Fc Receptors For IgM Research Articles

Anti-PEG IgM and complement system are required for the association of second doses of PEGylated liposomes with splenic marginal zone B cells

The accelerated blood clearance (ABC) phenomenon makes it crucial to use PEGylated liposomes and micelles to deliver drugs. The ABC phenomenon is an immune response against an initial dose of PEGylated liposome, which causes subsequent doses to be rapidly cleared by macrophages in the liver. We recently found that in the early phase of the ABC phenomenon, subsequent doses of PEGylated liposomes were associated with splenic marginal zone (MZ)-B cells and were transported from the MZ to the follicle (FO). In this study, we investigated the underlying mechanisms behind the association of subsequent doses of PEGylated liposomes with MZ-B cells in the spleen. Serum factors, anti-PEG IgM and complement system, were crucial to the association of PEGylated liposomes with MZ-B cells, while the sensitization of MZ-B cells by the first dose of PEGylated liposomes was not significant. It was the complement receptors (CRs) on the MZ-B cells, rather than either the PEG-specific B-cell receptors or the IgM Fc receptors, that were the main contributors to the association between PEGylated liposomes and MZ-B cells. It appeared that anti-PEG IgM would bind to PEGylated liposomes and causes subsequent complement activation, resulting in the formation of immune complexes of PEGylated liposome-anti-PEG IgM-complement. The MZ-B cells then recognized these immune complexes via their CRs. Such an association via CRs might have triggered the transport of the immune complex by MZ-B cells to the FO in the spleen. The information obtained in this study might be useful in the development of an efficient antigen delivery system to usher PEGylated nanoparticles into FO dendritic cells.

Unique ligand-binding property of the human IgM Fc receptor.

The IgM Fc receptor (FcμR) is the newest FcR, and coligation of FcμR and Fas/CD95 on Jurkat cells with agonistic IgM anti-Fas mAb was shown to inhibit Fas-induced apoptosis. The ligand-binding activity of human FcμR was further examined. FcμR-mediated protection from apoptosis was partially blocked by addition of 10(4) molar excess of IgM or its soluble immune complexes, but it could be inhibited by addition of 10-fold excess of IgM anti-CD2 mAb. This suggests that FcμR binds more efficiently to the Fc portion of IgM reactive with plasma-membrane proteins than to the Fc portion of IgM in solution. The former interaction occurred in cis on the same cell surface, but not in trans between neighboring cells. This cis engagement of FcμR resulted in modulation of Ca(2+) mobilization via CD2 on Jurkat cells or BCRs on blood B cells upon cross-linkage with the corresponding IgM mAbs. Several functional changes were observed with FcμR mutants: 1) significant increase in IgM ligand binding in the cytoplasmic tail-deletion mutant, 2) enhanced cap formation in FcμR upon IgM binding at 4°C with a point mutation of the transmembrane His to Phe, and 3) less protective activity of FcμR in IgM anti-Fas mAb-mediated apoptosis assays with a point mutation of the membrane-proximal Tyr to Phe. These findings show the importance of the cis engagement of FcμR and its critical role in receptor function. Hence, FcμR on B, T, and NK cells may modulate the function of surface proteins recognized by natural or immune IgM Abs on the shared membrane cell surface.

Monoclonal antibodies specific for human IgM Fc receptor inhibit ligand-binding activity.

A panel of six different murine hybridoma clones secreting IgG monoclonal antibodies (MAbs) specific for the human IgM Fc receptor (FcμR) was generated. All MAbs specifically precipitated a major protein of ∼60 kDa from membrane lysates of FcμR-bearing, but not FcμR-negative, cells as did IgM-ligands. Pre-incubation of membrane lysate of FcμR-bearing cells with these MAbs completely removed the ∼60 kDa IgM-reactive protein. By using recombinant human/mouse chimeric FcμR proteins, the epitope recognized by HM7 and HM10 MAbs was mapped to the Ig-like domain of human FcμR, whereas the other MAbs recognized the stalk region. Pre-incubation of FcμR(+) cells with the Ig-like domain-specific MAbs, but not with others, markedly inhibited subsequent IgM-ligand binding. A similar, but much weaker, inhibition was also observed when the incubation order was reversed. When FcμR(+) cells were simultaneously incubated with both IgM-ligands and MAbs, HM7 MAb efficiently competed with IgM for FcμR binding. Unlike control Jurkat cells, FcμR-bearing cells were resistant to apoptosis induced by agonistic IgM anti-Fas MAb (CH11); however, addition of the HM7 MAb inhibited the interaction of the Fc portion of CH11 MAb with FcμR, thereby promoting apoptosis of FcμR-bearing Jurkat cells. The variable regions of the HM7 MAb were composed of Ighv14-3, Ighd1-2, and Ighj2 for the γ2b heavy chain and Igk3-4 and Igkj2 for the κ light chain. These findings suggest that HM7 MAb efficiently blocks the ligand-binding activity of FcμR.

Physiopathologie de l’urticaire

Urticaria is a dermal edema resulting from vascular dilatation and leakage of fluid into the skin in response to molecules released from mast cells. The major mediator responsible for urticaria is histamine. However, the clinical spectrum and pattern of lesions indicate that other molecules, including prostaglandins, leukotrienes, cytokines, and chemokines, produced at different times after mast cell activation contribute to the polymorphism of this symptom and the variable evolution of this disease. It is a common practice to distinguish immunological and nonimmunological urticaria. Immunological urticaria is a hypersensitivity reaction mediated by antibodies and/or T-cells that results in mast cell activation. Although immunoglobulin (Ig) E-mediated type I hypersensitivity (HS) was long postulated to be the major immunological pathway associated with mast cell activation, interaction between IgEbound mast cells and allergens is unlikely to be the mechanism by which urticaria develops in most patients. It is now well established that urticaria may result from the binding of IgG auto-antibodies to IgE and/or to the receptor for IgE molecules on mast cells, thus corresponding to a type II HS reaction. These auto-immune urticarias represent up to 50 % of patients with chronic urticaria. Mast cell activation can also result from type III HS through the binding of circulating immune complexes to mast cell-expressing Fc receptors for IgG and IgM. Finally, under certain circumstances, T-cells can induce activation of mast cells, as well as histamine release (type IV HS). Nonimmunological urticarias result from mast cell activation through membrane receptors involved in innate immunity (e.g., complement, Toll-like, cytokine/chemokine, opioid) or by direct toxicity of xenobiotics (haptens, drugs). In conclusion, urticaria may result from different pathophysiological mechanisms that explain the great heterogeneity of clinical symptoms and the variable responses to treatment.

Increased serum IgA in Fcα/μR-deficient mice on the (129 x C57BL/6) F1 genetic background

Fcα/μR (CD351) is an Fc receptor for both IgA and IgM, which is abundantly expressed in the small intestine. However, the role of Fcα/μR in the intestinal tissue is largely unknown. Here, we found that Fcα/μR is highly expressed on follicular dendritic cells (FDCs) in Peyer's patches (PP) in the small intestine. Fcα/μR-deficient mice on the (129 x C57BL/6) F1 background showed increased serum, but not fecal, IgA level in response to gut-oriented antigens. IgA+ B cells were increased in PP, but not in the lamina propria, of Fcα/μR-deficient mice, which was attenuated after reduction of commensal microbiota by oral treatment with antibiotics. Analyses of bone marrow chimeric mice, in which either FDCs or blood cells or both lack the expression of Fcα/μR, suggested that FDCs, but not blood cells, were responsible for the increased serum IgA concentration in Fcα/μR-deficient mice. Moreover, Fcα/μR-deficient mice showed enhanced germinal center formation against commensal microbiota in PP. Thus, serum IgA production against gut-oriented antigens is negatively regulated by Fcα/μR on FDCs in the F1 mice.

Enhanced auto-antibody production and Mott cell formation in FcμR-deficient autoimmune mice

The IgM-Fc receptor (FcμR) is involved in IgM homeostasis as evidenced by increased pre-immune serum IgM and natural auto-antibodies of both IgM and IgG isotypes in Fcmr-deficient C57BL/6 (B6) mice. To determine the impact of Fcmr-ablation on autoimmunity, we introduced the Fcmr null mutation onto the Fas-deficient autoimmune-prone B6.MRL Fas (lpr/lpr) mouse background (B6/lpr). Both IgM and IgG auto-antibodies against dsDNA or chromatin appeared earlier in FcμR(-) B6/lpr than FcμR(+) B6/lpr mice, but this difference became less pronounced with age. Splenic B2 cells, which were 2-fold elevated in FcμR(+) B6/lpr mice, were reduced to normal B6 levels in FcμR(-) B6/lpr mice, whereas splenic B1 cells were comparable in both groups of B6/lpr mice. By contrast, marginal zone (MZ) B cells were markedly reduced in FcμR(-) B6/lpr mice compared with either FcμR(+) B6/lpr or wild type (WT) B6 mice. This reduction appeared to result from rapid differentiation of MZ B cells into plasma cells in the absence of FcμR, as IgM antibody to a Smith (Sm) antigen, to which MZ B cells are known to preferentially respond, was greatly increased in both groups (B6/lpr and B6) of FcμR(-) mice compared with FcμR(+) B6/lpr or B6 mice. Mott cells, aberrant plasma cells with intra-cytoplasmic inclusions, were also increased in the absence of FcμR. Despite these abnormalities, the severity of renal pathology and function and survival were all indistinguishable between FcμR(-) and FcμR(+) B6/lpr mice. Collectively, these findings suggest that FcμR plays important roles in the regulation of auto-antibody production, Mott cell formation and the differentiation of MZ B cells into plasma cells in B6.MRL Fas (lpr/lpr) mice.

The long elusive IgM Fc receptor, FcμR.

IgM exists as both a monomer on the surface of B cells and a pentamer secreted by plasma cells. Both pre-immune "natural" and antigen-induced "immune" IgM antibodies are important for protective immunity and for immune regulation of autoimmune processes by recognizing pathogens and self-antigens. Effector proteins interacting with the Fc portion of IgM, such as complement and complement receptors, have thus far been proposed but fail to fully account for the IgM-mediated protection and regulation. A major reason for this deficit in our understanding of IgM function seems to be lack of data on a long elusive Fc receptor for IgM (FcμR). We have recently identified a bona fide FcμR in both humans and mice. In this article we briefly review what we have learned so far about FcμR.

Novel IgM-derived antibody-drug conjugate selectively kills chronic lymphocytic leukemia cells through binding of Fcμ-receptor leading to rapid internalization and intracellular cytotoxic payload delivery (VAC3P.953)

Abstract The IgM Fc receptor (FcμR) is a previously unexploited target for monoclonal antibody therapy and antibody drug conjugate (ADC) therapy of B-cell chronic lymphocytic leukemia (CLL). While anti-FcμR IgG binding does not cause FcμR internalization, IgM bound to FcμR are rapidly internalized (>80% internalized within 5 minutes). FcμR is overexpressed in CLL with only limited expression on normal B, T, and NK cells. In our study, 29 out of 30 CLL patient samples (97%) evaluated by flow cytometry had high FcμR expression by CLL cells (mean(SD)=89.1(7.1)%) and the remaining patient had moderate CLL FcμR expression (57.9%). In contrast, FcμR expression by T cells (mean(SD)=8.9(5.2)%) was significantly lower (p<0.0001). We developed a FcμR targeting ADC consisting of the CH2-CH3-CH4 IgM regions conjugated to monomethyl auristatin F. Flow cytometric analyses of CLL patient samples showed potent ADC mediated killing of CLL cells (mean(SD)=78.8(10.1)%, p< 0.0001 versus scaffold treated control) but not T cells (mean(SD)=8.4(3.8)%, p=0.3210 versus scaffold treated control). The ADC also greatly reduced tumor burden in NOD/SCID/IL-2Rγnull mice xenografted with 4 CLL patient samples (mean reduction(SD)=74(20)% in blood and 65(19)% in spleen, n=30). In summary, FcμR is a promising new target for therapy of CLL and possibly other malignancies. Our preclinical results provide translational evidence for the clinical utility of IgM-derived protein scaffolds to target FcμR expressing cells.

The science and arts of antibodies.

Recently there have been major developments in understanding the basic mechanisms of actions of natural and adaptive antibodies, especially with regard to their anti-inflammatory and immunoregulatory properties, and the art of their clinical application in a variety of inflammatory and autoimmune disorders. This was the major theme of the International Forum of Immunoglobulin Research, held in Miami Florida on October 3–6, 2013. There have been several recent developments in the understanding of the role of natural autoantibodies in immune homeostasis, tolerance, and antimicrobial activity. Natural IgM antibodies to apoptotic-cell membrane determinants represent a distinct subset of antibodies that potentiate the phagocytic clearance of apoptotic cells and apoptotic bodies. Furthermore, these autoantibodies display potent antiinflammatory activities both in vitro and in vivo, which are mediated by MAPK phosphatase 1 signaling pathway (Caroline Gronwell, New York, USA). A role of natural IgM to be protective in human diseases comes from patients with systemic lupus erythematosus in which high levels of natural IgM to phosphocholine correlate with less active lupus disease, less organ damage, and lower risk of cardiovascular events. Srini Kaveri (Paris, France) and associates have identified and characterized the natural autoantibodies of the IgG isotype directed against the human Fc receptors. They show that the F(ab′)2 of IVIG recognizes the FcγRIII (CD16) and FcγRII (CD32). Furthermore, the immunopurified anti-FcγIII and anti-FcγII antibodies present in IVIG recognized soluble and membrane-bound FcR. These observations provide further evidence of the existence of natural autoantibodies towards a wide range of self-motifs, including those present in apoptotic bodies in immunoglobulin preparations that may participate in regulation of the immune response. Peter Lobo (Virginia, USA), using in vitro and murine knock out models, demonstrated an anti-inflammatory role of natural IgM antileucocyte antibodies (ALA). This anti-inflammatory effect of IgM is mediated, in part, by inhibiting TLR-4-induced translocation of NF-κB into the nucleus and inhibiting differentiation of naive Th0 cells into Th1 and Th17 cells. He also showed that intra-peritoneal administration of IgM prevents NODmice from developing autoimmune insulitis, which also involves Th1 and Th17 cells. Therefore, anti-inflammatory and immunoregulatory properties of natural antibodies may justify the use of polyclonal IgM preparations and/or development of monoclonal IgM for the treatment of inflammatory and autoimmune disease. Although a role of both ‘natural’ and ‘immune’ IgM antibodies in immune homeostasis, removal of self-antigen containing apoptotic cells and apoptotic bodies, and in protective immunity against pathogens is well-recognized, the mechanisms of IgM-mediated protection against microbes and immune-regulation; however, are not completely understood, Recent recognition and characterization of long since elusive Fc receptor for IgM (FcμR), in both humans and mice have shed light on some of these mechanisms. Hiromi Kubagawa (Alabama, USA) presented data on the cellular distribution of FcμR and potential functions. FcμR knock out mice are unable to secrete IgM; however, express surface IgM and other Ig isotypes on B cells, and secrete other immunoglobulins. Studies of these mice have reinforced the importance of both ‘natural’ and ‘immune’ IgM in microbial defense and in the regulation of autoimmunity. The sugar moieties attached to the IgG constant fragment (Fc) are essential for IgG functionality such as binding to cellular Fc receptors and complement activation. Presence or absence of distinct residues such as fucose and sialic acid can S. Gupta (*) University of California at Irvine, Irvine, CA, USA e-mail: sgupta@uci.edu

The old but new IgM Fc receptor (FcμR).

IgM is the first Ig isotype to appear during phylogeny, ontogeny and the immune response. The importance of both pre-immune "natural" and antigen-induced "immune" IgM antibodies in immune responses to pathogens and self-antigens has been established by studies of mutant mice deficient in IgM secretion. Effector proteins interacting with the Fc portion of IgM, such as complement and complement receptors, have thus far been proposed, but fail to fully account for the IgM-mediated immune protection and regulation of immune responses. Particularly, the role of the Fc receptor for IgM (FcμR) in such effector functions has not been explored until recently. We have identified an authentic FcμR in humans using a functional cloning strategy and subsequently in mice by RT-PCR and describe here its salient features and the immunological consequences of FcμR deficiency in mice. Since the FcμR we cloned was identical to Toso or Fas inhibitory molecule 3 (FAIM3), there have been spirited debates regarding the real function of FcμR/Toso/FAIM3 and we will also comment on this topic.

Complement-Activating IgM Enhances the Humoral but Not the T Cell Immune Response in Mice

IgM antibodies specific for a certain antigen can enhance antibody responses when administered together with this antigen, a process believed to require complement activation by IgM. However, recent data show that a knock-in mouse strain, Cμ13, which only produces IgM unable to activate complement, has normal antibody responses. Moreover, the recently discovered murine IgM Fc receptor (FcµR or TOSO/FAIM3) was shown to affect antibody responses. This prompted the re-investigation of whether complement activation by specific IgM is indeed required for enhancement of antibody responses and whether the mutation in Cµ13 IgM also caused impaired binding to FcµR. The results show that IgM from Cµ13 and wildtype mice bound equally well to the murine FcµR. In spite of this, specific Cμ13 IgM administered together with sheep red blood cells or keyhole limpet hemocyanine was a very poor enhancer of the antibody and germinal center responses as compared with wildtype IgM. Within seconds after immunization, wildtype IgM induced deposition of C3 on sheep red blood cells in the blood. IgM which efficiently enhanced the T-dependent humoral immune response had no effect on activation of specific CD4+ T cells as measured by cell numbers, cell division, blast transformation, or expression of the activation markers LFA-1 and CD44 in vivo. These observations confirm the importance of complement for the ability of specific IgM to enhance antibody responses and suggest that there is a divergence between the regulation of T- and B-cell responses by IgM.

Involvement of Fcα/μR (CD351) in autoantibody production

Antibody exerts various immune responses via binding to Fc receptors expressed on immune cells. Although several reports have demonstrated that IgM prevents autoantibody production, the role of IgM Fc receptors is largely unknown. To analyze the involvement of Fcα/μR (CD351), an Fc receptor for IgM and IgA expressed on B cells and follicular dendritic cells (FDCs), in IgM-mediated suppression of autoantibody production, we generated mice deficient in Fcα/μR on the background of MRL/MpJ-Faslpr/lpr (Fcamr−/−Faslpr/lpr) mice. Fcamr−/−Faslpr/lpr mice showed significantly lower titers of IgG autoantibodies against double strand (ds) DNA, histone and cardiolipin in the sera than did Fcamr+/+Faslpr/lpr mice. Moreover, Fcamr−/−Faslpr/lpr mice showed higher survival rate at the ages of 28, 32 and 40 weeks old, compared with Fcamr+/+Faslpr/lpr mice. These results suggest that Fcα/μR enhances, rather than suppresses, autoantibody production.

Is Toso/IgM Fc receptor (FcμR) expressed by innate immune cells?

Lang et al. have recently published that innate immune cells, including bone-marrow granulocytes and splenic macrophages, express the plasma-membrane protein Toso as a unique regulator of innate immune responses during bacterial infection and septic shock (1). In contrast, we and others have previously reported that the IgM Fc receptor (FcμR), originally designated as Toso or Fas apoptosis inhibitory molecule 3, is expressed only by B-lineage cells in mice and is involved in IgM homeostasis, B-cell survival, and humoral immune responses (2, 3). Because of these conflicting results, we have extensively reexamined the cellular distribution of Toso/FcμR and wish to provide some new results and comments.

Molecular characterization of the dimer formation of Fcα/μ receptor (CD351)

Fcα/μR (CD351) is an Fc receptor for both IgA and IgM and forms an atypical dimer that is resistant to reduction by 2-mercaptoethanol or boiling. We previously demonstrated that the cytoplasmic portion of Fcα/μR is required for dimer formation and for its efficient cell-surface expression. However, the biochemical nature of these phenomena has not been determined. By using a BW5147 mouse cell line expressing deletion mutants of the cytoplasmic region of Fcα/μR, we found that the region spanning amino acids 504–523 was required for efficient cell-surface expression, whereas the region spanning amino acids 481–490 was required for dimmer formation. Immunoblotting analyses of transfectants simultaneously expressing Flag-tagged Fcα/μR and hemagglutinin-tagged Fcα/μR suggested that Fcα/μR does not form homodimers. Instead, our data suggest that Fcα/μR forms heterodimers with an as-yet-unknown molecule with a molecular weight of 60–70kDa.

Involvement of Toso in activation of monocytes, macrophages, and granulocytes

Rapid activation of immune responses is necessary for antibacterial defense, but excessive immune activation can result in life-threatening septic shock. Understanding how these processes are balanced may provide novel therapeutic potential in treating inflammatory disease. Fc receptors are crucial for innate immune activation. However, the role of the putative Fc receptor for IgM, known as Toso/Faim3, has to this point been unclear. In this study, we generated Toso-deficient mice and used them to uncover a critical regulatory function of Toso in innate immune activation. Development of innate immune cells was intact in the absence of Toso, but Toso-deficient neutrophils exhibited more reactive oxygen species production and reduced phagocytosis of pathogens compared with controls. Cytokine production was also decreased in Toso(-/-) mice compared with WT animals, rendering them resistant to septic shock induced by lipopolysaccharide. However, Toso(-/-) mice also displayed limited cytokine production after infection with the bacterium Listeria monocytogenes that was correlated with elevated presence of Listeria throughout the body. Accordingly, Toso(-/-) mice succumbed to infections of L. monocytogenes, whereas WT mice successfully eliminated the infection. Taken together, our data reveal Toso to be a unique regulator of innate immune responses during bacterial infection and septic shock.

Critical role of the IgM Fc receptor in IgM homeostasis, B-cell survival, and humoral immune responses

IgM antibodies have been known for decades to enhance humoral immune responses in an antigen-specific fashion. This enhancement has been thought to be dependent on complement activation by IgM-antigen complexes; however, recent genetic studies render this mechanism unlikely. Here, we describe a likely alternative explanation; mice lacking the recently identified Fc receptor for IgM (FcμR) on B cells produced significantly less antibody to protein antigen during both primary and memory responses. This immune deficiency was accompanied by impaired germinal center formation and decreased plasma and memory B-cell generation. FcμR did not affect steady-state B-cell survival but specifically enhanced the survival and proliferation induced by B-cell receptor cross-linking. Moreover, FcμR-deficient mice produced far more autoantibodies than control mice as they aged, suggesting that FcμR is also required for maintaining tolerance to self-antigens. Our results thus define a unique pathway mediated by the FcμR for regulating immunity and tolerance and suggest that IgM antibodies promote humoral immune responses to foreign antigen yet suppress autoantibody production through at least two pathways: complement activation and FcμR.

Altered Ig levels and antibody responses in mice deficient for the Fc receptor for IgM (FcμR)

Cell surface Fc receptor for IgM antibody (FcμR) is the most recently identified member among FcRs. We determined the cellular distribution of mouse FcμR and the functional consequences of Fcmr disruption. Surface FcμR expression was restricted to B-lineage cells, from immature B to plasma cells, except for a transient down-modulation during germinal center reactions. Fcmr ablation had no significant effect on overall B- and T-cell development, but led to a reduction of marginal zone B cells and an increase in splenic B1 B cells. Preimmune serum IgM in mutant mice was significantly elevated as were natural autoantibodies. When immunized with live attenuated pneumococci, mutant mice mounted robust antibody responses against phosphorylcholine, but not protein, determinants compared with wild-type mice. By contrast, upon immunization with a hapten-carrier conjugate, nitrophenyl-coupled chicken γ-globulin (NP-CGG), the mutant mice had a diminished primary IgG1 response to both NP and CGG. These findings suggest that FcμR has an important role in IgM homeostasis and regulation of humoral immune responses.

The IgM Fc receptor, FCMR, promotes B cell development and modulates antigen-driven immune responses (176.9)

Abstract FCMR is a Fc receptor specific for pentameric IgM expressed at high levels by B cells. Although circulating IgM has profound effects on responses to pathogens, autoimmunity and B cell homeostasis, the biologic consequences of its binding to FCMR are poorly understood. We interrogated FCMR contributions to B cell function by studying mice lacking FCMR. FCMR transcripts are expressed at highest levels by follicular (FO) B cells and at much lower levels by developing B cells and other mature B cell subsets. FCMR-deficient mice have reduced numbers of developing B cells, splenic FO and peritoneal B-2 cells, but increased levels of peritoneal B-1a cells and autoantibodies. Following immunization, germinal center B cell and plasma cell numbers are increased. FCMR-deficient B cells are resistant to apoptosis induced by BCR ligation. Our studies demonstrate that FCMR functions are critical for B cell homeostasis, the prevention of autoreactive B cells and responsiveness to antigenic challenge.

Altered humoral immune responses in mice deficient for the Fc receptor for IgM (FcμR) (172.37)

Abstract Unlike humans, FcμR expression in mice is restricted to B cells with a hierarchy of cell surface expression level: follicular > marginal zone (MZ) > newly formed in spleen and B-2 = B-1a > B-1b in peritoneal cavity. To define the in vivo function of FcμR, Fcmr-deficient (KO) mice were generated by gene targeting. While total splenic CD19+ B and CD3+ T cell-numbers were comparable in KO and littermate control (WT) mice, MZ B cells were reduced ~4 fold and B-1 cells were increased ~2 fold in KO as compared with WT mice. Both serum IgM and IgG3 antibody (Ab) levels in naïve animals were significantly elevated in KO than WT mice. IgM and IgG3 natural Abs reacting with the nucleus or cytoplasm of HEp-2 cells were also elevated in KO mice. When immunized i.p. with a live avirulent strain of S. pneumoniae (R36A), much more robust and long-lasting IgM, IgG3 and IgA Ab-responses to phosphorylcholine developed in KO at low doses of R36A compared with WT. By contrast, when immunized i.p. with nitrophenyl (NP)-coupled chicken γ-globulin in alum, primary IgM anti-NP responses were comparable in both groups of mice, but secondary IgM responses were significantly impaired in KO mice. In contrast, the primary IgG1 anti-NP responses were impaired in KO, however secondary IgG1 responses were indistinguishable in both KO and WT mice irrespective of the antigen dose. These results show that Fcmr ablation results in altered Ab responses to both T-independent and -dependent antigens.

FcμR (TOSO/FAIM3) Is An O-Glycosylated Endocytic Receptor That Shuttles IgM From the Cell Surface to the Lysosome and Whose Expression Is Regulated by TLR Signaling

Abstract 2828TOSO/FAIM3 has recently been identified as the long sought after Fc receptor for IgM (FcμR). Previous studies have shown that FcμR is selectively overexpressed in chronic lymphocytic leukemia (CLL) cells as compared to normal B-cells or other B cell malignancies. In this study, we extend the characterization of FcμR and identify the mechanisms regulating its trafficking and expression. In CLL cells, analysis of FcμR protein expression by immunoblotting revealed one major band around 60 kDa, a series of fainter migrating species, and a band at 41 kDa corresponding to its predicted molecular weight. By contrast, normal B cells express predominantly the 60 kDa form. Inspection of FcμR sequence revealed numerous potential O-glycosylation sites in the extracellular domain and treatment with a general inhibitor of O-glycosylation, benzyl-N-acetyl- α-galactosaminide, confirmed that FcμR is indeed heavily O-glycosylated. By immunofluorescence confocal microscopy, we determined that O-glycosylation is critical for trafficking of FcμR to the cell surface as mutations in the predicted O-glycosylation sites led to intracellular retention of FcμR in HeLa cells transfected with the appropriate expression vectors. In addition, pharmacologic inhibition of O-glycosylation decreased FcμR expression on CLL cells. Next, we used biotinylated IgM and streptavidin DyLight 488 to determine the fate of IgM bound to FcμR. Addition of IgM to CLL cells prompted rapid internalization of both IgM and FcμR, reaching half maximal internalization of cell bound IgM within one minute and virtually complete internalization within 5 minutes. By contrast, a monoclonal antibody specific for FcμR was not internalized. Using immunofluorescence confocal microscopy and co-staining with transferrin and anti-LAMP-1 antiserum, we followed IgM trafficking. FcμR transported IgM through the endocytic pathway to the lysosome, where it was degraded. This last step was chloroquine but not bortezomib sensitive, confirming the role of the lysosome in FcμR degradation. Using a series of FcμR deletion mutants, we identified a proline-rich domain that is essential for cell surface expression of FcμR and a second domain, containing a YXX Φ motif, that controls internalization.Of particular interest, but so far elusive, is a possible functional role of FcμR in CLL pathogenesis. Our data suggest that a major physiologic role of FcμR may be internalization of IgM bound cargo into cells. Given the broad reactivity of IgM such cargo likely includes a variety of infectious agents as well as cellular debris that will be transported into the lysosome and thereby brought into contact with intracellular Toll-like Receptors (TLRs). To address a possible interaction between FcμR and TLRs, we investigated the effect of TLR activation on FcμR expression. There was a striking downregulation of FcμR on CLL cells in response to activation of TLR7 (using imiquimod) or TLR9 (using CpG-ODN) that involved both inhibition of transcription as well as degradation of FcμR through the lysosome. Following CpG stimulation, FcμR mRNA decreased with a half life of approximately 3 hours; that is at the same rate as after actinomycin D treatment. At 24 hours, FcμR protein was greatly reduced in response to either CpG or imiquimod, but interestingly there was less response to CpG in IGHV unmutated than in IGHV mutated CLL samples (p<0.001). In summary, FcμR is an O-glycosylated endocytic receptor that shuttles IgM from the cell surface to the lysosome. In vivo, FcμR could play a role in presenting IgM opsonized immune complexes to intracellular TLRs resulting in B-cell activation. Further studies are needed to clarify a potential contribution of FcμR to the activation of CLL cells in vivo. In addition, owing to the rapid internalization of IgM, we are pursuing FcμR as a potential pathway for the delivery of therapeutic antibody-drug conjugates into CLL cells (to be presented in a separate abstract).This work was supported by the Intramural Research Program of the National, Heart, Lung and Blood Institute. Disclosures:No relevant conflicts of interest to declare.