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Receptor-Facilitated Antigen Presentation Requires the Recruitment of B Cell Linker Protein to Ig¹

Karyn Siemasko^*, Brian J. Skaggs^*, Shara Kabak^*, Edward Williamson, Bruce K. Brown, Wenxia Song and Marcus R. Clark^2,*,

^* Section of Rheumatology, Departments of Medicine and Pathology, University of Chicago, Chicago, IL 60637; and Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742 ³ Abbreviations used in this paper: BCR, B cell Ag receptor; AMCA, 7-amino-4-methylcoumarin-3-acetic acid; BLNK, B cell linker protein; GFP, green fluorescent protein; ITAM, immunoreceptor tyrosine-based activation motif; Lamp-1, lysosome-associated membrane protein-1; MIIC, MHC class II-enriched compartment; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PLC, phospholipase C; PVDF, polyvinylidene difluoride; SH2, Src homology 2; SNARE, soluble N-ethylmalemide-sensitive factor attachment protein receptor; TfR, transferrin receptor.

	Abstract

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Ags that cross-link the B cell Ag receptor are preferentially and rapidly delivered to the MHC class II-enriched compartment for processing into peptides and subsequent loading onto MHC class II. Proper sorting of Ag/receptor complexes requires the recruitment of Syk to the phosphorylated immunoreceptor tyrosine-based activation motif tyrosines of the B cell Ag receptor constituent Ig. We postulated that the Ig nonimmunoreceptor tyrosine-based activation motif tyrosines, Y₁₇₆ and Y₂₀₄, contributed to receptor trafficking. Ig(YF_176,204)/Ig receptors were targeted to late endosomes, but were excluded from the vesicle lumen and could not facilitate the presentation of Ag to T cells. Subsequent analysis demonstrated that phosphorylation of Y₁₇₆/Y₂₀₄ recruited the B cell linker protein, Vav, and Grb2. Reconstitution of Ig(YF_176,204)/Ig with the B cell linker protein rescued both receptor-facilitated Ag presentation and entry into the MHC class II-enriched compartment. Thus, aggregation accelerates receptor trafficking by recruiting two separate signaling modules required for transit through sequential checkpoints.

	Introduction

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In the periphery, the selection, expansion, and maturation of Ag-specific B cell clones depend upon the quality of signals delivered by the B cell Ag receptor (BCR)³ (1) and the availability of cognate T cells (2). T lymphocyte recognition of B cell-presented peptides is required to initiate responses in primary follicles and to mature and restrict these responses in germinal centers (3, 4). These two MHC class II-restricted checkpoints ensure that the initiation and maturation of a humoral response is Ag-specific and coordinated with T cell activation. The transition between the capture of Ag and the presentation of immunogenic peptides can occur in as little as 20 min (4, 5, 6), indicating that activated B lymphocytes use unique strategies to process endocytosed Ags rapidly.

Indeed, the endocytic compartments preferentially targeted by the BCR are specialized for Ag processing. The best described of these is the MHC class II-enriched compartment (MIIC), which in murine B cells is multivesicular (7), lysosome-associated membrane protein-1⁺ (Lamp-1⁺), MHC class II⁺ (8), H2-M⁺, transferrin receptor (TfR)^-, and M6PR^- (9, 10, 11, 12, 13). In this compartment, MHC class II molecules loaded with specific peptides are first detected following antigenic recognition by the BCR (8). Peptide loading has also been described in an early endocytic compartment bearing the TfR termed the CIIV (14). It is thought that the MIIC, CIIV, and potentially other compartments can be used within a particular cell line to process Ag (15). The MIIC, however, might be preferred because the low pH of this compartment enhances the activity of many hydrolases and may increase the activity of H2-M (16, 17, 18).

The MIIC is not static, but its biochemical and physical properties are responsive to BCR-initiated signals (19, 20). First, receptor activation induces an accumulation of newly synthesized MHC class II within the MIIC (12, 21, 22). This may be facilitated by protein kinase C-dependent phosphorylation of invariant chain that augments late endosomal targeting (19). Second, receptor activation increases the acidity of the MIIC (12). Finally, BCR aggregation can induce the coalescence and fusion of MIIC vesicles to form large central vesicles of >1 µm in diameter (12). These results suggest that signals transduced through the BCR induce changes in the environment of the MIIC that enhance Ag processing and peptide loading onto MHC class II.

In addition to regulating the MIIC, BCR cross-linking greatly accelerates the internalization and targeting of antigenic complexes to it. The endocytic route through which the receptor traffics is not changed (23). However, aggregation of the BCR decreases the time spent by the bulk of receptor complexes in early endosomes, while hastening the appearance of MHC class II-peptide complexes within the MIIC (5, 23, 24, 25). The accelerated delivery of multivalent Ags favors the presentation of both high- and low-affinity Ags captured from the membranes of cells such as follicular dendritic cells (24). Receptor aggregation enhances the presentation of high affinity Ags by as much as 50-fold (23) and may allow initiation of immune responses to low-affinity Ags (24).

Aggregation of the BCR induces the phosphorylation of several tyrosines in the cytoplasmic tails of the receptor-associated chains Ig and Ig (26). Receptor-associated kinases phosphorylate these immunoreceptor tyrosine-based activation motif (ITAM) tyrosines upon engagement with a polyvalent ligand (27, 28, 29). Phosphorylation of the Ig ITAM serves both to recruit and activate the tyrosine kinase Syk (30, 31). One of the most proximal substrates of Syk, and possibly of other receptor-associated kinases, is the adaptor molecule B cell linker protein (BLNK) (32), which is required for the activation of phospholipase C (PLC) and c-Jun N-terminal kinase (33). Phosphorylation of BLNK forms a scaffold for the assembly of Grb2, Vav, Nck, and PLC2 at the plasma membrane (32).

Many of the molecules involved in signal initiation have been implicated in trafficking of the aggregated receptor complex. The cytoplasmic tails of Ig and Ig are both necessary and sufficient to recapitulate the trafficking of the BCR (34). Mutation of the Ig ITAM tyrosines (Y₁₈₂ and Y₁₉₃) to phenylalanines inhibits both Ag presentation and sorting to the MIIC (34). Syk also plays a crucial role in sorting receptors to late endosomes (35, 36). The mechanisms by which Syk, or any other molecules that may bind the phospho-Ig ITAM, mediates receptor trafficking are unknown. In addition, Ig contains two non-ITAM tyrosines at residues Y₁₇₆ and Y₂₀₄. One study indicated that BLNK might bind to phosphorylated Y₂₀₄; however, the contribution of this association to signaling or receptor trafficking was not investigated (37).

In this study, we report that phosphorylation of the non-ITAM Ig tyrosines is necessary for efficient delivery of aggregated receptor complexes into late endosomes. Both mutant receptor complexes (Ig(YF_176,204)/Ig) and wild-type Ig/Ig sort efficiently to late endosomes. However, the transport vesicles carrying mutant complexes did not enter the MIIC endosomes. These excluded receptors were compromised in their ability to facilitate the presentation of pulsed Ag to T cells. Subsequent experiments demonstrated that phosphorylation of Y₂₀₄ served to recruit BLNK, Vav, and Grb2 to the receptor. Reconstituting Ig(YF_176,204)/Ig with BLNK rescued the ability of the receptor both to enter late endosomes and to facilitate the presentation of Ag. These data reveal an unexpected mechanism by which the direct recruitment of BLNK to the BCR mediates the rapid delivery of antigenic complexes into late endocytic processing compartments.

	Materials and Methods

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Cell lines

The murine B cell lymphoma A20IIA1.6 (IgG2a⁺, FcR^-, I-Ad⁺, I-Ed⁺) (2) was cultured in IMDM (Life Technologies, Grand Island, NY) containing 10% FCS (HyClone, Logan, UT), 2 mM of glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in 7.5% CO₂. The myoglobin-specific T cell clone (Ach 8.2; A. Sant, University of Chicago, Chicago, IL) was maintained in DMEM (Life Technologies) supplemented as above for IMDM.

Platelet-derived growth factor receptor (PDGFR) chimera

The basic construction, expression, and activation of the PDGFR chimeras have been previously described (38, 39). Tyrosine to phenylalanine mutations were engineered using complementary primers and PCR. To fuse BLNK to the C terminus of Ig(YF_176,204), BLNK was first PCR-amplified from murine cDNA using primers complementary to the entire open reading frame. The 5' primers contained an EcoRI site, and the 3' primer contained a XhoI site. Using these sites, the cDNA was cloned to the 3' end of a cDNA encoding PDGFR/Ig(YF_176,204) and then inserted into the retroviral vector MIGR1. Virus was packaged in the GP-293 packaging cell line (Clontech Laboratories, Palo Alto, CA), and supernatants were used to infect A20IIA1.6 cells expressing PDGFR/Ig. Cells were sorted by FACS for green fluorescent protein (GFP) expression. All other cDNAs were cloned into pMuTKneo that contains the thymidine kinase promoter and the µ enhancer. Plasmids were cotransfected with plasmids encoding PDGFR/Ig into A20IIA1.6 cells and clonal transfectants selected with G418 (Life Technologies). To determine surface expression of each chimeric molecule, or the BCR, cells were first stained with mouse anti-PDGFR (Genzyme, Cambridge, MA; catalog number 1264-00) and anti-PDGFR Abs (R&D Systems, Minneapolis, MN; catalog number MAB1263), followed by FITC-conjugated anti-IgG1 (Zymed Laboratories, San Francisco, CA; catalog number 61-0100), or with goat anti-mouse IgG2a (Southern Biotechnology Associates, Birmingham, AL; catalog number 1080-01), followed by donkey anti-goat IgG FITC (Jackson ImmunoResearch Laboratories, West Grove, PA; catalog number 705-095-147). Samples were then examined by flow cytometry (FACScan; BD Biosciences, Bedford, MA).

Ag presentation

Preparation of the myoglobin/goat anti-rabbit IgG conjugate (final concentration of 20 ng/ml) has been previously described (34). Myoglobin was targeted to the chimeric complexes on each transfectant as a pulse by incubating the transfected cells on ice consecutively with PDGF-BB (100 ng/ml; Sigma-Aldrich, St. Louis, MO) for 5 min, mouse anti-human PDGFR (5 µg/ml) for 3 min, and rabbit anti-mouse IgG1 (5 µg/ml) for 10 min. The cells were washed and then incubated with the noted dilutions of myoglobin-conjugated goat anti-rabbit IgG for 15 min on ice. Cells were then incubated at 37°C for 30 min and washed, and 1 x 10⁵ cells/sample were incubated with 1 x 10⁵ myoglobin-specific T cell clones (Ach 8.2) for 24 h at 37°C. Supernatants were then harvested for IL-2 analysis by ELISA. Briefly, rat anti-mouse IL-2 (1 µg/ml; BD PharMingen, San Diego, CA; catalog number 18161D) was coated in binding buffer (0.1 M of Na₂HPO₄ (pH 9)) onto an Immunolon microtiter plate (Dynatech Laboratories, Chantilly, VA) at 4°C overnight. Plates were washed with PBS/Tween, blocked with 1% BSA/PBS for 30 min at room temperature, and then washed. Supernatants (100 µl) were incubated on coated wells for 2 h at room temperature, then washed with PBS/Tween. The IL-2 standard was purchased from R&D Systems. Biotin rat anti-mouse IL-2 (1 µg/ml; BD PharMingen; catalog number 554426) in blocking buffer was added to wells for 1 h at room temperature and then washed. Peroxidase-conjugated streptavidin (1 µg/ml; Jackson ImmunoResearch Laboratories; catalog number 016-030-084) was added for 30 min, and the plates were then washed. DAKO (Carpinteria, CA) served as the substrate, and the plates were read on an ELISA reader (Corixa, Hialeah, FL) at 650 nm. The data reported are representative of that obtained in three separate experiments.

Confocal microscopy

For BCR or chimera/Lamp-1 costaining, transfectants were incubated with either goat anti-mouse IgG2a (Southern Biotechnology Associates), followed by donkey anti-goat IgG FITC (Jackson ImmunoResearch Laboratories) or PDGF-BB ligand, mouse anti-PDGFR Ab as described above, and then with FITC-labeled (Zymed Laboratories) rabbit anti-mouse IgG1 (Jackson ImmunoResearch Laboratories) or rabbit anti-mouse IgG1, followed by 7-amino-4-methylcoumarin-3-acetic acid (AMCA)-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories; catalog number 711-155-152). Cells were placed at 37°C for 30 min and then fixed with 3% paraformaldehyde/3% sucrose in PBS, or in the case of GFP-expressing cells, fixed with PBS containing 2% formaldehyde and 0.05% glutaraldehyde (both from Sigma-Aldrich). Following fixation, cells were permeabilized with 0.05% saponin and stained for Lamp-1, as described previously (34). Confocal sections (0.75–1 µm) were acquired using a Zeiss (Oberkochen, Germany) 410 confocal microscope. Images are displayed by pseudocoloring using LSM software.

Immunoelectron microscopy

Cells were incubated with PDGF-BB ligand, mouse anti-human PDGFR, and rabbit anti-mouse IgG1, followed by 5-nm gold particle-conjugated goat anti-rabbit IgG (British Biocell International, Cardiff, U.K.; catalog number 15725) on ice, stimulated for 30 min at 37°C, then fixed in 8% paraformaldehyde/250 mM of HEPES (pH 7.2). Cells were washed in PBS, infiltrated with 2.3 M of sucrose, frozen in liquid nitrogen, and sectioned at -110°C. To immunostain sections for the late endosomal marker Lamp-1, samples were incubated in 10% FCS/0.12% glycine/PBS at room temperature for 30 min, then incubated with ID4B (Developmental Studies Hybridoma Bank; University of Iowa, Iowa City, IA) for 30 min at room temperature. The grids were washed in PBS/0.12% glycine and then stained with 10-nm gold particle-conjugated goat anti-rat IgG (catalogue 15771). Grids were then washed with PBS/glycine and then distilled water, followed by incubation with 1.8% methyl cellulose/0.3% uranyl acetate. A 100 CX transmission electron microscope (JEOL, Peabody, MA) was used at an accelerating voltage of 60 kV to view the sample (40). Quantitation of the micrographs was analyzed by blind scoring.

Internalization

Internalization was quantitated as previously described (34). Briefly, cells were stimulated via the chimera receptor, as described above, except the anti-mouse IgG1 Ab was HRP-labeled (Zymed Laboratories; catalog number 61-0120). After washing, 2.5 x 10⁷ cells were placed at 37°C for 5, 10, or 30 min, and then put on ice to prevent further endocytosis and then washed again. The concentration of noninternalized chimera labeled with HRP-conjugated Ab was identified colorimetrically by the addition of 5 mM of O-phenylenediamine HCl in 0.15 M of phosphate buffer (pH 6) with 0.15% H₂O₂ for 15 min at room temperature (41). The total HRP activity for the individual clones was determined by solubilizing a duplicate sample with PBS/1% Nonidet P-40 for 10 min at room temperature and then adding substrate, as outlined above for 15 min. Peroxidase reactions were terminated by the addition of 6 N of HCl and centrifugation at 20,000 x g for 10 min at 4°C. For a given experiment, total HRP activity varied <20% between clones.

Peptide precipitations

Peptides were synthesized at the University of Chicago peptide facility. The sequence of the peptide encompassing Y₁₇₆ was MPDDYEDENLY, and that encompassing Y₂₀₄ was LQGTYQDVGNL (using the single letter amino acid code). The peptides were synthesized with or without a phosphate group on the fifth tyrosine of the peptide. The peptides were covalently coupled to normal human serum-activated Sepharose (Amersham Biosciences, Piscataway, NJ). For precipitations, 2 x 10⁶ cells were washed and resuspended in 400-µl serum-free medium. Cells were either left unstimulated or stimulated with 25 µg/ml rabbit anti-mouse IgG (Jackson ImmunoResearch Laboratories; catalog number 315-005-045) for 2 min at 37°C. Cells were lysed by the addition of 2x lysis buffer (1% Nonidet P-40, 150 mM of NaCl, 10 mM of Tris, 0.4 mM of Na₄P₂O₇, 0.4 mM of EDTA, 10 mM of PMSF, and 1 µg/ml aprotinin, leupeptin, and -1-antitrypsin final). Lysates were clarified by centrifugation and rotated overnight at 4°C with Sepharose-coupled peptide beads. Precipitations were then washed, resolved by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membrane, and immunoblotted with 4G10 and anti-BLNK Abs.

Immunoprecipitations

Anti-Syk (sc-929), anti-Grb2 (sc-255), and anti-Vav (sc-132) Abs were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA), 4G10 anti-phosphotyrosine (catalog number 05-321) was purchased from Upstate Biotechnology (Lake Placid, NY), and polyclonal rabbit antisera were made to a GST fusion of the BLNK Src homology 2 (SH2) domain, while the anti-Ig rabbit antisera has been described previously (39). For each immunoprecipitation, 1 x 10⁷ cells were either left untreated or stimulated by incubating on ice with either rabbit anti-mouse IgG (Jackson ImmunoResearch Laboratories) at 25 µg/ml or PDGF-BB, mouse anti-human PDGFR, and rabbit anti-mouse IgG1, as described above, and then incubated at 37°C for 3 min. Cells were then washed and lysed in modified RIPA buffer (1% Nonidet P-40, 150 mM of NaCl, 10 mM of Tris, 0.1% SDS, 0.5% deoxycholate, 0.4 mM of Na₄P₂O₇, 0.4 mM of EDTA, 10 mM of NaF, 1 mM of PMSF, and 1 µg/ml aprotinin, leupeptin, and -1-antitrypsin). Lysates were clarified by centrifugation and precleared by incubating with 150 µl of a mixture of protein A- and protein G-Sepharose (Amersham Biosciences). The resulting supernatants were immunoprecipitated by incubation overnight at 4°C with the indicated Abs prebound to protein A-Sepharose. Immunoprecipitations were washed extensively, boiled in loading buffer, loaded onto SDS-PAGE gels, transferred to PVDF membranes (Millipore, Bedford, MA), and Western blotted with the indicated Abs.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Receptor-facilitated Ag presentation requires Ig Y₁₇₆ and Y₂₀₄

Within the complex structure of the BCR, the cytoplasmic tails of the Ig/Ig heterodimer determine the primary trafficking and signaling capacities of the receptor (34, 36, 42). To define the specific functional domains within the tails necessary for receptor trafficking, we used an approach that allowed different receptor complexes containing the cytoplasmic tails of Ig and Ig to be assembled on the cell surface. Chimeric molecules were constructed and expressed, in the FcR-deficient B cell lymphoma A20IIA1.6, in which the extracellular and transmembrane domains of the human PDGFR or were fused to the cytoplasmic tails of Ig or Ig (Fig. 1) (39). PDGFR and have equal affinity for PDGF-BB, therefore adding this ligand to cells expressing two different chimeras (PDGFR/Ig and PDGFR/Ig) forms Ig/Ig heterodimers that can then be aggregated first with mouse mAbs to the extracellular domain of PDGFR and then by rabbit anti-mouse IgG1.

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Construction and expression of chimeras containing Ig/Ig. A, Schematic of PDGFR chimeras. cDNA fragments were constructed encoding for chimeric molecules containing the extracellular and transmembrane domains of either human PDGFR or fused to the indicated cytoplasmic tails of Ig or Ig. PDGFR chimeras containing either wild-type or mutant Ig tails were coexpressed in B cell lymphoma cell line A20IIA1.6 with PDGFR/Ig. The addition of PDGF-BB induced heterodimers to form on the surface of each cell, which were then aggregated with anti-receptor Abs. B, Flow cytometric analysis of each A20IIA1.6 clone expressing BCR (left column) or wild-type and mutant PDGFR chimeras (right column). Expression of surface IgG2a, PDGFR (black), or PDGFR (dark gray) as well as an IgG1 isotype control (shaded) are shown.

We first examined whether Ig/Ig required Ig Y₁₇₆ and/or Y₂₀₄ to facilitate the presentation of Ag to specific T cell clones. Chimeric receptors on each transfectant were aggregated by sequential incubation on ice with PDGF-BB, by mouse anti-PDGFR Abs, and then by rabbit anti-mouse IgG1. These aggregated receptor complexes were pulsed with Ag by incubation with myoglobin conjugated to goat anti-rabbit IgG at 37°C for 30 min. In separate samples, Ag was targeted to the endogenous BCR on each transfectant, first by ligating the receptor with rabbit anti-mouse IgG and then by pulsing with myoglobin conjugated to goat anti-rabbit IgG. In both cases, cells were then washed and used as APCs in assays with the T cell clone Ach 8.2 (34). IL-2 production was assayed by ELISA after 24 h. Ag targeted either to the chimeric complexes containing wild-type Ig/Ig cytoplasmic tails or to the BCR was presented efficiently (Fig. 2). As expected, the magnitude of IL-2 produced correlated with the amount of available Ag. Although the A20IIA1.6 cell line constitutively secretes low levels of IL-2, no increase in IL-2 was detected in the absence of T cells nor in the absence of Ag (data not shown). In contrast, Ig(YF176,204)/Ig was incapable of effectively facilitating the presentation of Ag. Concurrent cross-linking of the endogenous BCR with anti-IgG2a Abs did not rescue Ig(YF_176,204)/Ig-facilitated Ag presentation, suggesting that the defect is intrinsic to Ig(YF176,204)/Ig (data not shown).

View larger version (24K): [in this window] [in a new window]   FIGURE 2. The presentation of pulsed Ag requires the Ig tyrosines Y176 and Y204. Aggregated BCR () or chimeric receptor complexes () were first formed on ice, and then myoglobin conjugated to goat anti-rabbit IgG was added. Cells pulsed with different dilutions of myoglobin-conjugated Ab were placed at 37°C for 30 min, washed, and then cocultured with the T cell clone Ach 8.2 for 24 h. IL-2 production was measured by ELISA. Each graph shows the mean values of samples done in triplicate from a representative single experiment. The experiment shown is representative of three separate experiments. Additional independently derived clones gave similar results (data not shown).

Entry into late endosomes requires Ig Y₁₇₆ and Y₂₀₄

To begin to examine the receptor-mediated processes dependent upon Y₁₇₆ and Y₂₀₄, we first determined whether efficient chimeric complex internalization depends upon either tyrosine. For these assays, HRP-coupled goat anti-rabbit IgG was used to label the aggregated receptor complexes, and internalization was allowed to proceed 5, 10 (data not shown), or 30 min (Fig. 3A). The percentage of total HRP activity still remaining on the cell surface was assayed colorimetrically. There was no significant difference in the degree of internalization between the wild-type and mutant chimeric complexes at any of the time points examined (Fig. 3A) (34) (data not shown). Accordingly, we next examined whether any of the chimeric complexes were aberrant in their ability to target late endosomes.

View larger version (14K): [in this window] [in a new window]   FIGURE 3. Ig/Ig and Ig(YF176,204)/Ig are internalized and target late endosomes. A, Chimeric molecules were stimulated with HRP-conjugated Abs at 37°C for 30 min. Samples were split, and nonlysed or lysed samples were assayed colorimetrically for HRP activity. Internalization percentage was calculated by: 100% x ((OD492solubilized - OD492nonsolubilized)/OD492solubilized). B, Ig/Ig and Ig(YF176,204)/Ig target late endosomes. Ig/Ig targeted to late endosomes as well as induced their translocation and fusion to form a large perinuclear complex (large arrows) (34 ). In contrast, Ig(YF176,204)/Ig appears to traffic to late endosomes, but does not induce their translocation to form a perinuclear complex (arrowheads). Ig(YF176,204)/Ig sometimes appeared to be in proximity with late endosomes rather than coinciding with them (small arrow). In these assays, cells were stimulated with BB ligand, mouse anti-human PDGFR, and then FITC-coupled rabbit anti-mouse IgG1 Abs for 30 min, and then fixed, permeabilized, and stained with rat anti-mouse Lamp-1 (ID4B), followed by PE-conjugated anti-rat IgG. C, Ig(YF176)/Ig and Ig(YF204)/Ig target to late endosomes. Following surface aggregation, Ig/Ig chimeric complexes traffic to, and induce the perinuclear translocation of, Lamp-1+ late endosomes (large arrows) (row 1). In contrast, Ig(YF176,204)/Ig complexes were detected in proximity of late endosomes, but colocalize poorly with them (small arrows) (row 2). There was no significant translocation of late endosomes to the perinuclear area. The phenotypes of the Ig(YF176)/Ig (rows 3 and 4) and Ig(YF204)/Ig (rows 5 and 6) chimeras appeared to be intermediate. In some cases, receptor targeting and colocalization occurred (arrowheads), while in other instances only coregionalization with peripherally distributed Lamp-1+ vesicles was evident (small arrows). For these assays, cells were stimulated with BB ligand, mouse anti-human PDGFR, and then FITC-labeled rabbit anti-mouse IgG1 for 30 min, and then fixed, permeabilized, and stained with rat anti-mouse Lamp-1 (ID4B), followed by Texas Red-conjugated anti-rat IgG.

Each clone was stimulated through either the BCR or the chimera with FITC-coupled Abs for 30 min and then fixed, permeabilized, and stained for Lamp-1 (ID4B, red). Upon stimulation of the clones through the BCR or the Ig/Ig chimera for 30 min (Fig. 3B), receptor complexes colocalized with a large perinuclear aggregate of Lamp-1⁺ vesicles (large arrows). In contrast, stimulation of Ig(YF_176,204)/Ig did not induce the perinuclear coalescence of Lamp-1-bearing late endosomal vesicles. The majority of endocytosed Ig(YF_176,204)/Ig mutant chimeric complexes did colocalize with peripherally distributed endosomes (arrowheads), but some receptor complexes appeared to be adjacent to Lamp-1⁺ vesicles rather than actually occupying the same region (small arrow), suggesting that Ig(YF_176,204)/Ig might be excluded from their lumen. Analysis of cells expressing Ig(YF₂₀₄)/Ig revealed a similar defect in receptor trafficking, while in those expressing Ig(YF₁₇₆)/Ig, receptor entry into Lamp-1⁺ vesicles appeared to be partially inhibited (Fig. 3C). To further characterize the defect in mutant receptor trafficking, we used immunoelectron microscopy.

The Ig/Ig or mutant chimeric complexes were stimulated with Abs conjugated with 5-nm gold particles for 30 min at 37°C. Cells were then fixed, and frozen sections were prepared and stained with ID4B and 10-nm gold-conjugated secondary Abs. Fig. 4 shows representative immunoelectron micrographs of Ig/Ig and Ig(YF176,204)/Ig. As observed when the BCR was similarly stimulated and analyzed (12), >80% of internalized Ig/Ig chimeric complexes were detected within Lamp-1⁺ vesicles (arrows), often within the intraluminal multivesicular bodies thought to be derived from sorted transport vesicles (45) (Fig. 4A). Many of the targeted Lamp-1⁺ vesicles were large, often >1 µm in diameter (Fig. 4B). In contrast, <5% of internalized Ig(YF176,204)/Ig chimeric complexes were found within Lamp-1⁺ vesicles. Approximately 70% of these complexes were located within proximity to Lamp-1⁺ vesicles (Fig. 4, C–E). All of the observed Lamp-1⁺ vesicles were small (<0.5 µm), indicating that translocation and homotypic fusion between late endosomal vesicles did not occur. Analysis of cells expressing Ig(YF₂₀₄)/Ig revealed a similar defect in entry, while in cells expressing Ig(YF₁₇₆)/Ig, labeled complexes were found both outside and inside of the Lamp-1⁺ vesicles (Fig. 5). These results indicate that mutations in Y₂₀₄, and to a lesser extent Y₁₇₆, inhibit the ability of Ig/Ig to enter late endosomes.

View larger version (113K): [in this window] [in a new window]   FIGURE 4. The Ig(YF176,204)/Ig chimera is excluded from late endosomes. A, Although abundant Ig/Ig receptor complexes are found within the lumen of large Lamp-1+ vesicles (A and B), Ig(YF176,204)/Ig receptor complexes are excluded from small Lamp-1+ vesicles (C–E). Bar equals 0.3 µm for A, and C–E, and 0.4 µm for B. Shown are immunoelectron micrographs of cells stimulated through either Ig/Ig or Ig(YF176,204)/Ig with Abs conjugated to 5-nm gold particles (arrows) for 30 min. Cells were then counterstained with ID4B and 10-nm gold particle-conjugated secondary Abs.

View larger version (148K): [in this window] [in a new window]   FIGURE 5. Both Ig Y176 and Y204 contribute to entry into late endosomes. Within 30 min of stimulation, abundant Ig/Ig receptor complexes were found within the lumen of large Lamp-1+ vesicles (upper left). In contrast, Ig(YF176,204)/Ig receptor complexes were excluded from Lamp-1+ vesicles (upper right). Receptor complexes of Ig(YF176)/Ig were found both outside and inside the Lamp-1+ vesicles (middle row), while Ig(YF204)/Ig were primarily excluded (lower row). Indicated chimeras were stimulated with Abs conjugated to 5-nm gold particles (arrows) for 30 min. Cells were then counterstained with ID4B and 10-nm gold particle-conjugated secondary Abs. All bars represent 0.2 µm.

BLNK, Vav, and Grb2 are recruited upon phosphorylation of Ig Y₂₀₄

Examination of the amino acid sequences flanking Y₁₇₆ and Y₂₀₄ revealed that Y₂₀₄ was part of a motif (Y-hydrophilic-philic-phobic) capable of binding group I SH2 domains (46). In contrast, Y₁₇₆ was not predicted to bind SH2 or any other phosphotyrosine binding domains. To determine which molecules bound to Y₁₇₆ or Y₂₀₄, we synthesized phosphorylated and nonphosphorylated peptides corresponding to the regions encompassing each tyrosine and coupled them to Sepharose beads. Precipitations from the lysates of unstimulated or stimulated wild-type A20IIA1.6 cells were resolved by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with anti-phosphotyrosine Abs (4G10). As seen in Fig. 6A, a single tyrosine-phosphorylated band of 65 kDa was detected in phospho-Y₂₀₄ precipitations from the lysates of stimulated cells. Of the molecules known to be tyrosine phosphorylated following BCR aggregation, the linker protein BLNK has a molecular mass of 65 kDa. Stripping and reprobing with anti-BLNK sera identified the band as BLNK. BLNK was also efficiently precipitated by the phospho-Y₂₀₄ peptide from the lysates of unstimulated cells.

View larger version (50K): [in this window] [in a new window]   FIGURE 6. Association of BLNK, Vav, and Grb2 with Ig/Ig. A, Phosphorylated and unphosphorylated peptides corresponding to the sequences surrounding Y176 and Y204 were used in precipitations from the lysates of unstimulated and BCR-stimulated cells. Precipitations were Western blotted sequentially with anti-phosphotyrosine (4G10) and anti-BLNK Abs. B, Cells were either left unstimulated (U) or stimulated through the BCR (B) or the indicated chimera (C). Immunoprecipitations with anti-BLNK, anti-Vav, or anti-Grb2 Abs were Western blotted with 4G10, anti-Ig (corresponding position of immunoreactivity on 4G10 blot indicated with arrowhead), or the primary immunoprecipitating Ab. C, In the upper set of panels, Syk immunoprecipitations were Western blotted with 4G10 and then anti-Syk and anti-Ig. In the lower panel, unstimulated or stimulated whole cell lysates (WCL) were Western blotted with 4G10.

Following BCR aggregation, Syk rapidly phosphorylates BLNK to form a scaffold at the plasma membrane for the assembly and activation of a variety of molecules including Vav and Grb2 (32, 47, 48). Therefore, we next examined whether some of these molecules associated with Ig/Ig. Cells were stimulated as before, and lysates were immunoprecipitated with Abs to Vav. After SDS-PAGE, the membrane was first immunoblotted with 4G10 and then stripped, divided, and immunoblotted with either anti-Vav or anti-Ig Abs (Fig. 6B, middle panels). As was observed in the BLNK immunoprecipitations, the Ig/Ig chimera coprecipitated with Vav, and this coassociation depended upon receptor aggregation and Y₂₀₄ and/or Y₁₇₆. Although mutation of either tyrosine was sufficient to disrupt the detectable association of Vav with Ig/Ig, mutation of both tyrosines was required to uncouple chimera aggregation from Vav phosphorylation. The apparent discordance between association and function is probably due to the stringent conditions under which our immunoprecipitations were performed. As with both BLNK and Vav, Grb2 associated with Ig/Ig in an aggregation- and Y₁₇₆/Y₂₀₄-dependent manner (Fig. 6B, lower panels).

The recruitment and activation of Syk depend on the ITAMs in the Ig/Ig cytoplasmic tails (43, 44). To ensure that mutation of Y₁₇₆/Y₂₀₄ did not perturb this function, immunoprecipitations of Syk from the lysates of cells stimulated through either chimeras of Ig/Ig, Ig(YF176,204)/Ig, or Ig(YF_182,193)/Ig were resolved by SDS-PAGE and transferred to PVDF membrane. These membranes were immunoblotted with 4G10, followed by stripping and reimmunoblotting with either anti-Syk or anti-Ig Abs. As seen in Fig. 6C, aggregation of both Ig/Ig and Ig(YF_176,204)/Ig equally induced the recruitment of Syk. As expected, mutation of the Ig ITAM tyrosines prevented this interaction.

As illustrated in Fig. 6B, the inductive tyrosine phosphorylation of BLNK and Vav is dependent upon Y₁₇₆ and Y₂₀₄. To examine whether mutation of these tyrosines globally compromised receptor-induced tyrosine phosphorylation, we stimulated each receptor complex, and total cell lysates from each sample were Western blotted with 4G10 (Fig. 6C, lower panel). Total inductive tyrosine phosphorylation was similar whether cells were stimulated through either the chimeras or through the endogenous BCR expressed on each cell line. From these data, we conclude that mutation of Y₁₇₆ and Y₂₀₄ results in a selective defect in Ig/Ig-dependent BLNK phosphorylation and recruitment.

BLNK mediates receptor entry into late endosomes

BLNK serves as the primary scaffold on which Vav and Grb2 assemble (32, 48). Furthermore, the BLNK SH2 domain can bind directly to phosphorylated Y₂₀₄ (Fig. 6A) (37, 68). Therefore, we examined whether adding BLNK back to Ig(YF_176,204)/Ig restored normal receptor trafficking. A cDNA was constructed encoding a chimera in which the 3' sequence of PDFGR/Ig(YF_176,204) was linked in frame to a cDNA-encoding murine BLNK. This cDNA was cloned into the bicistronic GFP-expressing retroviral vector MIGR1. A20IIA1.6 cells expressing PDGFR/Ig were infected with chimera-encoding retrovirus, and GFP-expressing cells were isolated by FACS. Expression of PDGFR/Ig(YF_176,204)/BLNK was confirmed both by flow cytometry and by immunoblotting with anti-BLNK and anti-PDGFR Abs. Surface expression of the chimera, as measured by flow cytometry, was 2-fold less than that of PDFGR/Ig(YF176,204) (data not shown).

The PDGFR chimeras or the BCR on cells expressing either Ig(YF176,204)/Ig or Ig(YF176,204)BLNK/Ig were pulsed with myoglobin, and their ability to present to the T cell clone Ach 8.2 was determined. The presence of BLNK restored the ability of Ig(YF176,204)/Ig to facilitate the presentation of Ag (Fig. 7A). We next determined whether BLNK allowed entry into the MIIC. The chimeras on cells expressing PDGFR/Ig with either Ig(YF_176,204) or Ig(YF_176,204)BLNK were aggregated, as described previously, and labeled with AMCA-coupled Abs for 30 min. Cells were then fixed and stained with rat anti-mouse Lamp-1 (ID4B), followed with Texas Red-conjugated anti-rat IgG. In contrast to Ig(YF_176,204)/Ig, Ig(YF_176,204)BLNK/Ig colocalized with, and induced the coalescence of, Lamp-1⁺ late endosomes (Fig. 7B). These data demonstrate that the recruitment of BLNK by Ig is required for the delivery of receptor-bound antigenic complexes to late endosomal processing compartments.

View larger version (15K): [in this window] [in a new window]   FIGURE 7. BLNK rescues mutant receptor trafficking and facilitated Ag presentation. A, A chimeric molecule containing Ig(YF176,204)/Ig cannot facilitate Ag presentation (). In contrast, a chimeric complex in which BLNK was fused to the C terminus of Ig(YF176,204) (Ig(YF176,204)BLNK/Ig) was capable of restoring Ag presentation (•). Ag presentation assays were conducted as described in Fig. 2, and are expressed as a percentage of maximal BCR response. Raw values that were used to calculate the percentage of maximum response for the tandem (at the 1/50 Ag dose) were as follows. Uncorrected IL-2 ELISA ODs at 650 nm were 2.529 and 1.989 for Ag targeted through the tandem chimera or BCR, respectively (yielding an uncorrected percentage of maximum response of 135% for the tandem). After subtracting background (stimulation minus Ag), OD values were 0.895 and 1.006, respectively, yielding the displayed value of 89%. B, Ig(YF176,204)/Ig target late endosomes, but do not colocalize (large arrows). In contrast, Ig(YF176,204)BLNK/Ig targeted and colocalized with late endosomes (small arrow). In addition, Ig(YF176,204)BLNK/Ig induced the coalescence of Lamp-1+ late endosomes. For these confocal experiments, cells were stimulated, as described in Materials and Methods, using AMCA-coupled Abs for 30 min, and then fixed, permeabilized, and stained with rat anti-mouse Lamp-1 (ID4B), followed with Texas Red-conjugated anti-rat IgG.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In addition to being required for receptor trafficking, the non-ITAM tyrosines were necessary for receptor-mediated BLNK and Vav phosphorylation. Phosphorylation was associated with recruitment to Ig/Ig, indicating that these two processes were interrelated. It is known that BLNK is rapidly phosphorylated following receptor ligation, and that Syk may be the responsible kinase (32). In vivo, the main determinant of BLNK binding, Y₂₀₄, is phosphorylated following receptor ligation. Furthermore, the SH2 domain of BLNK can bind directly to this phosphotyrosine (37, 68). These data indicate that BLNK is recruited directly to Ig, the same chain to which Syk is recruited and activated, providing a mechanism to ensure rapid phosphorylation. Ig has been demonstrated to be capable of activating signaling pathways downstream of BLNK, including PLC2 (43, 50, 51, 52). However, the cytoplasmic domain of Ig contains only the two tyrosines of a single ITAM, and therefore does not have a potential recruitment site for the SH2 domain of BLNK. Furthermore, BLNK does not coprecipitate with Ig alone following receptor ligation (data not shown). Therefore, it is likely that Ig uses different proximal mechanisms to initiate signaling.

The incorporation of BLNK into the activated receptor complex raises the possibility that it, or a molecule that it binds, such as Grb2 or Vav, acts to chaperone the receptor into late endosomes rather than as a linker to activate a permissive signaling pathway. In support of this possibility, inhibition of many of the signaling pathways distal to BLNK, including intracellular calcium mobilization (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate acid and EGTA), protein kinase C (Calphostin C), and Rac (expression of N¹⁷-Rac), had no effect on trafficking of the BCR (data not shown). Furthermore, providing a concurrent signal in trans through the BCR fails to rescue the presentation of Ag bound to Ig(YF_176,204)/Ig. However, BCR ligation does allow entry of this receptor complex into late endosomes (K. Siemasko, unpublished results). These data indicate that direct binding of BLNK to Ig serves as both a signaling complex and a chaperone to facilitate Ag presentation. To determine the mechanisms underlying these complex functions will require identification of the specific molecules and domains responsible for receptor trafficking. As deletion of BLNK impairs B cell development (53, 54, 55), studies in deficient mice are likely to be unrevealing.

Our data provide a model in which trafficking to late endosomes proceeds through a series of sequential checkpoints, each regulated by one or more signaling molecules bound to the Ig cytoplasmic tail (Fig. 8). The demonstration of sequential sorting and entry steps in the delivery of BCR complexes to late endosomes is reminiscent of endocytic trafficking mechanisms defined in yeast (56, 57, 58). Endocytosed yeast membrane proteins are sorted in multivesicular bodies for transport to specific subcellular compartments (59). Sorting is determined by specific motifs present in the cytoplasmic tails of endocytosed proteins (60, 61). To enter a compartment, transport vesicles must first tether, then fuse to the limiting membranes of the targeted vesicles. Rab proteins probably mediate tethering (62, 63, 64), while fusion requires the interaction of vesicle soluble N-ethylmalemide-sensitive factor attachment protein receptors (SNAREs) on the donor compartment with target SNAREs on the target or acceptor membranes (65). Mammalian structural and functional homologues of both the Rab and SNARE yeast proteins have been characterized in several cell types, including B lymphocytes (62, 56), indicating that the general mechanisms of endocytic trafficking are widely conserved.

View larger version (35K): [in this window] [in a new window]   FIGURE 8. Proposed model of BCR facilitated Ag processing, in which BCR trafficking to late endosomes proceeds through discrete sorting and entry checkpoints. Sorting through early endosomes (EE) requires Ig and the ITAM tyrosines of Ig, which serve to recruit Syk. Subsequent entry into late endosomes requires the phosphorylation of Ig Y204 and the recruitment of BLNK. The direct coupling of receptor signaling to trafficking ensures that polyvalent Ags are preferentially processed for presentation to T cells.

	Acknowledgments

 
The ID4B Ab was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences. We thank Andrea Sant for many helpful discussions.

	Footnotes

 
¹ K.S. is supported by a postdoctoral fellowship from the Cancer Research Institute. M.R.C. is supported by National Institutes of Health Grants GM56187 and GM52736.

² Address correspondence and reprint requests to Dr. Marcus R. Clark, Department of Medicine, University of Chicago, 5841 South Maryland Avenue, MC0930, Chicago, IL 60637. E-mail address: mclark{at}medicine.bsd.uchicago.edu

Received for publication August 21, 2001. Accepted for publication December 21, 2001.

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