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CD86 Regulates IgG1 Production via a CD19-Dependent Mechanism Nicholas W. Kin and Virginia M. Sanders This information is current as J Immunol 2007; 179:1516-1523; ; of September 23, 2021. doi: 10.4049/jimmunol.179.3.1516 http://www.jimmunol.org/content/179/3/1516 Downloaded from References This article cites 52 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/179/3/1516.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CD86 Regulates IgG1 Production via a CD19-Dependent Mechanism1

Nicholas W. Kin2 and Virginia M. Sanders3

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. The Journal of Downloaded from Immunology, 2007, 179: 1516–1523.

D86 was cloned in 1993 as a counterreceptor for both murine B cell increases the rate of mature IgG1 transcription, as CD28 and CTLA-4 that are expressed on T cells (1–3). determined by nuclear run-on analysis (15), the level of murine

CD86 is expressed primarily on APCs, such as B cells, IgG1 (8, 16), and human IgG4 protein (17), anti-apoptotic factors C 4 macrophages, and dendritic cells (DC) (2, 3), and functions as an ␬

(18), CD80 expression (19), NF- B (p50/p65) activation (15), Oct-2 http://www.jimmunol.org/ important costimulatory molecule. Resting B cells express CD86 expression, and binding to the 3Ј-IgH enhancer, as determined by at low levels (2), but rapidly up-regulate CD86 following activa- chromatin immunoprecipitation assay (15) and 3Ј-IgH enhancer ac- tion of the BCR (2), CD40 (4), IL-4R (5), LPS receptor (6, 7), and tivity, as shown using transiently and stably transfected B cell lines ␤ 2-adrenergic receptor (8, 9). CD86 on a B cell interacts with CD28 (20). Recently, the signaling pathway activated by CD86 proximal to on a T cell to activate a signaling pathway in the T cell that increases NF-␬B activation in a B cell was identified to involve PI3K/PDK1/ cell activation and expression of CD40 ligand (CD40L) and secretion Akt and PLC␥2/PKC␣␤ (20). CD86 signaling is also activated in DC of IL-4 (10), which are essential molecules that interact with CD40 exposed to CD28/Ig or CTLA-4/Ig to stimulate CD86. In these cells, and the IL-4R on a B cell, respectively, to induce class switch recom- CD86 stimulation increases the level of p38 MAPK activation, the bination to IgG (11, 12). CD86- and CD28-deficient mice produce by guest on September 23, 2021 1 production of IL-6, IFN-␣, and IDO (21, 22), but only in DC-express- less IgG following immunization with a T cell-dependent Ag when 1 ing CD19 (21), suggesting that CD19 may be necessary for CD86 to compared with wild-type (WT) mice (13, 14), likely due to the loss of function. However, the question remains as to the mechanism by the CD86/CD28 interaction, which is required for costimulation of the which CD86 stimulation activates PI3K, particularly because CD86 T cell to increase CD40L and IL-4 expression during the generation fails to express tyrosine residues in the cytoplasmic domain that are of an optimal T cell-dependent Ab response. essential for PI3K activation. However, an alternative explanation for the results using CD86- and CD28-deficient mice would be that CD86 signaling to the B CD19 is a coreceptor primarily expressed on the B cell and cell was lost, and that this signal was required to regulate the level plays an important role in B cell development, as well as BCR- and CD21-induced signal transduction (23). CD19-deficient mice of IgG1 produced directly. Recent studies revealed that stimulation of CD86 with either an anti-CD86 Ab or CD28/Ig on an activated 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 Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio CD21/CD81/Leu13 complex (26, 27), the BCR (28), CD180 State University, Columbus, OH 43210 (RP105) (29), and MHC class II (30). The cytoplasmic tail of Received for publication March 10, 2007. Accepted for publication May 14, 2007. CD19 contains nine tyrosine residues, that when phosphorylated, The costs of publication of this article were defrayed in part by the payment of page allow for the interaction with the SH2 domain-containing proteins, charges. This article must therefore be hereby marked advertisement in accordance including Lyn, Vav, Grb2, and p85␣ PI3K (reviewed in Ref. 23). with 18 U.S.C. Section 1734 solely to indicate this fact. Lyn is one protein tyrosine kinase (PTK) that phosphorylates 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 Insti- CD19 (31) and is reported to play both a positive and negative role tutes of Health Grant T32 AI55411. in regulating B cell activation (32). Thus, CD19 and Lyn work 2 This research is part of the dissertation research conducted by Nicholas W. Kin who together as important signaling intermediates to mediate the acti- is a predoctoral student in the Integrated Biomedical Science Graduate Program, The Ohio State University, Columbus, OH 43210. vation of PI3K in B cells following BCR and CD21 stimulation and, therefore, may also serve as potential signaling intermediates 3 Address correspondence and reprint requests to Dr. Virginia M. Sanders, 2194 Graves Hall, 333 West 10th Avenue, Columbus, OH 43210. E-mail address: to mediate PI3K activation following CD86 stimulation. [email protected] In the current study, we show that the addition of CD28/Ig to 4 Abbreviations used in this paper: DC, dendritic cells; WT, wild type; PTK, protein CD40L/IL-4-activated B cells on WT, but not CD86- or CD19- tyrosine kinase; KLH, keyhole limpet hemocyanin; CD40L, CD40 ligand. deficient, B cells increased the level of phosphorylation for Lyn Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 and CD19, as well as the amount of Lyn, Vav, and PI3K proteins www.jimmunol.org The Journal of Immunology 1517

FIGURE 1. CD86-deficient B cells produce less ϩ IgG1 and Oct-2 in vivo. WT CD4 T cells and WT B cells (WT) or WT CD4ϩ T cells and CD86-deficient B cells (CD86Ϫ/Ϫ) were adoptively transferred to RAG2- deficient mice. One week later, all mice received one i.p. injection of 100 ␮g TNP-KLH in alum. The percentage of total splenic B cells (B220ϩ) and CD4ϩ T cells (CD4ϩ) in the WT (A) and CD86Ϫ/Ϫ (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 Downloaded from total splenocytes from mice receiving WT B cells (Ⅺ) or CD86-deﬁcient B cells (f) using real-time PCR. D, The expression level of IL-4 in the CD4ϩ T cells from WT (24.6%) and CD86Ϫ/Ϫ (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 http://www.jimmunol.org/ total splenocytes on day 6 following immunization and the level of Oct-2 and actin was determined by Western p Ͻ 0.05 compared with the CD86Ϫ/Ϫ ,ء .blot analysis group. by guest on September 23, 2021

that immunoprecipitated with CD19. Serum IgG1 levels were de- stimulation of CD86. All reagents used were negative for the presence of creased in mice receiving CD86-deficient B cells when compared endotoxin, as determined by Etoxate (Sigma-Aldrich), a Limulus lysate assay with a level of detection Ͻ0.1 U/ml. 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 In vivo cell transfer and immunization mRNA and protein, but a normal level of germline IgG1 mRNA. Thus, our findings suggest that CD86 plays a key role in regulating Seven days before immunization, 5 ϫ 106 CD4ϩ T cells and either 20 ϫ the level of IgG produced in vitro and in vivo, independently of 106 WT B cells or CD86-deficient B cells were adoptively transferred into 1 ␮ class switch recombination. RAG2-deficient animals in a volume of 100 l PBS i.v. in the lateral tail vein. One week following the adoptive transfer of cells, mice were admin- istered 100 ␮g TNP-keyhole limpet hemocyanin (KLH) in alum i.p., and

Materials and Methods serum samples were collected 7 and 14 days later. The level of serum IgG1 Animals was determined in various dilutions of serum samples using ELISA. Female BALB/c and CD19-deficient (CD19Ϫ/Ϫ) mice were purchased from Taconic Farms. CD86-deficient (CD86Ϫ/Ϫ) mice were provided by Western blot Dr. A. Sharpe (Brigham and Woman’s Hospital, Boston, MA). Mice were Western blot analysis was performed as described previously (20). In brief, bred and housed within the pathogen-free facility at Taconic Farms. C3H/ ␮ Ϫ/Ϫ following activation, B cells were lysed, protein samples (5–20 g) were HeJ and TLR 4-deficient mice (TLR4 ) were purchased from The Jack- resolved by electrophoresis on 10% polyacrylamide gels, transferred to son Laboratory. Immobilon-P polyvinylidene difluoride membranes (Millipore), probed Resting B cell isolation and activation with either anti-CD19, anti-phospho-CD19, anti-Vav, anti-Lyn, anti- phospho-src family (Cell Signaling Technology), and anti-p85 PI3K (Up- Resting B cell isolation and activation was performed as described previ- state Biotechnology), detected with HRP-labeled secondary Abs, and ously (20). In brief, resting B cells were isolated using the AutoMacs developed with the LumiGlo Detection Kit (Cell Signaling Technology). machine (Miltenyi Biotec) following the manufacturer’s instructions and were activated with CD40L-expressing Sf9 cells at a B cell to Sf9 cell ratio Immunoprecipitation 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 Immunoprecipitations were performed using the Profound Mammalian Co- Systems) was added at a final concentration of 1 ␮g/ml. Pharmacologic immunoprecipitation Kit (Pierce). In brief, B cells were activated as de- inhibitors SU6656 and PP2 (Calbiochem) were added 30 min before the scribed above, protein isolated, immunoprecipitated with either an anti-Lyn 1518 CD86 SIGNALING IN B CELLS

FIGURE 2. CD19 expression is required for CD86 signaling. A–D,WT(Ⅺ), CD86-deﬁcient (u), and CD19-deﬁcient (f) 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) Ϯ SEM from three independent experiments. The level of Oct-2 mRNA on

day2(B) and mature IgG1 mRNA on day 5 (C) was determined using real-time PCR analysis and normal- ization to total actin. Data represent the mean Ϯ 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 Ϯ IgG1) SEM from three independent experiments. p Ͻ 0.05 compared with the CD40L/IL-4 alone ,ء group. Downloaded from http://www.jimmunol.org/

or anti-CD19 (Cell Signaling Technology) Ab following the manufactur- ng/ml. A secondary Ab, goat anti-mouse IgG1-alkaline phosphatase (BD er’s direction. The precipitates were analyzed using Western blot analysis Biosciences) was used for detection. p-Nitrophenyl phosphate (Sigma-Al- as described above. drich) was added, and color development was determined on a Spectramax Plus microplate reader (Molecular Devices) at a wavelength of 405 nm. Flow cytometry Statistics The number of B220ϩ and CD4ϩ cells was determined by FACS analysis as described previously (16). In brief, total splenocytes were collected on Data with three or more groups were analyzed by a one-way ANOVA day 6 following immunization and stained with PE-conjugated rat anti- followed by post hoc analysis, while data with two groups were analyzed mouse B220, FITC-conjugated rat anti-mouse CD4, and allophycocyanin- by a two-tailed paired t test to determine whether an overall statistically conjugated rat anti-mouse IL-4 (BD Biosciences). For intracellular FACS significant change existed. Statistically significant results were determined by guest on September 23, 2021 analysis, total splenocytes on day 6 were stimulated for 4 h with PMA (50 by a p value of Ͻ0.05. ng/ml; Sigma-Aldrich) and ionomycin (500 ng/ml; Sigma-Aldrich) in the presence of brefeldin A (1 ␮g/ml; BD Biosciences). Cells were then Results washed, fixed, and permeabilized using the Cytofix/Cytoperm (BD Bio- CD86-deficient B cells produce less IgG and Oct-2 in vivo sciences) kit according to the manufacturer’s directions and stained with a 1 rat anti-mouse IL-4 (clone BVD6–24G2) (eBioscience). All samples were Although the in vitro data to date show that direct stimulation of analyzed using a FACSAria flow cytometer (BD Biosciences). The data CD86 on a B cell regulates the amount of IgG produced, without were analyzed using FlowJo software (Tree Star). 1 affecting class switch recombination, animal models are required PI3K ELISA to prove in vivo relevance. A previous study from our laboratory used scid mice reconstituted with resting B cells alone that were The PI3K ELISA was performed as described previously (20). In brief, B cells were activated as described above, and protein was isolated and im- activated by the i.p. injection of both an anti-CD40 Ab and rIL-4 munoprecipitated with a rabbit anti-mouse PI3K Ab (Upstate Biotechnol- with or without an anti-CD86 Ab. Serum levels of IgG1 on day 14 ogy) for1hat4°C. Protein A/G-agarose beads in PBS were added for 1 h following activation were ϳ3-fold higher in the presence of an at 4°C, and samples were collected and washed. The immunoprecipitated anti-CD86 Ab as compared with anti-CD40/IL-4 alone (15), sug- proteins were used to analyze the PI3K activity using the PI3K ELISA (Echelon BioSciences) following the manufacturer’s directions. gesting that CD86 stimulation on some CD86-expressing cells affected the level of IgG1 produced. However, testing whether CD86 Quantitative real-time PCR signals directly in a B cell when T cells are present to provide B Quantitative real-time PCR was performed as described previously (20). cell help during a T cell-dependent IgG1 response in vivo proved The following primers were used: ␤-actin 5Ј-TACAGCTTCACCACC more difficult, because any attempt to stimulate CD86 would in- ACAGC-3Ј and 5Ј-AAGGAAGGCTGGAAAAGAGC-3Ј (annealing tem- terfere with T cell costimulation. Therefore, we established an perature, 60°C, 206-bp product); Oct-2 5Ј-ATCAAGGCTGAAGAC adoptive transfer model system in which only the B cell would CCCAGTG-3Ј and 5Ј-TGGAGGAGTTGCTGTATGTCCC-3Ј (annealing Ј lack CD86, while all other APCs, namely DC and macrophages, temperature, 60°C, 128-bp product); mature IgG1 transcript 5 -TATG GACTACTGGGGTCAAG-3Ј and 5Ј-CCTGGGCACAATTTTCTTGT-3Ј would express CD86 normally and allow for optimal T cell acti- ϩ (annealing temperature, 63°C, 205-bp product). vation to occur. CD4 T cells and resting B cells from WT and/or CD86-deficient mice were adoptively transferred to Rag2-deficient IgG ELISA 1 mice, resulting in mice that expressed WT-T/WT-B or WT-T/

For in vitro IgG1 determination, B cell culture supernatants were collected CD86-deficient B cells. To determine whether equal numbers of T on days 4–7, and for in vivo IgG1 determination, serum samples were and B cells were localized in the spleens of these mice 6 days collected and frozen immediately at Ϫ80°C until analysis. Costar 96-well following immunization with TNP-KLH, flow cytometry was per- flexiplates (Fisher Scientific) were coated with goat anti-mouse IgG (2 ϩ ϩ ␮ formed and the percentage of B220 and CD4 cells was found to g/ml; BD Biosciences), and a standard curve for IgG1 was prepared using ␮ known quantities of recombinant IgG1 protein in a range of 1 g/ml to 1 be equivalent between the mice receiving WT-T/WT-B (Fig. 1A) The Journal of Immunology 1519

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 CD4ϩ 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 germ-

line 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. When the reconstituted Rag2-deficient mice were immunized with the T cell-dependent Ag, TNP-KLH, mice receiving CD86- ϳ deficient B cells produced 2-fold less of both serum IgG1 (Fig.

1E) and splenocyte-mature IgG1 mRNA (Fig. 1C), but equivalent Downloaded from amounts of germline IgG when compared with mice that received FIGURE 3. CD86 stimulation on TLR4-deficient B cells produces sim- 1 ilar results to WT B cells. TLR4-deficient B cells were activated with WT B cells. Similarly, total splenocyte Oct-2 mRNA (Fig. 1C) and CD40L/IL-4 in the absence or presence of CD28/Ig and supernatants were protein (Fig. 1F), which were reported to increase in CD86-stim- ϳ collected on day 7. The level of total IgG1 was determined using ELISA. The ulated B cells (15), were also 2-fold less in the mice receiving data represent the mean (ng/ml IgG1) Ϯ SEM from three independent exper- CD86-deficient B cells as compared with WT B cells. Taken to- iments. B, The level of Oct-2 mRNA on day 2 and mature IgG mRNA on day 1 gether, these findings in vivo suggest that CD86 stimulation on a http://www.jimmunol.org/ 5 was determined from WT B cells activated with CD40L/IL-4 in the absence B cell during a T cell-dependent Ab response is needed to activate (Ⅺ) or presence (f) of CD28/Ig using real-time PCR analysis. The data rep- p Ͻ 0.05 com- an intracellular signaling pathway in the B cell to up-regulate the ,ء .resent mean Ϯ SEM from three independent experiments pared with the CD40L/IL-4 alone group. by guest on September 23, 2021

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 Ϯ 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␣ PI3K, and Lyn by Western blot analysis. One representative gel from three independent experiments is shown. 1520 CD86 SIGNALING IN B CELLS

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-␬B activation was recently identified to include PI3K/PDK1/Akt and PLC␥2/PKC␣␤ (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 ϳ3-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 Downloaded from 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 http://www.jimmunol.org/ exposed to CD28/Ig increased the level of Oct-2 mRNA ϳ2-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 ϳ2-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/ by guest on September 23, 2021

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 FIGURE 5. Lyn kinase plays a role in CD86 signaling. A, Western blot of Oct-2 and mature IgG1 mRNA (Fig. 3B) and protein (Fig. 3A) analysis of phospho-CD19 and total actin in B cells activated with CD40L/ induced by addition of CD28/Ig was similar to that seen in WT IL-4 for 16 h and then pretreated with an src-family kinase inhibitor, cells (Fig. 2, D and E). Thus, collectively, these data show that SU6656 (100 nM), for 30 min before the addition of CD28/Ig. Band den- CD86 stimulation on an activated B cell requires the presence of sities were determined using densitometry, and the data represent the mean CD19 to increase the level of PI3K activity, Oct-2 expression, and fold increase in phospho-CD19 normalized to total actin Ϯ SEM from three independent experiments. One representative gel from three indepen- IgG1 produced, suggesting that CD19 is a potential link between CD86 stimulation and the activation of PI3K in a B cell. dent 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 CD19 is activated by CD86 stimulation mean fold increase in Oct-2 mRNA normalized to actin Ϯ SEM from three independent experiments. C, Protein samples from resting, CD40L/IL-4- Because CD86 failed to signal in a CD19-deficient B cell, the activated, and CD40L/IL-4/CD28/Ig-activated B cells were immunopre- question remained as to the mechanism by which CD86 activated cipitated (IP) with an anti-Lyn Ab, and the precipitates were analyzed for CD19. CD19 contains nine tyrosine residues in the cytoplasmic the presence of phospho-Lyn and total Lyn by Western blot analysis. One p Ͻ ,ء .domain that are phosphorylated to promote CD19 interaction with representative gel from three independent experiments is shown a number of SH2 domain-containing proteins, including Lyn, Vav, 0.05 compared with the CD40L/IL-4 alone group. Grb2, and p85␣ 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 phopho- Because tyrosine phosphorylation of the CD19 cytoplasmic do- rylation of CD19 (Tyr513), which is the critical residue for CD19 main promotes the binding of SH2 domain-containing proteins, activation of PI3K (34), was increased at 5 and 15 min following such as PI3K, it was possible that CD86 activated CD19 to bind addition of CD28/Ig to WT B cells (Fig. 4A), but not when added and activate PI3K. B cells were activated as described above, and to CD86-deficient B cells (Fig. 4B). When TLR4-deficient B cells total protein was isolated and immunoprecipitated with an anti- were used, the data showed that the level of phosphorylated CD19 CD19 Ab, after which time the precipitates were analyzed by was increased to a similar level as seen using WT B cells (Fig. 4C). Western blot analysis. The amount of Lyn, Vav, and p85␣ PI3K The Journal of Immunology 1521 that immunoprecipitated with CD19 increased following the addi- of DC that expressed CD19 responded to CD86 stimulation (21), tion of CD28/Ig (Fig. 4D). The blots were also probed for the suggesting that CD19 may be required for CD86 signaling to occur presence of CD86, but failed to show an interaction with CD19 in DC. The present findings using WT and CD19-deficient B cells (data not shown), suggesting that CD86 activates CD19 through a showed that CD86 stimulation on an activated WT B cell increased mechanism other than direct interaction. Thus, these results sug- the phosphorylation and activation of CD19, whereas the activa- gest that CD86 stimulation on a B cell activates CD19 to poten- tion of CD19-deficient B cells did not, strengthening our hypoth- tially mediate the activation of PI3K. esis that CD19 expression is required for CD86 to function in a B cell. Therefore, because CD86 signaling has been shown in two CD86 stimulation activates the PTK Lyn different cell types to involve CD19 and NF-␬B, it is becoming Lyn is a src-family PTK reported to be capable of phosphorylating more likely that CD86 is able to signal directly and can no longer CD19 (31). However, Lyn kinase must first be activated through be considered simply a costimulatory molecule for CD28 on a tyrosine phosphorylation. Y397, which is located in the kinase T cell. domain of Lyn, positively regulates activity, while Y508, which is Yet, because CD19-deficient B cells have been reported to re- located in the regulatory domain of Lyn, negatively regulates Lyn spond differently to activation stimuli, it was possible that the in- activity (reviewed in Ref. 32). To determine whether CD86 stim- ability of CD19-deficient B cells to respond to CD28/Ig may have ulation requires a PTK to positively regulate the phosphorylation been due to an inherent defect of CD40 signaling in these cells, as of CD19, we designed three experiments. First, B cells were ac- opposed to a defect in CD19-dependent CD86 signaling. This pos- tivated as described above, and two different src-family kinase sibility was particularly relevant because CD40 stimulation is re- inhibitors, SU6656 and PP2, were added 30 min before addition of ported to be associated with CD19 activation (35). For example, Downloaded from CD28/Ig. Pretreatment with either one of the src-family kinase the ability of CD19-deficient B cells to proliferate upon CD40 inhibitors prevented the CD86-induced increase in phospho-CD19 stimulation was reported to be less than that in WT B cells (36), (Fig. 5A). Second, to further confirm that a PTK was involved in while another report showed that the proliferation remained un- the CD86-induced signaling pathway, B cells were activated as changed (25). One possible explanation for these contrasting re- described above in the presence or absence of a src-family kinase sults is that the ligands used to stimulate CD40 differed, i.e., anti- inhibitor. In the presence of the src-family kinase inhibitor, CD40 Ab decreased proliferation in CD19-deficient B cells when http://www.jimmunol.org/ CD28/Ig failed to increase the level of Oct-2 mRNA (Fig. 5B) compared with WT B cells (36), while recombinant CD40L in- above that induced by CD40L/IL-4 alone. And third, because the duced no change (25). If these two CD40-stimulating reagents ac- PTK Lyn was immunoprecipitated with CD19 (Fig. 4C), it was tivate different signaling intermediates, as has been reported pre- possible that CD86 stimulation regulated the phosphorylation of viously (37), then it is possible that each signaling intermediate Lyn. To test this possibility, B cells were activated as described affected proliferation differently. Our findings using recombinant above, and total protein was isolated and immunoprecipitated with CD40L expressed on the surface of Sf9 cells would support the an anti-Lyn Ab. The level of phospho-Lyn (Y397) that positively finding of Rickert et al. (25) and expand it to the IgG1 response regulates Lyn activity was increased at 5 min following CD86 because WT and CD19-deficient B cells activated with CD40L/ by guest on September 23, 2021 stimulation on WT B cells (Fig. 5C). Thus, these results suggest IL-4 produced comparable levels of IgG1 (Fig. 2D). This latter that Lyn is the PTK that is activated by CD86 stimulation to in- finding was also true for CD40/IL-4-activated B cells that made crease the phosphorylation of CD19. IgE (38). Another possible explanation is that proliferation results may be misleading when interpreting Ab results. For example, Discussion proliferation in the B cell was measured at 72 h, while regulation

The goal of the present study was to identify the mechanism by of the role of mature IgG1 transcription was measured on day 5, at which CD86 activated PI3K, and to confirm the relevance of CD86 a time when any change in proliferation that may have occurred at stimulation on a B cell responding to a T cell-dependent Ag in 72 h may not be relevant to the IgG1 response that occurs days vivo. Using an adoptive transfer model, the present data show that later. Taken together, the loss of CD86 signaling in a CD19-defi- the loss of CD86 expression on a B cell alone prevents the gen- cient B cell appears to be due to a lack of CD19, and not to an eration of an optimal IgG1 response, even in the presence of a inherent defect in the CD19-deficient B cell to make Ab after normal IL-4 and germline IgG1 response. We also show, for the CD40 stimulation. first time, that CD86 stimulation on a CD40L/IL-4-activated WT, Previous data showed that CD86 stimulation increases PI3K ac- but not CD86- or CD19-deficient, B cell in vitro increases the tivity (20), although the mechanism by which this occurs remained phosphorylation of Lyn and CD19, as well as the amount of Lyn, unknown because CD86 lacks expression of a tyrosine residue in Vav, and p85␣ PI3K that immunoprecipitated with CD19. Thus, the cytoplasmic domain that is required for PI3K activation. If our findings suggest that CD86 on a B cell plays a key role in previous reports are correct that CD19 stimulation activates PI3K regulating the level of IgG1 produced in vitro and in vivo, inde- (39, 40), and if our present finding is correct that CD86 stimulation pendently of class switch recombination, and that Lyn and CD19 activates CD19, then CD19 may be the link between CD86 stim- may serve as the signaling intermediates activated by CD86 prox- ulation and PI3K activation. However, what remains unclear is imal to PI3K. how CD86 mediates the activation of CD19. One possibility may Initially, CD86 was thought to be a costimulatory molecule that be that CD86 activates CD19 through a direct physical interaction. was unable to signal itself. However, a number of studies to date For example, CD21/CD81 are reported to activate CD19 via a in both B cells and DC have suggested that CD86 is able to signal direct physical interaction using their extracellular and transmem- directly. For example, stimulation of CD86 with CD28/Ig on either brane domains (26, 41). In contrast, MHC class II (30) and CD180 a B cell or DC increased NF-␬B-mediated gene transcription, as (RP105) (29) are reported to activate CD19 without a detectable was shown using transient transfection systems (20, 22), suggest- physical interaction. Our unpublished findings using coimmuno- ing that CD86 on either a B cell or dendritic cell activate at least precipitation suggest that CD86 and CD19 may not physically in- one common signaling intermediate. This finding also suggests teract with each other. Although this finding does not preclude the that the signaling intermediates activated proximal to NF-␬B may possibility that CD86 and CD19 formed a weak interaction that also be the same. A recent study in DC suggested that only a subset was undetectable using our immunoprecipitation protocol, it does 1522 CD86 SIGNALING IN B CELLS suggest that some mechanism would need to exist other than direct requirement of T cell costimulation. The present in vivo results physical interaction. We propose, based on the present data, that addressed this issue and suggest that expression of CD86 on the B this mechanism involves the phosphorylation of Lyn, phospho- cell alone is required for an optimal IgG1 response to occur. An- Lyn binding to CD19, and subsequent phosphorylation of the other study also attempted to determine the role of CD86 signaling CD19 cytoplasmic domain. However, the mechanism by which in vivo using a mixed chimeras model system and found that CD40 CD86 might activate Lyn directly remains unknown, but may be expression on a B cell was essential for class switch recombination similar to the mechanism used by other B cell-associated receptors to IgG1, but that CD80/86 expression on the B cell was not (33). that also activate CD19. For example, crosslinking of the BCR The present data agree with this finding, because no change was

(42) or CD21/CD19/CD81 (43) is known to recruit these receptor detected in germline IgG1 mRNA between WT and CD86-defi- complexes into lipid raft domains where Lyn is selectively en- cient B cells. However, in contrast to the mixed chimeras model riched (44). If CD86 uses a similar mechanism, then it also would system, our adoptive transfer model system showed that B cells ϳ need to be recruited into a lipid raft domain following either lacking CD86 expression produced 2-fold less mature IgG1 CD40L/IL-4 and/or CD86 stimulation, providing a localized re- mRNA and protein. The reason for the discrepancy about the role gion in which CD86 would cluster with Lyn and/or CD19. In sup- of CD86 in mature IgG1 production in the two model systems is port of this hypothesis are the findings that crosslinking of either not yet evident, but our finding suggests that the expression of CD86 or CD40 on mature DC recruited CD86 to lipid raft domains CD86 on a B cell may play a crucial role in establishing the level

(45). If the mechanism by which CD86 signals in a DC is similar of IgG1 produced in vivo. to a B cell, which may involve recruitment of CD86 into lipid raft Human and murine CD86 share ϳ70% gene homology, with a domains and clustering with signaling intermediates to allow for number of differences located in the cytoplasmic domain. Both Downloaded from CD19 activation, then this clustering may be the mechanism by human and murine cytoplasmic domains contain putative PKC which CD86 activates Lyn, CD19, and PI3K in a B cell. phosphorylation sites, i.e., serine and threonine residues, but the Previous studies used an anti-CD86 Ab to stimulate CD86 on human CD86 cytoplasmic domain contains a greater number of the B cell surface. However, we recently switched to using potential phosphorylation sites. This suggests that similar mecha- CD28/Ig to more closely mimic the endogenous ligand found on T nisms may be used for CD86 signal transduction in both species,

cells, i.e., CD28. Because studies in DC using CD28/Ig suggested but that humans may use additional phosphorylation sites to reg- http://www.jimmunol.org/ a role for both CD80 and CD86 signaling in some of the functional ulate signal intensity. However, the question remains as to whether responses measured (22), and because CD28/Ig can bind to both the findings in mice about CD86 signaling reflect the mechanism CD80 and CD86 (46), the possibility existed that the loss of CD86 by which human CD86 might function. First, CD86 stimulation on in a CD86-deficient B cell prevented CD80/CD86 heterodimeriza- human tonsillar B cells in vitro was reported to increase the pro- tion and, therefore, might explain the loss of CD28/Ig-induced duction of IgG4 (17), suggesting that human CD86 might also signaling when CD86-deficient B cells were used. We think that signal directly within a human B cell to regulate the level of IgG4 this possibility is unlikely because fluorescence resonance electron produced. The ability of both human and murine CD86 to regulate transfer technology showed that CD80 exists primarily as a ho- the level of IgG4/IgG1 produced by a B cell suggests that similar modimer on the B cell surface, while CD86 exists primarily as a signaling mechanisms might be used intracellularly. Interestingly, by guest on September 23, 2021 monomer (47). Likewise, the chemical properties of the potential one report in humans showed that a polymorphism in the human dimer interfaces of CD80 and CD86 were shown to be very dif- CD86 cytoplasmic domain of an alanine to threonine change at ϩ ferent. CD80 expresses a hydrophobic interface that would pro- 1057 position (50), which introduced an additional potential phos- mote dimerization, while CD86 expresses a hydrophilic interface phorylation site, was associated with a lower incidence of acute that would make it less prone to dimerization (48). Therefore, it is liver transplant rejection (51). This finding suggested that the in- unlikely that CD80 and CD86 interact on the cell surface to me- troduction of an additional phosphorylation site in the cytoplasmic diate the CD28/Ig-induced effects and, therefore, make it unlikely domain of human CD86 might have altered the signaling capabil- that the loss of an effect when using CD86-deficient B cells was ities of CD86 and/or the ability to costimulate a T cell. More in- due to the loss of dimerization with CD80. Nonetheless, CD80- terestingly, a number of studies in mice (8, 20) and humans (17) deficient B cells will be used in future experiments to further rule have suggested that prior activation of the B cell is required for out this possibility. Another reason why we think CD80 is not CD86 signaling to activate an intracellular signaling pathway. If involved in the signaling induced by CD28/Ig in a B cell is the fact true, potentially the CD86 cytoplasmic domain requires phosphor- that to induce the signaling intermediates measured in the present ylation to become competent to signal. In support of this hypoth- study within the first 24 h of B cell activation, both CD80 and esis, one study reported that the murine CD86 cytoplasmic domain CD86 would need to be concomitantly expressed during this time was phosphorylated only after cell activation, suggesting that period. However, the kinetics of CD80 and CD86 expression are phosphorylation may play a role in the ability of CD86 to signal in very different following B cell activation. CD86 surface expression a murine B cell (52). Taken together, both human and mouse data is detectable at 6 h and maximal at 24–48 h after activation (Ref. suggest that CD86 stimulation on a B cell regulates the level of a 7 and data not shown), while CD80 surface expression is detect- T cell-dependent Ab response similarly, but the role played by the able by 24 h and maximal at 48–72 h (Ref. 49 and data not shown). potential phosphorylation sites in the cytoplasmic domain of hu- This difference in expression kinetics makes it unlikely that CD80 man and murine CD86 remain unknown. 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 argu- Acknowledgments ments make it unlikely that CD80 and CD86 interact with each We gratefully acknowledge Drs. William Lafuse (The Ohio State Univer- sity, Columbus, OH) and Kerry Campbell (Fox Chase Cancer Center, Phil- other on a B cell at the time of addition of CD28/Ig to induce an adelphia, PA) for helpful discussions regarding the research. 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 Disclosures cell-dependent Ab response in vivo has been a challenge due to the The authors have no financial conflict of interest. The Journal of Immunology 1523

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