Syk Tyrosine Kinase and B Cell Antigen Receptor (BCR) Immunoglobulin-α Subunit Determine BCR-mediated Major Histocompatibility Complex Class II–restricted Antigen Presentation

B Lymphoma Cell Lines.

The B lymphoma IIA1.6 is an FcγR-defective variant of A20 B lymphoma cells (23). The anti-TNP A20 cells were obtained by transfection of genomic clones encoding the light and the heavy μ chains of the BCR specific for TNP (24). These cell lines were cultured in RPMI 1640 containing 10% FCS, 10 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μM 2-ME, and 5 mM sodium pyruvate (GIBCO BRL, Paisley, UK). FcγR/Ig-α and FcγR/Ig-β chimeras were constructed by adding the DNA sequences encoding the cytoplasmic domain of the Ig-α or Ig-β subunits to cDNA encoding the extracellular and transmembrane domains of mouse FcγRII (22). cDNAs encoding FcγR/Ig-α chimeras with simple or double mutations of tyrosine residues, contained in the ITAM, were constructed using PCR with alanine in place of tyrosine. All of these constructs have been described previously (25). The resulting constructions were inserted in an expression vector bearing a neomycin gene resistance, sequenced, and stably expressed in the mouse B cell line IIA1.6. Cell surface expression of FcγR chimeras was measured with the rat anti–mouse FcγR mAb 2.4G2, and was revealed by FITC-coupled mouse anti–rat antibodies (26). The samples were analyzed with a FACScan^® flow cytometer (Becton Dickinson, San Jose, CA).

Stable Expression of Syk Dominant Negative Mutant.

The syk dominant negative mutant was constructed by joining the EcoRI-KpnI fragment of rat syk cDNA (27) to a KpnI-XbaI PCR fragment amplified from a hemagglutinin (HA)-tag–containing plasmid. The sequence coding for the HA-tag is recognized by the mAb 12CA5. The resulting truncated syk has been sequenced, and corresponded to the first 260 amino acids of the rat syk cDNA fused to the sequence GSGYSYDVPDYA. This construct was then inserted in an Sr-driven expression vector bearing the puromycin gene resistance. After linearization with ScaI restriction enzyme, 50 μg of DNA was used to electroporate B cells expressing FcγR/Ig-α or FcγR/Ig-β chimeras as described (26). After 48 h, the transfected cells were resuspended at 10³ cells/ml in a culture medium containing 2 μg/ml puromycin (Sigma Chemical Co., St. Louis, MO). This cell suspension was distributed in 96-well plates at 100 μl/well and kept at 37°C for several weeks. Growing cells were selected for the expression of the truncated syk using FITC-coupled 12CA5 anti–HA-tag antibody (Boehringer Mannheim Corp., Indianapolis, IN). Cells were fixed with 3% paraformaldehyde in PBS, then incubated for 10 min in 100 mM glycine in PBS. Cells were permeabilized with 0.05% saponin (Sigma Chemical Co.) in PBS, then further incubated for 30 min at room temperature with 20 μg/ml FITC-coupled 12CA5 in 0.05% saponin, 0.2% BSA in PBS. After washing, the cells were resuspended in PBS, and the samples were analyzed by FACScan^® (Becton Dickinson). Positive cells were further characterized by Western blotting using an affinity-purified rabbit antibody raised against the amino acids 13–31, mapping in the first src homology 2 (SH2) domain of the human syk (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The sequence of this peptide is identical in rat and mouse syk. 5 × 10⁵ cells were lysed with 0.5% Triton X-100, then run on 10% polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). The filters were incubated for 1 h with 0.1 μg/ml of the anti-syk antibody, washed three times, then further incubated with a horseradish peroxidase (HRP)-coupled goat anti–rabbit antiserum (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). Chemiluminescence was detected using a commercial kit (Boehringer Mannheim Corp.). The G2 and C2 clones for the FcγR/Ig-α–expressing cells, and the E8 and E10 clones for the FcγR/Ig-β–expressing cells were selected because they expressed high levels of truncated syk (30 kD), and because they expressed similar levels of FcγR chimera (detected with the rat anti–mouse FcγR antibody 2.4G2) as parental cells.

T Cell Hybridomas.

T cell hybridomas and cells used in antigen presentation assays were cultured in RPMI 1640 containing 10% FCS, 10 mM glutamine, penicillin (100 U/ml), streptomycin (100 μg/ml), and 2β-ME (5 × 10⁻⁵ M). The specificity of all CD4⁺ T cell hybridomas is shown in Table 1. The C1 λ repressor–specific hybridomas 24.4, A128, 26.1, and 4G2 were characterized previously (28–30). The anti-HEL T cell hybridomas B9.1 and CAB43 and the anti-OVA T cell hybridomas 3DO 54.8 and 3DO18.3 were described previously (31, 32).

Assays for Antigen Presentation.

Antigen presentation was assessed by culturing transfected IIA1.6 cells together with specific T cell hybridomas for 18–20 h in the presence of various concentrations of antigens complexed or not with specific antibodies. The λ repressor was complexed with the two mAbs 22D and 51F (33), the HEL (Sigma Chemical Co.) was complexed with the mAbs F10.6.14 and F9.13.7 (34), and the OVA was complexed with the IgG fraction of a rabbit anti-OVA antiserum (Sigma Chemical Co.). The complexes were preformed by incubating different concentrations of purified λ repressor, HEL, or OVA (from 30,000 to 0.5 ng) with 15 μg/ml of each mAb or 50 μg/ml of rabbit IgG at 37°C for 15 min. The release of IL-2 by the T cell hybridoma was determined by a CTL.L2 proliferation assay (35). Each point represents the average of duplicate samples, which varied by <5%.

Kinetics analysis was performed as described previously (16). In brief, a dose of antigen (3 μg/ml) was used for which antigen presentation was strictly dependent on immune complex (IC) formation with the mAbs. IC were preformed at 37°C by mixing the purified λ repressor with 51F and 22D mAbs to make a 10× mix in the culture medium. 30 μl of preformed IC was added to 270 μl of APCs adjusted to 2 × 10⁶ cells/ml and incubated at 37°C for different times. To assess antigen presentation by A20 cells expressing TNP-specific IgM, TNP-coupled λ repressor (10 TNP per molecule of λ repressor) were incubated under similar conditions. The cells were then washed twice with PBS, fixed with 0.05% glutaraldehyde for 20 min on ice, and washed again twice. Fixed cells in duplicate samples of 100 μl were added to 50 μl of T cell hybridomas and adjusted to 2 × 10⁶ cells/ml. After 24 h, IL-2 production was tested as above. When indicated, APCs were preincubated for 3 h at 37°C with 10 μg/ml cycloheximide diluted from a stock solution at 10 mg/ml in water. In this case, cycloheximide was also present during incubation with preformed IC before fixation with glutaraldehyde.

The binding of OVA IC to FcγRs was confirmed by immunofluorescence and FACScan^® analysis. The complexes were made by mixing OVA (15 μg/ml; Sigma Chemical Co.) and the IgG fraction of a rabbit anti-OVA antiserum (50 μg/ml; Sigma Chemical Co.). For costimulation experiments, APCs were incubated with or without OVA IC for 18 h and then fixed as described above. Fixed cells in duplicate samples of 100 μl were added to 50 μl of T cell hybridomas 24.4 and 26.1 (adjusted to 2 × 10⁶ cells/ml) and various concentrations of C1 λ repressor 12–26 peptides. In another set of experiments, APCs were incubated either with λ repressor (30 μg/ml) and preformed OVA IC to stimulate FcγR, or with preformed λ repressor IC (as described above) and F(ab′)₂ fragments of specific goat anti–mouse IgG2a antibodies (15 μg/ml; Southern Biotechnology Associates, Inc., Birmingham, AL), which do not cross-react with IgG1 anti–λ repressor mAbs 51F and 22D, and specifically stimulate endogenous membrane IgG2a on IIA1.6 cells. After 18 h, the cells were fixed and incubated with C1 λ repressor–specific T cell hybridomas 24.4 and 26.1. T cell stimulation was assessed using a CTL.L2 proliferation assay.

Detection of Tyrosine Kinase Activation.

Cells were preincubated at 4°C with or without 20 μg/ml of 2.4G2 for 30 min, and then washed twice with RPMI 1640. 5 × 10⁶ cells were then stimulated with prewarmed F(ab′)₂ fragments of mouse anti–rat antiserum (50 μg/ml) at 37°C for 2 min. As a positive control, untreated cells were stimulated with prewarmed F(ab′)₂ fragments of anti-IgG antibodies (30 μg/ml) for 2 min at 37°C. The stimulated cells were washed and resuspended in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Triton X-100) containing a cocktail of protease inhibitors and tyrosine phosphatase inhibitors (5 mM NaF, 5 mM EDTA, 1 mM Orthovanadate). Phosphoproteins were immunoprecipitated with agarose-coupled antiphosphotyrosine antibodies PT-66 (Sigma Chemical Co.) or a rabbit antiserum raised against the 11 COOH-terminal amino acids from human syk coupled with KLH. The phosphoproteins were then run on 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride membrane (Millipore Corp.) to be detected with the antiphosphotyrosine mAb Py20 coupled with HRP (ICN Biomedicals, Orsay, France). Chemiluminescence was detected with a commercial kit (Boehringer Mannheim Corp.) by exposure of the filters to X-omat film (Eastman Kodak Co., Rochester, NY). The filters were then stripped by a 30-min incubation at 50°C in 50 mM Tris, pH 6.8, 2% SDS, 100 mM β-mercaptoethanol, incubated for 1 h with 0.1 μg/ml of the anti-syk antibody (Santa Cruz Biotechnology, Inc.), washed three times, then further incubated with an HRP-coupled goat anti– rabbit antiserum. For the kinase assay, 1 μg of glutathione S-transferase (GST)-HS1 fusion protein served as a specific substrate for syk immunoprecipitated (36) in 30 μl of kinase buffer (30 mM Hepes, pH 7, 10 mM MgCl₂, 5 mM MnCl₂, 70 mM Orthovanadate). It was constructed by joining a BamHI-EcoRI PCR fragment containing the HSI peptide motif (EQEDEPEGDYEEVLE-Stop) in-frame into the polylinker of the pGEK-2TK vector.

IC Internalization.

The cells were washed once in internalization buffer (RPMI 1640, 5% FCS, 10 mM glutamine, 5 mM sodium pyruvate, 50 mM 2-ME, and 20 mM Hepes, pH 7.4) and incubated with HRP–anti-HRP IC for 2 h at 4°C (10⁷ cells/ml). IC were prepared as a 10× solution in internalization buffer (HRP 50 μg/ml, and a polyclonal rabbit anti-HRP antibody at 400 μg/ml) for 30 min at 37°C. After fixation of HRP IC, the cells were washed three times in internalization buffer and incubated at 37°C for various times (2 × 10⁶ cells/ml). Internalization was stopped by adding cold internalization buffer, and the cells were washed once in PBS. Duplicates for each time point were either left in PBS at 4°C to measure cell surface HRP IC, or incubated in Triton X-100 (0.1%) for 5 min at room temperature to measure the total amount of HRP IC. The HRP was revealed by adding substrate buffer (0.5 mg/ml OPD [Sigma Chemical Co.] and 0.12% H₂O₂ in 0.05 M phospho-citrate buffer, pH 5.0) at 4°C. The reaction was stopped with 6 N HCl, and the change in color was determined spectrophotometrically at 492 nm.

BCR-mediated Antigen Uptake Induces the Presentation of Numerous T Cell Epitopes.

The binding of exogenous antigens to the BCR potentiates antigen uptake and degradation in B lymphocytes. To determine if BCR-mediated antigen uptake has any consequences for the presentation of various T cell epitopes, we compared antigen presentation after fluid phase or BCR-mediated antigen uptake using A20 B cells expressing or not expressing TNP-specific IgM. The λ phage repressor (C1) was used as antigen. TNP-coupled (3 μg/ml) or -uncoupled C1 (30 μg/ml) was incubated for various times with A20 cells expressing anti-TNP IgM (A20 anti-TNP). The cells were then fixed, and C1 presentation was detected with two T hybridomas specific for the same peptide in association with IA or IE of the H2^d haplotype: the hybridoma 24.4, which recognized a dominant T cell epitope (IA^d 12-26), and the hybridoma 26.1, which recognized a cryptic T cell epitope (IE^d 12-26) (28). These two T cell epitopes were detected on anti-TNP A20 cells after 2 h of incubation with TNP-coupled antigens (Fig. 1 a). The incubation of presenting cells with the protein synthesis inhibitor cycloheximide for 3 h before and during incubation with TNP-coupled C1 completely abolished BCR-mediated antigen presentation (Fig. 1 a). In contrast, fluid phase uptake of uncoupled C1 (Fig. 1 a) only led to the presentation of the IA^d 12-26 epitope after a lag time of 4 h. Under these conditions, antigen presentation was partially resistant to the cycloheximide treatment. Therefore, BCR-mediated antigen internalization has a dual function in antigen presentation: by addressing antigens to newly synthesized MHC class II molecules, BCR induces the presentation of T cell epitopes which are not presented after fluid phase antigen uptake.

The cytoplasmic tail of the BCR Ig-α subunit is able to target antigen to newly synthesized MHC class II (16). Therefore, we investigated whether the direct targeting of C1 through the cytoplasmic tail of Ig-α is able to induce the presentation of the dominant, IA^d 12-26, and the cryptic, IE^d 12-26, T cell epitopes. IIA1.6 B cells (FcγR-defective variant of A20 B cells) expressing FcγR chimeras containing the cytoplasmic tail of Ig-α or Ig-β (FcγR/Ig-α and FcγR/Ig-β, respectively [22]) were incubated with C1 (3 μg/ml) complexed with two anti-C1 mAbs (15 μg/ml each) to target C1 on FcγR chimeras. After various periods of culturing at 37°C, the presenting cells were fixed and incubated with the two T cell hybridomas 24.4 and 26.1. The presentation of IA^d 12-26 and IE^d 12-26 T cell epitopes occurred with similar kinetics on FcγR/Ig-α–expressing cells (Fig. 1 b) and A20 anti-TNP (Fig. 1 a). In addition, the incubation of presenting cells with cycloheximide inhibited antigen presentation induced by direct targeting of C1 through the Ig-α cytoplasmic tail (Fig. 1 b). Surprisingly, similar experiments with FcγR/Ig-β–expressing cells revealed that endosomal targeting of C1 to recycling class II by the Ig-β cytoplasmic tail (16) was not able to induce presentation of the cryptic IE^d 12-26 T cell epitope, whereas the dominant IA^d 12-26 T cell epitope was efficiently and quickly presented by recycling class II molecules (no effect of cycloheximide treatment; Fig. 1 b). Using HRP–anti-HRP rabbit IgG IC as a multivalent ligand of FcR chimeras, we observed that both Ig-α and Ig-β chimeras internalized IC with similar efficiency and kinetics (Fig. 1 c). Therefore, the incapacity of the Ig-β chimera to induce presentation of the IE^d 12-26 epitope was not due to inefficient ligand internalization. These results suggested that the BCR induced presentation of a dominant and a cryptic T cell epitope by newly synthesized class II molecules through a targeting signal contained in the cytoplasmic tail of Ig-α.

Therefore, a function of the BCR in antigen presentation might be to induce the presentation of a large spectrum of T cell epitopes by targeting antigens to newly synthesized class II. To address this question, we used a panel of T cell hybridomas specific for various peptides deriving from C1, HEL, or OVA, restricted by the IA or IE H2^d class II molecules. The efficiency of presentation of IgG-complexed antigens was compared in FcγR/Ig-α– and FcγR/Ig-β–expressing cells (Table 1). The targeting of these different proteins through the cytoplasmic tail of Ig-α induced the presentation of all epitopes tested, i.e., C1 IA^d 48-64 and IA^d 80-102, HEL IE^d 108-116 and IA^d 44-62, and the IA^d-restricted OVA epitopes recognized by the 3DO54.8 and the 3DO18.3 T cell hybridomas. In contrast, the processing of the same IC by FcγR/Ig-β–expressing cells led only to the presentation of some of these T cell epitopes (Table 1). Antigen targeting by the Ig-α or Ig-β cytoplasmic tails through newly synthesized or recycling class II molecules, respectively, did not lead to the presentation of the same peptides. These results raised the question, What is the intracellular mechanism of specific presentation of these T cell epitopes by Ig-α and BCR-mediated antigen internalization?

B Cell Activation Is Not Sufficient for Presentation of the Cryptic Epitope.

The Ig-α and Ig-β cytoplasmic tails induce the activation of different signaling pathways (22) and interact with distinct cytoplasmic effectors (21). Therefore, the selective presentation of epitopes by B cells expressing anti-TNP–specific IgM or FcγR/Ig-α chimera might be either a direct consequence of a different intracellular targeting of antigen, or an indirect effect of B cell activation which might induce surface expression of putative costimulatory molecules required for the efficient activation of T cell hybridomas.

To distinguish between these possibilities, the FcγR/ Ig-α– and FcγR/Ig-β–expressing cells were incubated with or without irrelevant IC (OVA, 15 μg/ml; anti-OVA rabbit IgG, 50 μg/ml), to induce B cell activation (not shown). After 24 h, the cells were then fixed and incubated with increasing concentrations of 12-26 peptide and either the 24.4 or the 26.1 T cell hybridoma. The peptide was presented to both T cells with similar efficiency by the unstimulated and the stimulated cells (Fig. 2 a). Similar results were obtained when the cells were fixed after 6 or 12 h incubation with IC (not shown). Therefore, the signaling pathways induced through Ig-α and Ig-β did not modify the efficiency of presentation of the 12-26 peptide by IA^d and IE^d molecules.

Nevertheless, the BCR or Ig-α cytoplasmic tail could induce signaling events that modified the composition of processing compartments, leading to the presentation of the IE^d 12-26 epitope. The effect of Ig-α–induced cell activation on intracellular processing of C1 was assessed by coincubating FcγR/Ig-α–expressing cells with irrelevant IC (as shown in Fig. 2 a) in order to trigger cell activation through the Ig-α cytoplasmic tail, and with soluble C1 (30 μg/ml), which typically only induced the presentation of the IA^d 12-26 epitope (see Fig. 1 a and Fig. 3 b). After 24 h, the cells were fixed and incubated either with 24.4 or 26.1 T cells (Fig. 2 b). B cell signaling through Ig-α did not lead to the presentation of the IE^d 12-26 epitope and did not increase the presentation of the IA^d 12-26 epitope after the fluid phase uptake of the C1 (Fig. 2 b).

However, fluid phase antigen uptake is not very efficient in B cells, and could be a limiting step that leads to failure to generate enough peptides to induce the presentation of the IE^d 12-26 epitope. To test this possibility, the FcγR/ Ig-β–expressing cells were simultaneously incubated with IgG1-complexed C1 (to allow efficient generation of 12-26 peptide) and with specific anti-IgG2a antibodies (to induce B cell activation through endogenous mIg). The cells were then fixed and incubated with the two T cell hybridomas. BCR stimulation did not induce FcγR/Ig-β–expressing cells to present IE^d 12-26 epitope and did not increase presentation of the IA^d 12-26 epitope (Fig. 2 c). Therefore, the stimulation of the whole BCR was not sufficient to allow presentation of the cryptic IE^d 12-26 epitope after C1 internalization through the Ig-β cytoplasmic tail; however, the 12-26 peptide was efficiently generated since it was presented by IA^d molecules, although the direct targeting of C1 to the TNP-specific BCR was able to induce presentation of the same T cell epitope (Fig. 1 a). In addition, the failure of the Ig-β chimera to induce presentation of cryptic IE^d 12-26 epitope was not due to a lower sensitivity of the T cell hybridoma, as suggested by the results obtained for fixed presenting cells (shown in Fig. 2 a), since similar results were obtained with unfixed cells (Table 1). Indeed, on unfixed cells, the two T cell hybridomas detected the IA^d 12-26 and IE^d 12-26 T cell epitopes with similar sensitivity (see Fig. 3 c, and data not shown). These data suggested that the BCR Ig-α subunit contains a signal that can recruit proteins involved in the antigen targeting toward endosomal compartments, where the 12-26 peptide may be generated and associated to IA^d and IE^d MHC class II molecules.

Tyrosine Residues Contained in the Ig-α Cytoplasmic Tail Are Required for Receptor-mediated Presentation of the Cryptic IE^d 12-26 T Cell Epitope.

The two major functions assigned to the Ig-α/Ig-β heterodimer are intracellular signaling and receptor internalization, both dependent on ITAMs contained in their cytoplasmic tails. To discriminate between these two functions of the BCR subunits, we analyzed antigen presentation through FcγR/Ig-α chimeras containing mutated tyrosine residues, previously shown to prevent the activation of tyrosine kinases (25).

First, we analyzed IC internalization by the FcγR/Ig-α chimera in which tyrosine residues of Ig-α ITAM were replaced by alanines (FcγR/Ig-α Y23-A Y34-A). HRP– anti-HRP rabbit IgG IC were bound to wild-type and mutated FcγR/Ig-α–expressing cells at 4°C, then the cells were cultured at 37°C for various times to allow receptor internalization. Internal and cell surface fractions of IC were determined by an enzymological assay. As shown in Fig. 3 a, both wild-type and mutated Ig-α chimeras internalized IC with similar kinetics. Tyrosine residues of Ig-α ITAM, as for Ig-β (37), did not appear to be required for the internalization of multivalent ligands.

Therefore, we evaluated whether mutation of these tyrosine residues, which inhibit B cell activation, altered the presentation of the dominant, IA^d 12-26, and the cryptic, IE^d 12-26, T cell epitopes. Cells were incubated with various concentrations of C1 complexed or not with two anti-C1 mAbs (15 μg/ml each) and the two T cell hybridomas. Although wild-type and double-mutated FcγR/Ig-α chimeras were able to induce presentation of the IA^d 12-26 epitope at low concentrations of IgG-complexed C1, no presentation of the cryptic IE^d 12-26 epitope was detected when IC were internalized through the FcγR/Ig-α Y23-A Y34-A chimera (Fig. 3 b). We verified that stimulation of the 24.4 and 26.1 T cell hybridomas was obtained at similar concentrations of 12-26 peptides with FcγR/Ig-α– or FcγR/Ig-α Y23-A Y34-A–expressing cells (Fig. 3 c). In addition, we obtained similar results with B cells expressing Ig-α chimera where the first (FcγR/Ig-α Y23-A) or the second (FcγR/Ig-α Y34-A) tyrosine residue of Ig-α ITAM was mutated to alanine (Table 1). The mutation of tyrosine residues of the Ig-α ITAM also affected the presentation of IA^d 48-62 HEL epitopes, but had no effect on the IE^d 108-116 T cell epitope derived from HEL when the antigen was complexed with specific antibodies (Table 1). Therefore, we concluded that mutations that abolished the ability of the Ig-α cytoplasmic tail to induce B cell activation also blocked the intracellular targeting of antigens, determining the presentation of some, but not all, T cell epitopes.

The Recruitment of Syk Tyrosine Kinase Is Required for BCR-mediated Antigen Presentation.

After BCR stimulation, tyrosine kinases phosphorylate ITAM tyrosine residues, enabling them to associate with the two SH2 domains of syk tyrosine kinase (18, 19). Since mutation of the tyrosine of Ig-α ITAM inhibits the generation of some T cell epitopes, we further evaluated the role of syk tyrosine kinase in antigen presentation through the BCR Ig-α subunit.

First, we tested whether the double mutation of both tyrosine residues contained in Ig-α ITAM affected syk activation through the cytoplasmic tail of Ig-α. Wild-type and mutated FcγR/Ig-α–expressing cells were stimulated for 2 min at 37°C through FcγRs (using the anti-FcγR mAb, 2.4G2), or through endogenous mIgG2a (using F(ab′)₂ fragments of rabbit anti–mouse IgG), or, in a control experiment, with irrelevant F(ab′)₂ fragments of mouse anti– rat IgG. Cell lysates were prepared, and phosphotyrosine proteins were immunoprecipitated with agarose-coupled PT66 and analyzed by immunoblotting with HRP-coupled antiphosphotyrosine mAb Py20 or rabbit anti-syk antisera. As shown in Fig. 4 a, B cell stimulation through endogenous BCR or the Ig-α chimera induced the phosphorylation of similar proteins, among them the syk tyrosine kinase (Fig. 4 b). Mutated Ig-α chimera lacked the ability to induce phosphorylation of syk as well as of other kinases substrates, although endogenous mIgs were functional in these cells (Fig. 4). In addition, the kinase activity of syk was increased after FcγR/Ig-α stimulation but not after cross-linking of the tyrosine-mutated Ig-α chimera as measured by phosphorylation of a specific substrate of syk kinase, a GST fusion protein containing HS1 polypeptide (reference 36; Fig. 4 b). Therefore, the Ig-α cytoplasmic tail is able to activate syk tyrosine kinase and to mediate the presentation of numerous T cell epitopes via a targeting signal located in the Ig-α ITAM.

To further evaluate the role of syk tyrosine kinase in antigen presentation by B cells, we devised a strategy to specifically block the recruitment and activation of syk by the Ig-α subunit of the BCR. For this purpose, an inactive form of syk was stably expressed in FcγR/Ig-α–expressing B cells. A cDNA encoding the two SH2 domains of syk tagged with an HA peptide but lacking its kinase domain was inserted in an expression vector bearing the puromycin resistance gene. After transfection, expressing cells were selected with puromycin and cloned. The clones were screened for the expression of HA-tag by intracellular FACScan^® analysis using FITC-coupled anti-HA mAb (not shown). Positive clones were further characterized by Western blotting using a rabbit antiserum specific for a peptide contained in the NH₂ portion of the syk SH2 domains present both in dominant negative mutant (32 kD) and in endogenous syk (70 kD; Fig. 5 a). The activation of endogenous syk kinase through the Ig-α cytoplasmic tail was then analyzed in the A2 and G2 clones.

For this purpose, the two clones were left unstimulated or stimulated through the Ig-α chimera or endogenous mIgG2a, then phosphoproteins were immunoprecipitated and analyzed by Western blotting using Py20 and anti-syk antibodies. Overexpression of the syk dominant negative mutant prevented the phosphorylation of various intracellular proteins, as well as endogenous syk kinase, induced through the Ig-α cytoplasmic tail. Although global tyrosine phosphorylation was affected, syk phosphorylation was only marginally decreased after B cell stimulation through endogenous mIg (Fig. 5 b). Similarly, we found that overexpression of syk SH2 domains led to a substantial inhibition of kinase activity in syk immunoprecipitates obtained after cell stimulation through the Ig-α cytoplasmic tail, whereas only a marginal effect was found after endogenous mIg stimulation, although the same quantity of syk protein was immunoprecipitated (Fig. 5 c). These results suggested that syk could be activated through an Ig-α–dependent and –independent pathway.

To verify this hypothesis, we examined the effect of overexpression of syk dominant negative mutant on syk activation through the cytoplasmic tail of Ig-β. Clones overexpressing the syk dominant negative mutant were obtained in B cells expressing the FcγR/Ig-β chimera. The E8 and E10 clones, shown in Fig. 6 a, were analyzed. Syk kinase activity and phosphorylation could not be inhibited by overexpression of syk SH2 domains after stimulation of the Ig-β chimera (Fig. 6 b). In conclusion, both subunits of the Ig-α/Ig-β heterodimer were able to activate syk tyrosine kinase, but only syk activation through Ig-α was inhibited by the syk dominant negative mutant. This model allowed us to evaluate the role of syk kinase in MHC class II–restricted antigen presentation through the subunits of the BCR.

Using HRP–anti-HRP rabbit IgG IC, we first established that the overexpression of the syk dominant negative mutant did not affect the internalization of cross-linked Ig-α chimera (Fig. 7 a) or Ig-β chimera (not shown). The presentation of C1 was next analyzed in cells overexpressing the syk dominant negative mutant. No presentation of the cryptic IE^d 12-26 epitope was detected when IC were internalized through the FcγR/Ig-α chimera in cells overexpressing the two syk SH2 domains, whereas presentation of the dominant IA^d 12-26 epitope was only slightly reduced (Fig. 7 b). No effect of the syk dominant negative mutant was observed with FcγR/Ig-β–expressing cells (Fig. 7 b), although the cells stimulated the 24.4 and 26.1 T cell hybridomas at similar concentrations of 12-26 peptide (Fig. 7 c). In addition, overexpression of the syk dominant negative mutant slowed down presentation of the IA^d 12-26 epitope after the internalization of IgG-complexed C1 through the Ig-α chimera (Fig. 7 d  ). Therefore, the overexpression of the syk dominant negative mutant seemed able to modulate the presentation of T cell epitopes which are specifically generated by antigen targeting to newly synthesized, such as the cryptic IE^d 12-26, T epitope issuing from the C1. The syk tyrosine kinase, a major effector of the BCR-induced signaling pathway, therefore, is also involved in BCR-mediated antigen presentation by newly synthesized MHC class II molecules.