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TOSO/FAIM3 has recently been identified as the long sought after Fc receptor for IgM (FcµR). FcµR is expressed on human CD19+ B cells, CD4+/CD8+ T cells, and CD56+/CD3− NK cells, and has been shown to be overexpressed in chronic lymphocytic leukemia (CLL) cells. CLL is a malignancy of mature IgM+ B lymphocytes that display features of polyreactive, partially anergized B cells related to memory B cells. Herein, we report that FcµR is O-glycosylated in its extracellular domain and identify the major sites of O-glycosylation. By employing immunofluorescence confocal microscopy, we found that FcµR localized to the cell membrane, but also found that large pools of FcµR accumulate in the trans-Golgi network. Aggregation of FcµR on CLL cells by IgM prompted rapid internalization of both IgM and FcµR, reaching half maximal internalization of cell bound IgM within one minute. Upon internalization, FcµR transported IgM through the endocytic pathway to the lysosome, where it was degraded. Using a series of FcµR deletion mutants, we identified a proline-rich domain essential for cell surface expression of FcµR and a second domain, containing a YXXΦ motif, that controls internalization. While it has been reported, that BCR activation increases FcµR expression; we found that activation of TLRs strongly downregulated FcµR at both the mRNA and protein levels. Through internalization of IgM bound immune complexes, FcµR may play a role in immune surveillance and contribute to B-cell activation. In addition, FcµR deserves study as a potential pathway for the delivery of therapeutic antibody-drug conjugates into CLL cells.
Chronic lymphocytic leukemia (CLL) is an incurable malignancy of IgM+, IgD+ mature B lymphocytes (1). CLL can be divided into two main subgroups with distinct clinical behavior based on the presence or absence of acquired somatic mutations in the IGHV gene expressed by the leukemic B cells (1). Patients whose tumor cells express an IGHV gene carrying somatic mutations (M-CLL) have a more indolent disease and longer overall survival than do patients whose tumors express an IGHV gene in germline or “unmutated” configuration (UM-CLL). The cell of origin is still unclear; however, the BCR of many CLL cells share characteristics with natural antibody producing B cells that recognize microbial antigens as well as self-antigens, suggesting that antigen selection plays a role in the ontogeny of CLL. Indeed, CLL cells display many characteristics of antigen experienced B-cells (reviewed in (1)), including a skewed use of immunoglobulin heavy chain (IGHV) genes, expression of BCRs with structurally very similar antigen-binding pockets, cell surface expression of activation markers, and a characteristic gene expression profile similar to memory B cells (2). In support of antigen signaling as a key pathway propagating the clonal expansion of CLL, we recently demonstrated BCR activation on CLL cells in the lymph node microenvironment (3).
Depending on the immunoglobulin isotype specificity and cellular expression pattern, FcRs play a role in a variety of cellular functions, including, phagocytosis, antibody dependent cellular cytotoxicity, antibody secretion, antigen presentation, mast cell degranulation, and cell proliferation (4, 5). Distinct FcRs have been identified for IgG (FcγRI, FcγRII, FcγRIII), IgE (FcεR), and IgA (FcαR); however, until recently no FcR for IgM had been identified (6, 7). TOSO was first described as an inhibitor of FAS-mediated apoptosis in T cells; however (8), it has newly been show to be the long-sought after FcR for IgM and consequently has been renamed FcµR (9). Recent studies have shown that FcµR does not inhibit FAS-mediated apoptosis directly, but rather, an inhibitory effect was only apparent when an anti-FAS antibody of the IgM isotype was used, but not when antibodies of the IgG isotype were used (9). FcµR is a transmembrane protein containing an extracellular Ig-like domain with homology to two other FcRs that can bind IgM, Fcα/µR and the polymeric Ig receptor (pIgR), both of which can bind IgM and IgA but are not expressed on T or B cells. In contrast to other FcRs, FcµR is expressed on B and T cells but not on phagocytes or myeloid cells and selectively binds IgM immunoglobulin with very high affinity.
We, and others, became interested in TOSO/FcµR because of the observation that it is selectively overexpressed in CLL as compared to other B-cell malignancies (10–12). Although its function on CLL has not been defined, it has been shown that B cell receptor stimulation increases FcµR expression on CLL cells (9, 11). FcµR expression could thus be a reflection of ongoing BCR activation of CLL cells. Consistent with this view is the relatively higher FcµR expression on CLL cells in patients with a more aggressive disease course, specifically in the UM-CLL subtype, and in patients with high leukemic cell count, advanced clinical stage, and need for chemotherapy. In addition, relatively higher FcµR mRNA levels have also been detected in normal memory B cells. Thus, FcµR expression in CLL is also consistent with the malignant clone originating from a memory B cell.
In the present study, we extend the characterization of FcµR, and determine its subcellular localization and trafficking. We demonstrate that FcµR efficiently internalizes IgM and transports it through the endocytic pathway to the lysosome for degradation. The importance of these findings is twofold. First, it indicates a possible role for FcµR in immune surveillance and B-cell activation. Second, it suggests that FcµR could be a valuable therapeutic target for the treatment of CLL.
With written informed consent samples were collected from treatment naïve CLL patients. Peripheral blood mononuclear cells (PBMC) were isolated by gradient centrifugation (Lymphocyte Separation Media, MP Biomedicals, Irvine, CA) and cultured in RPMI 1640 medium (Gibco, Long Island, NY) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 2 mM L-glutamine, 1000 units/mL penicillin G and 100 µg/mL streptomycin (Gibco). CD19+ selection was performed by using CD19 microbeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instruction. The IGHV mutational status of the CLL cells was determined by reverse transcription-PCR (RT-PCR) followed by DNA sequencing as previously described (13). Lymphoma cell lines were grown in RPMI and HeLa cells in DMEM (Lonza, Walkersville, MD).
Anti-FcµR mAb was purchased from Abnova Corporation (Taiwan, China) and ATPase p97 from Fitzgerald Industries International (Concord, MA). Horseradish peroxidase linked anti-mouse secondary antibody was purchased from Amersham Biosciences (Little Chalfont, England). Antibodies used for immunofluorescence studies were as follows: rabbit polyclonal anti-TGN46 (Novus Biologicals, Littleton, CO), Alexa 568 conjugate of transferrin from human serum (TF-A568, Invitrogen, Carlsbad, CA) and rabbit polyclonal anti-LAMP-1 (Abcam, Cambridge, MA). Unconjugated primary antibodies were visualized with goat anti-mouse Alexa 488 (Invitrogen), donkey anti-rabbit Texas Red and donkey anti-rabbit Cy5 (Jackson ImmunoResearch, West Grove, PA). DNA was labeled with Hoechst 3358 (Invitrogen).
Benzyl-2-acetamido-2-deoxy-a-D-galactopyranoside (used at 2.5 mM) and actinomycin D (used at 10 µM) were purchased from Calbiochem (San Diego, CA). Tunicamycin (used at 5 µg/ml), phenylarsine oxide (used at 30 µM), and chloroquine (used at 20 µM) were purchased from Sigma (St. Louis, MO). Bortezomib (used at 10 nM) was purchased from Millennium pharmaceuticals (Cambridge, MA). Single-strand, phosphorothioate-modified CpG-oligodeoxynucleotides (CpG-ODN) were synthesized by Integrated DNA Technologies (IDT, Coralville, IA). The sequence of CpG53 (5’-TCGTCGCTGTCTCCG-3’) has been reported. CpG-ODN was used at 1 µM. Imiquimod was purchased from Invivogen (San Diego, CA).
The prediction of N- and O-glycosylation sites was performed by analyzing the full length sequence of FcµR on the NetNGlyc1.0 (www.cbs.dtu.dk/services/netNglyc) and NetOGlyc3.1 (www.cbs.dtu.dk/services/netNglyc) server (Central for Biological Sequence Analysis, Technical University of Denmark, Lyngby, Denmark) (14). The prediction of N-glycosylation sites was based on identification of Asn-Xaa-Ser/Thr consensus sequences.
FcµR cDNA clone (NM_005449.3) was obtained from Origene Technologies, Inc. (Rockville, MD). It was amplified with forward primer 5’-TCTAGAAAGCTTGCCACCATGGACTTCTGGCTTTGGCCAC-3’ and reverse primer 5’-TCTAGAGAATTCCTACTTATCGTCGTCATCCTTGTAATCCGCCGCGGCAGGAACATTGATGTAGTCATC-3’, in order to add a Flag tag at the C-terminus, digested with HindIII and EcoRI and cloned into pcDNA3.1 (Invitrogen). Point mutations of the putative O-glycosylated residues and truncation mutants of the cytoplasmic portion of FcµR were generated using the QuickChange site-directed mutagenesis method according to the manufacturer protocol (Stratagene, Santa Clara, CA). pCNA3.1-FcµR-Flag was used as a template. The primers used to obtain O-glycosylation mutants are as follows: G1, 5’-TCTTCCAAATTCGTAACCAGAGTTGCCGCACCAGCTCAAAG-3’; G2, 5’-AGTTCACCACGCCGCCCCCGCCGCCCAAATCGCCCACCGCCC-3’; G3, 5’-GGTGACAAGCCCCGAGCCTTCCTGCCAT-3’. Altered nucleotides are underlined. The primers used for the truncation mutants are as follows: D1, sense 5’-GGGCTGGTGGTGGGAGCGGACGCT-3’, antisense 5’-AGCGTCCGCTCCCACCACCAGCCC-3’; D2, sense, 5’-CGCGGCGCGCTCGTTCTCTGAAGACC-3’, antisense 5’-GGTCTTCAGAGAACGAGCGCGCCGCG-3’; D3, sense, 5’-CCTGGCTCCATGCCCCAGCGGCGGAT-3’, antisense, 5’-ATCCGCCGCTGGGGCATGGAGCCAGG-3’. All constructs were confirmed by DNA sequencing.
Total RNA was isolated from cell pellets using the RNeasy mini-kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Total RNA was reversed transcribed into cDNA. FcµR transcript expression level was assessed by quantitative RT-PCR using the ABI Prism 7700 sequence detector (Applied Biosystems, Carlsbad, CA). Transcript levels were normalized to the housekeeping gene beta-2-microglobulin and compared against a standard curve. Primers used to analyze FcµR in this study have been previously described (15). The primer/probe set for beta-2-microglobulin was purchased from Applied Biosystems (TaqMan Gene Expression Assays). All samples were run in triplicate.
10–20×106 cells were harvested and lysed in RIPA buffer (50–200 µL) containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, sodium deoxycholate 0.25%, NP40 1%, protease and phosphatase inhibitors (Roche Applied Science, Indianapolis, IN). Protein concentrations were determined by Bradford assay. 50 µg of proteins (cell lines) and 25–60 µg of proteins (primary cells) were separated on a SDS-acrylamide gel (4–12%), transferred to PVDF membranes (Invitrogen) and subsequently subjected to immunoblot analysis using indicated antibodies. Membranes were developed with enhanced chemiluminescence substrate (Super Signal, Pierce, Rockford, IL), and the signal was revealed using a Fujifilm LAS-4000 imager and quantified by using Multi Gauge V3.0 software (Fujifilm Life Science, Tokyo, Japan).
To detect FcµR expression, 2×105 CLL cells were incubated with 1 µg of anti-FcµR mAb antibody or isotype control. Samples were washed with phosphate-buffered saline (PBS)/1% bovine serum albumin (BSA) and incubated with a 1:100 dilution (predetermined) of GAM-PE (Immunotech, France). In addition, cells were labeled using an anti-CD19 FITC Ab to enable gating of B cells. Samples were acquired using the FACSCanto flow cytometer (BD Biosciences). Data were analyzed with the FACS evaluation program FlowJo (version 7.5, Tree Star, Inc., Stanford, CA). The mean fluorescence intensity (MFI) was used for surface marker expression.
IgM internalization was assessed by flow cytometry. Briefly, CLL cells were incubated at 4°C for 30 min with IgM-biotin. Cells were then washed and maintained on ice as a control for maximal binding or incubated at 37°C for the indicated times to allow internalization. After incubation at 37°C, cells were placed on ice, washed and labeled using DyLight488-streptavidin conjugate (Jackson ImmunoResearch) to reveal the remaining IgM present on the cell surface of CLL cell. CD19+ B cells were gated for analysis. Flow cytometry and analysis was done as described.
HeLa cells were grown on coverslips and transfected with the indicated constructs using Fugene 6 (Roche) according to the manufacturer’s protocol. Non adherent cells (Mino and CD19+ selected CLL cells) were plated on alcian blue (SIGMA) treated coverslips and allowed to adhere for 15 min at 37°C. Cells were fixed for 15 min at RT in 3% paraformaldehyde (Electron Microscopy Sciences) then washed with PBS and incubated in Staining Buffer (0.05% saponin, 10mM glycine, 5% FBS, PBS) for 15 min. In the case of CLL cells, permeabilization was performed in the presence of 10% human serum AB (Gemini Bio-Products, West Sacramento, CA) to reduce non specific protein binding. Cells were incubated with primary antibody for 1 hour, washed twice with PBS and then incubated with secondary antibody for 1 hour. The absence of cross-reactivity for the donkey anti-rabbit and goat anti-mouse secondary antibodies were confirmed (supplementary Fig. 1A and B). Each antibody was diluted with Staining Buffer. Following immunostaining, cells were washed twice and labeled with 1 µg/mL of Hoechst 3358 diluted in Staining Buffer for 5 min at RT. Coverslips were washed twice and mounted on slide with Fluoromount-G (SouthernBiotech). For mAb internalization experiments, cells were incubated for 15 min at 4°C with anti-FcµR mAb, washed with ice-cold PBS and incubated at 37°C with DMEM media supplemented with human transferrin conjugated to Alexa 568 (TF-A568). After the indicated incubation times, cells were fixed with 3% paraformaldehyde and stained. For IgM uptake assays, transfected HeLa cells were incubated with biotin-conjugated IgM (Jackson ImmunoResearch) for 30 min at 4°C, washed twice with ice-cold PBS and incubated with DyLight488-streptavidin conjugate for an additional 30 min at 4°C. After two washes in PBS, cells were incubated for the indicated times at 37°C with DMEM media supplemented with TF-A568, fixed with 3% paraformaldehyde and stained. Images were acquired using a Leica TCS SP5 laser scanning confocal microscope (LAS AF software) using the HCX PLAPO 63X objective (numerical aperture: 1.4). Images were processed using Adobe Photoshop using only level and contrast adjustment (without manipulating the gamma function). Each group of image was processed and analyzed using the same settings.
We analyzed FcµR protein expression in normal and malignant B cells by Western blotting (Fig. 1A). PBMC were collected from healthy individuals, patients with CLL, leukemic mantle cell lymphoma (MCL), and leukemic follicular lymphoma (FL). Consistent with previous studies (10–12), CLL cells showed increased expression of FcµR compared to normal B cells (Fig. 1A, left and middle panel). In MCL, FcµR was not expressed or was only present at low levels, and in a case of leukemic follicular lymphoma FcµR expression was similar to normal B cells (Fig. 1A, middle panel). Of the MCL cell lines tested only Mino expressed FcµR, while HBL-2, and Jeko were negative (Fig. 1A, right panel). Several other non-Hodgkin lymphoma cell lines likewise did not express FcµR (data not shown). To further confirm the overexpression of FcµR in CLL cells compared to normal B cells, we measured FcµR protein expression by flow cytometry (Fig. 1B). The results indicate that CLL cells express high levels of FcµR on the cell surface in comparison to normal B cells.
The predicted molecular weight of FcµR is 41 kDa. However, Western-blots stained with an anti-FcµR mAb displayed additional bands of higher molecular weight that often made up the bulk of FcµR expression (Fig. 1A). In CLL samples, FcµR mAb identified one major band around 60 kDa, a series of fainter migrating species ranging from 60 to 41 kDa, and a band at 41 kDa likely corresponding to the predicted molecular weight of the protein. In contrast, normal B cells predominantly expressed the 60 kDa form. These observations indicate that FcµR is subject to posttranslational modifications with some apparent differences between CLL cells and normal B cells.
Cellular and membrane proteins are often modified by glycosylation, which depending on the type of linkage between the oligosaccharide moiety and the protein backbone is identified as N- or O-glycosylation. Computer modeling revealed the absence of canonical N-glycosylation sites but identified 8 residues (T164, T165 S178, S179, T181, T182, T185 T203) in the extracellular domain of FcµR that could be sites for O-glycosylation (Fig. 1C) (16). To determine whether FcµR is indeed O-glycosylated, we first used a general inhibitor of O-glycosylation, benzyl-N-acetyl-α-galactosaminide (BAG) that competitively inhibits O-glycan chain extension (17, 18). HeLa cells that do not express FcµR were transfected with an FcµR expression vector or empty vector control. 24 hours after transfection, HeLa cells were treated with BAG or the N-glycosylation inhibitor tunicamycin for an additional 24 hours. The cells were then lysed and FcµR expression was analyzed using Western blotting. Transfected HeLa cells expressed FcµR protein with a size distribution similar to what was observed for the endogenous protein in CLL cells; one band at the predicted molecular weight of 41 kDa, and further bands of higher molecular weight (Fig. 1D). Treatment with BAG induced a complete disappearance of the high molecular weight bands. In contrast, treatment with tunicamycin, had no effect on the mobility of FcµR on SDS-PAGE. Next, we tested whether the endogenous FcµR is O-glycosylated in Mino and CLL cells (Fig. 1E). Whereas untreated cells contained both 41 and 60 kDa forms of FcµR, treatment with BAG resulted in the disappearance of the 60 kDa form and persistence of a band in the 45 kDa range. In contrast, tunicamycin treatment had no effect on the size distribution of FcµR. Why the lower band migrates slightly higher after BAG treatment is not defined but likely indicates reduced but not fully absent glycosylation. Also, overall expression of FcµR was reduced in BAG treated cells, consistent with disturbed post-translational processing and accelerated degradation of the aberrant protein. Taken together, these results indicate that FcµR is heavily O- but not N-glycosylated in Mino and CLL cells.
Next, we investigated the structural requirements for O-glycosylation using a set of FcµR expression constructs with engineered mutations of the major predicted O-glycosylation sites. Threonines (T164, T165) in G1, serines/threonines (S178, S179, T181, T182, T185) in G2, and the threonine (T203) in G3 were replaced by alanines (Fig. 2A). These constructs were transfected into HeLa cells and protein expression was analyzed by Western blotting. The G1 and G2 mutants resulted in a significant diminution of the higher molecular weight FcµR species (glycosylated FcµR) (Fig. 2B). The mutant G3 did not affect the molecular size of FcµR suggesting that this threonine residue is not glycosylated (not shown). Double mutation in both the G1 and G2 sites virtually abolished apparent glycosylation.
O-glycosylation can be involved in the control of a protein’s subcellular localization and trafficking and plays an important role in the expression of immnuoreceptors such as DR6 and others (19). As a previous study has shown that the mouse homologue of FcµR is mainly localized to the cell surface, we sought to determine whether differences in glycosylation affect its subcellular localization (20). To do this, we visualized FcµR expression in transfected HeLa cells by immunofluorescence (Fig. 2C). Cells transfected with the Flag-tagged wild-type construct showed FcµR cell surface expression as expected. Surprisingly, we also detected substantial protein accumulation in an intracellular compartment and a punctuate staining in the cytoplasm of transfected cells. Indeed, the majority of expressed FcµR appeared to be localized intracellularly. Mutations of the glycosylation sites G1 and G3 had no effect on the subcellular expression of FcµR. In contrast, mutation of the G2 site resulted in intracellular retention of FcµR and complete absence of cell surface expression. To confirm that inhibition of O-glycosylation inhibits transport of FcµR to the cell surface in primary cells, CLL cells were treated for 48 hours with BAG and expression of FcµR was analyzed by flow cytometry (Fig. 2D). Treatment of two primary CLL samples with BAG decreased the cell surface levels of FcµR. Taken together, these results show that O-glycosylation is critical for normal trafficking of FcµR to the cell surface. Furthermore, we also discovered that FcµR accumulates in large quantities in an intracellular compartment.
Next, we sought to identify the subcellular location where the bulk of FcµR was found to be expressed. To this end, HeLa cells transfected with Flag-tagged wild-type FcµR were analyzed by confocal microscopy using antibodies against select markers of intracellular organelles. FcµR colocalized extensively with TGN46 (Fig. 3A, magnification Z1 and supplementary Fig. 1D), a marker protein of the trans-Golgi network (TGN) (21), but not or less so with markers for early endosomes (transferrin receptor, TFR) or late endosomes (LAMP-1) (Fig. 3B, magnification Z2). As a control, we confirmed the subcellular localization of FcµR to the TGN using an anti-Flag antibody directed against the fused peptide tag (supplementary Fig. 1C).
To rule out that overexpression of FcµR contributed to accumulation in the TGN, we confirmed the location of endogenous FcµR in the MCL cell line Mino and in CD19+ selected CLL cells. The subcellular distribution of FcµR in Mino and in CLL cells was similar to that observed in transfected HeLa cells, thus we were able to confirm localization of endogenous FcµR to the TGN (Fig. 3C, D). By comparing permeabilized to non-permeabilized CLL cells we demonstrated that FcµR is primarily localized intracellularly (Fig. 3D). These results demonstrate for the first time in primary CLL cells that the bulk of FcµR protein resides intracellularly in the TGN and in small vesicles that are probably sorting endosomes.
One of the major functions of the TGN is to insure sorting of proteins destined for the plasma membrane, endosomal compartments or specialized secretory granules (22, 23); however, retrograde transport in the endocytic route to the TGN has also been characterized for several proteins (24). As shown in Fig. 3, we detected an accumulation of FcµR in the Golgi complex. In order to determine whether FcµR is stocked in the TGN and then delivered to the plasma membrane or recycled from the cell surface back to the TGN, we analyzed the internalization dynamics of FcµR by confocal microscopy. HeLa cells transiently transfected with FcµR were incubated for 15 minutes at 4°C with FcµR mAb to label cell surface protein. Cells were then rapidly warmed to 37°C in the presence of human transferrin conjugated to Alexa 568 (TF-A568) in order to monitor the internalization kinetics and to label the early endosomal compartment. The internalization was stopped at the indicated times by fixing cells with paraformaldehyde (PFA). At 4°C, FcµR staining was restricted to the cell surface, reflecting efficient binding of the mAb (Fig. 4A). After a 15 minute incubation at 37°C, a small fraction of FcµR-associated antibody was internalized and co-localized with transferrin-positive early endosomes (Fig. 4B, magnification Z1). After 45 minutes, the internalized FcµR mAb partially co-localized with LAMP-1, a specific marker of late endosomes/lysosomes, as indicated in the merged images (Fig. 4C, magnification Z2). By contrast, co-staining with a mAb against TGN46 did not reveal any translocation of internalized FcµR to the TGN (data not shown). Interestingly, even after 45 minutes, only a minor fraction of FcµR mAb had entered the endocytic pathway, suggesting that, in the absence of its natural ligand, basal FcµR recycling and degradation from the plasma membrane is slow. Thus, FcµR does not recycle from the plasma membrane to the TGN but, once internalized, follows the endocytic pathway.
Thus far, our studies have utilized only mAbs for the evaluation of FcµR localization and internalization. We next sought to investigate the effect of IgM binding to FcµR. We found that IgM was rapidly internalized into CLL cells (Fig. 5A). Using biotinylated IgM and DyLight488-streptavidin, we monitored IgM cell surface binding and determined the internalization kinetics by flow cytometry. Strikingly, almost 60% of cell surface bound IgM was internalized within a minute reaching complete internalization by 5 minutes (Fig. 5B). This observation indicates that, following ligand binding, FcµR is immediately internalized in contrast to its slow basal recycling. To confirm these results and further characterize the internalization, CLL cells were incubated at 37°C for 60 minutes in presence or absence of phenylarsine oxide (PAO), a well-characterized inhibitor of clathrin-dependent endocytosis (25, 26). Flow cytometric analysis showed that the addition of PAO reduced IgM internalization by more than half, indicating that IgM is at least partially internalized into CLL cells by a clathrin-dependent mechanism (Fig. 5C).
Following ligand binding and internalization, cell surface receptors may traffic to lysosomal vesicles for degradation or recycle back to the cell surface. In order to visualize internalization and trafficking of IgM, FcµR transfected HeLa cells were labeled with IgM-biotin, followed by DyLight488-streptavidin on ice before being warmed up to 37°C for the indicated times. As expected at 4°C, IgM-biotin/DyLight488-streptavidin complexes formed clusters at the cell surface of the cell (Fig. 5D). After 5 minutes at 37°C, transferrin and FcµR were both internalized, albeit into different vesicles. This observation implies that FcµR segregates in different membrane domains and was internalized into pre-endosomal vesicles distinct from the ones used by TFR. After 45 minutes, IgM was mainly found to be expressed in early endosomes where it now co-localized with transferrin; however, a fraction had already reached the lysosome, as identified by LAMP-1 staining. After 60 minutes, IgM staining was decreased and almost exclusively concentrated in the lysosome.
To characterize the structural requirements for rapid internalization of FcµR upon IgM binding, we inspected the 118-amino acid sequence of the FcµR cytoplasmic tail. Three canonical domains associated with trafficking and endocytosis were apparent: an arginine-rich domain, a proline-rich domain, and 3 YXXΦ motifs (where Φ is a bulky hydrophobic residue). The proline rich domain (PRD) is capable of binding Src Homology 3 domain (SH3) containing proteins (27). Such proteins are implicated in regulating protein localization, enzymatic activities, and multicomponent signaling complexes (28). Arginine-rich domains (ARD) are positively charged and can be crucial for the sorting function of flanking PRDs and can also be essential for certain protein-protein interactions (29, 30). Finally, YXXΦ motif are known to mediate internalization and lysosomal targeting of several transmembrane proteins including the TFR through recruitment of adaptor proteins (31, 32). In order to test whether any of these regions could be involved in the trafficking of FcµR, we generated a series of deletion mutants and transfected them into HeLa cells (Fig. 6A). As shown in Fig. 6B, IgM bound to FcµR expressing HeLa cells and the ligand receptor pair co-localized at the cell surface. As expected, following 45 minutes of internalization, FcµR and IgM localized primarily in intracellular structures and were partially degraded, consistent with trafficking to lysosome.
Deletion of the ARD domain (construct D1, Fig. 6) had no effect on IgM binding to the cell surface, indicating that FcµR expression and trafficking between TGN and cell surface was not impaired by this mutation. Deletion of the PRD domain (construct D2, Fig. 6) resulted in sequestration of FcµR within an intracellular compartment, possibly the TGN and its complete absence from cell surface, as shown by both IgM and anti-FcµR staining. However, it is possible that this phenotype is not entirely due to the absence of the PRD domain as a regulatory structure as it could also be a consequence of missfolding of the mutant protein. Deletion of the domain containing the YXXΦ motifs (construct D3, Fig. 6) did not affect FcµR expression or IgM binding at the cell surface but completely abolished its internalization. Thus, this domain is essential for FcµR dependent endocytosis of IgM from the cell surface.
IgM binds strongly to natural antigens and constitutes a first line of defense against encapsulated bacteria and viruses (33–35). Thus, in vivo IgM is often loaded with antigenic molecules that can then be internalized by FcµR expressing cells. As we have shown here, FcµR bound IgM is rapidly internalized and shuttled to the lysosome. There, certain Toll-like receptors (TLR), a component of the innate immune response, can be activated by pathogen derived molecules such as single strand RNA (binding to TLR7) or unmethylated DNA (binding to TLR9).
To investigate interactions between the TLR system and FcµR expression, we studied the effect of the TLR7 agonist imiquimod, and the TLR9 agonist CpG oligodeoxynucleotides (CpG-ODN) on FcµR expression. CLL cells were incubated with imiquimod and CpG-ODN and FcµR expression was assessed by Western blotting and quantitative real-time RT-PCR. Immunoblotting showed a striking downregulation of FcµR protein in CLL cells within 24 hours of exposure to either TLR ligand (Fig. 7A). Downregulation of FcµR protein was observed in all twelve CLL samples tested, albeit with some differences between the two major CLL subtypes. Interestingly, in the case of CpG-ODN, FcµR downregulation was significantly more pronounced in IGHV mutated patients (81% reduction in IGHV mutated versus 57% reduction in IGHV unmutated cases, p<0.05; Fig. 7B). In contrast, there were no differences between the two CLL subtypes in their response to imiquimod.
We next investigated whether FcµR downregulation occurs at the transcriptional level. FcµR mRNA expression was quantified by real-time PCR and normalized to beta-2-microgobulin. As shown in Fig. 7C, FcµR mRNA expression was also greatly reduced in mutated samples but less so in unmutated samples after 24 hours of treatment with CpG-ODN. Marked downregulation of FcµR mRNA in response to imiquimod was seen in both CLL subtypes and paralleled the effect observed on protein expression. To test whether the CpG-ODN triggered reduction in FcµR mRNA levels was due to a reduction in FcµR gene expression and/or a decrease in the half-life of FcµR transcripts, CLL cells were also treated with actinomycin D, a widely used transcriptional inhibitor. After both CpG-ODN and actinomycin D treatments, the half-life of FcµR mRNA was approximately 3 hours, indicating that TLR9 activation inhibits FcµR transcription (Fig. 7D).
Finally, we determined the role of proteasomal or lysosomal protein degradation in the regulation of FcµR expression. CLL cells were pretreated for 1 hour with the proteasome inhibitor bortezomib or with chloroquine, an inhibitor of lysosomal acidification, and then incubated for an additional 16 hours with or without CpG-ODN (Fig. 7E). Whereas FcµR protein levels remained unchanged in the presence of bortezomib, we observed a significant accumulation of FcµR in cells exposed to chloroquine. Upon CpG-ODN stimulation (CpG-ODN treatment), bortezomib could not prevent the decrease in FcµR protein. In contrast, pretreatment with chloroquine induced a further increase in FcµR protein level. These data indicate that FcµR is degraded through a chloroquine-sensitive pathway consistent with its shuttling and degradation in the lysosome.
In this study we demonstrate that FcµR is an O-glycosylated endocytic receptor that shuttles IgM from the cell surface to the lysosome, where it is degraded. FcµR is highly expressed on CLL cells and promotes rapid uptake of IgM into these cells. Activation of the TLR system in CLL cells lead to a dramatic downregulation of FcµR expression. In vivo, FcµR could thus transport IgM opsonized immune complexes into the lysosome where depending on the type of cargo carried by the IgM molecules, TLR activation may ensue.
Our work and the work of others (11, 12) identify FcµR expression as a characteristic feature of CLL cells. FcµR is expressed only at low levels in normal peripheral blood B cells, and absent in other B-cell malignancies, including MCL, diffuse-large-B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, and Hodgkin’s lymphoma. In CLL cells, we observed two forms of FcµR a 41 kDa form, corresponding to its predicted molecular weight and a 60 kDa form, whereas normal B cells predominantly expressed the 60 kDa form. We also demonstrate for the first time that the majority of FcµR protein remains in an intracellular compartment and is preferentially localized to the TGN. One of the major functions of the TGN is to ensure terminal glycosylation and sorting of cell surface and secreted proteins (36, 37). We therefore investigated the glycosylation state of FcµR and identified three sites of extensive O-glycosylation explaining the higher molecular weight of the predominant 60 kDa form. Using a series of mutants we found that proper O-glycosylation is critical for trafficking of FcµR to the cell surface.
The finding that the majority of FcµR is present in the TGN was surprising and raises the question about a possible functional role of this intracellular pool. One possible explanation could be that the TGN serves as an intracellular storage site and that under certain conditions FcµR may be rapidly released to the cell surface. Alternatively, FcµR might function as an intracellular chaperone aiding Ig assembly as has recently been suggested for the FcR-like A (FcRLA) protein that associates with intracellular IgG and IgM in B cells (38). While, our preliminary data suggested that FcµR can bind IgM in the TGN (data not shown), it seems unlikely that FcµR plays a major role in IgM expression given, that several IgM expressing B-cell malignancies such as MCL and diffuse-large-B-cell lymphoma do not express FcµR.
Our data identify FcµR as an endocytic receptor that, once internalized, shuttles to the lysosome, where it is degraded. This conclusion is based both on the visualization of internalized FcµR by immunofluorescence where it co-localized with the lysosomal marker LAMP-1, as well as on the increase in FcµR protein levels in cells exposed to chloroquine. Chloroquine prevents acidification of the lysosome and thereby blocks lysosomal protein degradation. Inhibition of the proteasome on the other hand had no effect on FcµR protein levels. FcµR loaded with soluble IgM was rapidly internalized, reaching half maximal levels within 1 minute and virtually complete internalization within 5 minutes. Similarly, HeLa cells expressing FcµR have been shown to internalize IgM-conjugated microbeads (39). FcµR could thus play a role in the regulation of the circulating IgM pool and contribute to the disposal of IgM bound pathogens or cellular debris. Finally, we identified that a C-terminal YXXΦ motif is essential for FcµR internalization. This is consistent with the role described for YXXΦ motifs in AP2 recruitment and clathrin dependent endocytosis (40, 41).
A major physiologic role of FcµR then appears to be the internalization of IgM into cells of the adaptive immune system and the shuttling of IgM bound cargo to the lysosome. Given the broad reactivity of IgM such cargo likely includes a variety of infectious agents as well as cellular debris (34). While FcRs play a role in phagocytosis and antigen presentation, transport of IgM bound cargo into the lysosome of mature lymphocytes will bring pathogen specific molecular structures in contact with intracellular TLRs. In particular TLR7 and TLR9 recognizing single stranded RNA and unmethylated DNA, respectively, are an important part of the immune surveillance network and are expressed in CLL cells (42). Cooperation between TLR signals and immune receptor signaling, in particular with the BCR, can amplify the immune response and overcome anergy (43). Furthermore, there is recent evidence that cooperation of TLR and BCR signaling can provide non-redundant survival signals in lymphoma (44). This raises the intriguing question whether CLL cells also integrate signals from these two immunoreceptor pathways. Indeed, in a previous study analyzing the contribution of the tumor microenvironment to CLL activation in vivo, we found evidence for not only BCR but also TLR activation in the lymph node (3). In light of these considerations, FcµR could be a crucial link in the activation of CLL cells. To address a possible interaction between FcµR and TLR, we investigated the effect of TLR activation on FcµR expression. There was a striking downregulation of FcµR on CLL cells stimulated with TLR7 or TLR9 ligands that involved both inhibition of transcription as well as degradation of FcµR through the lysosome. Further studies are needed to clarify the potential role of FcµR and TLR signaling in the pathogenesis and progression of CLL. Equally interesting will be to study whether FcµR plays a physiologic role in host defense. This latter question will likely be answered in FcµR knockout mice that reportedly are viable and show no gross abnormalities (39).
The selective expression on CLL tumor cells and its ability to rapidly internalize IgM make FcµR a promising target for the delivery of therapeutic antibody-drug conjugates (ADC). ADCs combine the unique specificity of a monoclonal antibody linked to a potent cytotoxic drug through chemical linkers (45). Two ADCs, brentuximab vedotin (SGN-35, Seattle Genetics), an anti-CD30 mAb conjugated to monomethylauristatin E (46), and trastuzumab-DM1 (Genentech), an anti-HER2 mAb conjugated to maytansinoid 1 (47), are in late-stage clinical trials. Gemtuzumab ozogamicin, an anti-CD33 mAb, has been approved by the FDA in 2000 for the treatment of patients with acute myeloid leukemia but had to be withdrawn recently due to safety concerns (45). Our data show that upon binding to its receptor, IgM is rapidly internalized (Fig. 5B) and delivered to the lysosome (Fig. 5C). This route of intracellular trafficking represents an excellent opportunity for the selective activation of ADC in the targeted cells as it is possible to construct an ADC in the form of an inactive prodrug that is only activated upon intracellular proteolysis (48). Taken together, these features suggest that FcµR may represent an efficient and selective mechanism to deliver cytotoxic agents into the malignant cells. Ongoing studies aim to develop this approach as a novel therapeutic option for CLL patients.
We are grateful to Keyvan Keyvanfar for his help with flow cytometry setup and to Christoph Rader and Sarah E. M. Herman for helpful discussions and critical reading of the manuscript.
This research was supported by the Intramural Research Program of the National, Heart, Lung and Blood Institute, NIH.