on the cell surface to mediate the CD28/Ig-induced effects and, therefore, make it unlikely that the loss of an effect when using CD86-deficient B cells was due to the loss of dimerization with CD80. Nonetheless, CD80-deficient B cells will be used in future experiments to further rule out this possibility. Another reason why we think CD80 is not involved in the signaling induced by CD28/Ig in a B cell is the fact that to induce the signaling intermediates measured in the present study within the first 24 h of B cell activation, both CD80 and CD86 would need to be concomitantly expressed during this time period. However, the kinetics of CD80 and CD86 expression are very different following B cell activation. CD86 surface expression is detectable at 6 h and maximal at 24–48 h after activation (Ref. 7 and data not shown), while CD80 surface expression is detectable by 24 h and maximal at 48–72 h (Ref. 49 and data not shown). This difference in expression kinetics makes it unlikely that CD80 would be expressed to any detectable level on the B cell to interact with CD86 when CD28/Ig was added to our culture system 16 h following CD40L/IL-4 activation. Taken together, these two arguments make it unlikely that CD80 and CD86 interact with each other on a B cell at the time of addition of CD28/Ig to induce an intracellular signal and, therefore, we conclude that the activation of Lyn, CD19, and PI3K is likely due to CD86 stimulation alone.

Determining the role of CD86 signaling in a B cell during a T cell-dependent Ab response in vivo has been a challenge due to the requirement of T cell costimulation. The present in vivo results addressed this issue and suggest that expression of CD86 on the B cell alone is required for an optimal IgG1 response to occur. Another study also attempted to determine the role of CD86 signaling in vivo using a mixed chimeras model system and found that CD40 expression on a B cell was essential for class switch recombination to IgG1, but that CD80/86 expression on the B cell was not (33). The present data agree with this finding, because no change was detected in germline IgG1 mRNA between WT and CD86-deficient B cells. However, in contrast to the mixed chimeras model system, our adoptive transfer model system showed that B cells lacking CD86 expression produced ~2-fold less mature IgG1 mRNA and protein. The reason for the discrepancy about the role of CD86 in mature IgG1 production in the two model systems is not yet evident, but our finding suggests that the expression of CD86 on a B cell may play a crucial role in establishing the level of IgG1 produced in vivo.

Human and murine CD86 share ~70% gene homology, with a number of differences located in the cytoplasmic domain. Both human and murine cytoplasmic domains contain putative PKC phosphorylation sites, i.e., serine and threonine residues, but the human CD86 cytoplasmic domain contains a greater number of potential phosphorylation sites. This suggests that similar mechanisms may be used for CD86 signal transduction in both species, but that humans may use additional phosphorylation sites to regulate signal intensity. However, the question remains as to whether the findings in mice about CD86 signaling reflect the mechanism by which human CD86 might function. First, CD86 stimulation on human tonsillar B cells in vitro was reported to increase the production of IgG4 (17), suggesting that human CD86 might also signal directly within a human B cell to regulate the level of IgG4 produced. The ability of both human and murine CD86 to regulate the level of IgG4/IgG1 produced by a B cell suggests that similar signaling mechanisms might be used intracellularly. Interestingly, one report in humans showed that a polymorphism in the human CD86 cytoplasmic domain of an alanine to threonine change at + 1057 position (50), which introduced an additional potential phosphorylation site, was associated with a lower incidence of acute liver transplant rejection (51). This finding suggested that the introduction of an additional phosphorylation site in the cytoplasmic domain of human CD86 might have altered the signaling capabilities of CD86 and/or the ability to costimulate a T cell. More interestingly, a number of studies in mice (8, 20) and humans (17) have suggested that prior activation of the B cell is required for CD86 signaling to activate an intracellular signaling pathway. If true, potentially the CD86 cytoplasmic domain requires phosphorylation to become competent to signal. In support of this hypothesis, one study reported that the murine CD86 cytoplasmic domain was phosphorylated only after cell activation, suggesting that phosphorylation may play a role in the ability of CD86 to signal in a murine B cell (52). Taken together, both human and mouse data suggest that CD86 stimulation on a B cell regulates the level of a T cell-dependent Ab response similarly, but the role played by the potential phosphorylation sites in the cytoplasmic domain of human and murine CD86 remain unknown.


   Acknowledgments
 
We gratefully acknowledge Drs. William Lafuse (The Ohio State University, Columbus, OH) and Kerry Campbell (Fox Chase Cancer Center, Philadelphia, PA) 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 Grant AI37326. N.W.K. is a recipient of a training grant award from National Institutes of Health Grant T32 AI55411. Back

2 This research is part of the dissertation research conducted by Nicholas W. Kin who is a predoctoral student in the Integrated Biomedical Science Graduate Program, The Ohio State University, Columbus, OH 43210. Back

3 Address correspondence and reprint requests to Dr. Virginia M. Sanders, 2194 Graves Hall, 333 West 10th Avenue, Columbus, OH 43210. E-mail address: Sanders.302{at}osu.edu Back

4 Abbreviations used in this paper: DC, dendritic cells; WT, wild type; PTK, protein tyrosine kinase; KLH, keyhole limpet hemocyanin; CD40L, CD40 ligand. Back

Received for publication March 10, 2007. Accepted for publication May 14, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Freeman, G. J., A. S. Freedman, J. M. Segil, G. Lee, J. F. Whitman, L. M. Nadler. 1989. B7, a new member of the Ig superfamily with unique expression on activated and neoplastic B cells. J. Immunol. 143: 2714-2722. [Abstract]
  2. Freeman, G. J., J. G. Gribben, V. A. Boussiotis, J. W. Ng, V. A. Restivo, Jr, L. A. Lombard, G. S. Gray, L. M. Nadler. 1993. Cloning of B7-2: a CTLA-4 counter-receptor that costimulates human T cell proliferation. Science 262: 909-911. [Abstract/Free Full Text]
  3. Azuma, M., D. Ito, K. Okumara, J. H. Phillips, L. L. Lanier, C. Somoza. 1993. B70 antigen is a second ligand for CTLA-4 and CD28. Nature 366: 76-79. [Medline]
  4. Roy, M., A. Aruffo, J. Ledbetter, P. Linsley, M. Kehry, R. Noelle. 1995. Studies on the interdependence of gp39 and B7 expression and function during antigen-specific immune responses. Eur. J. Immunol. 25: 596-603. [Medline]
  5. Stack, R. M., D. J. Lenschow, G. S. Gray, J. A. Bluestone, F. W. Fitch. 1994. IL-4 treatment of small splenic B cells induces costimulatory molecules B7-1 and B7-2. J. Immunol. 152: 5723-5733. [Abstract]
  6. Hathcock, K. S., G. Laszlo, H. B. Dickler, J. Bradshaw, P. Linsley, R. J. Hodes. 1993. Identification of an alternative CTLA-4 ligand costimulatory for T cell activation. Science 262: 905-907. [Abstract/Free Full Text]
  7. Lenschow, D. J., G. H. T. Su, L. A. Zuckerman, N. Nabavi, C. L. Jellis, G. S. Gray, J. Miller, J. A. Bluestone. 1993. Expression and functional significance of an additional ligand for CTLA-4. Proc. Natl. Acad. Sci. USA 90: 11054-11058. [Abstract/Free Full Text]
  8. Kasprowicz, D. J., A. P. Kohm, M. T. Berton, A. J. Chruscinski, A. Sharpe, V. M. Sanders. 2000. Stimulation of the B cell receptor, CD86 (B7-2), and the beta 2-adrenergic receptor intrinsically modulates the level of IgG1 and IgE produced per B cell. J. Immunol. 165: 680-690. [Abstract/Free Full Text]
  9. Kohm, A. P., A. Mozaffarian, V. M. Sanders. 2002. B cell receptor- and beta-2-adrenergic receptor-induced regulation of B7-2 (CD86) expression in B cells. J. Immunol. 168: 6314-6322. [Abstract/Free Full Text]
  10. Linsley, P. S., J. A. Ledbetter. 1993. The role of the CD28 receptor during T cell responses to antigen. Ann. Rev. Immunol. 11: 191-212. [Medline]
  11. Lin, S. C., H. H. Wortis, J. Stavnezer. 1998. The ability of CD40L, but not lipopolysaccharide, to initiate immunoglobulin switching to immunoglobulin G1 is explained by differential induction of NF-{kappa}B/Rel proteins. Mol. Cell. Biol. 18: 5523-5532. [Abstract/Free Full Text]
  12. Warren, W. D., K. L. Roberts, L. A. Linehan, M. T. Berton. 1999. Regulation of the germline immunoglobulin C{gamma}1 promoter by CD40 ligand and IL-4: dual role for tandem NF-{kappa}B binding sites. Mol. Immunol. 36: 31-44. [Medline]
  13. Shahinian, A., K. Pfeffer, K. P. Lee, T. M. Kundig, K. Kishihara, A. Wakeham, K. Kawai, P. S. Ohashi, C. B. Thompson, T. W. Mak. 1993. Differential T cell costimulatory requirements in CD28-deficient mice. Science 261: 609-612. [Abstract/Free Full Text]
  14. Borriello, F., M. P. Sethna, S. D. Boyd, A. N. Schweitzer, E. A. Tivol, D. Jacoby, T. B. Strom, E. M. Simpson, G. J. Freeman, A. H. Sharpe. 1997. B7-1 and B7-2 have overlapping, critical roles in immunoglobulin class switching and germinal center formation. Immunity 6: 303-313. [Medline]
  15. Podojil, J. R., N. W. Kin, V. M. Sanders. 2004. CD86 and beta2-adrenergic receptor signaling pathways, respectively, increase Oct-2 and OCA-B expression and binding to the 3'-IgH enhancer in B cells. J. Biol. Chem. 279: 23394-23404. [Abstract/Free Full Text]
  16. Podojil, J. R., V. M. Sanders. 2003. Selective regulation of mature IgG1 transcription by CD86 and beta2-adrenergic receptor stimulation. J. Immunol. 170: 5143-5151. [Abstract/Free Full Text]
  17. Jeannin, P., Y. Delneste, S. Lecoanet-Henchoz, J.-F. Gauchat, J. Ellis, J.-Y. Bonnefoy. 1997. CD86 (B7-2) on human B cells: a functional role in proliferation and selective differentiation into IgE- and IgG4-producing cells. J. Biol. Chem. 272: 15613-15619. [Abstract/Free Full Text]
  18. Suvas, S., V. Singh, S. Sahdev, H. Vohra, J. A. Agrewala. 2002. Distinct role of CD80 and CD86 in the regulation of the activation of B cell and B cell lymphoma. J. Biol. Chem. 277: 7766-7775. [Abstract/Free Full Text]
  19. Sahoo, N. C., K. V. Rao, K. Natarajan. 2002. CD80 expression is induced on activated B cells following stimulation by CD86. Scand. J. Immunol. 55: 577-584. [Medline]
  20. Kin, N. W., V. M. Sanders. 2006. CD86 stimulation on a B cell activates the phosphatidylinositol 3-kinase/Akt and phospholipase C{gamma}2/protein kinase C{alpha}beta signaling pathways. J. Immunol. 176: 6727-6735. [Abstract/Free Full Text]
  21. Baban, B., A. M. Hansen, P. R. Chandler, A. Manlapat, A. Bingaman, D. J. Kahler, D. H. Munn, A. L. Mellor. 2005. A minor population of splenic dendritic cells expressing CD19 mediates IDO-dependent T cell suppression via type I IFN signaling following B7 ligation. Int. Immunol. 17: 909-919. [Abstract/Free Full Text]
  22. Orabona, C., U. Grohmann, M. L. Belladonna, F. Fallarino, C. Vacca, R. Bianchi, S. Bozza, C. Volpi, B. L. Salomon, M. C. Fioretti, et al 2004. CD28 induces immunostimulatory signals in dendritic cells via CD80 and CD86. Nat. Immunol. 5: 1134-1142. [Medline]
  23. Del Nagro, C. J., D. C. Otero, A. N. Anzelon, S. A. Omori, R. V. Kolla, R. C. Rickert. 2005. CD19 function in central and peripheral B-cell development. Immunol. Res. 31: 119-131. [Medline]
  24. Engel, P., L. J. Zhou, D. C. Ord, S. Sato, B. Koller, T. F. Tedder. 1995. Abnormal B lymphocyte development, activation, and differentiation in mice that lack or overexpress the CD19 signal transduction molecule. Immunity 3: 39-50. [Medline]
  25. Rickert, R. C., K. Rajewsky, J. Roes. 1995. Impairment of T-cell-dependent B-cell responses and B-1 cell development in CD19-deficient mice. Nature 376: 352-355. [Medline]
  26. Matsumoto, A. K., J. Kopicky-Burd, R. H. Carter, D. A. Tuveson, T. F. Tedder, D. T. Fearon. 1991. Intersection of the complement and immune systems: a signal transduction complex of the B lymphocyte-containing complement receptor type 2 and CD19. J. Exp. Med. 173: 55-64. [Abstract/Free Full Text]
  27. Bradbury, L. E., G. S. Kansas, S. Levy, R. L. Evans, T. F. Tedder. 1992. The CD19/CD21 signal transducing complex of human B lymphocytes includes the target of antiproliferative antibody-1 and Leu-13 molecules. J. Immunol. 149: 2841-2850. [Abstract]
  28. Carter, R. H., D. T. Fearon. 1992. Lowering the threshold for antigen receptor stimulation of B lymphocytes. Science 256: 105-107. [Abstract/Free Full Text]
  29. Yazawa, N., M. Fujimoto, S. Sato, K. Miyake, N. Asano, Y. Nagai, O. Takeuchi, K. Takeda, H. Okochi, S. Akira, et al 2003. CD19 regulates innate immunity by the toll-like receptor RP105 signaling in B lymphocytes. Blood 102: 1374-1380. [Abstract/Free Full Text]
  30. Bobbitt, K. R., L. B. Justement. 2000. Regulation of MHC class II signal transduction by the B cell coreceptors CD19 and CD22. J. Immunol. 165: 5588-5596. [Abstract/Free Full Text]
  31. Fujimoto, M., J. C. Poe, P. J. Jansen, S. Sato, T. F. Tedder. 1999. CD19 amplifies B lymphocyte signal transduction by regulating Src-family protein tyrosine kinase activation. J. Immunol. 162: 7088-7094. [Abstract/Free Full Text]
  32. Xu, Y., K. W. Harder, N. D. Huntington, M. L. Hibbs, D. M. Tarlinton. 2005. Lyn tyrosine kinase: accentuating the positive and the negative. Immunity 22: 9-18. [Medline]
  33. Lumsden, J. M., J. A. Williams, R. J. Hodes. 2003. Differential requirements for expression of CD80/86 and CD40 on B cells for T-dependent antibody responses in vivo. J. Immunol. 170: 781-787. [Abstract/Free Full Text]
  34. Wang, Y., S. R. Brooks, X. Li, A. N. Anzelon, R. C. Rickert, R. H. Carter. 2002. The physiologic role of CD19 cytoplasmic tyrosines. Immunity 17: 501-514. [Medline]
  35. Gardby, E., X. J. Chen, N. Y. Lycke. 2001. Impaired CD40-signalling in CD19-deficient mice selectively affects Th2-dependent isotype switching. Scand. J. Immunol. 53: 13-23. [Medline]
  36. Sato, S., A. S. Miller, M. C. Howard, T. F. Tedder. 1997. Regulation of B lymphocyte development and activation by the CD19/CD21/CD81/Leu 13 complex requires the cytoplasmic domain of CD19. J. Immunol. 159: 3278-3287. [Abstract]
  37. Baccam, M., G. A. Bishop. 1999. Membrane-bound CD154, but not CD40-specific antibody, mediates NF-{kappa}B-independent IL-6 production in B cells. Eur. J. Immunol. 29: 3855-3866. [Medline]
  38. Pongratz, G., J. W. McAlees, D. H. Conrad, R. S. Erbe, K. M. Haas, V. M. Sanders. 2006. The level of IgE produced by a B cell is regulated by norepinephrine in a p38 MAPK- and CD23-dependent manner. J. Immunol. 177: 2926-2938. [Abstract/Free Full Text]
  39. Buhl, A. M., C. M. Pleiman, R. C. Rickert, J. C. Cambier. 1997. Qualitative regulation of B cell antigen receptor signaling by CD19: selective requirement for PI3-kinase activation, inositol-1,4,5-trisphosphate production and Ca2+ mobilization. J. Exp. Med. 186: 1897-1910. [Abstract/Free Full Text]
  40. Tuveson, D. A., R. H. Carter, S. P. Soltoff, D. T. Fearon. 1993. CD19 of B cells as a surrogate kinase insert region to bind phosphatidylinositol 3-kinase. Science 260: 986-989. [Abstract/Free Full Text]
  41. Matsumoto, A. K., D. R. Martin, R. H. Carter, L. B. Klickstein, J. M. Ahearn, D. T. Fearon. 1993. Functional dissection of the CD21/CD19/TAPA-1/Leu-13 complex of B lymphocytes. J. Exp. Med. 178: 1407-1417. [Abstract/Free Full Text]
  42. Petrie, R. J., P. P. Schnetkamp, K. D. Patel, M. Awasthi-Kalia, J. P. Deans. 2000. Transient translocation of the B cell receptor and Src homology 2 domain-containing inositol phosphatase to lipid rafts: evidence toward a role in calcium regulation. J. Immunol. 165: 1220-1227. [Abstract/Free Full Text]
  43. Cherukuri, A., P. C. Cheng, H. W. Sohn, S. K. Pierce. 2001. The CD19/CD21 complex functions to prolong B cell antigen receptor signaling from lipid rafts. Immunity 14: 169-179. [Medline]
  44. Pierce, S. K.. 2002. Lipid rafts and B-cell activation. Nat. Rev. Immunol. 2: 96-105. [Medline]
  45. Meyer zum Bueschenfelde, C. O., J. Unternaehrer, I. Mellman, K. Bottomly. 2004. Regulated recruitment of MHC class II and costimulatory molecules to lipid rafts in dendritic cells. J. Immunol. 173: 6119-6124. [Abstract/Free Full Text]
  46. Freeman, G. J., F. Borriello, R. J. Hodes, H. Reiser, J. G. Gribben, J. W. Ng, J. Kim, J. M. Goldberg, K. Hathcock, G. Laszlo, et al 1993. Murine B7-2, an alternative CTLA4 counter-receptor that costimulates T cell proliferation and interleukin 2 production. J. Exp. Med. 178: 2185-2192. [Abstract/Free Full Text]
  47. Zhang, X., J. C. Schwartz, S. C. Almo, S. G. Nathenson. 2003. Crystal structure of the receptor-binding domain of human B7-2: insights into organization and signaling. Proc. Natl. Acad. Sci. USA 100: 2586-2591. [Abstract/Free Full Text]
  48. Bhatia, S., M. Edidin, S. C. Almo, S. G. Nathenson. 2005. Different cell surface oligomeric states of B7-1 and B7-2: implications for signaling. Proc. Natl. Acad. Sci. USA 102: 15569-15574. [Abstract/Free Full Text]
  49. Razi-Wolf, Z., G. J. Freeman, F. Galvin, B. Benacerraf, L. M. Nadler. 1992. Expression and function of the murine B7 antigen, the major costimulatory molecule expressed by peritoneal exudate cells. Proc. Natl. Acad. Sci. USA 89: 4214-4210.
  50. Delneste, Y., R. Bosotti, G. Magistrelli, J. Y. Bonnefoy, J. F. Gauchat. 2000. Detection of a polymorphism in exon 8 of the human CD86 gene. Immunogenetics 51: 762-763. [Medline]
  51. Marin, L. A., M. R. Moya-Quiles, M. Miras, M. Muro, A. Minguela, J. Bermejo, P. Ramirez, A. M. Garcia-Alonso, P. Parrilla, M. R. Alvarez-Lopez. 2005. Evaluation of CD86 gene polymorphism at +1057 position in liver transplant recipients. Transplant Immunol. 15: 69-74. [Medline]
  52. Lenschow, D. J., T. L. Walunas, J. A. Bluestone. 1996. CD28/B7 system of T cell costimulation. Ann. Rev. Immunol. 14: 233-258. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
B. Kretschmer, K. Luthje, S. Schneider, B. Fleischer, and M. Breloer
Engagement of CD83 on B Cells Modulates B Cell Function In Vivo
J. Immunol., March 1, 2009; 182(5): 2827 - 2834.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
N. W. Kin, D. M. Crawford, J. Liu, T. W. Behrens, and J. F. Kearney
DNA Microarray Gene Expression Profile of Marginal Zone versus Follicular B Cells and Idiotype Positive Marginal Zone B Cells before and after Immunization with Streptococcus pneumoniae
J. Immunol., May 15, 2008; 180(10): 6663 - 6674.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kin, N. W.
Right arrow Articles by Sanders, V. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kin, N. W.
Right arrow Articles by Sanders, V. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS