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
) 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
). 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
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 () and delivered to the lysosome (). 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.