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The Journal of Immunology, 1999, 162: 2732-2740.
Copyright © 1999 by The American Association of Immunologists

Regulation of B Cell Receptor-Mediated MHC Class II Antigen Processing by Fc{gamma}RIIB11

Neelam M. Wagle, Anne E. Faassen, Julie H. Kim and Susan K. Pierce2

Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The processing and presentation of Ag by Ag-specific B cells is highly efficient due to the dual function of the B cell Ag receptor (BCR) in both signaling for enhanced processing and endocytosing bound Ag. The BCR for IgG (Fc{gamma}RIIB1) is a potent negative coreceptor of the BCR that blocks Ag-induced B cell proliferation. Here we investigate the influence of the Fc{gamma}RIIB1 on BCR-mediated Ag processing and show that coligating the Fc{gamma}RIIB1 and the BCR negatively regulates both BCR signaling for enhanced Ag processing and BCR-mediated Ag internalization. Treatment of splenic B cells with F(ab')2 anti-Ig significantly enhances APC function compared with the effect of whole anti-Ig; however, whole anti-Ig treatment is effective when binding to the Fc{gamma}RIIB1 was blocked by a Fc{gamma}RII-specific mAb. Processing and presentation of Ag covalently coupled to anti-Ig were significantly decreased compared with Ag coupled to F(ab')2anti-Ig; however, the processing of the two Ag-Ab conjugates was similar in cells that did not express Fc{gamma}RIIB1 and in splenic B cells treated with a Fc{gamma}RII-specific mAb to block Fc binding. Internalization of monovalent Ag by B cells was reduced in the presence of whole anti-Ig as compared with F(ab')2 anti-Ig, but the internalized Ag was correctly targeted to the class II peptide loading compartment. Taken together, these results indicate that the Fc{gamma}RIIB1 is a negative regulator of the BCR-mediated Ag-processing function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Ag-dependent activation of B lymphocytes to proliferate and differentiate into Ab-producing plasma cells requires the collaborative interaction of B cells with Ag-specific Th cells 1 . The interaction of B and T cells is dependent on the B cell’s processing and presentation of Ag with the MHC class II molecules. In the absence of specific foreign Ags, assembly of peptide-class II complexes occurs constitutively in B cells; however, binding of specific Ag to B cells leads to a concerted effort to fill class II molecules with peptides derived from the Ag bound to the BCR.3 BCR-mediated processing differs from the constitutive processing of Ag in at least two ways. The BCR serves to concentrate and target Ag to the subcellular endocytic compartments in which the actual processing occurs (reviewed in Refs. 2 and 3), and cross-linking the BCR signals for enhanced Ag processing (reviewed in 4 .

The transport function of the BCR was first suggested by the greatly increased efficiency with which B cells processed Ag bound to the BCR compared with fluid phase pinocytosis. From studies showing that Ag-specific B cells maximally presented Ag at 1/103 to 1/104 the concentration of Ag required by nonspecific B cells 5, 6, 7 it was concluded that the BCR endocytosed Ag and delivered it to the intracellular sites of Ag processing. The isolation and characterization of the subcellular compartments in which peptide-class II complexes are formed made it possible to directly demonstrate the trafficking of the BCR and bound Ag from the plasma membrane to the peptide loading compartments 8, 9, 10 . Recent results indicate that the transport and signaling functions of the BCR are interrelated and that signaling through the BCR influences the Ag transport function of the BCR.

In addition to its Ag transport function, we previously showed that the BCR also provides signals that result in enhanced APC function 4 . The effect of the BCR signaling appeared to be on the Ag processing and presentation function of the B cell rather than on the costimulatory function because the Ag-specific T cell hybrid used to assess Ag presentation was demonstrated not to be dependent on either B7.1 (CD80) or B7.2 (CD86) costimulatory function for its activation or sensitive to up-regulation of costimulatory function in B cells 11 . The relationship between the BCR-initiated signals that influence Ag processing and those required to induce proliferation is not known, although cell proliferation is not required for enhanced APC function.

The signals initiated by cross-linking the BCR affect several aspects of B cell Ag processing. Early studies showed that cross-linking the BCR enhanced both the processing of Ag taken up by fluid phase pinocytosis as well as the presentation of a peptide that does not require processing 12 . Thus, BCR signaling appears to function in part to influence processing independently of Ag internalization. Subsequently, we showed the BCR signaling also affects the targeting of the BCR and Ag to the peptide loading compartments. Although the BCR constitutively transports Ag from the plasma membrane through endosomes to the class II peptide loading compartment, the cross-linking of the BCR increased the rate of internalization and accelerated the intracellular targeting of the BCR to the peptide loading compartment without altering the transport pathway 9 . The observed affect of BCR cross-linking on its transport, but not internalization, was the result of signaling through the BCR rather than simple physical aggregation of the BCR, as shown by the ability of kinase inhibitors that blocked BCR signaling to prevent the accelerated transport of cross-linked BCR and bound Ag 13 . The molecular mechanisms by which BCR signaling influenced BCR-mediated Ag processing have not been completely delineated. However, we showed that BCR signaling at the plasma membranes results in rapid and transient biochemical changes in the class II peptide loading compartment, including changes in the patterns of the associated phosphoproteins and small m.w. GTPases that play a role in intracellular transport 14 . We speculate that these biochemical changes may be important in mediating the effect of BCR signaling. At present, the relationship between signaling for enhanced APC function and signaling for accelerated BCR transport remains to be elucidated.

The B cell receptor for IgG (Fc{gamma}RIIB1) has been shown to be a negative coreceptor of the BCR (reviewed in 15, 16). The Fc{gamma}RIIB1 binds to immune complexes and modulates B cell activation triggered by the BCR, resulting in inhibition of Ab responses. Presumably this pathway serves as a feedback mechanism to regulate B cell activation when sufficient Ab has been produced to result in immune complex formation. Immune complexes or whole Ig-specific Abs which simultaneously cross-link the surface Fc{gamma}RIIB1 with the BCR inhibit the activation of splenic B cells to proliferate 17 . The inhibition of B cell activation is reversed in the presence of anti-Fc{gamma}RII Abs which prevent the binding of Fc portion of Ig-specific Abs to Fc{gamma}RIIB1. Coligation of the Fc{gamma}RIIB1 aborts B cell activation triggered through cross-linking the BCR by a mechanism which requires a 13 amino acid sequence in the cytoplasmic domain of the Fc{gamma}RIIB1 named ITIM for immunoreceptor tyrosine-based inhibition motif 18 . Coligation of Fc{gamma}RIIB1 with the BCR results in premature termination of phosphoinositide hydrolysis and Ca2+ mobilization and inhibits B cell proliferation 15, 16 . Although the precise mechanism by which the BCR signaling pathway is blocked is not completely understood, current evidence suggests that the ITIM of the Fc{gamma}RIIB1, once phosphorylated, functions to recruit SH2-containing phosphotyrosine phosphatases to the BCR 19 . Very recent evidence indicates that Fc{gamma}RIIB1 inhibition of BCR-mediated B cell activation is integrated by the dephosphorylation of CD19 leading to inactivation of PI 3 kinase activity 20 .

Here we investigate the effect of the Fc{gamma}RIIB1 on B cell APC function. We provide evidence that coligating of the BCR and Fc{gamma}RIIB1 by whole anti-Ig regulates both signaling through the BCR for enhanced processing activity as well as the BCR-mediated internalization of Ag. These results indicate that in addition to its effect on BCR signaling for B cell proliferation, the Fc{gamma}RIIB1 modulates BCR activation by regulating Ag presentation to Th cells.


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

Adult CBA/J mice, 6–8 wk old, were purchased from The Jackson Laboratory (Bar Harbor, ME).

Ags, Abs, and cell lines

Pigeon cytochrome c (Pc) and conjugates of Pc covalently coupled to whole rabbit Abs specific for the heavy and light chains of mouse IgM and IgG (Pc-anti-Ig) and F(ab')2 fragments of rabbit Abs specific for heavy and light chains of mouse Ig (Pc-F(ab')2anti-Ig) were prepared as previously described 21, 22 . The molar ratios of Pc to Ab for both the Pc-anti-Ig and Pc-F(ab')2anti-Ig conjugates were approximately 2.6:1. Rabbit Abs specific for mouse IgM(anti-Ig) and F(ab')2 and Fab fragments of goat Abs specific for mouse IgM (F(ab')2anti-Ig and (Fab)anti-Ig, respectively) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). The Fc{gamma}RII-specific mAb 2.4G2 23 , class II Ek-specific mAb 17.3.3s 24 , and anti-Thy 1 mAb were purified from culture supernatants of hybridomas obtained through the American Type Culture Collection (Manassas, VA). B7.1(CD80)- and B7.2(CD86)-specific mAbs were purchased from PharMingen (San Diego, CA).

CH27 is an IgM+ Fc{gamma}R- B cell lymphoma 25 . The mouse T cell hybridoma TPc9.1, generated in this laboratory, is specific for Pc presented by Ek-expressing APC and cross-reacts with tobacco hornworm moth cytochrome c (THMc) 26 . TPc9.1 secretes IL-2 upon activation. The CTLL-2 cell line is an IL-2-dependent cell line and was obtained from American Type Culture Collection 27 .

APC assay

B cells were prepared from RBC-depleted spleen cell suspensions as previously described 12 by treatment with anti-Thy-1 mAbs and guinea pig complement (Life Technologies, Grand Island, NY). B cells or CH27 cells were cocultured with TPc9.1 cells (5 x 104) at 37°C for 24 h as described previously 11 . When indicated, B cells were fixed (5 x 106/ml) by treatment in 0.15% paraformaldehyde (Sigma, St. Louis, MO) as previously described 12 or were treated with mitomycin C (Sigma) for 20 min before addition of TPc9.1 cells. The IL-2 content of the culture supernatant was determined by the ability to maintain the growth of the IL-2-dependent CTLL cell line as measured by [3H]TdR incorporation.

Measurement of BCR-mediated internalization

(Fab)anti-Ig was labeled with 125I as previously described 9, 28 . Briefly (Fab)anti-Ig (25 µg) was incubated for 10 min at 4°C with 1 mCi of [125I]Na in 1 M Tris containing 0.33 M NaCl and 0.11 µM ICl. The reaction was quenched with an equal volume of 5 mg/ml NaI in PBS, and the iodinated protein was chromatographed on a Sephadex G-25 column (Isolab, Akron, OH) with PBS containing 1% BSA and 5 mg/ml NaI. Splenic B cells were assayed for the internalization and degradation of 125I-labeled (Fab)anti-Ig as previously described 9 . Briefly, splenic B cells (2.4 x 108/ml) were washed in DMEM containing 6 mg/ml BSA and 20 mM MOPS and incubated at 4°C for 1 h with 125I-labeled (Fab)anti-Ig (1 µg/ml) in the presence or the absence of whole anti-Ig or F(ab')2anti-Ig (10 µg/ml) or Fc{gamma}RII-specific mAb 2.4G2 (10 µg/ml). Cells were washed in DMEM containing 6 mg/ml BSA and 20 mM MOPS, resuspended at 5 x 106 cells/ml, and incubated at 37°C for increasing lengths of time. After incubation, cells were pelleted, and the radioactivity present in the supernatant (released fraction), that removed from the cell by acid treatment (surface fraction), and the remaining cell-associated radioactivity after acid treatment (internal fraction) were quantified by gamma counting (Micromedic Systems, Horsham, PA) as detailed previously 9 .

DAB chemical cross-linking and MHC class II biosynthesis

CH27 cells were metabolically labeled with [35S]Met by incubation in Met-free DMEM containing 5% dialyzed FCS for 30 min at 37°C, then pulsed for 15 min with [35S]Met (100 µCi/ml) at 37°C. During the [35S]Met pulse, horseradish peroxidase (HRP)-coupled to anti-Ig (HRP-anti-Ig; 20 µg/ml; Vector Laboratories, Burlingame, CA) was added to the culture medium. After washing, the cells were incubated in 15% culture medium at 37°C for varying lengths of chase time. The diaminobenzidine (DAB) chemical cross-linking assay was conducted as previously detailed 9 . Briefly, at the end of each chase point, ice-cold HBSS was added to the cells. The cells were washed in HBSS and resuspended in DAB reaction buffer (0.5 mg/ml DAB and 0.003% H2O2 in HBSS). The cells were incubated at 4°C for 45 min in the dark. In the control reaction, H2O2 was omitted from the DAB reaction buffer. In the presence of H2O2 and HRP, DAB cross-links proteins to form large insoluble polymers that can be removed from solution by centrifugation 29 . The cells were washed in HBSS and lysed with the Nonidet P-40 lysis buffer, and cross-linked proteins were removed from the lysate by centrifugation at 100,000 x g for 30 min at 4°C. The radiolabeled class II Ek molecules from the lysate supernatant were immunoprecipitated as described using the Ek-specific mAb 17.3.3s 30 and were analyzed by SDS-PAGE without reducing or boiling the samples.

Flow cytometry

B cells (1 x 106 cells/50 µl) were incubated for 45 min on ice in PBS containing 5% FCS and 0.02% NaN3 containing FITC-labeled 17.3.3s mAb specific for I-Ek. Cells were washed in cold PBS-FACS and fixed in 1% formaldehyde. The cells were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Coligation of the BCR and the Fc{gamma}RIIB1 down-regulates BCR-mediated signaling for enhanced Ag processing and presentation

Previous studies have shown that whole anti-Ig is a poor inducer of B cell signaling for proliferation due to its ability to coligate the Fc{gamma}RIIB1 and BCR 17 . The addition of Fc{gamma}RII-specific Abs blocks Fc binding to the Fc{gamma}RIIB1, resulting in augmented B cell proliferation. As shown, whole anti-Ig and anti-Fc{gamma}RII have the predicted effect on induction of B cell proliferation. B cells proliferate in response to anti-Ig in a dose-dependent fashion as measured after 72 h in culture, and the response to anti-Ig is greatly increased in the presence of anti-Fc{gamma}RII (Fig. 1Go). The time course of the response shows that significant proliferation is not measurable before 24 h of culture (Fig. 1Go).



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  		FIGURE 1. Fc{gamma}RIIB1 down-regulates BCR-induced B cell proliferation. Right panel, Mouse splenic B cells were incubated with graded concentrations of whole anti-Ig in the presence or the absence of the Fc{gamma}RII-specific mAb (2.4G2) (10 µg/ml) for 72 h at 37°C. The cells were pulsed with [3H]TdR and were harvested 6 h later. Left panel, B cells were incubated with whole anti-Ig (5 µg/ml) in the presence or the absence of the Fc{gamma}RII-specific mAb 2.4G2 (10 µg/ml) at 37°C for increasing lengths of time. At the end of each time period the cells were pulsed with [3H]TdR and harvested 6 h later. The proliferative responses were expressed as counts per minute incorporated by 2 x 105 B cells.

 
To determine the effect of coligation of the Fc{gamma}RIIB1 and BCR on BCR signaling for enhanced Ag presentation, B cells were tested for their ability to activate a Pc-specific T cell hybrid, TPc9.1, to secrete IL-2 in cultures containing Pc and whole anti-Ig, whole anti-Ig plus the Fc{gamma}RII-specific mAb 2.4G2, or F(ab')2 anti-Ig. Treatment with whole anti-Ig resulted in a small increase in the ability of B cells to process and present Pc (Fig. 2GoA). Addition of the anti-Fc{gamma}RII mAb to anti-Ig resulted in enhanced processing equivalent to that of B cells treated with F(ab')2 anti-Ig. At a constant concentration of anti-Ig, the effect of the Fc{gamma}RII-specific mAb on Ag-processing activity was dose dependent (Fig. 2GoB). Similarly, at a constant concentration of the Fc{gamma}RII-specific mAb, the effect of the anti-Ig on Ag processing was dose dependent (Fig. 2GoC).



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  		FIGURE 2. Fc{gamma}RIIB1 down-regulates BCR-induced augmentation of APC function. A, Graded concentrations of Pc were added to cultures containing splenic B cells (2 x 105) and TPc9.1 cells in the presence of anti-Ig (5 µg/ml), anti-Ig (5 µg/ml) plus anti-Fc{gamma}R (10 µg/ml), or F(ab')2anti-Ig (5 µg/ml). B, Splenic B cells (2 x 105) and TPc9.1 cells (5 x 104) were cultured with Pc (2 µM) and increasing concentrations of anti-Fc{gamma}R with or without anti-Ig (1.25 µg/ml). C, Splenic B cells (2 x 105) and TPc9.1 cells (5 x 104) were cultured with Pc (2 µM) and increasing concentrations of anti-Ig alone or in the presence of anti-Fc{gamma}RII (5 µg/ml) or an isotype-matched control mAb (5 µg/ml). D, B cells were treated with mitomycin C for 20 min or were not treated (E), then washed and cultured with TPc9.1 cells and increasing concentrations of Pc in the presence of anti-Ig or anti-Ig plus anti-Fc{gamma}R. In all cases culture supernatants were tested for their IL-2 content after 24 h as described in Materials and Methods, and the results are expressed as counts per minute of [3H]TdR incorporated by IL-2-dependent CTLL-2 cells.

 
To determine whether the effect of anti-Fc{gamma}RII on anti-Ig-induced enhancement of Ag processing was due to the effect of the anti-Fc{gamma}RII on anti-Ig-induced proliferation, B cells were treated with mitomycin C to block proliferation and were tested for their ability to process and present Pc. The effects of anti-Ig and anti-Fc{gamma}RII on the processing and presentation of mitomycin C-treated and untreated cells were similar (Fig. 2Go, D and E), indicating that the effect of anti-Fc{gamma}RII on anti-Ig enhancement of Ag processing is not attributable to the effect of anti-Fc{gamma}RII on anti-Ig-induced proliferation. In addition, the enhancement of Ag processing did not depend on B7.1 (CD80) and B7.2 (CD86), as addition of Abs to B7.1 and B7.2 had no effect (data not shown).

We earlier showed that treatment of B cells with F(ab')2anti-Ig resulted in enhanced presentation of peptides that did not require processing 12 . To determine whether this effect of BCR cross-linking was also regulated by the Fc{gamma}RIIB1, B cells were incubated with either anti-Ig alone or with anti-Ig and anti-Fc{gamma}RII mAb for 4, 6, and 18 h; washed; fixed; and tested for their ability to present the peptide THMc81–103 to TPc9.1 cells. Presentation of the peptide THMc81–103 was significantly more pronounced in B cells treated with both anti-Fc{gamma}R and anti-Ig than in B cells treated with anti-Ig alone (Fig. 3GoA). The effect of BCR cross-linking on peptide presentation was first observed after 6 h of treatment with anti-Ig in the presence of anti-Fc{gamma}R and was clearly apparent after 18 h of treatment. At 18 h there was no significant difference in the expression of the class II I-Ek molecules on the B cells that were untreated or treated with anti-Ig alone or anti-Ig plus anti-Fc{gamma}R (Fig. 3GoB). In addition, the enhanced presentation ability of the anti-Ig- plus anti-Fc{gamma}R-treated cells did not depend on proliferation, as similar results were obtained using B cells treated with mitomycin C to block proliferation (data not shown). Taken together, these results indicate that the coligation of the Fc{gamma}RIIB1 and the BCR, which blocks the B cell signaling required for proliferation, blocks the B cell signaling required for enhanced Ag processing and presentation.



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  		FIGURE 3. Fc{gamma}RIIB1 down-regulates BCR-induced enhancement of peptide presentation but does not affect class II expression. A, Splenic B cells were cultured for 4, 6, or 18 h at 37°C in medium alone or in medium containing anti-Ig (5 µg/ml) or anti-Ig (5 µg/ml) plus anti-Fc{gamma}R (10 µg/ml). The cells were washed and fixed with 0.15% paraformaldehyde and tested for their ability to present the peptide THMc81–103 to a T cell hybrid by coculturing the B cells with TPc9.1 cells (5 x 104) for 24 h in the presence of graded concentrations of the peptide, THMc81–103. The IL-2 content of culture supernatants was measured 24 h later, and the results are expressed as counts per minute of [3H]TdR incorporated by the IL-2-dependent CTLL-2 cells. B, Splenic B cells cultured for 18 h as described in A were stained with a fluorescently labeled I-Ek-specific mAb and analyzed by flow cytometry.

 
BCR-mediated Ag processing is down-regulated by Fc{gamma}RIIB1

To determine whether engagement of the Fc{gamma}RIIB1 and BCR affects the processing of BCR-bound Ag, B cells were tested for their ability to process and present conjugates of Pc covalently coupled to whole anti-Ig (Pc-anti-Ig) or to F(ab')2anti-Ig (Pc-F(ab')2anti-Ig) to the Pc-specific T cell hybrid TPc9.1. The molar ratio of Pc to anti-Ig was equivalent in these two conjugates (2.6:1), and the conjugates showed equivalent binding to splenic B cells (data not shown). B cells presented both Pc-anti-Ig and Pc-F(ab')2anti-Ig at 1/100th the Pc concentrations required for processing of Pc alone (Fig. 4Go). However, maximal presentation of the Pc-F(ab')2anti-Ig conjugate was greater than that of Pc-anti-Ig by approximately 30%, suggesting that the presence of the Fc fragment in the Pc-anti-Ig conjugates had a negative effect on BCR-mediated Ag processing.



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  		FIGURE 4. Maximal presentation of Pc-anti-Ig is reduced compared with that of Pc-F(ab')2 anti-Ig. Graded concentrations of Pc-anti-Ig, Pc-F(ab')2anti-Ig, or Pc were added to cultures containing splenic B cells (2 x 105) and TPc9.1 cells (5 x 104) and incubated at 37°C for 24 h. Culture supernatants were tested for their IL-2 content, and the results are expressed as counts per minute of [3H]TdR incorporated by CTLL-2 cells.

 
To further characterize the effect of the Fc{gamma}RIIB1 on processing of the Pc conjugates, the time course of Ag processing was investigated. Our previous results showed that BCR signaling results in the accelerated targeting of the BCR to the peptide loading compartment 9 ; thus, it was of interest to determine whether coligating the Fc{gamma}RIIB1 and the BCR affected the time course of Ag processing and presentation. Splenic B cells were cultured with Pc-anti-Ig or Pc-F(ab')2anti-Ig for 4 or 6 h, washed, fixed, and tested for their ability to stimulate TPc9.1 cells. B cells processed and presented Pc-F(ab')2anti-Ig significantly earlier than Pc-anti-Ig (Fig. 5Go). After 4-h incubation with the Pc-F(ab')2anti-Ig, splenic B cells were stimulatory to the TPc9.1 cells, while the presentation by B cells incubated with Pc-anti-Ig was barely measurable (Fig. 5Go). The processing and presentation of Pc- F(ab')2anti-Ig increased by 6 h, remaining greater than that of Pc-anti-Ig. The difference in the processing of the two conjugates could be attributed to the ability of Pc-anti-Ig to engage the Fc{gamma}RIIB1 rather than to an inherent difference in the two Pc-Ab conjugates, because the B cell lymphoma CH27, which does not express the Fc{gamma}RIIB1, processed Pc-anti-Ig and Pc-F(ab')2anti-Ig nearly equivalently (Fig. 5Go).



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  		FIGURE 5. The time dependency of processing and presentation of Pc-anti-Ig and Pc-F(ab')2anti-Ig. Splenic B cells (left panels) or CH27 cells (right panels) were cultured with Pc-anti-Ig or Pc-F(ab')2anti-Ig for 4 or 6 h, washed, and fixed with 0.15% paraformaldehyde. Graded numbers of fixed cells were incubated with TPc9.1 cells (5 x 104) at 37°C, and the IL-2 content of the culture supernatant was measured 24 h later. Results are expressed as counts per minute of [3H]TdR incorporated by CTLL-2 cells.

 
To further examine whether the reduced processing and presentation of Pc-anti-Ig relative to Pc-F(ab')2anti-Ig was due to the binding of Fc portion of Ig in the Pc-anti-Ig conjugate to the Fc{gamma}RIIB1, B cells were tested for their ability to process and present Pc-anti-Ig and Pc-F(ab')2anti-Ig in the presence of the Fc{gamma}RII-specific mAb 2.4G2, which blocks Fc-Fc{gamma}RIIB1 interactions. The addition of the 2.4G2 mAb resulted in a significant increase in the processing and presentation of Pc-anti-Ig (Fig. 6Go), while the addition of an isotype-matched control mAb had no effect (data not shown). The anti-Fc{gamma}RII mAb did not completely restore the processing of Pc-anti-Ig to the levels of Pc-F(ab')2 anti-Ig. The interaction of the Fc portion of Pc-anti-Ig with the Fc{gamma}RIIB1 may be difficult to block completely during the entire time course of the processing experiment. The Pc-anti-Ig may also have a competitive advantage in binding to the Fc{gamma}RIIB1 compared with the 2.4G2 mAb, as the conjugate is concentrated on the plasma membrane by its binding to the BCR, while the 2.4G2 mAb is free in solution. Taken together, these findings indicate that coligation of the Fc{gamma}RIIB1 and BCR results in a reduced processing of BCR-bound Ag.



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  		FIGURE 6. Blocking Fc{gamma}RIIB1/Fc interactions augments processing of Pc-anti-Ig. Splenic B cells were cultured with Pc-anti-Ig or Pc-F(ab')2anti-Ig for 6 h in the presence or the absence of anti-Fc{gamma}R (10 µg/ml). The cells were washed and fixed with 0.15% paraformaldehyde. Graded numbers of fixed cells were incubated with TPc9.1 cells (5 x 104), and the IL-2 contents of the culture supernatants were measured 24 h later. Results are expressed as counts per minute of [3H]TdR incorporated by CTLL-2 cells.

 
Anti-Ig and F(ab')2anti-Ig induce different patterns of BCR internalization

The initial steps in BCR-mediated Ag processing involve the binding of Ag to the BCR and subsequent internalization of the BCR and bound Ag. We earlier showed that the BCR is constitutively internalized and trafficks to the class II peptide loading compartment, and that cross-linking the BCR results in an increase in the rate of internalization and transport to this compartment 9 . The accelerating trafficking, but not internalization, was dependent on BCR signaling 13 . To determine the effect of Fc{gamma}R-BCR coligation on BCR internalization, the internalization of monovalent 125I-labeled (Fab)anti-Ig was investigated after cross-linking with either whole anti-Ig or F(ab')2anti-Ig. Splenic B cells were incubated with either 125I-labeled Fab-anti-Ig alone or in the presence of whole anti-Ig or F(ab')2anti-Ig for 30 min at 4°C, washed, and warmed to 37°C for increasing periods of time. The radioactivity released from the cells into the supernatant (release), on the cell (surface), or inside the cell (internal) was measured (Fig. 7Go). Upon warming the 125I-labeled F(ab)anti-Ig alone was internalized poorly, representing the constitutive level of internalization in the absence of BCR cross-linking with whole anti-Ig increased internalization, and cross-linking with F(ab')2anti-Ig resulted in still greater internalization of 125I-labeled F(ab)anti-Ig. Treatment with both whole anti-Ig and Fc{gamma}RII-specific mAb 2.4G2 together increased the rate and amount of internalization of 125I-labeled F(ab')anti-Ig to that achieved by cross-linking with F(ab')2anti-Ig. A similar pattern was observed monitoring the percent of the 125I-labeled F(ab)anti-Ig that remained on the surface after warming. Taken together, these results indicate that coligation of the Fc{gamma}RIIB1 and BCR decreases internalization of the BCR and bound Ag.



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  		FIGURE 7. Blocking Fc{gamma}RIIB1/Fc interactions increases anti-Ig-induced internalization of the BCR. Splenic B cells were assayed for the internalization of 125I-labeled F(ab)anti-Ig as described in Materials and Methods. Briefly, splenic B cells (2.4 x 108 cells/ml) were incubated at 4°C for 1 h with 125I-labeled F(ab)anti-Ig (1 µg/ml) alone or in the presence of whole anti-Ig (10 µg/ml), whole anti-Ig (10 µg/ml) plus anti-Fc{gamma}R (10 µg/ml), F(ab')2anti-Ig (10 µg/ml), or F(ab')2anti-Ig (10 µg/ml) plus anti-Fc{gamma}R (10 µg/ml). Cells were washed and incubated (1 x 107 cells/ml) at 37°C for the times indicated. The radioactivity in the released, cell surface, and internal fractions was measured. The data are reported as a percentage of the total counts per minute per time point for duplicate samples representative of two experiments.

 
Coligation of the Fc{gamma}RIIB1 does not result in mistargeting of internalized BCR

To determine the effects of coligation of the Fc{gamma}RIIB1 and BCR on transport of the internalized BCR to the class II peptide loading compartment, a nondisruptive protein cross-linking method was employed. In this method, splenic B cells were allowed to internalize HRP-anti-Ig. In the presence of the membrane-permeable cross-linking reagent DAB and H2O2, HRP catalyzes the cross-linking of proteins, resulting in large insoluble polymers. Thus, the proteins in any subcellular compartment into which HRP-anti-Ig has entered will be cross-linked following the addition of DAB and H2O2. Using this method, we previously showed that 30 min after internalization, HRP-anti-Ig bound to the BCR enters the dense subcellular fractions that contain the class II peptide loading compartment 9 . Splenic B cells were pulsed with [35S]Met for 15 min during incubation with HRP-anti-Ig in the presence and the absence of the anti-Fc{gamma}RII mAb 2.4G2. Cells were washed and chased in complete medium without HRP-anti-Ig for varying periods of time up to 3 h. At the end of each time point, B cells were washed and incubated in a DAB reaction buffer containing DAB and H2O2 at 4°C to allow DAB-mediated cross-linking in HRP-containing compartments. In control reactions, H2O2 was omitted from the DAB reaction buffer. After the DAB reaction, the cells were lysed, cross-linked proteins were removed by centrifugation, and soluble class II molecules were immunoprecipitated from the lysate. The immunoprecipitates were analyzed by SDS-PAGE without reducing or boiling the samples, conditions under which peptide-bound heterodimers of class II molecules are stable. In B cells treated with anti-Fc{gamma}R SDS-stable class II dimers were first detected after approximately 1–2 h of chase and continued to accumulate through 3 h (Fig. 8Go, A and B). The stable dimers were cross-linked by HRP in the presence of H2O2 and removed from the sample for the first 1–2 h of chase and by 3 h were less accessible to HRP, presumably having exited the peptide loading compartment and trafficked to the plasma membrane. This observation was similar to our previously published result obtained using Fc{gamma}R-negative CH27 cells 9 . In the absence of the anti-Fc{gamma}RII mAb (Fig. 8Go, A and B) the pattern was similar, in that SDS-stable class II molecules that appear between 1 and 3 h of chase were cross-linked by HRP in the presence of H2O2. Thus, in the presence or the absence of Fc{gamma}RIIB1 coligation to the BCR, the internalized BCR was correctly transported to the class II peptide loading compartment. However, there were differences between the anti-Fc{gamma}RII-treated and untreated cells. Firstly, fewer SDS-stable class II molecules formed in cells in which Fc{gamma}RIIB1 was coligated to the BCR even though total class II biosynthesis was not affected. Secondly, the SDS-stable class II molecules in cells in which Fc{gamma}RIIB1 was coligated to the BCR remained sensitive to HRP cross-linking for a shorter period of time.



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  		FIGURE 8. Effect of Fc{gamma}RIIB1 on trafficking of the BCR to the IIPLC. Splenic B cells (1.6 x 108) were incubated with [35S]Met (100 µCi/ml) in Met- media at 37°C for 15 min in the presence of HRP-anti-Ig alone (20 µg/ml) or with anti-Fc{gamma}R (10 µg/ml), washed, and incubated in complete medium at 37°C for various chase times. At the end of each chase time, cells were washed and incubated in DAB reaction buffer containing DAB and H2O2. In control experiments, H2O2 was omitted. Cells were lysed and centrifuged to remove cross-linked protein polymers, and soluble class II molecules were immunoprecipitated from the supernatant. The immunoprecipitates were analyzed by SDS-PAGE without boiling or reducing the sample, conditions under which peptide filled class II dimers are stable. Radiolabeled proteins were detected by fluorography. A, A representative autoradiograph is shown. B, The region of gel containing the 60-kDa dimers was scanned by densitometry, and the results of two independent experiments are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been well established that the Fc{gamma}RIIB1 expressed by B cells functions as a negative coreceptor of the BCR (reviewed in Refs. 15 and 16). Passively transferred Ab and Ag Ab complexes had been shown previously to inhibit Ab responses in vivo 31 . Recent studies showing enhanced Ab responses in Fc{gamma}RII-deficient mice 32 support a role for Fc{gamma}RIIB1 in the negative feedback regulation of Ab responses in vivo. A highly conserved 13-amino acid ITIM sequence in the cytoplasmic domain of the Fc{gamma}RIIB was shown to be both necessary and sufficient to inhibit BCR-induced B cell activation 18 . Although the precise step in the B cell signaling cascade at which the coligated Fc{gamma}RIIB1 aborts the BCR signal is not completely delineated, the Fc{gamma}RIIB1 has been shown to block BCR-induced phosphoinositide hydrolysis and calcium mobilization 33, 34 , indicating a block before inositol ligand hydrolysis in the BCR signaling cascade. Phosphorylation of the tyrosine within the ITIM of the Fc{gamma}RIIB1 results in binding of SH2-containing tyrosine phosphatases 19 , suggesting that Fc{gamma}RIIB1 may function as a negative coreceptor by recruiting phosphatases to the BCR, reducing the magnitude of BCR signaling. Recent studies indicated that phosphorylated CD19 may be an important substrate for these phosphatases 20 .

Here we investigated whether the Fc{gamma}RIIB1 when coligated to the BCR influences BCR-mediated Ag processing. Our previously published results provided evidence for two possibly overlapping functions for the BCR in Ag processing. The first was to signal for enhanced Ag processing 12 and for accelerated transport of the BCR to the peptide loading compartment 9 ; the second was to transport Ag to the peptide loading compartment (reviewed in 3 . Cross-linking the BCR results in signals that augment the processing and presentation of Ag taken up by fluid phase pinocytosis and the presentation of peptides that do not require processing 12 . Enhanced processing following BCR cross-linking was shown to be dependent on kinase activity 13, 14 ; however, the precise step in the BCR signaling pathway required to induce enhanced processing remains to be elucidated. The results presented here show that the coligation of the Fc{gamma}RIIB1 with the BCR blocks BCR-induced enhancement of APC function. Our previous results showed that BCR cross-linking also directly influenced BCR internalization, accelerating the rate of internalization of the BCR from the plasma membrane and increasing the amount of BCR internalized 9 . The enhanced internalization following BCR cross-linking was not dependent on signaling as shown by the inability of kinase inhibitors, which block BCR signaling, to block enhanced internalization 13 . Here we show that coligation of the Fc{gamma}RIIB1 and BCR reduced both the rate of internalization and the maximal amount of BCR internalized. Because accelerated internalization did not require signaling, this effect of the Fc{gamma}RIIB1 is most likely attributed to the physical retention of the BCR on the cell surface by the Fc{gamma}RIIB1. The Fc{gamma}RIIB1 is not an internalizing receptor, does not cluster in coated pits, and has an enhanced association with the cytoskeleton that restricts its movement to the plasma membrane 35, 36 . Thus, any BCR that becomes coligated to the Fc{gamma}RIIB1 may be retained on the cell surface, blocking its ability to transport Ag to the peptide loading compartment. Treatment of B cells with whole anti-Ig did not completely block internalization of 125I-labeled F(ab)anti-Ig, suggesting that coligation of the BCR and Fc{gamma}RIIB1 was incomplete, permitting unligated BCR to be internalized. In support of this conclusion, while this manuscript was under review, Minskoff et al. 37 published evidence that the Fc{gamma}RIIB1 when coligated to the BCR acts to retain the BCR on the cell surface.

Concerning regulation of transport of the BCR to the peptide loading compartment, we showed here that in cells in which the Fc{gamma}RIIB1 and BCR were coligated, the portion of the BCR that was internalized was correctly targeted to the class II peptide loading compartment. This indicates that the coligation of Fc{gamma}RIIB1 and BCR did not block the BCR-induced signals required for proper targeting to the peptide loading compartment. The coligation of Fc{gamma}RIIB1 and BCR did reduce the effectiveness of BCR to mediate the processing and presentation of Ag-anti-Ig conjugates. Because the enhanced processing of Ag bound to the BCR has been shown in part to be dependent on kinase activity 13 , this effect of Fc{gamma}RIIB1 may be due to its ability to block BCR signaling. In addition, less BCR and Ag were internalized into cells in which Fc{gamma}RIIB1 and BCR were coligated, which would also contribute to the reduced processing of the Ag bound to the BCR.

The results presented here indicated some subtle effects of coligating the Fc{gamma}RIIB1 and BCR on trafficking in the peptide loading compartment. In experiments using a nondisruptive chemical cross-linking method to colocalize the BCR and newly synthesized class II molecules, we observed that newly formed SDS-stable class II molecules remained sensitive to HRP-cross-linking for a longer period of time in cells in which the Fc{gamma}RIIB1 and BCR were unligate compared with coligated cells. This suggests that the class II molecules were retained longer in the peptide loading compartment after binding peptide. Alternatively, it is possible that BCR and bound HRP-anti-Ig were present not only in the peptide loading compartment but also in other endocytic compartments through which the class II molecules traffick to the cell surface.

An understanding of the molecular mechanisms by which coligating the Fc{gamma}RIIB1 and the BCR regulates BCR-mediated Ag processing awaits a more detailed understanding of the components of the BCR that are necessary for correct targeting of the BCR to the peptide loading compartment and which components are necessary to generate signal transduction cascades that influence processing. The BCR is a complex of the membrane Ig and the disulfide-linked heterodimer of Ig{alpha} and Igß subunits that contain the immunoreceptor tyrosine-based activation motifs (reviewed in 38 . Current evidence indicates that the Ig{alpha}/Igß heterodimer is necessary for Ag uptake and presentation (reviewed in 3 . Mutagenesis of the transmembrane region of membrane Ig that allowed surface expression in the absence of Ig{alpha} and Igß abolished all signaling functions and much of the capacity to endocytose Ag 39, 40 . These functions could be restored by the fusion of the cytoplasmic domain of Igß. In other studies a point mutation in the transmembrane domain of Ig abolished the Ag-presenting function of the BCR even though Ag uptake and delivery to processing compartments appeared normal 41 . This finding suggested that the BCR is specifically targeted to subcellular compartments for processing and that these compartments cannot be reached by a default pathway following cross-linking. However, how the Ig{alpha}/Igß heterodimer functions in targeting to the peptide loading compartments and how the transport function is modified by signaling is not known.

A possible biochemical link between Fc{gamma}RIIB1 and the Ag processing pathway was suggested by the recent observation that the effect of Fc{gamma}RIIB1 on BCR signaling was integrated by CD19 dephosphorylation, resulting in decreased phosphoinositol 3-kinase activity 20 . We previously showed that wortmannin, a phosphoinositol 3-kinase inhibitor, blocked Ag processing 42 . Wortmannin treatment was shown to block the formation of SDS-stable class II dimers, and here we observed a reduction in the number of SDS-stable dimers in cells in which the Fc{gamma}RIIB1 was coligated to the BCR. Thus, it is possible that wortmannin and the Fc{gamma}RIIB1 have similar effects on the processing pathway. There are many forms of PI-3 kinase that function in both signal transduction and membrane transport in the endocytic system (reviewed in 43 , and it will be of interest to further explore the effect of Fc{gamma}RIIB1 and BCR coligation on these.

Based on the results presented here, we would place the Fc{gamma}RIIB1 among a group of receptors and conditions that regulate the outcome of Ag processing in B cells. The receptors that have been identified include CD40, which when ligated to CD40 ligand reduces B cell APC function 44 ; CD19, which when cross-linked reduces B cell Ag presentation (our unpublished observation); and the class II molecules themselves, which when cross-linked signal for enhanced processing 11 . The effect on processing appears selective in that signaling through several receptors has no effect on Ag processing, including LPS, CD22, CD45, B7.1, and B7.2 4 . We have also provided evidence that the induction of the universal stress response results in augmented APC function in B cells 45 . The developmental state of the B cell dictated the ability of B cells to process and present Ag in that neonatal B cells that express adult levels of class II molecules and BCR are deficient in Ag processing 46 . Lastly, infection of B cells with influenza virus was shown to block APC function 47 . How this array of factors serves to modulate the B cell APC function remains to be determined and awaits further progress on several fronts, including a more complete understanding of the BCR signaling pathway, an elucidation of the discrete steps in the BCR-mediated Ag processing pathway, and a more detailed picture at the molecular level of the assembly of peptide-class II complexes.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI27957, AI18939, and AI40309 and by fellowships from the Myasthenia Gravis Foundation and the Arthritis Foundation (to N.M.W.). Back

2 Address correspondence and reprint requests to Dr. Susan K. Pierce, Department of Biochemistry, Molecular Biology, and Cell Biology, 2153 N. Campus Dr., Hogan 3-120, Northwestern University, Evanston, IL 60208. E-mail address: Back

3 Abbreviations used in this paper: BCR, B cell Ag receptor; anti-Ig, rabbit Abs specific for mouse Ig heavy and light chains; Pc, pigeon cytochrome c; Pc-anti-Ig, Pc covalently coupled to rabbit Abs specific for mouse Ig heavy and light chains; Pc-F(ab')2anti-Ig, Pc covalently coupled to F(ab')2 of goat Abs specific for mouse Ig; (Fab)anti-Ig, Fab of goat Abs specific for mouse Ig; F(ab')2anti-Ig, F(ab')2 of goat Abs specific for mouse Ig; THMc1–103, peptide fragment of THMc residues 81–103; DAB, diaminobenzidine; HRP, horseradish peroxidase. Back

Received for publication March 10, 1998. Accepted for publication November 30, 1998.


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 Introduction
 Materials and Methods
 Results
 Discussion
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