Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

Antigen-presenting cells (APCs), to stimulate CD4⁺ helper T lymphocytes, must capture and process exogenous antigens into antigenic peptides which associate with MHC class II molecules in the endocytic pathway (1, 2). The formation of peptide-class II complexes classically occurs in endolysosomal compartments (called MIIC or CIIV), which accumulate newly synthesized class II molecules (3), or in recycling endosomes, which contain mature class II molecules internalized from the cell surface and then recycled back to the plasma membrane (6, 7). Therefore, peptide-class II complexes may be generated at any point throughout the endocytic pathway (8). This study delineates how antigen receptors might determine the delivery of antigens to different compartments and thus could modulate MHC class II-restricted antigen presentation.

B lymphocytes mediate efficient class II-restricted antigen presentation first by facilitating the uptake of soluble antigens through clonally distributed receptors (BCRs)¹ (9). These receptors are multichain complexes composed of a ligand-binding module, the membrane immunoglobulin (mIg), and a transducing module, the Ig-/Ig- heterodimer (10, 11), both chains containing a conserved peptidic motif located in their cytoplasmic tails. This immunoreceptor tyrosine-based activation motif (ITAM [12, 13]) consists of conserved amino acid residues (D2xY2xL7xY2xL) which couple receptors to intracellular effectors, leading to cell activation (11) and antigen internalization (14). Antigen recognition by mIg triggers the activation of Src family phosphotyrosine kinases (PTKs), resulting in tyrosine phosphorylation of Ig-/Ig- ITAM (17) and the recruitment of PTK Syk by phosphorylated ITAM (18, 19), which turns on different signaling pathways (11). Concomitantly, the BCR facilitates the endocytosis of soluble antigens and their access to endosomal/lysosomal compartments, where the antigens are degraded into peptides which then associate to MHC class II molecules to be presented to T lymphocytes (20). Therefore, another important function of the BCR is to address soluble antigens to the intracellular sites of peptide loading on class II molecules (5, 15). To understand the role of the BCR in antigen presentation, a critical question should therefore be addressed: Do signal transduction effectors, involved in BCR-mediated B cell activation, determine the presentation of peptide-MHC class II complexes through BCR-mediated antigen internalization?

The functions of BCR subunits in the intracellular targeting of the BCR partially answer this question. Indeed, the BCR is involved in the delivery of mIg-bound antigens to newly synthesized MHC class II molecules accumulating in endosomal compartments (5). The association of the Ig-/Ig- sheath to mIg may account for the ability of the BCR to induce both specific endosomal targeting and efficient antigen presentation (14, 15). In the complex structure of the BCR, both Ig- and Ig- cytoplasmic domains contain internalization signals (16), but only the Ig- cytoplasmic tail was able to target antigens to newly synthesized MHC class II, whereas Ig- cytoplasmic tail contains targeting signals that direct antigens towards recycling class II (16). In addition, the cytoplasmic tails of Ig- and Ig- were reported to interact with distinct cytoplasmic effectors (21) and to trigger different signaling pathways (22). Thus, the cytoplasmic domain of Ig- interacts with specific cytoplasmic proteins that might be necessary for the intracellular targeting of the antigen-bound BCR to MHC class II compartments. Two related questions were addressed specifically: Does BCR-mediated antigen targeting to class II compartments have any immunological consequences for the selection of specific T cell epitopes, and what are the cytoplasmic effectors of this particular endosomal sorting of antigen-bound BCR to class II compartments? We show that the delivery of mIg-bound antigens, through Ig-, to newly synthesized MHC class II molecules induced the presentation of a large spectrum of T cell epitopes derived from  repressor (C1), hen egg lysozyme (HEL), or ovalbumin (OVA). In contrast, antigen targeting to recycling class II through the Ig- cytoplasmic tail led to the presentation of only some of these epitopes. We then identified a signal in the Ig- cytoplasmic tail that was responsible for the presentation of these T cell epitopes and the activation of syk tyrosine kinase. Endogenous syk activation and presentation of T cell epitopes, induced through the Ig- cytoplasmic tail, were abolished by the overexpression of a syk mutant devoid of kinase domain, which had no effect on antigen presentation induced by the Ig- cytoplasmic tail. Therefore, the recruitment of syk tyrosine kinase by the Ig- BCR subunit in the endosomal pathway may be an important step in potentiating secondary B cell immune response by addressing antigens to MHC class II compartments.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

B Lymphoma Cell Lines.

Stable Expression of Syk Dominant Negative Mutant.

T Cell Hybridomas.

5

Assays for Antigen Presentation.

Detection of Tyrosine Kinase Activation.

IC Internalization.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

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  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.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   The BCR and BCR Ig- subunit determined antigen presentation by newly synthesized class II molecules. (a) A20 cells expressing anti-TNP-specific mIg were incubated with TNP-coupled (top, circles) or uncoupled (bottom, squares)  repressor (3 and 30 µg/ml, respectively) for various times with (+) or without () cycloheximide (Cx; 10 µg/ml). The cells were then fixed with glutaraldehyde before incubation with the T cell hybridoma 24.4, specific for the IAd 12-26 dominant epitope, or with the T cell hybridoma 26.1, specific for the IEd 12-26 cryptic epitope. (b) IIA1.6 cells expressing either FcR/Ig- (top) or FcR/Ig- chimera (bottom) were incubated with C1  repressor-IgG complexes for various times with (+) or without () cycloheximide (Cx; 10 µg/ml). The cells were then fixed with glutaraldehyde before incubation with the same two T cell hybridomas for 24 h. T cell stimulation was evaluated by a CTL.L2 proliferation assay. Similar results were obtained in three independent experiments. (c) Internalization of the HRP-anti-HRP IC by FcR/Ig- or FcR/Ig- chimera was measured as described in Materials and Methods.

The cytoplasmic tail of the BCR Ig- 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- 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 (FcR-defective variant of A20 B cells) expressing FcR chimeras containing the cytoplasmic tail of Ig- or Ig- (FcR/Ig- and FcR/Ig-, respectively [22]) were incubated with C1 (3 µg/ml) complexed with two anti-C1 mAbs (15 µg/ml each) to target C1 on FcR 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 FcR/Ig--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- cytoplasmic tail (Fig. 1 b). Surprisingly, similar experiments with FcR/Ig--expressing cells revealed that endosomal targeting of C1 to recycling class II by the Ig- 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- and Ig- chimeras internalized IC with similar efficiency and kinetics (Fig. 1 c). Therefore, the incapacity of the Ig- 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-.

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 FcR/Ig-- and FcR/Ig--expressing cells (Table 1). The targeting of these different proteins through the cytoplasmic tail of Ig- 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 FcR/Ig--expressing cells led only to the presentation of some of these T cell epitopes (Table 1). Antigen targeting by the Ig- or Ig- 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- and BCR-mediated antigen internalization?

The Ig- and Ig- 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 FcR/Ig- 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 FcR/ Ig-- and FcR/Ig--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- and Ig- did not modify the efficiency of presentation of the 12-26 peptide by IA^d and IE^d molecules.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   B cell activation per se does not induce presentation of the IEd-restricted epitope. (a) FcR/Ig- and FcR/Ig- chimera-expressing cells were incubated for 24 h with OVA-anti-OVA IC. The cells were then fixed with glutaraldehyde and incubated at increasing concentrations of 12-26 C1  repressor peptide and the T cell hybridomas specific for the dominant (IAd 12-26) or cryptic (IEd 12-26) epitopes. The secretion of IL-2 in the cell culture supernatants was measured. Activation of chimera-expressing cells with IC did not modify the efficiency of presentation of the 12-26 peptide. Similar results were obtained in two independent experiments. (b) Cell activation through FcR/Ig- chimera does not induce presentation of the cryptic epitope after antigen internalization by fluid phase uptake. FcR/Ig--expressing cells were incubated overnight in the presence of 30 µg/ml C1  repressor with or without irrelevant OVA-anti-OVA IC, in order to engage FcR/Ig- chimeric receptors. The cells were then fixed and reincubated overnight with T cell hybridomas specific for the dominant or the cryptic epitopes. Engagement of FcR/Ig- chimeric receptors did not induce presentation of the IEd-restricted cryptic epitope. (c) Cell activation through surface IgG does not induce the presentation of the C1  repressor cryptic epitope after IC internalization through FcR/Ig-. Cells expressing FcR/Ig- were incubated continuously in the presence of C1  repressor IC in the absence or presence of specific goat anti-mouse IgG2a F(ab')2 fragments, in order to induce cell activation through endogenous surface IgG2a. The cells were then fixed and reincubated overnight with T cell hybridomas specific for the dominant or the cryptic epitopes. Cell activation through surface IgG2a did not induce presentation of the cryptic epitope by FcR/ Ig- -expressing cells. Errors bars in b and c indicate the mean variation of T cell stimulation of three experiments.

Nevertheless, the BCR or Ig- 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--induced cell activation on intracellular processing of C1 was assessed by coincubating FcR/Ig--expressing cells with irrelevant IC (as shown in Fig. 2 a) in order to trigger cell activation through the Ig- 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- 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).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Tyrosine residues of Ig- ITAM are involved in the selective presentation of a cryptic epitope. (a) Internalization of the HRP-anti-HRP IC by FcR/Ig- chimera or FcR/Ig- Y23A Y34A mutant was measured as described in Materials and Methods. (b) IIA1.6 cells expressing either FcR/Ig- chimera or FcR/Ig- Y23A Y34A mutant were cultured in the presence of increasing concentrations of C1  repressor (fluid phase uptake, squares) or C1  repressor-IgG complexes (FcR-mediated endocytosis, circles). T cell hybridomas specific for a dominant epitope (IAd 12-26) or a cryptic epitope (IEd 12-26) from the C1  repressor were also included in the cultures. The secretion of IL-2 by the T cell hybrids was measured after an overnight incubation. (c) Both transfected cell lines presented the 12-26 peptide on IAd or IEd with identical efficiency. The data presented are representative of results obtained in three independent experiments.

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 FcR/ Ig--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 FcR/Ig--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- 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- 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- 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- 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-/Ig- 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 FcR/Ig- chimeras containing mutated tyrosine residues, previously shown to prevent the activation of tyrosine kinases (25).

First, we analyzed IC internalization by the FcR/Ig- chimera in which tyrosine residues of Ig- ITAM were replaced by alanines (FcR/Ig- Y23-A Y34-A). HRP- anti-HRP rabbit IgG IC were bound to wild-type and mutated FcR/Ig--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- chimeras internalized IC with similar kinetics. Tyrosine residues of Ig- ITAM, as for Ig- (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 FcR/Ig- 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 FcR/Ig- 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 FcR/Ig-- or FcR/Ig- Y23-A Y34-A-expressing cells (Fig. 3 c). In addition, we obtained similar results with B cells expressing Ig- chimera where the first (FcR/Ig- Y23-A) or the second (FcR/Ig- Y34-A) tyrosine residue of Ig- ITAM was mutated to alanine (Table 1). The mutation of tyrosine residues of the Ig- 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- 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.

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- 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- subunit.

First, we tested whether the double mutation of both tyrosine residues contained in Ig- ITAM affected syk activation through the cytoplasmic tail of Ig-. Wild-type and mutated FcR/Ig--expressing cells were stimulated for 2 min at 37°C through FcRs (using the anti-FcR 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- chimera induced the phosphorylation of similar proteins, among them the syk tyrosine kinase (Fig. 4 b). Mutated Ig- 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 FcR/Ig- stimulation but not after cross-linking of the tyrosine-mutated Ig- 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- 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- ITAM.

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Tyrosine residues of Ig- ITAM are required for activation of the syk tyrosine kinase through the BCR. IIA1.6 cells expressing either FcR/Ig- chimera or FcR/Ig- Y23A Y34A mutant were left unstimulated or stimulated through endogenous BCR with F(ab')2 fragments of rabbit anti-mouse IgG antibodies (15 µg/ml), or through Fc chimera with the rat anti-mouse FcR antibody 2.4G2 (20 µg/ml) then F(ab')2 fragments of mouse anti-rat IgG (30 µg/ml). In control lanes, the cells were incubated for the same time with only F(ab')2 fragments of mouse anti-rat IgG (30 µg/ml). The cells were then washed and lysed with 0.5% Triton X-100. Cell lysates were immunoprecipitated with antiphosphotyrosine antibodies (a) or rabbit anti-syk antibodies (b). (a) Phosphoproteins were detected by Western blotting using HRP-coupled antiphosphotyrosine antibody Py20 (top), or a rabbit antiserum specific for the NH2-terminal end of the syk tyrosine kinase (bottom). (b) The phosphorylation of syk in immunoprecipitates was detected by Western blotting using HRP-coupled antiphosphotyrosine antibody Py20 (top). The syk tyrosine kinase activity was measured in a kinase assay using a specific substrate, the GST-HS1 fusion proteins. The phosphorylation of GST- HS1 fusion proteins was detected by Western blotting using HRP-coupled antiphosphotyrosine antibody Py20 (middle). The rate of immunoprecipitated syk tyrosine kinase was controlled for each point on the same filter using a rabbit antiserum specific for the NH2-terminal end of the syk tyrosine kinase (bottom).

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- subunit of the BCR. For this purpose, an inactive form of syk was stably expressed in FcR/Ig--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- cytoplasmic tail was then analyzed in the A2 and G2 clones.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   A dominant negative mutant of syk inhibits the activation of endogenous syk tyrosine kinase through the BCR Ig- subunit. (a) A cDNA encoding the two SH2 domains of the rat syk tyrosine kinase was stably overexpressed in FcR/Ig- chimera cells. The clones G2 and C2 are representative of a series of 20 independent clones characterized by Western blotting using a rabbit antiserum specific for the NH2-terminal end of syk. Arrowheads, Endogenous syk tyrosine kinase (70 kD) and the dominant negative mutant (32 kD). FcR/Ig--positive cells overexpressing or not the dominant negative syk (G2 and C2 clones) mutant were left unstimulated or stimulated through the endogenous BCR or Fc chimera as described in the legend to Fig. 4. The cells were then washed and lysed with 0.5% Triton X-100. Cell lysates were immunoprecipitated with antiphosphotyrosine antibodies (b) or rabbit anti-syk antibodies (c). (b) Phosphoproteins (top) or syk tyrosine kinase (bottom) was detected by Western blotting as in Fig. 4. (c) The phosphorylation of GST-HS1 fusion proteins was detected, after a kinase assay, by Western blotting using HRP-coupled antiphosphotyrosine antibody Py20 (top). The quantity of immunoprecipitated syk was controlled for each point on the same filter using a rabbit antiserum specific for the NH2-terminal end of syk (bottom).

For this purpose, the two clones were left unstimulated or stimulated through the Ig- 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- 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- 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--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-. Clones overexpressing the syk dominant negative mutant were obtained in B cells expressing the FcR/Ig- 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- chimera (Fig. 6 b). In conclusion, both subunits of the Ig-/Ig- heterodimer were able to activate syk tyrosine kinase, but only syk activation through Ig- 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.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   The syk dominant negative mutant did not affect endogenous syk activation of endogenous syk tyrosine kinase through the BCR Ig- subunit. (a) A cDNA encoding the two syk SH2 domains was stably overexpressed in FcR/Ig- chimera-positive cells. The E8 and E10 clones are representative of a series of 10 independent clones characterized by Western blotting using a rabbit antiserum specific for the NH2-terminal end of syk. Arrowheads, Endogenous syk tyrosine kinase (70 kD) and the dominant negative mutant (32 kD). (b) FcR/Ig--positive cells overexpressing the dominant negative syk (E8 and E10 clones) mutant were left unstimulated or stimulated through the endogenous BCR or Fc chimera as in the legend to Fig. 4. The cells were then washed and lysed with 0.5% Triton X-100. Cell lysates were immunoprecipitated with rabbit anti-syk antibodies (top; the kinase activity was measured and controlled as in the legend to Fig. 4) or with antiphosphotyrosine antibodies (bottom; phosphorylated syk was blotted with an anti-syk antiserum as in the legend to Fig. 4).

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- chimera (Fig. 7 a) or Ig- 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 FcR/Ig- 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 FcR/Ig--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- 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.

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Overexpression of syk dominant negative mutant selectively affected presentation of the cryptic T cell epitope through the BCR Ig- subunit. (a) Internalization of the HRP-anti-HRP IC by the FcR/Ig- chimera was measured in G2 (  gray circles) and C2 (black circles) clones overexpressing the syk dominant negative mutant. (b) FcR/Ig- (circles) and FcR/Ig- (squares) chimera-positive cells overexpressing (black and gray) or not (white) syk dominant negative mutant were cultured in the presence of increasing concentrations of C1-IgG complexes (FcR-mediated endocytosis). T cell hybridomas specific for a dominant epitope (IAd 12-26) or a cryptic epitope (IEd 12-26) from the C1  repressor were also included in the cultures. The secretion of IL-2 by the T cell hybrids was measured after an overnight incubation. (c) All the transfected cell lines presented the 12-26 peptide on IAd or IEd with identical efficiency. (d) The FcR/Ig--positive presenting cells, overexpressing (black and gray) or not (white) syk dominant negative mutant, were incubated with C1- IgG complexes for various times, then fixed with glutaraldehyde before incubation with the IAd 12-26-specific T cell hybridomas for 24 h. T cell stimulation was evaluated by a CTL.L2 proliferation assay. Similar results were obtained in three independent experiments.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

Capture of antigens by BCRs is the first step in antigen entry into the endosomal pathway, where antigenic peptides are generated and associated with newly synthesized MHC class II molecules. We report here that antigen targeting to newly synthesized class II molecules through the BCR or Ig--associated subunit induces the presentation of a larger spectrum of T cell epitopes than antigen targeting to recycling class II molecules. Using mutagenesis analysis of the Ig- cytoplasmic tail and stable overexpression in B cells of a syk dominant negative mutant, we describe evidence that signal transduction effectors of B lymphocyte activation are involved in the generation of peptide-MHC class II complexes.

How do Ig- and Ig- cytoplasmic tails induce presentation of different T cell epitopes? Quantitative differences in the rate of antigen internalization can be excluded, since the Ig- chimera was expressed at a lower level than the Ig- chimera (22), and since both chimeric receptors mediated internalization of IC with similar kinetics and efficiency (Fig. 1 c [16]). In contrast, the kinetics of antigen presentation induced by the Ig- and Ig- cytoplasmic tails demonstrated that they target antigens to newly synthesized or recycling pools of MHC class II molecules, respectively. Analysis of various T cell epitopes provides new evidence to further distinguish these two antigen-presentation pathways. Thus, antigen internalization via the Ig-, but not the Ig-, chimera stimulated the DO18.3 hybridoma (Table 1), which recognizes an IA^d-restricted OVA T cell epitope specifically generated in invariant chain-positive presenting cells (32, 38). In contrast, the invariant chain- independent epitope recognized by DO54.8 hybridoma (32) is presented after antigen internalization through the Ig- or Ig- chimera. These data suggest that the Ig- cytoplasmic tail targets antigens to endosomal compartments, where class II molecules transiently accumulate through an invariant chain-dependent mechanism which characterizes newly synthesized class II molecules. In addition, the data also supported the hypothesis that some T cell epitopes cannot be generated in an alternative antigen-presentation pathway defined by compartments where class II molecules cycle with the cell surface (6, 7). Indeed, antigen targeting to recycling class II molecules through Ig- did not induce the stimulation of DO18.3 hybridoma or other T cell hybridomas specific for cryptic or subdominant epitopes derived from the  repressor and HEL. Therefore, the two MHC class II-restricted antigen pathways can be functionally distinguished on the basis of T cell epitopes they are able to efficiently generate. However, the complete BCR as well as the Ig- chimera address antigen to the newly synthesized pool of MHC class II molecules, presenting a large spectrum of peptides. Therefore, the Ig- subunit is dominant over Ig- in this process and accounts for the ability of the BCR to induce efficient antigen presentation during secondary in vivo immune responses. This conclusion raised a new question: What is the intracellular mechanism of BCR- or Ig--mediated antigen presentation?

Two distinct functions have been assigned to Ig- and Ig-: first, to address antigen to different pools of MHC class II molecules (16), and second, to induce different signaling pathways (22, 25). Therefore, the overall cell activation induced by Ig- or the complete BCR might be responsible for the ability of the Ig- chimera to induce stimulation of numerous T cells. However, this hypothesis is unlikely because, first, the stimulation of 12-26 IA^d- and IE^d-restricted T cell hybridomas by Ig- chimera-expressing cells was not modified by chimera cross-linking, and second, the overall cell activation induced by BCR cross-linking was not able to increase presentation of the IA^d 12-26 epitope or to induce presentation of the IE^d 12-26 epitope by Ig- chimera-expressing cells. These data indicated that potential alterations in antigen processing during cell activation do not account for induction of presentation of these T cell epitopes. In addition, we excluded the possibility that BCR or Ig- chimera cross-linking increased cell surface expression of MHC class II or costimulatory molecules such as B.7 or CD40 ligand (not shown). In contrast, mutagenesis analysis identifies, in the Ig- cytoplasmic tail, a targeting signal able to induce efficient antigen presentation of numerous peptides. Indeed, mutation of tyrosine residues contained in the Ig- ITAM, which completely abolish signal transduction (25, 39, 40), affects Ig--mediated antigen presentation, whereas similar mutation in Ig- ITAM did not affect BCR internalization and BCR-mediated antigen presentation (37). These results indicate that tyrosine residues of Ig- and Ig- ITAM are differently involved in the recruitment of cytoplasmic effectors mediating antigen presentation.

Tyrosine mutants of the Ig- ITAM also provide a new tool to dissociate the two major functions of the BCR, ligand internalization and signal transduction, since these mutations block activation of most of the tyrosine kinases but had no effect on receptor internalization. In addition, these mutations specifically alter the presentation of some, but not all, T cell epitopes and inhibited the phosphorylation and activation of syk tyrosine kinase. The role of the kinase activity of syk in BCR-mediated antigen presentation was investigated using B cells overexpressing the two SH2 domains of syk. This mutant protein may be recruited by tyrosine-phosphorylated ITAM and should act as a dominant negative mutant of ITAM function. Indeed, in cells overexpressing the syk mutant, the activation of endogenous syk kinase was blocked after Ig- chimera stimulation and was inhibited after BCR stimulation, whereas syk activation through Ig- was not affected by overexpression of the syk mutant. It is probable that the ITAMs of both BCR subunits activated syk by different mechanisms as suggested by previous data indicating that the Ig- and Ig- cytoplasmic tails interact with distinct cytoplasmic effectors (21) and activate different signaling pathways (22). An attractive hypothesis is that only the phosphorylated ITAM of Ig- directly interacts with the SH2 domains of syk and induce syk activation, whereas the activation of syk through the Ig- cytoplasmic tail is determined by the interaction of another molecule. Whatever the intracellular mechanism of syk activation through the two BCR subunits, our data clearly show that the overexpression of the syk mutant differentially affects antigen presentation through Ig- and Ig-. This is in agreement with previous data showing that the mutation of Ig- ITAM tyrosine residues did not affect Ig--mediated antigen presentation (37), whereas similar mutations in Ig- ITAM only affected the presentation of some, but not all, T cell epitopes (Table 1). Therefore, there is compelling evidence that functionally distinguishes classic and alternative pathways of antigen presentation. Different peptides associate with recycling and newly synthesized pools of class II molecules which are specifically targeted by signals contained in the cytoplasmic tails of antigen receptors. Although these signals are located in a similar ITAM, they can be functionally distinguished. Mutation of tyrosine residues in ITAM and overexpression of the syk dominant negative mutant did not affect antigen presentation using recycling class II molecules through the Ig- cytoplasmic tail, whereas both alter antigen presentation using newly synthesized class II through the Ig- cytoplasmic tail. These data indicate that although both Ig-- and Ig--phosphorylated ITAMs activate syk, the kinase domain of syk seems to be involved only in the targeting of antigen receptors to newly synthesized class II molecules which accumulated, before cell surface arrival, in endocytic compartments called CIIV (for Class II vesicles [3]).

What is the function of the kinase domain of syk in BCR-mediated antigen presentation? The phosphorylated ITAMs of the Ig-/Ig- sheath interact and activate syk as well as other cytoplasmic proteins (21). Recently, syk was shown to associate with or to activate the regulatory subunit of the phosphatidylinositol 3-kinase (PI3 kinase), p85 (41), which activates the catalytic subunit, p110 (42). The target of this enzyme is the inositol ring bond to the fatty acids constituting most cell membranes. The role of PI3 kinases in intracellular transport has been demonstrated by analysis of yeast mutants (vps 34, yeast equivalent of the PI3 kinase) deficient for transport to the vacuole (yeast equivalent of lysosome), and by using wortmannin, inhibitor of endosomal transport (42). The sequential recruitment of syk and PI3 kinase, after receptor aggregation and ITAM phosphorylation, could be a crucial step in endosomal sorting of the BCR, as demonstrated previously for PTK receptors. The kinase domain of the epidermal growth factor receptor (EGFR), is required in receptor sorting toward multivesicular endosomal compartments (43). In the case of the platelet growth factor receptor (PGFR), the sorting of the receptor toward lysosomes is determined by the interaction of its kinase domain with PI3 kinase (44). Although the precise mechanism by which syk kinase determines antigen presentation remains unclear, our results allow us to propose two nonexclusive hypotheses for syk kinase activity in endosomal sorting of antigen-bound BCR. Since the syk dominant negative mutant specifically affected antigen presentation using newly synthesized class II molecules, syk might be required for the endosomal sorting of antigen-bound BCR to class II compartments by the direct activation of the kinase domain of syk or by the recruitment of cytoplasmic effectors, such as PI3 kinases. In a second mechanism, the recruitment and activation of syk kinase might alter the structure and composition of endosomal compartments involved in antigen processing. In either case, it appears likely that syk has a novel and important function in intracellular trafficking and antigen presentation.