Activatory and Inhibitory Fcγ Receptors Augment Rituximab-mediated Internalization of CD20 Independent of Signaling via the Cytoplasmic Domain*

Background: Fcγ receptor (FcγR) IIb augments internalization of CD20 from the surface of B cells in response to rituximab treatment. Results: Activatory and inhibitory FcγR augment internalization, independent of the FcγR cytoplasmic domain. Conclusion: Active signaling is not required for FcγR-augmented internalization of CD20 in response to rituximab treatment. Significance: FcγR may play a structural role in augmenting CD20 internalization. Type I anti-CD20 mAb such as rituximab and ofatumumab engage with the inhibitory FcγR, FcγRIIb on the surface of B cells, resulting in immunoreceptor tyrosine-based inhibitory motif (ITIM) phosphorylation. Internalization of the CD20·mAb·FcγRIIb complex follows, the rate of which correlates with FcγRIIb expression. In contrast, although type II anti-CD20 mAb such as tositumomab and obinutuzumab also interact with and activate FcγRIIb, this interaction fails to augment the rate of CD20·mAb internalization, raising the question of whether ITIM phosphorylation plays any role in this process. We have assessed the molecular requirements for the internalization process and demonstrate that in contrast to internalization of IgG immune complexes, FcγRIIb-augmented internalization of rituximab-ligated CD20 occurs independently of the FcγRIIb ITIM, indicating that signaling downstream of FcγRIIb is not required. In transfected cells, activatory FcγRI, FcγRIIa, and FcγRIIIa augmented internalization of rituximab-ligated CD20 in a similar manner. However, FcγRIIa mediated a slower rate of internalization than cells expressing equivalent levels of the highly homologous FcγRIIb. The difference was maintained in cells expressing FcγRIIa and FcγRIIb lacking cytoplasmic domains and in which the transmembrane domains had been exchanged. This difference may be due to increased degradation of FcγRIIa, which traffics to lysosomes independently of rituximab. We conclude that the cytoplasmic domain of FcγR is not required for promoting internalization of rituximab-ligated CD20. Instead, we propose that FcγR provides a structural role in augmenting endocytosis that differs from that employed during the endocytosis of immune complexes.

Anti-CD20 mAb are classified as type I (rituximab (RTX)like) 3 or type II (tositumomab-like) based on functional differences that they mediate in various in vitro assays (1). Type I mAb cause redistribution of CD20 into lipid rafts, favoring potent complement dependent cytotoxicity, whereas Type II mAb are ineffective in these assays but more potently elicit homotypic adhesion and a nonapoptotic lysosomal form of cell death (2)(3)(4)(5)(6). We recently observed that in addition, type I anti-CD20 mAb mediate rapid internalization of CD20 from the cell surface, thereby reducing antibody efficacy, whereas type II mAb do not (7,8). We subsequently showed that internalization of type I anti-CD20 mAb was greatly augmented by their engagement with Fc␥RIIb on the cell surface via antibody bipolar bridging and that the rate of internalization positively correlated with cell surface expression of Fc␥RIIb (8). Higher expression of target cell Fc␥RIIb was associated with reduced survival or response in cancer patients treated with RTX therapy in two retrospective trials (8,9).
Previously, we proposed that in contrast to the treatment of cancer, CD20 internalization may be advantageous in the treatment of autoimmune disease (10), where rituximab therapy has proven beneficial (11). Its mechanism of action is still poorly understood, but it has been suggested that type I anti-CD20 mAb promote a regulatory B cell response that can suppress autoimmune responses (12). Fc␥RIIb is down-regulated on B cells of patients with systemic lupus erythematosis (13) but is up-regulated on a subset of regulatory B cells (14). Therefore, Fc␥RIIb-mediated internalization of CD20 in response to type I mAb ligation may result in preferential clearance of pathogenic Fc␥RIIb-low cells in systemic lupus erythematosis, while sparing Fc␥RIIb-high regulatory B cells. Thus, it is of great interest to elucidate the mechanism by which interaction between type I anti-CD20 mAb and Fc␥RIIb promotes internalization of the CD20⅐mAb⅐Fc␥RIIb complex to design strategies to inhibit the process and improve therapy in the treatment of malignancy or augment it in situations such as systemic lupus erythematosis where internalization may prove beneficial.
Given our initial observations that type I anti-CD20 mAb appeared to be unique in their ability to interact with and activate Fc␥RIIb in cis (8), we theorized that activation of the ITIM and signaling via the Fc␥R initiated the endocytic process, analogous to the interaction between Fc␥RIIb2 and immune complexes (15,16). Endocytosis of immune complex in the form of heat-aggregated human IgG (ahIgG) is dependent on the expression of a complete ITIM within the cytoplasmic domain of Fc␥RIIb (15,16) and is completely abrogated in cells expressing mutated forms of the receptor in which the ITIM has been truncated (15). Furthermore, ahIgG remains on the surface of cells expressing the b1 isoform of Fc␥RIIb because of an extra 19 amino acids in the cytoplasmic domain that excludes the receptor from clathrin-coated pits (16).
We have previously observed that both b1 and b2 isoforms of Fc␥RIIb are equally effective at augmenting internalization of RTX-ligated CD20 (10), raising the possibility that the mechanism of endocytosis is different from the internalization of immune complex. We have also found that the majority of mAb directed to a range of B cell receptors interact with and activate Fc␥RIIb via antibody bipolar bridging, with the extent of activation related to the level of mAb bound to the cell surface (10). The type II anti-CD20 mAb tositumomab also activated Fc␥RIIb, although to a much lesser extent than RTX (10). The presence of Fc␥RIIb failed to alter the rate of internalization of most mAb-ligated receptors, raising the question as to whether activation of Fc␥RIIb and signaling via the ITIM is indeed the mechanism by which type I anti-CD20 mAb augment internalization of CD20. Here we have investigated this question, as well as whether expression of other activatory Fc␥R can promote internalization of CD20 in response to type I anti-CD20 mAb ligation, and the underlying molecular mechanism.

EXPERIMENTAL PROCEDURES
Cell Lines-The Burkitt's lymphoma cell lines Ramos and Raji were obtained from the European Collection of Cell Cultures and maintained in complete cell culture media (RPMI 1640, 4 mM L-glutamine, 1 mM sodium pyruvate, and 10% (v/v) FCS (all from Invitrogen)).
Generation of WT and Mutant Human Fc␥R Constructs-Human Fc␥RIIb1, Fc␥RIIb2, and truncated Fc␥RIIb⌬cyt expression vectors were constructed previously (8,10). Human Fc␥RI and human Fc␥RIIIa V158 were amplified from complementary DNA obtained from primary human leukocytes using specific primers and cloned into the pIRES vector (Clontech) co-expressing the FcR common ␥-chain amplified from the same cells. Human Fc␥RIIa was amplified from human leukocytes using specific primers and cloned into the pCI PURO expression vector. The pCI PURO vector was constructed by subcloning the puromycin resistance gene from Ppuro (Clontech) into pCI-neo (Promega) via PuvII/BamHI sites. To generate a truncated mutant version of Fc␥RIIa lacking the intracellular domain (Fc␥RIIa⌬cyt), a stop mutation at residue 244 was introduced using the QuikChange multi site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Fc␥RIIb⌬cyt and Fc␥RIIa⌬cyt were used to construct TGI230 -232IAT and IAT224 -226TGI transmembrane mutants, respectively, by site-directed mutagenesis.
Monoclonal Antibody Production and Labeling-Rituximab was gifted by Southampton General Hospital oncology pharmacy. Nonradiolabeled tositumomab was gifted by Professor T. Illidge (University of Manchester, Manchester, UK). F(abЈ) 2 fragments of RTX were produced as described previously (17). AT10 was produced in-house (18) and used to generate F(abЈ) 2 fragments prior to labeling with Alexa Fluor 647 (A647) using the A647 labeling kit according to the manufacturer's instructions (Invitrogen). RTX, RTX F(abЈ) 2 , and tositumomab were labeled with A488-TFP ester according to the manufacturer's instructions (Invitrogen).
Preparation of Heat-aggregated Human IgG-Human IgG was treated at 62°C for 30 min to induce aggregation. Heataggregated human IgG was then separated from the monomeric fraction by size exclusion HPLC.
Antibody Internalization Assays-Internalization of A488labeled mAb was quantified as reported previously (7,8,10) using the following formula: % cell surface mAb remaining on B cells ϭ (mean fluorescence intensity (MFI) of unquenched cells Ϫ MFI of quenched cells)/MFI of unquenched cells ϫ 100. The MFI of unstained cells was subtracted as background. Internalization of ahIgG was quantified by treating cells with 20 g/ml ahIgG for 30 min at 4°C. After washing, cells were divided in to two fractions. One half was maintained at 4°C (time 0 fraction), whereas the other half was incubated at 37°C (internalized fraction) for the times indicated. Both fractions were then stained with A488-labeled polyclonal goat anti-human IgG (Jackson ImmunoResearch Laboratories), and the MFI was quantified by flow cytometry. Internalization was expressed as the proportion of ahIgG remaining at the cell surface compared with time 0 using the following formula: % cell surface ahIgG remaining ϭ (MFI of internalized fraction/MFI of time 0 fraction) ϫ 100.
Western Blotting-For the detection of phosphorylated Fc␥RIIb, cells were incubated with 5 g/ml RTX for 30 min or left untreated as described previously (8,10). Membranes were then probed with EP926Y (rabbit anti-human Fc␥RIIb (phospho-specific); Cambridge Bioscience). For the detection of total Fc␥RIIa/b, cells plated at 4 ϫ 10 6 /ml were preincubated at 37°C for 30 min before the addition of A488-labeled RTX (5 g/ml) or 20 g/ml ahIgG for the indicated times or left untreated. The cells were then washed in ice-cold PBS and resuspended in lysis buffer as described previously (10,19). Membranes were probed with MAB1330 (mouse anti-human Fc␥RII; R&D Systems) in TBS-Tween 0.05%, 5% BSA, 0.05% sodium azide at 4°C overnight and then with donkey anti-mouse IgG HRP-linked F(abЈ) 2 for 1 h at room temperature and washed, and the signal was visualized using ECL reagents and light-sensitive film (all from GE Healthcare Lifesciences). Where quantification was required, densitometry was employed using ImageJ software (National Institutes of Health). All quantities were normalized to ␣-tubulin to control for variations in protein loading.
Fc␥RIIa/b Internalization Assay by Cell Surface Biotinylation-Biotinylation of cell surface proteins was performed as described previously (20). Briefly, 4 ϫ 10 7 cells were resuspended in 10 ml of 0.25 mg/ml sulfo-NHS-SS-biotin (Thermo Scientific) for 1 h at 4°C. The cells were then washed twice with 25 mM L-lysine (Sigma-Aldrich), 5% FCS/PBS followed by 5% FCS/PBS and resuspended in full tissue culture medium. Cells were returned to 37°C and either left untreated or treated with A488-labeled RTX (5 g/ml). At each time point, cells were treated three times with 100 mM sodium 2-mercaptoethanesulfonate (MESNa), 50 mM Tris, 100 mM NaCl, pH 8.5 (Sigma-Aldrich) or buffer lacking MESNa, followed by two washes with 5 mg/ml iodoacetamide (Sigma-Aldrich) and a final wash with PBS. Cells were then lysed in lysis buffer and added to 85 l of NeutrAvidin-agarose beads (Thermo Scientific) and left at 4°C overnight on a rotator. The beads were washed five times with wash buffer (Thermo Scientific) and treated as for Western blotting described above. Blots were then probed for Fc␥R. Densitometry was employed, and internalization at each time point was quantified and expressed as the proportion of Fc␥RIIa/b present at 0 h using the following formula: % internalized Fc␥R ϭ (intensity of internalized band after MESNa treatment/intensity of non-MESNa-treated band at 0 h) ϫ 100.
Fc␥RIIa/b Recycling Assay by Cell Surface Biotinylation-Cells were biotinylated as described above and then treated with A488-labeled RTX (5 g/ml) for 30 min or 2 h as indicated. After treatment with 100 mM MESNa, 50 mM Tris, 100 mM NaCl, pH 8.5, as above, the cells were resuspended in 1 ml of full tissue culture medium and divided into two fractions. One fraction was treated immediately with MESNa for a second time or buffer lacking MESNa (0-h fraction). The second fraction was returned to 37°C for 2 h to allow recycling of proteins to the cell surface (2-h fraction). This fraction was then also treated with MESNa or buffer alone. Finally, the cells were lysed in lysis buffer and added to NeutrAvidin-agarose beads as above. Blots were then probed for Fc␥RII. Densitometry was employed, and the data were expressed as a proportion of the band intensity at 0 h using the following formula: relative density of Fc␥R (%) ϭ (intensity of band/intensity of band at 0 h (non-MESNa-treated)) ϫ 100. Blots were also probed for CD22 as a positive control (H-221, rabbit polyclonal IgG; Santa Cruz Biotechnology).
Confocal Microscopy-To determine the intracellular trafficking of RTX and Fc␥RIIa/b, cells were incubated with A488labeled RTX for the times indicated and then washed and fixed with 2% paraformaldehyde as described previously (8). For detection of Fc␥RIIa/b and LAMP-1, the cells were permeabilized with 0.3% saponin and incubated with A647-labeled AT10 F(abЈ) 2 and biotin-conjugated anti-human LAMP-1 (eBioscience), respectively. The cells were then washed, stained with streptavidin-A547 (Invitrogen), and transferred onto slides.
Statistical Analysis-Analyses were performed using the Mann-Whitney U test for unpaired samples and the Wilcoxon signed ranks test for paired samples with a two-tailed hypothesis using GraphPad Prism version 6.00 for Windows (GraphPad software).

The Intracellular Domain of Fc␥RIIb Is Not Required for
Fc␥RIIb-augmented Internalization of RTX-We previously observed a lack of correlation between the ability of cell surfacebound mAb to interact with and activate Fc␥RIIb via bipolar antibody bridging and increased internalization of the mAbligated receptor (10). This led us to question whether phosphorylation of the ITIM of Fc␥RIIb was necessary for augmenting CD20 internalization in response to RTX ligation.
To investigate this, we transfected Fc␥RIIbϪve Ramos cells with a truncated version of Fc␥RIIb (Fc␥RIIb⌬cyt) lacking the ITIM-containing cytoplasmic domain as used previously (10). Colonies expressing low, medium, or high levels of the receptor were selected (Fig. 1A), reflecting the expression of Fc␥RIIb on normal human B cells, primary lymphoma cells overexpressing the receptor, and an even higher (likely nonphysiological) level of Fc␥RIIb, respectively.
Western blots were conducted to confirm the lower molecular mass of Fc␥RIIb⌬cyt in transfected cells (Fig. 1B). Cells transfected with a low level of WT Fc␥RIIb2 (8,10) or empty vector were used as positive and negative controls, respectively. As expected, WT Fc␥RIIb2 had a molecular mass of ϳ32 kDa. Lysates prepared from Fc␥RIIb⌬cyt transfectants displayed reduced molecular mass bands at 27 kDa, consistent with the absence of the intracellular domain. The intensity of the Fc␥RIIb⌬cyt bands also reflected their differing surface expression. Transfectants were then treated with A488-labeled RTX and probed for phosphorylated Fc␥RIIb (Fig. 1C). As expected (8,10), treatment with RTX reliably activated Fc␥RIIb in lysates prepared from cells transfected with WT but not truncated transfectants, confirming the absence of the ITIM in Fc␥RIIb⌬cyt transfected cells.
To determine whether Fc␥RIIb⌬cyt could augment internalization of CD20 in response to RTX ligation, transfectants were cultured with A488-labeled RTX for 2 h, and the proportion of RTX remaining on the cell surface was quantified (Fig. 1D). Expression of Fc␥RIIb⌬cyt at a level normally seen on B cells (Fc␥RIIb⌬cyt low) resulted in a significant increase in internalized RTX compared with Fc␥RIIbϪve controls (p Ͻ 0.01), indi-cating that phosphorylation of the Fc␥RIIb ITIM is not required for this activity. Furthermore, there was no significant difference in the rate of RTX internalization mediated by Fc␥RIIb⌬cyt transfectants and cells expressing WT Fc␥RIIb2, suggesting that the absence of the ITIM had no effect on the rate of internalization mediated by the truncated receptor. As observed previously with Fc␥RII b1 (10) and b2 (8) transfected cells, there was a dose-dependent increase in the internalization of RTX in cells expressing higher levels of Fc␥RIIb⌬cyt (Fig. 1D).
Budde et al. (15) previously observed an absolute requirement for the Fc␥RIIb2 ITIM in the internalization of ahIgG, suggesting that Fc␥RIIb-augmented internalization of RTX occurs by an alternative mechanism to the endocytosis of immune complexes. However, these results were generated using transfectants of the A20 IIA1.6 mouse B cell line. Therefore, to confirm the importance of the ITIM in internalization of ahIgG in human B cells, we measured the rate of internaliza-tion of ahIgG in our Ramos Fc␥RIIb transfectants. Initially we attempted to quantify the rate of internalization of A488-labeled ahIgG using the same method as for Fig. 1D but saw incomplete quenching of the fluorescent signal at time 0, possibly because of the inability of the secondary anti-A488 Ab to fully penetrate the immune complex. Thus, we adopted an alternative method to quantify internalization in which we treated cells with unlabeled ahIgG and measured the level remaining on the cell surface over 60 min using a secondary A488-labeled anti-human IgG Ab (see "Experimental Procedures" and Fig. 1E). As expected (15), almost all ahIgG had been internalized from the surface of cells expressing WT Fc␥RIIb2 by 30 min, with only a low level remaining after 60 min (median; 11.04% of time 0 fraction). In contrast, a substantial proportion of ahIgG (median; 53.42% of time 0 fraction) remained on the cell surface of cells expressing Fc␥RIIb1. The level of internalization was further reduced in Fc␥RIIb⌬cyt transfectants, which retained the majority of ahIgG on the cell surface after Ϫ, not treated; ϩ, RTX-treated. D, Ramos transfectants were cultured with 5 g/ml A488-labeled RTX for 2 h. The proportion of total mAb remaining on the cell surface was assessed by flow cytometry after treatment of cells with anti-A488 to quench cell surface fluorescence. Transfectants were compared using the Mann-Whitney U test. NS, not significant, n ϭ 6 -7. E, Ramos transfectants were cultured with 20 g/ml ahIgG for 1 h. The proportion of total Ab remaining on the cell surface after 30 and 60 min was assessed by flow cytometry after treatment of cells with A488-labeled anti-human IgG, n ϭ 6. Horizontal bars represent the median. Activatory Fc␥R Augment the Internalization of CD20 in Response to Ligation by RTX-Having established that the intracellular ITIM-containing domain of Fc␥RIIb was dispensable for promoting internalization of CD20, we asked whether expression of other IgG-binding receptors, in particular activatory Fc␥R, could also augment CD20 internalization in response to RTX ligation. Although normal human B cells only express the inhibitory Fc␥R, Gamberale et al. (21) observed heterogeneous expression of Fc␥RIIa in malignant B cells from patients with chronic lymphocytic leukemia. Fc␥RIIa shares ϳ93% homology with Fc␥RIIb in the extracellular and transmembrane domains but differs substantially in the intracellular domain (22) because it contains an immunoreceptor tyrosinebased activatory motif (ITAM) rather than an ITIM (23). From the high degree of homology between the extracellular and transmembrane domains of Fc␥RIIa and Fc␥RIIb, we anticipated that Fc␥RIIa would also augment internalization of RTXligated CD20.
To investigate this, we transfected Fc␥RIIaϪve Ramos cells with WT Fc␥RIIa and selected colonies expressing a low level of Fc␥RIIa (WT Fc␥RIIa low), comparable to the level of Fc␥RIIb on WT Fc␥RIIb2 low transfectants and a colony expressing very high levels of expression (Fc␥RIIa high) ( Fig. 2A). Cells were cultured with A488-labeled RTX, and the proportion of mAb remaining on the surface quantified at 1 and 6 h. WT Fc␥RIIb2 low and empty vector transfected cells were included as positive and negative controls, respectively (Fig. 2B).
Expression of WT Fc␥RIIa at a low level resulted in a significant increase in the rate of CD20 internalization in response to RTX ligation at 1 and 6 h, compared with controls, but to a much lesser extent than cells expressing WT Fc␥RIIb2. There was a faster rate of internalization in cells expressing a high level of WT Fc␥RIIa, confirming that Fc␥RIIa was able to augment internalization of RTX-ligated CD20. However, the rate of internalization mediated by the high level of WT Fc␥RIIa was also slower than observed in Ramos cells expressing WT Fc␥RIIb1 at a comparably high level as demonstrated previously (10). As with cells expressing Fc␥RIIb, a slower rate of CD20 internalization was observed in response to ligation with the type II anti-CD20 mAb tositumomab, compared with RTX in cells expressing WT Fc␥RIIa (Fig. 2C).
Having established that Fc␥RIIa could augment the internalization of RTX from the cell surface, albeit less efficiently than Fc␥RIIb, to understand the underlying mechanism, we were interested to see whether other Fc-binding receptors had similar activity or whether it was restricted to Fc␥RIIa/b. Unlike Fc␥RIIa/b, Fc␥RI and Fc␥RIIIa do not express an intrinsic signaling domain (24). Instead, they associate with the ITAMcontaining ␥ chain via homologous sequences in the transmembrane domains (25). Association between Fc␥RI and Fc␥RIIIa and the ␥ chain in the endoplasmic reticulum protects the Fc␥R from degradation and is necessary for expression on the cell surface (26). We therefore transfected Ramos cells with Fc␥RI and Fc␥RIIIa alongside the ␥ chain but were unable to generate stable transfectants. We were also unable to detect expression of these receptors after transient transfection (data not shown), perhaps because co-association of the Fc␥R and the ␥ chain failed to occur, preventing cell surface expression.
We were able to successfully transiently transfect Raji cells with Fc␥RI or Fc␥RIIIa as determined by flow cytometry (Fig.  3A). 24 h after transfection, cells were cultured with A488-labeled anti-CD20 mAb for 2 h, and the proportion of mAb remaining on the cell surface was quantified and compared between Fc␥RϪve and Fc␥Rϩve cells (Fig. 3B).
We observed a significant increase in the rate of CD20 internalization in response to RTX ligation in Raji cells transiently expressing Fc␥RI and Fc␥RIIIa, demonstrating that the ability to mediate this activity is not restricted to cells expressing Fc␥RIIa/b. A lower rate of internalization was observed in response to RTX F(abЈ) 2 , indicating that the effect was dependent on the Fc-Fc␥R interaction as has been observed previously (8,10).
The Difference in Rate of CD20 Internalization Mediated by Fc␥RIIa and Fc␥RIIb in Response to RTX Ligation Is Due to Greater Degradation of Fc␥RIIa-The slower rate of CD20 internalization promoted by expression of Fc␥RIIa on Ramos cells compared with Fc␥RIIb2 was unexpected given the high degree of homology between the extracellular and transmembrane domains of Fc␥RIIa and Fc␥RIIb and the higher affinity of Fc␥RIIa for IgG1 (27). However, Zhang and Booth (22) recently demonstrated that subsequent to ahIgG binding and internalization, Fc␥RIIa and Fc␥RIIb were divergently sorted. Fc␥RIIa was degraded in the lysosome along with the bound ahIgG, whereas Fc␥RIIb was recycled back to the plasma membrane, leaving behind the ahIgG to be degraded in the lysosome. We have previously demonstrated co-internalization of Fc␥RIIb with CD20 upon ligation with type I anti-CD20 mAb (8,10) and anticipated that the same occurs with Fc␥RIIa. Given the greater efficacy of Fc␥RIIb, we hypothesized that Fc␥RIIa was degraded within the lysosome alongside CD20 following interaction with RTX, whereas a proportion of Fc␥RIIb was recycled back to the membrane as occurs in response to ahIgG, allowing further rounds of interaction with mAb-ligated CD20 on the cell surface.
Zhang and Booth (22) investigated the sorting of Fc␥RIIa and Fc␥RIIb in a transfected hamster fibroblast cell line and in human monocyte-derived macrophages, but not B cells. Therefore, to confirm whether Fc␥RIIa and Fc␥RIIb were also differentially degraded in human B cells, Ramos cells expressing equivalent levels of WT Fc␥RIIa or Fc␥RIIb2 were incubated with ahIgG, and the proportion of total Fc␥R remaining was quantified at 1, 2, and 6 h by Western blotting (Fig. 4). Although the reduction of Fc␥RIIa was less pronounced than that observed by Zhang and Booth (22), in agreement with their findings, by 6 h there was a lower proportion of Fc␥RIIa remaining (median; 67.53% of untreated cells), compared with Fc␥RIIb (median; 100.5% of untreated cells), which remained stable over the duration of the assay.
After establishing that Fc␥RIIa was also preferentially degraded after engagement of immune complex in human B cells, we were interested to determine whether the same was true after interaction with RTX-ligated CD20. Ramos cells expressing equivalent levels of WT Fc␥RIIb1, Fc␥RIIb2, or Fc␥RIIa were cultured with A488-labeled RTX and the proportion of total Fc␥R remaining was quantified at 1, 2, and 6 h by Western blotting (Fig. 5). In contrast to treatment with ahIgG, the proportion of Fc␥RIIb1, Fc␥RIIb2, and Fc␥RIIa was reduced over time in cells cultured with RTX, compared with untreated cells. Although the loss of Fc␥RIIa was slightly faster than that of Fc␥RIIb, it was not clear whether this difference was sufficient to explain the slower rate of CD20 internalization mediated by Fc␥RIIa compared with Fc␥RIIb.
Because the extracellular and transmembrane domains of Fc␥RIIa and Fc␥RIIb are ϳ93% identical, we decided to focus

Fc␥R Augment Internalization of RTX-ligated CD20
FEBRUARY 27, 2015 • VOLUME 290 • NUMBER 9 on whether the divergent intracellular domains were responsible for the differences in the rate of RTX internalization observed between cells expressing the two receptors. We have already observed that the intracellular domain of Fc␥RIIb is dispensable for promoting increased internalization of mAbligated CD20 (Fig. 1), so we next investigated whether the same was true for Fc␥RIIa. Furthermore, if the divergent intracellular domain was responsible for the slower rate of internalization mediated by Fc␥RIIa, we anticipated that removal of it would augment the rate of CD20 internalization in response to RTX ligation.
We transfected Fc␥RϪve Ramos cells with a truncated version of Fc␥RIIa (Fc␥RIIa⌬cyt) lacking the ITAM-containing cytoplasmic domain. A clone expressing a low level of receptor, comparable with that expressed on WT Fc␥RIIa low transfectants, was selected (Fig. 6B). WT Fc␥RIIa low, Fc␥RIIa⌬cyt low, WT Fc␥RIIb2 low, and Fc␥RIIb⌬cyt low cells were cultured with A488-labeled RTX for 6 h, and the proportion of cell surface mAb remaining was quantified. Ramos cells transfected with empty vector were included as negative controls (Fig. 6C).
As already observed, expression of WT Fc␥RIIa and WT Fc␥RIIb augmented internalization of RTX-ligated CD20, with WT Fc␥RIIa less effective than WT Fc␥RIIb. Mutated recep-tors lacking the intracellular domain also augmented internalization but loss of the intracellular domain from Fc␥RIIa failed to increase the rate to that observed in cells expressing Fc␥RIIb⌬cyt, with the difference between Fc␥RIIa⌬cyt and Fc␥RIIb⌬cyt remaining (Fig. 6C). These results confirmed that the intracellular domain of Fc␥RIIa was also dispensable for augmenting internalization of RTX-ligated CD20 and was not responsible for the slower rate observed, compared with Fc␥RIIb.
In addition to measuring the rate of internalization of RTX, we also measured the total Fc␥R remaining in cells expressing Fc␥RIIa⌬cyt and Fc␥RIIb⌬cyt by Western blotting after treatment with A488-labeled RTX for 1, 2 and 6 h (Fig. 7, A and B). In contrast to cells expressing WT Fc␥RIIa, the level of Fc␥RIIa⌬cyt was rapidly reduced by 1 h (median; 48.7% of untreated cells) in cells cultured with RTX, before increasing over the remainder of the experiment. Conversely, levels of Fc␥RIIb were maintained at 1 h (median; 101.5% of untreated cells) and were only slightly reduced by 6 h (median; 82.79% of untreated cells). These results suggested that similarly to Fc␥RIIb, Fc␥RIIa efficiently interacts with RTX on the surface of cells but is then rapidly internalized and degraded, in contrast to Fc␥RIIb. The difference in the rate of internalization of RTX-ligated CD20 mediated by Fc␥RIIa and Fc␥RIIb is consistent with divergent sorting of the two receptors within lysosomes after internalization as previously described (22). Zhang and Booth (22) demonstrated that divergent intracellular sorting of Fc␥RIIa and Fc␥RIIb occurred independently of the Fc-binding extracellular domains of the receptors, suggesting that differences between the transmembrane or intracellular domains were responsible. We have already demonstrated that the intracellular domains of Fc␥RIIa and Fc␥RIIb are dispensable for their ability to augment internalization of RTXligated CD20, so we focused on whether differences in the transmembrane domains were responsible for the slower rate of internalization mediated by Fc␥RIIa.
To investigate this possibility, we generated mutated versions of Fc␥RIIa⌬cyt and Fc␥RIIb⌬cyt, in which the transmembrane domains were exchanged between the two receptors. We transfected Ramos cells with these constructs to generate transfectants expressing either an Fc␥RIIa⌬cyt extracellular domain with an Fc␥RIIb transmembrane domain (IIa⌬cytTmIIb) or an Fc␥RIIb⌬cyt extracellular domain with an Fc␥RIIa transmembrane domain (IIb⌬cytTmIIa). Colonies were selected in which the expression level was comparable with those used previously (Fig. 6, A and B).
The cells were cultured with A488-labeled RTX for 6 h, and the proportion of total mAb remaining on the cell surface was quantified (Fig. 6C). Exchange of the transmembrane domains failed to reverse the difference observed between cells expressing Fc␥RIIa and Fc␥RIIb. Expression of the Fc␥RIIa transmembrane domain actually further augmented the rate of internalization mediated by IIb⌬cytTmIIA, whereas expression of the Fc␥RIIb transmembrane domain made no difference to the rate of internalization mediated by IIa⌬cytTmIIb. These results demonstrate that neither the intracellular nor transmembrane domains were responsible for the slower rate of CD20 internalization mediated by Fc␥RIIa in response to RTX ligation. Instead, the difference may be determined by subtle differences between the extracellular domains.
We measured the total Fc␥R remaining in cells expressing IIa⌬cytTmIIb and IIb⌬cytTmIIa by Western blotting after treatment with A488-labeled RTX for 1, 2, and 6 h (Fig. 7, C and  D). Levels of IIb⌬cytTmIIa were maintained over the duration of the assay, reaching their lowest levels at 1 h (median; 92.24% of untreated cells). In contrast, the majority of IIa⌬cytTmIIb was lost from cells by 6 h (median; 46.41% of untreated cells)    FEBRUARY 27, 2015 • VOLUME 290 • NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5431 but with delayed kinetics compared with cells expressing Fc␥RIIa⌬cyt (Fig. 7, A and B).

Fc␥R Augment Internalization of RTX-ligated CD20
The lack of any role for the cytoplasmic and transmembrane domains in determining the difference in the rate of internalization of RTX-ligated CD20 mediated by Fc␥RIIa and Fc␥RIIb suggested that the difference may not be due to divergent sorting of the two receptors after internalization as observed in cells treated with ahIgG (22), which was demonstrated to be independent of the extracellular domains. However, in an attempt to rule out this possibility, we adopted a reversible biotinylation strategy to specifically look at the internalization and recycling of Fc␥RIIa/b in response to RTX treatment. We treated Ramos cells transfected with WT Fc␥RIIb1, WT Fc␥RIIa, Fc␥RIIb⌬cyt, or Fc␥RIIa⌬cyt with membrane-impermeable sulfo-NHS-SS-biotin to biotinylate cell surface proteins including Fc␥RIIa/b and then cultured them in the presence or absence of A488-labeled RTX (Figs. 8 and 9).
Immediately following biotinylation of cell surface proteins (0 h), total cell surface Fc␥RIIa/b was immunoprecipitated with streptavidin-coated beads and quantified by Western blot (Figs.  8, A and C, and 9, A and C). Treatment with MESNa at this time point to reduce the disulfide bond present in the cell surface sulfo-NHS-SS-biotin, liberating the biotin component, demonstrated the reversibility of this process, resulting in almost total loss of Fc␥RIIa/b. Increases in Fc␥RIIa/b over time indicate a decrease in cell surface-accessible protein caused by internalization.
In untreated cells expressing WT Fc␥RIIb1 and WT Fc␥RIIa, the amount of Fc␥RII observed in the assay increased over the time course, indicating that the receptor was constitutively being internalized in resting cells (Fig. 8, B and D). The same was true in cells expressing Fc␥RIIb⌬cyt and Fc␥RIIa⌬cyt (Fig.  9, B and D). A lower molecular mass band appeared in cells expressing WT Fc␥RIIb1 and WT Fc␥RIIa, which became  more prominent over time. This band was absent from cells expressing the truncated receptors, suggesting that Fc␥RIIa/b was cleaved within or proximal to the cytoplasmic domain following internalization.
After treatment with RTX, there was a small increase in the level of internalized WT Fc␥RIIa/b1 compared with untreated cells at 1 h (Fig. 8), with a smaller increase at later time points compared with untreated cells, possibly because of increased protein degradation observed in response to RTX (Fig. 5). As in untreated cells, a lower molecular mass band appeared in response to RTX treatment. In cells expressing Fc␥RIIb⌬cyt or Fc␥RIIa⌬cyt, there was also an increased level of protein at early time points compared with untreated cells, suggesting that RTX treatment increases the rate of receptor internalization (Fig. 9). Interestingly, the rate of internalization was largely similar between cells regardless of whether they were expressing Fc␥RIIa or Fc␥RIIb, despite the different rates of internalization of RTX-ligated CD20 mediated by the two Fc␥R. Zhang and Booth (22) demonstrated that Fc␥RIIb2 is recycled back to the cell surface after internalization in response to ahIgG, in contrast to Fc␥RIIa, which is degraded in the lysosome. Thus, we used the reversible biotinylation strategy to specifically look at recycling of Fc␥RIIa/b. Ramos cells transfected with WT Fc␥RIIb1, WT Fc␥RIIa, Fc␥RIIb⌬cyt, and Fc␥RIIa⌬cyt were treated with sulfo-NHS-SS-biotin and then cultured in the presence of A488-labeled RTX to stimulate internalization of the Fc␥R. Following treatment with MESNa to remove cell surface biotin, cells were returned to 37°C to allow recycling of proteins to the cell surface, followed by a second treatment with MESNa (Fig. 10). Any decrease in the level of Fc␥RIIa/b detected by Western blotting after the second MESNa treatment represents an increase in cell surfaceaccessible protein caused by recycling.
In cells expressing WT Fc␥RIIb1 and Fc␥RIIa, there was a reduction in the level of Fc␥R detected after 2 h prior to MESNa treatment in response to RTX (Fig. 10, A and B), consistent with

Fc␥R Augment Internalization of RTX-ligated CD20
FEBRUARY 27, 2015 • VOLUME 290 • NUMBER 9 degradation of the receptors after internalization (Fig. 5). This coincided with an increase in the level of the lower molecular mass form of the receptor, suggesting continued cleavage following internalization. There was a further decrease in both WT Fc␥RIIb1 and Fc␥RIIa after MESNa treatment, suggesting that a proportion of both receptors were recycled back to the cell surface. Interestingly, the lower molecular mass form of the receptors appeared to be preferentially recycled compared with the higher molecular mass band (Fig. 10A), possibly accounting for the low levels present in untreated cells (Fig. 8, A and C). Similar to the WT receptors, there was a decrease in the level of Fc␥RIIb⌬cyt after 2 h prior to MESNa treatment in response to RTX (Fig. 10, C and D), consistent with degradation of the receptor after internalization (Fig. 7). In contrast, the level of Fc␥RIIa⌬cyt was maintained over time. After treatment with MESNa, there was a decrease in the level of both Fc␥RIIb⌬cyt and Fc␥RIIa⌬cyt, suggesting recycling of both receptors to the cell surface independently of the cytoplasmic domain (Fig. 10, C and D). Once again, there was little difference in the proportion of recycled Fc␥R between cells expressing Fc␥RIIa and Fc␥RIIb. Recycling of CD22 was also measured in cells as a positive control (Fig. 10E), which is constitutively endocytosed and recycled (20), illustrating the validity of our assay.
The lack of a large difference in the rate of internalization and recycling between Fc␥RIIa and Fc␥RIIb in response to RTX as measured using reversible biotinylation led us to theorize that in contrast to divergent sorting of the CD20⅐RTX⅐Fc␥RIIa and CD20⅐RTX⅐Fc␥RIIb trimeric complexes after internalization, Fc␥RIIa may be internalized and traffic to lysosomes independently of RTX-ligated CD20 after interaction within the plasma membrane. To investigate this theory, we treated Ramos cells transfected with WT Fc␥RIIb1, WT Fc␥RIIa, Fc␥RIIb⌬cyt, and Fc␥RIIa⌬cyt with A488-labeled RTX. After 1 and 5 h, cells were fixed, permeabilized, and stained with A647-labeled AT10 F(abЈ) 2 and biotinylated LAMP-1 followed by streptavidin-labeled A547 to follow the trafficking of RTX-ligated CD20, Fc␥RII, and lysosomes, respectively, by confocal microscopy ( Fig. 11). At both the 1-and 5-h time points, RTX staining was highly punctate in cells transfected with WT, and Fc␥RIIb⌬cyt as has been demonstrated previously in primary CLL cells expressing Fc␥RIIb (7,8). AT10 F(abЈ) 2 staining was similarly punctate, with all Fc␥RIIb completely co-localizing with RTX, suggesting close interaction between the two, as demonstrated previously (8). After 5 h, there was also some co-localization between RTX⅐AT10 F(abЈ) 2 and LAMP-1 staining, consistent with CD20, RTX, and Fc␥RIIb being internalized as a trimeric complex and trafficking to lysosomes.
In contrast to cells transfected with Fc␥RIIb, RTX staining was less punctate and more diffuse in cells transfected with WT and Fc␥RIIa⌬cyt (Fig. 11). There was some co-localization between RTX and AT10 F(abЈ) 2 in cells transfected with WT Fc␥RIIa after 1 and 5 h, suggesting the occurrence of antibody bipolar bridging. However, a large degree of the AT10 F(abЈ) 2 staining was co-localized with LAMP-1 after 1 h, with almost complete co-localization by 5 h. RTX staining was absent from many of the areas in which AT10 F(abЈ) 2 and LAMP-1 were co-localized, particularly after 5 h, suggesting that WT Fc␥RIIa may traffic to the lysosomes independently of RTX, which remains in the plasma membrane. In cells expressing Fc␥RIIa⌬cyt, the majority of the AT10 F(abЈ) 2 staining co-localized with RTX in the plasma membrane. Intensity of the AT10 F(abЈ) 2 staining was reduced, consistent with the rapid reduction in total Fc␥RIIa⌬cyt as measured by Western blotting (Fig. 7A). The intensity of AT10 F(abЈ) 2 staining was increased in Fc␥RIIa⌬cyt-expressing cells after 5 h (Fig. 11), consistent with increasing levels of total Fc␥RIIa (Fig. 7A). However, AT10 F(abЈ) 2 remained co-localized with RTX in these cells, with little co-localization with LAMP-1.

DISCUSSION
We previously observed that most mAb directed at receptors expressed on the surface of B cells interact with and activate Fc␥RIIb expressed in cis via antibody bipolar bridging, with the level of activation related to the amount of mAb bound to FIGURE 11. Fc␥RIIa traffics to the lysosomes independently of RTX-ligated CD20. Ramos cells transfected with WT Fc␥RIIb1, WT Fc␥RIIa, Fc␥RIIb⌬cyt, and Fc␥RIIa⌬cyt were cultured with 5 g/ml A488-labeled RTX. After 1 (A) or 5 (B) h, cells were fixed, permeabilized, and stained with A647-labeled AT10 F(abЈ) 2 to label Fc␥RII and biotin-conjugated LAMP-1 followed by A547-labeled streptavidin to label lysosomes. the cell surface. However, we saw a lack of correlation between the ability of mAb to activate Fc␥RIIb and the rate of internalization of the receptor⅐mAb complex (10), with the majority of mAb unaffected by the presence of Fc␥RIIb. This suggested that activation of Fc␥R was insufficient to augment internalization, leading us to question whether phosphorylation of the ITIM was required for Fc␥RIIb-augmented internalization of CD20 in response to type I anti-CD20 mAb ligation.
Using truncated variants of Fc␥RIIb, we have demonstrated here that the cytoplasmic domain is not required for mediating this activity, which explains our previous data showing no difference in the ability of the b1 and b2 isoforms of Fc␥RII to augment internalization of mAb-ligated CD20 (10). These data clearly indicate that Fc␥RIIb functions differently in augmenting the internalization of CD20 compared with endocytosis of ahIgG.
The finding that active signaling via the ITIM was not required for Fc␥RIIb-mediated internalization of mAb-ligated CD20 led us to consider whether ITAM-containing activatory Fc␥R could also promote internalization. Transient expression of Fc␥RI and Fc␥RIIIa on Raji cells demonstrated that these activatory Fc␥R augmented internalization of CD20 in response to RTX. We also found that expression of the activatory receptor Fc␥RIIa augmented internalization of RTX-ligated CD20, but at a much slower rate than Fc␥RIIb. This may help to explain the heterogeneity observed in the rate of RTX-mediated internalization observed between patients with CLL (8,10). There is a correlation between the rate of internalization of RTX-ligated CD20 and cell surface Fc␥RII expression as measured by staining with the pan Fc␥RII mAb AT10, but there is still substantial variation between patients that express the receptor at equivalent levels (8). AT10 binds to both Fc␥RIIa and Fc␥RIIb, and because both Fc␥RIIa and Fc␥RIIb may be expressed on CLL cells (21), expression of Fc␥RIIa would be predicted to mediate a slower rate of internalization than the equivalent expression of Fc␥RIIb.
We initially assumed that divergence in the cytoplasmic domains would be responsible for the different rates of CD20 internalization observed between cells expressing Fc␥RIIa and Fc␥RIIb in response to RTX ligation. This assumption was based on studies demonstrating the importance of the cytoplasmic domain in intracellular sorting of Fc␥RIIa and Fc␥RIIb (22) and in determining the ability of the b1 and b2 isoforms of Fc␥RIIb to mediate endocytosis of immune complexes (16). However, all these studies utilized immune complexes in the form of ahIgG to activate the Fc␥R. Previously, we surmised that the interaction between type I anti-CD20 mAb and Fc␥R via antibody bipolar bridging was analogous to that mediated by immune complexes. However, the results of this study suggest that this is not the case. Consistent with this conclusion, we found that the difference in rate of CD20 internalization mediated by cells expressing Fc␥RIIa and Fc␥RIIb was independent of differences between the Fc␥R cytoplasmic domains. We then focused on whether differences in the transmembrane domains were responsible for the slower rate of internalization mediated by Fc␥RIIa. Although there are only three adjacent amino acids that differ between the transmembrane regions of the two receptors, mutations within the transmembrane region of both Fc␥RIIa and Fc␥RIIb have been associated with the ability of the receptors to translocate to lipid rafts (28,29). However, although the differential ability to enter lipid rafts may have been involved in the reduced ability of Fc␥RIIa to augment internalization of RTX-ligated CD20, the exchange of transmembrane domains between Fc␥RIIa and Fc␥RIIb suggested that differences between the two receptors in the extracellular domains were responsible.
Fc␥RIIa⌬cyt was rapidly degraded upon RTX treatment, suggesting that it was internalized independently of RTX-ligated CD20, which was internalized relatively slowly. This conclusion is supported by the high degree of Fc␥RIIa⅐lysosomal co-localization in cells transfected with WT Fc␥RIIa, suggesting that despite the slower rate of degradation, WT Fc␥RIIa traffics rapidly to the lysosomes upon engagement with RTX, whereas RTX-ligated CD20 remains in the plasma membrane. In contrast, WT and Fc␥RIIb⌬cyt remained colocalized with RTX, consistent with it remaining as a trimeric CD20⅐RTX⅐Fc␥RIIb complex as suggested previously (8). Detachment and internalization of Fc␥RIIa from the CD20⅐RTX complex may explain the slower rate of CD20 internalization mediated by this receptor.
It is unclear why the rate of degradation was so varied between the WT, truncated and transmembrane mutant forms of Fc␥RIIa in response to RTX stimulation. One possibility is that they were all internalized at approximately the same rate upon interaction with RTX-ligated CD20 but were degraded at different rates within the lysosome. This would explain why WT Fc␥RIIa was detectable within lysosomes at 1 h, because of the continued presence of intact protein, whereas Fc␥RIIa⌬cyt, which was degraded much quicker, was only detectable in the plasma membrane. This is supported by the reversible biotinylation experiments demonstrating only minor differences in the rate of internalization between the WT and truncated forms of Fc␥RIIa in response to RTX.
It could be argued that the presence of significant amounts of only WT Fc␥RIIa within the lysosomes was due to divergent sorting of Fc␥RIIa and Fc␥RIIb by the mechanism described by Zhang and Booth (22) subsequent to internalization of trimeric CD20⅐RTX⅐Fc␥RII complexes and not due to independent internalization of the Fc␥R. However, several lines of evidence argue against this possibility. First, Zhang and Booth (22) described co-localization of both ahIgG and Fc␥RIIa within lysosomes, suggesting that they were internalized as a dimeric complex. In contrast, we observed WT Fc␥RIIa in lysosomes without co-localized RTX, suggesting that it was internalized independently of RTX-ligated CD20. Second, the rapid rate of degradation of Fc␥RIIa⌬cyt contrasts with the slow rate of RTX internalization in these cells. These data suggest that rapid internalization of Fc␥RIIa⌬cyt occurred, despite it being unobservable within lysosomes by confocal microscopy. Third, the differences between Fc␥RIIa and Fc␥RIIb observed by Zhang and Booth (22) were due to differences between the cytoplasmic or transmembrane domains of the two Fc␥R, in contrast to our data, in which the differences were due to variation between the extracellular domains. Finally, using a reversible biotinylation approach, we observed little difference in internalization and recycling between Fc␥RIIa and Fc␥RIIb in response to RTX treatment.
Given the high degree of homology with Fc␥RIIb, and the higher affinity of Fc␥RIIa for IgG1, we initially predicted that Fc␥RIIa would promote a faster rate of CD20 internalization than cells expressing Fc␥RIIb in response to RTX ligation. Fc␥R affinities were determined by surface plasmon resonance using Fc␥R immobilized on a solid surface with monomeric or ahIgG added in solution (27). However, this experimental set up may be more representative of trans-interactions between Fc␥R on the surface of cells and IgG in solution and does not necessarily represent cis interactions between mAb and Fc␥R interacting within a phospholipid membrane on the same cell surface. Although this is pure speculation, it is possible that Fc␥RIIa may have a lower affinity for IgG present in cis. Lower affinity might explain why Fc␥RIIa detaches from RTX-ligated CD20 prior to internalization and degradation, resulting in the slower rate of CD20 internalization than cells expressing Fc␥RIIb.
The observation that active signaling via Fc␥R is not required to augment endocytosis of the CD20⅐mAb⅐Fc␥R complex suggests that Fc␥R may play a more physical/structural role in the process that is absent in response to ligation of cell surface receptors by most other mAb. We are now investigating alternative mechanisms by which antibody bipolar bridging may augment internalization of CD20 that are independent of Fc␥Rmediated signaling. We have observed here and in previous studies (8,10) that RTX-ligated CD20 is internalized faster than tositumomab-ligated CD20 even in B cells that do not express Fc␥R, suggesting that the type I mAb inherently initiate endocytosis independently of Fc␥R interaction. The function of Fc␥R expression may be to augment this process by binding to and recruiting RTX-ligated CD20 to sites within the membrane where endocytosis has already been initiated. Thus, Fc␥R may increase the amount of CD20 that is internalized per endocytic event, as opposed to increasing the rate of endocytosis per se. If Fc␥RIIa has a lower affinity for RTX than Fc␥RIIb within the plasma membrane, it may be less able to recruit RTX-ligated CD20 to sites of endocytosis, resulting in less endocytosis of the receptor overall. This may also explain why RTX staining remains more diffuse in cells expressing Fc␥RIIa, compared with Fc␥RIIb.
Another potential mechanism involves lipid rafts. The raft redistributing properties of CD20 were first described by Deans and co-workers (30 -32), and we subsequently demonstrated that type I, but not type II, anti-CD20 mAb cause CD20 to translocate to these domains (3). This activity also corresponds with their ability to mediate internalization of the CD20⅐ mAb⅐Fc␥RIIb complex. Fc␥RIIb can be found localized to both raft and nonraft regions of the plasma membrane in untreated B cells, but the proportion of raft-associated receptor is increased upon co-engagement with the BCR (33). Redistribution of CD20 and Fc␥RIIb to lipid rafts may initiate endocytosis, as has been observed for other receptor⅐ligand complexes and viruses (34). Although most mAb interact with Fc␥RIIb (10), it is possible that only interaction between mAb and Fc␥RIIb within lipid rafts is sufficient to initiate endocytosis of the mAb⅐receptor complex and that interaction in nonraft regions may be insufficient to augment internalization. This may explain why type II anti-CD20 mAb do not mediate internalization of CD20 and why tositumomab stimulates phosphorylation of Fc␥RIIb to a lesser extent than RTX (10). By failing to mediate redistribution of CD20 to lipid rafts, interaction with Fc␥RIIb may be restricted to nonraft regions, whereas RTX may interact with both raft and nonraft fractions. Fc␥RIIa also translocates to lipid rafts upon cross-linking (35), so if this mechanism is important, it is unclear why Fc␥RIIa mediates a slower rate of CD20 internalization than Fc␥RIIb. The rate and extent of Fc␥RIIa and Fc␥RIIb to redistribute to lipid rafts have not been directly compared, so it is possible that there is a difference between these two Fc␥R. Alternatively, if Fc␥RIIa binds to RTX with lower affinity in the plasma membrane, it may detach from RTX after recruitment to raft fractions or partition to different raft microdomains than CD20, explaining why the receptor is internalized independently of the RTX-ligated receptor. We are currently investigating the importance of raft redistribution of both CD20 and Fc␥RIIb in promoting the internalization of CD20 in response to type I anti-CD20 mAb ligation.
In conclusion, we have demonstrated that both inhibitory and activatory Fc␥R can augment internalization of CD20 in response to ligation by type I anti-CD20 mAb, independent of signaling via the cytoplasmic domain. This verifies our previous conclusions that screening of potential therapeutic mAb for their ability to activate Fc␥R expressed in cis is insufficient to predict whether a mAb will remain cell surface-localized. Alternatively, Fc␥R may play a structural role in augmenting internalization of type I anti-CD20 mAb-ligated CD20, possibly involving recruitment of CD20 to sites of endocytosis or via redistribution of CD20 to lipid raft domains.