Altered Ig levels and antibody responses in mice deficient for the Fc receptor for IgM (Fc?R).
ABSTRACT: Cell surface Fc receptor for IgM antibody (Fc?R) is the most recently identified member among FcRs. We determined the cellular distribution of mouse Fc?R and the functional consequences of Fcmr disruption. Surface Fc?R 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 ?-globulin (NP-CGG), the mutant mice had a diminished primary IgG1 response to both NP and CGG. These findings suggest that Fc?R has an important role in IgM homeostasis and regulation of humoral immune responses.
Project description:FcR specific for pentameric IgM (FCMR) is 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 that lack FCMR. FCMR transcripts are expressed at different levels by various B cell subsets. FCMR-deficient mice have reduced numbers of developing B cells, splenic follicular and peritoneal B-2 cells, but increased levels of peritoneal B-1a cells and autoantibodies. After immunization, germinal center B cell and plasma cell numbers are increased. FCMR-deficient B cells are sensitive to apoptosis induced by BCR ligation. Our studies demonstrate that FCMR is required for B cell differentiation and homeostasis, the prevention of autoreactive B cells, and responsiveness to antigenic challenge.
Project description:Previous studies with mice lacking secreted IgM (sIgM) due to a deletion of the ?s splice region (?s-/- ) had shown sIgM involvement in normal B cell development and in support of maximal Ag-specific IgG responses. Because of the changes to B cell development, it remains unclear to which extent and how sIgM directly affects B cell responses. In this study, we aimed to explore the underlying mechanisms of sIgM-mediated IgG response regulation during influenza virus infection. Generating mice with normally developed ?s-deficient B cells, we demonstrate that sIgM supports IgG responses by enhancing early Ag-specific B cell expansion, not by altering B cell development. Lack of Fc?R expression on B cells, but not lack of Fc?/?R expression or complement activation, reduced antiviral IgG responses to the same extent as observed in ?s-/- mice. B cell-specific Fcmr-/- mice lacked robust clonal expansion of influenza hemagglutinin-specific B cells early after infection and developed fewer spleen and bone marrow IgG plasma cells and memory B cells, compared with controls. However, germinal center responses appeared unaffected. Provision of sIgM rescued plasma cell development from ?s-/- but not Fcmr-/- B cells, as demonstrated with mixed bone marrow chimeric mice. Taken together, the data suggest that sIgM interacts with Fc?R on B cells to support early B cell activation and the development of long-lived humoral immunity.
Project description:The FcR for IgM (FcµR) is the newest member of the FcR family, selectively expressed by lymphocytes, and distinct from FcRs for switched Ig isotypes that are expressed by various immune cell types and non-hematopoietic cells. From studies of <i>Fcmr</i>-ablated mice, FcµR was shown to have a regulatory function in B-cell tolerance, as evidenced by high serum titers of autoantibodies of the IgM and IgG isotypes in mutant mice. In our previous studies, both cell-surface and serum FcµR levels were elevated in patients with chronic lymphocytic leukemia (CLL), where antigen-independent self-ligation of BCR is a hallmark of the neoplastic B cells. This was assessed by sandwich ELISA using two different ectodomain-specific mAbs. To determine whether the serum FcµR is derived from cleavage of its cell-surface receptor (shedding) or its alternative splicing to skip the transmembrane exon resulting in a 70-aa unique hydrophilic C-terminus (soluble), we developed a new mouse IgG1κ mAb specific for human soluble FcμR (solFcμR) by taking advantages of the unique nature of transductant stably producing His-tagged solFcµR and of an <i>in vivo</i> differential immunization. His-tagged solFcμR attached to exosomes and plasma membranes, allowing immunization and initial hybridoma screening without purification of solFcμR. Differential immunization with tolerogen (membrane FcμR) and immunogen (solFcμR) also facilitated to generate solFcμR-specific hybridomas. The resultant solFcμR-specific mAb reacted with serum FcµR in subsets of CLL patients. This mAb, along with another ectodomain-specific mAb, will be used for verifying the hypothesis that the production of solFcµR is the consequence of chronic stimulation of BCR.
Project description:Receptors interacting with the constant domain of immunoglobulins (Igs) have a number of important functions in vertebrates. They facilitate phagocytosis by opsonization, are key components in antibody-dependent cellular cytotoxicity as well as activating cells to release granules. In mammals, four major types of classical Fc receptors (FcRs) for IgG have been identified, one high-affinity receptor for IgE, one for both IgM and IgA, one for IgM and one for IgA. All of these receptors are related in structure and all of them, except the IgA receptor, are found in primates on chromosome 1, indicating that they originate from a common ancestor by successive gene duplications. The number of Ig isotypes has increased gradually during vertebrate evolution and this increase has likely been accompanied by a similar increase in isotype-specific receptors. To test this hypothesis we have performed a detailed bioinformatics analysis of a panel of vertebrate genomes. The first components to appear are the poly-Ig receptors (PIGRs), receptors similar to the classic FcRs in mammals, so called FcRL receptors, and the FcR ? chain. These molecules are not found in cartilagous fish and may first appear within bony fishes, indicating a major step in Fc receptor evolution at the appearance of bony fish. In contrast, the receptor for IgA is only found in placental mammals, indicating a relatively late appearance. The IgM and IgA/M receptors are first observed in the monotremes, exemplified by the platypus, indicating an appearance during early mammalian evolution. Clearly identifiable classical receptors for IgG and IgE are found only in marsupials and placental mammals, but closely related receptors are found in the platypus, indicating a second major step in Fc receptor evolution during early mammalian evolution, involving the appearance of classical IgG and IgE receptors from FcRL molecules and IgM and IgA/M receptors from PIGR.
Project description:Although Fc receptors (FcRs) for switched immunoglobulin (Ig) isotypes have been extensively characterized, FcR for IgM (FcmuR) has defied identification. By retroviral expression and functional cloning, we have identified a complementary DNA (cDNA) encoding a bona fide FcmuR in human B-lineage cDNA libraries. FcmuR is defined as a transmembrane sialoglycoprotein of approximately 60 kD, which contains an extracellular Ig-like domain homologous to two other IgM-binding receptors (polymeric Ig receptor and Fcalpha/muR) but exhibits an exclusive Fcmu-binding specificity. The cytoplasmic tail of FcmuR contains conserved Ser and Tyr residues, but none of the Tyr residues match the immunoreceptor tyrosine-based activation, inhibitory, or switch motifs. Unlike other FcRs, the major cell types expressing FcmuR are adaptive immune cells, including B and T lymphocytes. After antigen-receptor ligation or phorbol myristate acetate stimulation, FcmuR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcmuR expression during B and T cell activation. Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcmuR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis.
Project description:Fc?R is a high-affinity receptor for the Fc portion of human IgM. It participates in B cell activation, cell survival and proliferation, but the full range of its functions remains to be elucidated. The receptor has an extracellular immunoglobulin (Ig)-like domain homologous to those in Fc?/?R and pIgR, but unlike these two other IgM receptors which also bind IgA, Fc?R exhibits a binding specificity for only IgM-Fc. Previous studies have suggested that the IgM/Fc?R interaction mainly involves the C?4 domains with possible contributions from either C?3 or C?2. To define the binding site more precisely, we generated three recombinant IgM-Fc proteins with specific mutations in the C?3 and C?4 domains, as well as a construct lacking the C?2 domains, and analyzed their interaction with the extracellular Ig-like domain of Fc?R using surface plasmon resonance analysis. There is a binding site for Fc?R in each IgM heavy chain. Neither the absence of the C?2 domains nor the quadruple mutant D340S/Q341G/D342S/T343S (in C?3 adjacent to C?2) affected Fc?R binding, whereas double mutant K361D/D416R (in C?3 at the C?4 interface) substantially decreased binding, and a single mutation Q510R (in C?4) completely abolished Fc?R binding. We conclude that glutamine at position 510 in C?4 is critical for IgM binding to Fc?R. This will facilitate discrimination between the distinct effects of Fc?R interactions with soluble IgM and with the IgM BCR.
Project description:OBJECTIVE:Fc? receptor IIb (Fc?RIIb) is an essential negative regulator of B cells that blocks B cell receptor (BCR) signaling and triggers c-Abl-dependent apoptosis of B cells. Fc?RIIb-deficient mice display splenomegaly with expansion of B cells, leading to lupus. Fc?RIIb-I232T is a hypofunctional polymorphism associated with lupus susceptibility in humans, an autoimmune disease linked to diminished deletion of autoreactive B cells. In the context of the Fc?RIIb-I232T polymorphism, we investigated the role of Fc?RIIb in the deletion of low-affinity germinal center (GC) B cells, an important mechanism for preventing autoimmunity. METHODS:We generated Fc?RIIb232T/T mice to mimic human Fc?RIIb-I232T carriers and immunized mice with chicken gamma globulin (CGG)-conjugated NP, a T cell-dependent antigen, to examine the response of GC B cells. RESULTS:Compared to wild-type (WT) mice, Fc?RIIb232T/T mice showed increased numbers of low-affinity NP-specific IgG and NP-specific B cells and plasma cells; additionally, the expression of a somatic mutation (W33L) in their VH 186.2 genes encoding high-affinity BCR was reduced. Notably, Fc?RIIb232T/T mice had a higher number of GC light zone B cells and showed less apoptosis than WT mice, despite having equivalent follicular helper T cell numbers and function. Moreover, phosphorylation of c-Abl was reduced in Fc?RIIb232T/T mice, and treatment of WT mice with the c-Abl inhibitor nilotinib during the peak of GC response resulted in reduced affinity maturation reminiscent of Fc?RIIb232T/T mice. CONCLUSION:Our findings provide evidence of a critical role of Fc?RIIb/c-Abl in the negative selection of GC B cells in Fc?RIIb232T/T mice. Importantly, our findings indicate potential benefits of up-regulating Fc?RIIb expression in B cells for treatment of systemic lupus erythematosus.
Project description: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 (Fc?R). We have recently identified a bona fide Fc?R in both humans and mice. In this article we briefly review what we have learned so far about Fc?R.
Project description:Monoclonal antibodies find broad application as therapy for various types of cancer by employing multiple mechanisms of action against tumors. Manipulating the Fc-mediated functions of antibodies that engage immune effector cells, such as NK cells, represents a strategy to influence effector cell activation and to enhance antibody potency and potentially efficacy. We developed a novel approach to generate and ascertain the functional attributes of Fc mutant monoclonal antibodies. This entailed coupling single expression vector (pVitro1) antibody cloning, using polymerase incomplete primer extension (PIPE) polymerase chain reaction, together with simultaneous Fc region point mutagenesis and high yield transient expression in human mammalian cells. Employing this, we engineered wild type, low (N297Q, NQ), and high (S239D/I332E, DE) FcR-binding Fc mutant monoclonal antibody panels recognizing two cancer antigens, HER2/neu and chondroitin sulfate proteoglycan 4. Antibodies were generated with universal mutagenic primers applicable to any IgG1 pVitro1 constructs, with high mutagenesis and transfection efficiency, in small culture volumes, at high yields and within 12?days from design to purified material. Antibody variants conserved their Fab-mediated recognition of target antigens and their direct anti-proliferative effects against cancer cells. Fc mutations had a significant impact on antibody interactions with Fc receptors (FcRs) on human NK cells, and consequently on the potency of NK cell activation, quantified by immune complex-mediated calcium mobilization and by antibody-dependent cellular cytotoxicity (ADCC) of tumor cells. This strategy for manipulation and testing of Fc region engagement with cognate FcRs can facilitate the design of antibodies with defined effector functions and potentially enhanced efficacy against tumor cells.
Project description:The Fc receptors (FcRs) and their interactions with immunoglobulin and innate immune opsonins, such as C-reactive protein, are key players in humoral and cellular immune responses. As the effector mechanism for some therapeutic monoclonal antibodies, and often a contributor to the pathogenesis and progression of autoimmunity, FcRs are promising targets for treating autoimmune diseases.This review discusses the nature of different FcRs and the various mechanisms of their involvement in initiating and modulating immunocyte functions and their biological consequences. It describes a range of current strategies in targeting FcRs and manipulating their interaction with specific ligands, while presenting the pros and cons of these approaches. This review also discusses potential new strategies including regulation of FcR expression and receptor crosstalk.FcRs are appealing targets in the treatment of inflammatory autoimmune diseases. However, there are still knowledge limitations and technical challenges, the most important being a better understanding of the individual roles of each of the FcRs and enhancement of the specificity in targeting particular cell types and specific FcRs.