Cutting Edge: FcγRII-B1 Regulates the Presentation of B Cell Receptor-Bound Antigens¹

Murine B lymphocyte FcγRII-B1 negatively regulates activation of B cells by multiple mechanisms to help maintain clonal specificity and proper feedback control of secreted Abs. First, FcγRII-B1 mediates negative signal transduction events that counteract activation signals triggered by B cell Ag receptor (BCR)⁵ cross-linking (reviewed in 1 . Established effects resulting from cocross-linking of FcγRII-B1 with the surface Ig (sIg) include inhibition of IP3 production (2), calcium influx (3, 4, 5, 6), blast formation (7), and proliferation (8). More recent studies have begun to reveal the mechanism of this FcγRII-B1-mediated inhibition. Cocross-linking of FcγRII-B1 with sIg results in phosphorylation of the tyrosine residue in the FcγRII-B1 immunoreceptor tyrosine-based inhibitory motif (ITIM) (5), followed by recruitment of the protein tyrosine phosphatases SHP-1 and SHP-2 (9, 10) and the inositol phosphatase SHIP (10, 11, 12). Indeed, B cells from motheaten mice, which are defective in SHP-1 (13, 14, 15), exhibit a loss of FcγRII-B1-mediated negative regulation (9, 16). FcγRII-B1 cross-linking with sIg also results in reduced tyrosine phosphorylation of CD19 (17, 18), inhibition of the ras pathway (19, 20), and apoptosis (12, 21, 22). Furthermore, an increase in Ig production occurs in FcγRII-B1 knockout mice (23), consistent with a decrease in the capacity for B cells to be negatively regulated in vivo.

FcγRII-B1 also helps to maintain specificity of B cell activation. The BCR binds a specific Ag via sIg, which is then internalized by endocytosis for processing and ultimately for presentation to T helper cells. In contrast, FcγRII-B1 binds any Ag, as long as it is contained within an IgG-immune complex. The FcγRII-B1-bound Ags are not internalized. As shown in the murine A20 B cell lymphoma, the binding of immune complexes to endogenous FcγRII-B1 fails to result in efficient Ag presentation, instead resulting in the formation of caps of immune complex and FcγRII-B1 on the cell surface (3). In A20 cells transfected with the endocytosis-competent murine macrophage FcγRII-B2, the FcγRII-B2 isoform does internalize and efficiently present Ags bound in IgG-immune complexes (3). Although murine FcγRII-B1 and -B2 are encoded by the same structural gene, the -B1 isoform results from an alternative mRNA-splicing event that places a unique 47-amino acid cytoplasmic tail insertion that lies upstream of the ITIM and inhibits endocytosis by preventing accumulation in clathrin-coated pits (3, 24, 25). FcγRII-B1-mediated negative regulation of B lymphocyte activation is therefore dependent upon multiple sequences contained within the cytoplasmic tail domain.

In this study, we were interested in further elucidating the role of FcγRII-B1 in the regulation of B lymphocyte function. We find that the receptor, by virtue of its ability to block endocytosis when cross-linked to BCR, can act as a dominant-negative inhibitor of BCR-mediated Ag processing. Thus, by inhibiting Ag presentation, FcγRII-B1 can inhibit Ab responses by indirectly inhibiting Ag-dependent T cell stimulation.

The A20 murine B lymphoma cell line, which expresses endogenous FcγRII-B1 and cell surface IgG, and the A6B9 cell line, which expresses stably transfected murine FcγRII-B2 (3), were used for this study. A6B9 cells were derived from transfecting an FcγRII-B1-negative A20 cell line, IIA1.6, that maintains the cell surface IgG. FcγRII-B1 mutants containing a deletion or point mutation in the cytoplasmic tail were prepared by PCR mutagenesis (primers are available upon request) and transfected into IIA1.6 cells as previously described (3). The murine T cell hybridoma 2R.50, which recognizes peptides (specificity unknown) derived from F(ab′)₂ fragments of rabbit Ig in the context of I-A^d (26), was used in the Ag presentation assay.

The Ag presentation assay used was based on a previously published assay (27). A20 or A6B9 cells suspended at 2 × 10⁶ cells/ml in RPMI 1640 complete medium (10% FBS, 50 μM β-mercaptoethanol (Sigma, St. Louis, MO), 1.0 mM sodium pyruvate (Life Technologies, Gaithersburg, MD), 0.1 mM nonessential amino acids (Life Technologies), and 4 mM glutamine (Life Technologies)) were incubated with various concentrations of Ag (F(ab′)₂ fragments of rabbit anti-mouse IgG (Cappel, Durham, NC)), either alone or in preformed soluble immune complexes with intact goat anti-rabbit IgG (Cappel) for 3 h at 37°C. Immune complexes were prepared by incubating intact goat anti-rabbit IgG with Ag in RPMI 1640 complete medium for 30 min at 37°C at a molar ratio of 2.5:1, respectively. Following the 3-h incubation of A20 and A6B9 cells with Ag, cells were fixed with 0.5% paraformaldehyde for 30 min at room temperature, washed, and resuspended with 2R.50 cells (2 × 10⁶ cells/ml). After an 18-h incubation at 37°C, the supernatants were collected and assayed for IL-2 by ELISA (Endogen, Woburn, MA).

The endocytosis assay used to analyze internalization of sIg- and Fc receptor-bound Ag was based on a previously published protocol (3) with the following changes included. A20 or A6B9 cells suspended at 1 × 10⁷ cells/ml in RPMI 1640 complete medium plus 10 mM HEPES, pH 7.4, were incubated with horseradish peroxidase (HRP)-tagged F(ab′)₂ fragments of rabbit anti-mouse IgG (20 μg/ml, Zymed) either alone or in preformed immune complexes with intact goat anti-rabbit IgG (50 μg/ml, Cappel) for 15 min on ice and then for various times at 37°C. Ice-cold RPMI 1640 complete medium with HEPES was added to stop the reaction, followed by two washes with cold PBS. Each sample was then split into two equal volumes that were incubated in PBS plus or minus 0.5% Triton X-100 for 15 min on ice, and the cell-associated HRP activity was determined. The percentage of internalized HRP was calculated for each time point. Endocytosis of Fc receptor-bound immune complexes was assayed as previously described (3).

Specific Ag bound to the BCR is endocytosed, processed within vesicles of the endocytic pathway, and resulting peptide-MHC II complexes then routed back to the cell surface for presentation to helper T cells. B cell FcγRII-B1, on the other hand, binds any Ag that is contained within IgG-immune complexes regardless of the Ag specificity of the BCR. Since FcγRII-B1 is unable to mediate endocytosis, Ag bound in this way is not internalized and therefore is not presented to T cells (3). Since the presence of excess IgG Ab may cross-link Ag bound to the BCR to FcγRII-B1, we asked if the formation of a cross-linked complex between BCR and FcγRII-B1 might prevent processing and presentation of BCR-bound Ag.

We utilized A20 cells, which express endogenous FcγRII-B1 and cell surface IgG in the BCR. F(ab′)₂ fragments of rabbit anti-mouse IgG were used as the Ag, since the antigenic epitope of the A20 surface IgG is unknown. A20 cells were incubated with various concentrations of Ag, either alone or in preformed soluble immune complexes with intact goat anti-rabbit IgG, and then assayed for their ability to present peptide to 2R.50 cells. As shown in Figure 1 A, presentation increased with increasing concentrations of Ag to a higher degree when rabbit F(ab′)₂ fragments were added alone compared with presentation of Ag that was added to cells in the form of immune complexes. This was consistent with the F(ab′)₂ fragments added alone being bound and internalized by the BCR whereas preformed complexes of rabbit F(ab′)₂ and goat-anti-rabbit IgG would be bound to FcγRII-B1, with or without also binding to the BCR.

Results from the A20 cells were next compared with results obtained with A6B9 cells, an FcγRII-negative A20 cell derivative expressing transfected FcγRII-B2. The FcγRII-B2 isoform is endocytosis positive, and we have previously shown that nonspecific Ags in IgG-immune complexes can be internalized in A6B9 cells and can be presented to T cells (3). In the present study, A6B9 cells were assayed for presentation of F(ab′)₂ fragments of rabbit anti-mouse IgG added to cells either alone or in immune complexes, as above. In contrast to A20 cells, A6B9 cells demonstrated enhanced presentation with increasing concentrations of Ag at a similar rate, whether or not rabbit F(ab′)₂ fragments were added to cells in association with the immune complex Ab (Fig. 1 B). These results show that FcγRII-B1 inhibited presentation of a BCR-specific immune complex Ag, whereas FcγRII-B2 did not.

To ensure that inhibition of presentation of the BCR-specific immune complex Ag in A20 cells was indeed due to the Fc receptor, A20 and A6B9 cells were incubated with Ag, either alone or in the form of immune complexes, in the presence of the anti-Fc receptor Ab 2.4G2. Under the conditions used, the 2.4G2 Ab blocks immune complex binding to both FcγRII-B1 and FcγRII-B2. In A20 cells, inhibition of Ag presentation in the presence of 2.4G2 occurred to the same extent (shown as percent inhibition in the parentheses) whether or not Ag was added to cells alone or in immune complexes (Fig. 2). Since 2.4G2 cross-reacts with the rabbit anti-mouse IgG F(ab′)₂ fragments, this result was not surprising: addition of both 2.4G2 and the F(ab′)₂ would effectively form an immune complex that could bind directly to FcγRII-B1 or cross-link the BCR to FcγRII-B1 on these cells. Furthermore, we observed that 2.4G2 inhibits endocytosis but not the binding of the F(ab′)₂ fragments in cells expressing either FcγRII-B1 or -B2 (data not shown), which would result in decreased Ag presentation.

More informative, therefore, were experiments with A6B9 cells expressing the internalization-competent FcγRII-B2 isoform. Also as shown in Figure 2, A6B9-mediated presentation of Ag added to cells in the presence of 2.4G2 was inhibited more when the Ag was contained within immune complexes (84% inhibition) than when Ag was added alone (38% inhibition). Therefore, in A6B9 cells, presentation of immune complex Ag was clearly dependent upon both the BCR and FcγRII-B2. However, when immune complexes were given, presentation via FcγRII-B2 appeared to occur preferentially over presentation via the BCR, despite the fact that the immune complex contained an Ag that could bind to BCR. Taken together, these results show that the inhibition of immune complex Ag presentation observed in A20 cells (compared with Ag added to cells alone) was indeed due to the Fc receptor, FcγRII-B1.

To test whether the observed FcγRII-B1-mediated inhibition of presentation of a BCR-specific immune complex Ag was due to the endocytosis-negative phenotype of this Fc receptor isoform, A20 and A6B9 cells were examined for their ability to endocytose HRP-tagged Ag added to cells either alone or in immune complexes. HRP-tagged Ag was incubated with cells continuously to mimic conditions of the Ag presentation assay, and internalization of Ag as measured by HRP activity was monitored over time. As shown in Figure 3, A20 cells showed decreased endocytosis of immune complex Ag compared with internalization of Ag added to cells alone (Fig. 3,A), whereas A6B9 cells demonstrated similar rates of endocytosis with either form of Ag (Fig. 3 B). These results are presumably due to the endocytic competence of the particular cell surface Fc receptor, FcγRII-B1 being internalization-negative while FcγRII-B2 is internalization-positive. Therefore, we can conclude that inhibition of presentation of the BCR-specific immune complex Ag in A20 cells was mediated by FcγRII-B1 at the level of endocytosis.

The FcγRII-B1-unique cytoplasmic tail insertion inhibits endocytosis by preventing accumulation in clathrin-coated pits (3, 24, 25). To determine whether or not the entire insertion sequence was required for inhibition of endocytosis, FcγRII-B1 deletion mutants were assayed for internalization competence. FcγRII-B1 missing amino acid residues in the amino-terminal half of the cytoplasmic tail insertion (B1 (CTΔ7-31)) endocytosed bound immune complexes at a similar rate as the -B2 isoform (Fig. 4). In contrast, endocytosis of FcγRII-B1 with amino acid residues in the carboxyl-terminal half of the insertion omitted (B1 (CTΔ33-54)) occurred at the same low level as that of intact FcγRII-B1 (Fig. 4). Therefore, the amino-terminal half of the murine FcγRII-B1 cytoplasmic tail insertion contains sequences needed to inhibit endocytosis and the observed down-regulation of presentation of a BCR-specific immune complex Ag.

Finally, since tyrosine phosphorylation has been implicated in FcγRII function, we asked whether the single tyrosine reside found in the receptor’s amino-terminal half of the cytoplasmic tail insertion might play a role in regulating endocytosis in FcγRII-B1. As shown in Figure 4, this proved not to be the case. This result demonstrates that the ability of the FcγRII-B1 insertion to block endocytosis was not dependent on the presence of a phosphorylation site within the cytoplasmic tail insertion.

In this study, we observed that A20 cell FcγRII-B1 inhibited presentation of a sIg-specific, immune complex Ag to T cells. Moreover, this FcγRII-B1-mediated inhibition occurred at the level of immune complex endocytosis and, hence, was dependent upon the cytoplasmic tail insertion. Therefore, FcγRII-B1 not only inhibits B cell activation, it inhibits activation of T cells as well. In addition, it is possible that the relative affinity of an immune complex with a BCR-specific Ag for either the sIg or FcγRII-B1 helps to determine the outcome of a particular immune response, positive or negative.

The mechanism by which the FcγRII-B1 cytoplasmic tail insertion inhibits endocytosis is not completely understood. Previous work suggests that FcγRII-B1 associates with the cytoskeleton (25), and perhaps this association is responsible for preventing accumulation in clathrin-coated pits (3, 24, 25). We determined that the amino-terminal half of the FcγRII-B1 insertion contains sequences required to inhibit endocytosis. The human FcγRII-B1 isoform, which also mediates negative signaling in B cells but not endocytosis (28, 29), contains a cytoplasmic tail insertion that is highly homologous in sequence and length (19 amino acid residues) to the amino-terminal half of the murine B1 isoform, suggesting a conservation of function. With the absence of the cytoplasmic tail insertion sequence, murine FcγRII-B2 mediates efficient endocytosis in A6B9 cells (Fig. 4; 3 . As observed with FcγRII-B2-transfected MDCK cells (30, 31), FcγRII-B2-mediated endocytosis in A6B9 cells requires a di-leucine motif in the ITIM (data not shown). Furthermore, the -B2 isoform can mediate negative signaling upon cocross-linking with the sIg in A6B9 cells (3). It is possible that the amino-terminal half of the FcγRII-B1 insertion alters the conformation of the cytoplasmic tail domain such that the di-leucine motif is not available to mediate endocytosis but the ITIM still functions in negative signal transduction. FcγRII-B1 thus functions in a multifaceted manner to maintain a tight control on B cell activation and to maintain the Ag specificity of the humoral immune response.

We thank Dr. Richard Mitchell for providing the 2R.50 cells and Dr. Deborah Lazzarino for many helpful discussions.