Low-affinity IgG Research Articles

An unexpected protective role of low-affinity allergen-specific IgG through the inhibitory receptor FcγRIIb

Induction of allergen-specific IgG antibodies is a critical parameter for successful allergen-specific immunotherapy. IgG antibodies can inhibit IgE-mediated mast cell activation through direct allergen neutralization or through the inhibitory receptor FcγRIIb. The affinity of IgE antibodies to the allergen has been shown to be critical for cellular activation. Here we addressed the question of affinity thresholds of allergen-specific IgG antibodies for inhibition of mast cell activation using 2 different mAbs against the major cat allergen Fel d 1 both invitro and invivo in mice. Sequences of the 2 high-affinity mAbs were back-mutated to germline, resulting in low-affinity (10-7mol/L) antibodies of the exact same specificity. Using these newly generated recombinant antibodies, we demonstrate that low-affinity antibodies are still able to inhibit mast cell activation through FcγRIIb but do not neutralize the allergen. Antibody affinity dictates the mechanism of mast cell inhibition, and IgG antibodies triggering the inhibitory FcγRIIb pathway can show a broader cross-reactivity pattern than previously thought. This indicates that allergen-specific immunotherapy generates a larger protective umbrella of inhibitory IgG antibodies than previously appreciated.

Basophils from allergic patients are neither hyperresponsive to activation signals nor hyporesponsive to inhibition signals

Basophil activation contributes to inflammatory reactions, especially in allergy. It is controlled, both positively and negatively, by several mechanisms. High-affinity IgE receptors (FcεRI) generate a mixture of activation and inhibition signals when aggregated, the ratio of which depends on the concentration of allergen recognized by receptor-bound IgE. Low-affinity IgG receptors (FcγRIIA/B) generate inhibition signals when coengaged with FcεRI by allergen-antibody immune complexes. Commensal and probiotic bacteria, such as Lactobacillus paracasei, generate inhibition signals through still unclear mechanisms. We sought to investigate whether mechanisms that control, both positively and negatively, basophil activation, which were unraveled and studied in basophils from healthy donors, are functional in allergic patients. FcεRI and FcγRIIA/B expression, FcεRI-dependent activation, FcεRI-dependent inhibition, and FcγRIIB-dependent inhibition were examined in blood basophils incubated overnight with or without L paracasei and challenged under 10 experimental conditions. Basophils from healthy donors were compared with basophils from patients who consulted an allergology outpatient clinic over a period of 3months with respiratory allergy, anaphylaxis antecedents, chronic urticaria, and/or atopic dermatitis. Patients' basophils expressed neither more FcεRI nor less FcγRIIB than basophils from healthy donors. They were neither hyperreactive to positive regulation nor hyporeactive to negative regulation, irrespective of the receptors or mechanisms involved and the allergic manifestations of the patients. Regulatory mechanisms that control basophil activation are fully functional in allergic patients. Intrinsic defects in these mechanisms do not explain allergic manifestations. Based on these mechanisms, immune checkpoint modifiers can be developed as novel therapeutic tools for allergy.

Mucor circinelloides induces platelet aggregation through integrin αIIbβ3 and FcγRIIA

Thrombosis is a hallmark of the fatal fungal infection mucormycosis. Yet, the platelet activation pathway in response to mucormycetes is unknown. In this study we determined the platelet aggregation potential of Mucor circinelloides (M. circinelloides) NRRL3631, characterized the signaling pathway facilitating aggregation in response to fungal spores, and identified the influence of the spore developmental stage upon platelet aggregation potential. Using impedance and light-transmission aggregometry, we showed that M. circinelloides induced platelet aggregation in whole blood and in platelet-rich plasma, respectively. The formation of large spore-platelet aggregates was confirmed by light-sheet microscopy, which showed spores dispersed throughout the aggregate. Aggregation potential was dependent on the spore’s developmental stage, with the strongest platelet aggregation by spores in mid-germination. Inhibitor studies revealed platelet aggregation was mediated by the low affinity IgG receptor FcγRIIA and integrin αIIbβ3; Src and Syk tyrosine kinase signaling; and the secondary mediators TxA2 and ADP. Flow cytometry of antibody stained platelets showed that interaction with spores increased expression of platelet surface integrin αIIbβ3 and the platelet activation marker CD62P. Together, this is the first elucidation of the signaling pathways underlying thrombosis formation during a fungal infection, highlighting targets for therapeutic intervention.

A novel bispecific DARPin targeting FcγRIIB and FcεRI-bound IgE inhibits allergic responses.

Binding of allergen-specific IgE to its high-affinity receptor FcεRI on basophils and mast cells is a central event in the development of allergies. Exposure of these cells to allergens induces the release of soluble mediators causing allergic symptoms. The inhibitory low-affinity IgG Fc-receptor FcγRIIB is co-expressed on allergic effector cells and has been implicated in negative regulation of immediate hypersensitivity responses. In order to harvest the inhibitory function of this receptor, we aimed to select specific binders against FcγRIIB and to generate a bispecific molecule simultaneously targeting FcγRIIB and FcεRI-bound IgE on the surface of allergic effector cells. We selected FcγRIIB-specific binding molecules from a library of designed ankyrin repeat proteins using ribosome display technology. The bispecific binding modality was generated by molecular cloning and recombinant protein expression. We determined binding characteristics on molecular and cellular levels using SPR, ELISA, and flow cytometry. The inhibitory potential of the newly described molecules was assessed in different cellular degranulation assays ex vivo and in a mouse model of passive systemic anaphylaxis. We demonstrate that the selected DARPin® proteins recognize FcγRIIB with high affinity. Furthermore, the bispecific binding protein successfully interferes with allergen-induced cell degranulation and efficiently inhibits systemic anaphylaxis in vivo. Mechanistically, we report that FcγRIIB-mediated inhibition of effector cell activation requires direct ligation to an activating FcεRI receptor. The described bispecific DARPin protein has the ability to co-ligate FcγRIIB with FcεRI-bound IgE on allergic effector cells and represents an efficient dual-modality to interfere with allergic hypersensitivity reactions.

IL-3 up-regulates and activates human eosinophil CD32 and αMβ2 integrin causing degranulation.

Eosinophils contribute to the pathogenesis of multiple diseases, including asthma. Treatment with antibodies targeting IL-5 or IL-5 receptor α reduces the frequency of asthma exacerbations. Eosinophil receptors for IL-5 share a common ß-chain with IL-3 and GM-CSF receptors. We recently reported that IL-3 is more potent than IL-5 or GM-CSF in maintaining the ERK/p90S6K/RPS6 ribosome-directed signaling pathway, leading to increased protein translation. We aimed to determine disease-relevant consequences of prolonged eosinophil stimulation with IL-3. Human blood eosinophils were used to establish the impact of activation with IL-3 on IgG-driven eosinophil degranulation. When compared to IL-5, continuing exposure to IL-3 further induced degranulation of eosinophils on aggregated IgG via increased production and activation of both CD32 (low affinity IgG receptor) and αMß2 integrin. In addition, unlike IL-5 or GM-CSF, IL-3 induced expression of CD32B/C (FCGRIIB/C) subtype proteins, without changing CD32A (FCGRIIA) protein and CD32B/C mRNA expression levels. Importantly, these in vitro IL-3-induced modifications were recapitulated in vivo on airway eosinophils. We observed for the first time upregulation of CD32B/C on eosinophils, and identified IL-3 as a potent inducer of CD32- and αMß2-mediated eosinophil degranulation.

Humoral Immunity Provides Resident Intestinal Eosinophils Access to Luminal Antigen via Eosinophil-Expressed Low-Affinity Fcγ Receptors.

Eosinophils are native to the healthy gastrointestinal tract and are associated with inflammatory diseases likely triggered by exposure to food allergens (e.g., food allergies and eosinophilic gastrointestinal disorders). In models of allergic respiratory diseases and in vitro studies, direct Ag engagement elicits eosinophil effector functions, including degranulation and Ag presentation. However, it was not known whether intestinal tissue eosinophils that are separated from luminal food Ags by a columnar epithelium might similarly engage food Ags. Using an intestinal ligated loop model in mice, in this study we determined that resident intestinal eosinophils acquire Ag from the lumen of Ag-sensitized but not naive mice in vivo. Ag acquisition was Ig-dependent; intestinal eosinophils were unable to acquire Ag in sensitized Ig-deficient mice, and passive immunization with immune serum or Ag-specific IgG was sufficient to enable intestinal eosinophils in otherwise naive mice to acquire Ag in vivo. Intestinal eosinophils expressed low-affinity IgG receptors, and the activating receptor FcγRIII was necessary for Ig-mediated acquisition of Ags by isolated intestinal eosinophils in vitro. Our combined data suggest that intestinal eosinophils acquire lumen-derived food Ags in sensitized mice via FcγRIII Ag focusing and that they may therefore participate in Ag-driven secondary immune responses to oral Ags.

Neonatal Fc Receptor Regulation of Lung Immunoglobulin and CD103+ Dendritic Cells Confers Transient Susceptibility to Tuberculosis.

The neonatal Fc receptor (FcRn) extends the systemic half-life of IgG antibodies by chaperoning bound Fc away from lysosomal degradation inside stromal and hematopoietic cells. FcRn also transports IgG across mucosal barriers into the lumen, and yet little is known about how FcRn modulates immunity in the lung during homeostasis or infection. We infected wild-type (WT) and FcRn-deficient (fcgrt−/−) mice with Pseudomonas aeruginosa or Mycobacterium tuberculosis to investigate whether recycling and transport of IgG via FcRn influences innate and adaptive immunity in the lung in response to bacterial infection. We found that FcRn expression maintains homeostatic IgG levels in lung and leads to preferential secretion of low-affinity IgG ligands into the lumen. Fcgrt−/− animals exhibited no evidence of developmental impairment of innate immunity in the lung and were able to efficiently recruit neutrophils in a model of acute bacterial pneumonia. Although local humoral immunity in lung increased independently of the presence of FcRn during tuberculosis, there was nonetheless a strong impact of FcRn deficiency on local adaptive immunity. We show that the quantity and quality of IgG in airways, as well as the abundance of dendritic cells in the lung, are maintained by FcRn. FcRn ablation transiently enhanced local T cell immunity and neutrophil recruitment during tuberculosis, leading to a lower bacterial burden in lung. This novel understanding of tissue-specific modulation of mucosal IgG isotypes in the lung by FcRn sheds light on the role of mucosal IgG in immune responses in the lung during homeostasis and bacterial disease.

Mechanisms of anaphylaxis in human low-affinity IgG receptor locus knock-in mice

Anaphylaxis can proceed through distinct IgE- or IgG-dependent pathways, which have been investigated in various mouse models. We developed a novel mouse strain in which the human low-affinity IgG receptor locus, comprising both activating (hFcγRIIA, hFcγRIIIA, and hFcγRIIIB) and inhibitory (hFcγRIIB) hFcγR genes, has been inserted into the equivalent murine locus, corresponding to a locus swap. We sought to determine the capabilities of hFcγRs to induce systemic anaphylaxis and identify the cell types and mediators involved. hFcγR expression on mouse and human cells was compared to validate the model. Passive systemic anaphylaxis was induced by injection of heat-aggregated human intravenous immunoglobulin and active systemic anaphylaxis after immunization and challenge. Anaphylaxis severity was evaluated based on hypothermia and mortality. The contribution of receptors, mediators, or cell types was assessed based on receptor blockade or depletion. The human-to-mouse low-affinity FcγR locus swap engendered hFcγRIIA/IIB/IIIA/IIIB expression in mice comparable with that seen in human subjects. Knock-in mice were susceptible to passive and active anaphylaxis, accompanied by downregulation of both activating and inhibitory hFcγR expression on specific myeloid cells. The contribution of hFcγRIIA was predominant. Depletion of neutrophils protected against hypothermia and mortality. Basophils contributed to a lesser extent. Anaphylaxis was inhibited by platelet-activating factor receptor or histamine receptor 1 blockade. Low-affinity FcγR locus-switched mice represent an unprecedented model of cognate hFcγR expression. Importantly, IgG-related anaphylaxis proceeds within a native context of activating and inhibitory hFcγRs, indicating that, despite robust hFcγRIIB expression, activating signals can dominate to initiate a severe anaphylactic reaction.

Optimizing antiviral humoral responses the natural (killer) way

Abstract Many vaccine efforts to prevent deadly viral diseases aim to generate high-affinity, class-switched virus-specific antibodies, which arise mainly from germinal center (GC) reactions. However, vaccine-induced GCs are sometimes inadequate to prevent infections with major human pathogens, including HIV. An improved understanding of the mechanisms regulating the GC reaction will improve future vaccines. We discovered that natural killer (NK) cells suppress the generation of GC B cells and follicular helper T (Tfh) cells in a perforin-dependent manner following acute viral infection. Remarkably, a fraction of activated NK cells up-regulate the B-cell follicle homing receptor, CXCR5, which potentially facilitates interactions between cytotoxic NK cells and GC-inducing cells in or near B-cell follicles. We performed RNAseq analysis of this unique subset of NK cells to uncover potential mechanisms of humoral immune suppression. We also interrogated qualitative changes in antigen-specific B cells induced by NK cells following immunization with the hapten-carrier immunogen, NP-KLH. NK cells restricted the NP-KLH-induced GC response and number of NP-specific B cells. In the absence of NK cells, there was an increase in NP-specific plasma cells in the bone marrow at early time points and a higher ratio of high- to low-affinity NP-specific IgG in sera at late time points, suggesting that NK cells impair the quality of the humoral response that develops following immunization. To date, our data provide evidence that NK-cell suppression of GCs represents a previously underappreciated regulatory axis, potentially mediated by a unique subset of NK cells, which could be manipulated to enhance future vaccine efforts.

Diagnosis and treatment of autoimmune hemolytic anemia: classic approach and recent advances.

Diagnosis and treatment of autoimmune hemolytic anemia: classic approach and recent advances.

Impaired NK Cell Responses to Pertussis and H1N1 Influenza Vaccine Antigens in Human Cytomegalovirus-Infected Individuals.

NK cells contribute to postvaccination immune responses after activation by IL-2 from Ag-specific memory T cells or by cross-linking of the low-affinity IgG receptor, CD16, by Ag–Ab immune complexes. Sensitivity of NK cells to these signals from the adaptive immune system is heterogeneous and influenced by their stage of differentiation. CD56dimCD57+ NK cells are less responsive to IL-2 and produce less IFN-γ in response to T cell–mediated activation than do CD56bright or CD56dimCD57− NK cells. Conversely, NK cell cytotoxicity, as measured by degranulation, is maintained across the CD56dim subsets. Human CMV (HCMV), a highly prevalent herpes virus causing lifelong, usually latent, infections, drives the expansion of the CD56dimCD57+NKG2C+ NK cell population, skewing the NK cell repertoire in favor of cytotoxic responses at the expense of cytokine-driven responses. We hypothesized, therefore, that HCMV seropositivity would be associated with altered NK cell responses to vaccine Ags. In a cross-sectional study of 152 U.K. adults, with HCMV seroprevalence rate of 36%, we find that HCMV seropositivity is associated with lower NK cell IFN-γ production and degranulation after in vitro restimulation with pertussis or H1N1 influenza vaccine Ags. Higher expression of CD57/NKG2C and lower expression of IL-18Rα on NK cells from HCMV seropositive subjects do not fully explain these impaired responses, which are likely the result of multiple receptor–ligand interactions. This study demonstrates for the first time, to our knowledge, that HCMV serostatus influences NK cell contributions to adaptive immunity and raises important questions regarding the impact of HCMV infection on vaccine efficacy.

Modulation of IgE mediated reactions by FcgammaRII (HYP3P.406)

Abstract Cross-linking two high affinity IgE Receptors (FcεRI) by incubation with an anti-IgE antibody binding to the receptor bound IgE’s leads to a dose dependent bell-shaped activation of basophils. Besides of the high affinity IgE receptor basophils also express two isotypes of the low affinity IgG Receptor CD32, the activatory CD32a and the inhibitory CD32b. Previous work has shown that cross-linking of FcεRI and the inhibitory CD32b decreases cell activation, hinting at the Fc part of IgG antibodies taking part in basophil modulation. To characterize the interaction between FcεRI and CD32 we used two anaphylactogenic monoclonal anti-IgE antibodies. First we produced F(ab’)2 fragments which showed an increased maximal activation of basophils compared to the full length antibodies. Blocking the binding of IgG-Fc to CD32 with an anti-CD32 molecule results as well in a higher activation, indicating an effect of IgG-Fc. Then we humanized Le27 to increase the affinity to CD32. Humanized Le27 activated basophils to the same extend as the original Le27. To investigate whether the cross-linking of FcεRI with CD32 solely has an effect on basophil activation we produced a monovalent Le27 that can cross-link FcεRI and CD32, but not two FcεRI’s. Surprisingly, this monovalent antibody did activate basophils in whole blood, but not isolated basophils. Experiments will be performed with the monovalent Le27 to investigate if it can inhibit allergen-induced basophil activation.

Improved FcγRIIb Targeting Functionally Translates into Enhanced Inhibition of Basophil Activation

Background: Mast cells and basophils express the high-affinity IgE receptor, FcεRI, as well as the low-affinity IgG receptor, FcγRIIb. While FcεRI is responsible for IgE-dependent degranulation upon coaggregation with allergens, FcγRIIb has been shown to downregulate degranulation through cross-linking with FcεRI. A previously developed fusion protein consisting of an anti-IgE DARPin linked to the human IgG₁-Fc part (DE53-Fc) has been shown to simultaneously target FcεRI and FcγRIIb with low affinity and to thereby prevent basophil activation. The affinity of a ligand for its receptor is known to be critical for the functional consequences of the binding. So we generated two mutated DE53-Fc molecules with either an improved (DE53-Fc mut+) or a reduced (DE53-Fc mut-) binding to FcγRIIb and assessed their potential to inhibit IgE-dependent basophil activation. Methods: DE53-Fc was modified by introducing single site-directed point mutations in the Fc part. The mutated constructs were used to assess kinetic parameters as well as the inhibitory capacity on basophil activation and the production of leukotriene C₄ (LTC₄) and IL-13. Results: DE53-Fc mut+ showed increased affinity for FcγRIIb as well as an enhanced potential to inhibit IgG₁ binding to FcγRIIb, resulting in improved efficacy in functional assays. Furthermore, DE53-Fc mut+ decreased de novo-synthesized LTC₄ as well as the cytokine IL-13, suggesting that it might be an inhibitor of the allergic late-phase reaction. Conclusion: Our data suggest that improved binding to FcγRIIb at constant low-affinity binding to IgE leads to more efficient coaggregation of FcεRI-FcγRIIb and results in the enhanced inhibition of basophil activation.

Direct antiglobulin (“Coombs”) test-negative autoimmune hemolytic anemia: A review

We have reviewed the literature to identify and characterize reports of warm-antibody type, autoimmune hemolytic anemia in which the standard direct antiglobulin reaction was negative but a confirmatory test indicated that the red cells were opsonized with antibody. Three principal reasons account for the absence of a positive direct antiglobulin test in these cases: a) IgG sensitization below the threshold of detection by the commercial antiglobulin reagent, b) low affinity IgG, removed by preparatory washes not conducted at 4°C or at low ionic strength, and c) red cell sensitization by IgA alone, or rarely (monomeric) IgM alone, but not accompanied by complement fixation, and thus not detectable by a commercial antiglobulin reagent that contains anti-IgG and anti-C3. In cases in which the phenotype is compatible with warm-antibody type, autoimmune hemolytic anemia and the direct antiglobulin test is negative, an alternative method to detect low levels of IgG sensitization, use of 4°C, low ionic strength washes to prepare the cells for the direct antiglobulin test reaction to permit retention and identification of low affinity IgG antibodies, and, if the latter are uninformative, testing for sensitization with an anti-IgA, and, if necessary, an anti-IgM reagent identifies cases of warm-antibody type, immune hemolysis not verified by a commercial reagent.

Patient autoantibodies induce platelet destruction signals via raft-associated glycoprotein Ib and Fc RIIa in immune thrombocytopenia

Immune thrombocytopenia (ITP) is an acquired immune-mediated disorder characterized by thrombocytopenia in the absence of an underlying cause.1 The patho-physiology of ITP is multifactorial and includes the development of autoantibodies that trigger abnormal thrombopoiesis, enhanced platelet destruction, complement activation and T-cell mediated effects.1–3 Platelet autoantibodies are detected in approximately 50% of patients4 and generally target the fibrinogen receptor αIIbβ3 or the receptor for von Willebrand factor (VWF), the glycoprotein (GP) Ib-V-IX complex. Anti-αIIbβ3 antibodies (70–80% of cases) are thought to induce thrombocytopenia through increased platelet clearance by Fcγ receptor-bearing macrophages. Autoantibodies against GPIb-V-IX (20–40% of cases) often induce a more severe fall in platelet count5 that is less responsive to standard therapies, such as intravenous immunoglobulin G (IVIG).6 Thrombocytopenia induced by GPIb-V-IX autoantibodies has not been characterized in great detail. Some monoclonal antibodies against GPIbα are known to induce platelet activation7 that may lead to accelerated platelet destruction in ITP patients8 with autoantibodies against this receptor. Here we report how an autoantibody against GPIbα, obtained from a patient with ITP, induces recognition signals for macrophages through interplay between glycoprotein Ibα and the low affinity IgG receptor FcγRIIa in lipid rafts.

A report of a rare case of autoimmune haemolytic anaemia in a patient with Hodgkin's disease in whom routine serology was negative.

Autoimmune haemolytic anaemia (AIHA) is an acquired clinical condition which is characterised by the production of auto-antibodies that bind to the surface of circulating erythrocytes, leading to haemolysis and decreased survival of the red blood cells. The estimated yearly incidence of AIHA is 1–3 cases per 100,000 persons in the general population1. The direct antiglobulin test (DAT) is considered to be a cornerstone in establishing the diagnosis of AIHA, since there is uptake of autoantibodies and/or complement components onto the affected red blood cells. In suspected cases of AIHA, a positive DAT is predictive in 83% of patients, but not all cases of AIHA are DAT-positive2. Between 5% to 10% of all cases of AIHA are DAT-negative3. Three causes for this situation have been identified: (i) red blood cell bound IgG molecules, below the threshold of detection of the DAT, (ii) low-affinity IgG autoantibodies that are washed off the red cells during the washing phase for the test, and (iii) red cell bound IgA and rare warm IgM autoantibodies that are not detectable by the routine anti-human globulin reagent3. Given these different possibilities, a negative DAT must be weighed in the light of clinical suspicion. If there is clinical evidence of haemolysis, a thorough investigation with more specialised testing is needed. We report here a case of DAT-negative AIHA in a patient with Hodgkin’s lymphoma which was detected in our hospital blood bank laboratory.

Quorum Sensing Contributes to Activated IgM-Secreting B Cell Homeostasis

Maintenance of plasma IgM levels is critical for immune system function and homeostasis in humans and mice. However, the mechanisms that control homeostasis of the activated IgM-secreting B cells are unknown. After adoptive transfer into immune-deficient hosts, B lymphocytes expand poorly, but fully reconstitute the pool of natural IgM-secreting cells and circulating IgM levels. By using sequential cell transfers and B cell populations from several mutant mice, we were able to identify novel mechanisms regulating the size of the IgM-secreting B cell pool. Contrary to previous mechanisms described regulating homeostasis, which involve competition for the same niche by cells having overlapping survival requirements, homeostasis of the innate IgM-secreting B cell pool is also achieved when B cell populations are able to monitor the number of activated B cells by detecting their secreted products. Notably, B cell populations are able to assess the density of activated B cells by sensing their secreted IgG. This process involves the FcγRIIB, a low-affinity IgG receptor that is expressed on B cells and acts as a negative regulator of B cell activation, and its intracellular effector the inositol phosphatase SHIP. As a result of the engagement of this inhibitory pathway, the number of activated IgM-secreting B cells is kept under control. We hypothesize that malfunction of this quorum-sensing mechanism may lead to uncontrolled B cell activation and autoimmunity.

Adjuvant dependence of APS pathology-related low-affinity antibodies during secondary immune response to tetanus toxoid in BALB/c mice

One of the established animal models for autoimmune disease antiphospholipid syndrome (APS) is TTd hyperimmunization of mice. Tetanus toxoid (TTd) and plasma protein β2GPI share structural homology so that immunization with TTd induces appearance of cross-reactive antibodies. In this paper, we have investigated the presence and dynamic of fluctuation of specific (anti-TTd) and auto (anti-β2GPI) antibodies induced in BALB/c mice during secondary immune response after TTd immunization with alhydrogel or glycerol as adjuvants. In addition, we followed the induced reproductive pathology as a sign of autoimmune outcome. We show undoubtedly adjuvant dependance of (1) level of induced anti-TTd IgG antibodies, (2) changes in levels of low-affinity anti-β2GPI IgG antibodies, and (3) change in fecundity and fertility during secondary immune response. These findings once more indicate the importance of chosen adjuvants used for successful immunization and eventual autoantibody outcome, this time associated with the processes involving low affinity, natural antibodies.

The Receptor LMIR3 Negatively Regulates Mast Cell Activation and Allergic Responses by Binding to Extracellular Ceramide

Mast cells (MCs) are key effector cells in allergic reactions. However, the inhibitory mechanism that prevents excessive activation of MCs remains elusive. Here we show that leukocyte mono-immunoglobulin-like receptor 3 (LMIR3; also called CD300f) is a negative regulator of MC activation in vivo. LMIR3 deficiency exacerbated MC-dependent allergic responses in mice, including anaphylaxis, airway inflammation, and dermatitis. Both physical binding and functional reporter assays via an extracellular domain of LMIR3 showed that several extracellular lipids (including ceramide) and lipoproteins were possible ligands for LMIR3. Importantly, MCs were frequently surrounded by extracellular ceramide in vivo. Upon engagement of high-affinity immunoglobulin E receptor, extracellular ceramide-LMIR3 binding inhibited MC activation via immunoreceptor tyrosine-based inhibitory and switch motifs of LMIR3. Moreover, pretreatment with LMIR3-Fc fusion protein or antibody against either ceramide or LMIR3 interfered with this binding in vivo, thereby exacerbating passive cutaneous anaphylaxis. Thus, the interaction between extracellular ceramide and LMIR3 suppressed MC-dependent allergic responses.

Fcγ Receptors Inhibit Mouse and Human Basophil Activation

Besides high-affinity IgE receptors (FcεRI), human basophils express activating (FcγRIIA) and inhibitory (FcγRIIB) low-affinity IgG receptors. IgG receptors (FcγR) were also found on mouse basophils, but not identified. We investigated in this study FcγR and the biological consequences of their engagement in basophils of the two species. We found the following: (1) that mouse basophils also express activating (FcγRIIIA) and inhibitory (FcγRIIB) low-affinity FcγR; (2) that activating FcγR can activate both human and mouse basophils, albeit with different efficacies; (3) that negative signals triggered by inhibitory FcγR are dominant over positive signals triggered by activating FcγR, thus preventing both human and mouse basophils from being activated by IgG immune complexes; (4) that the coengagement of FcεRI with inhibitory and activating FcγR results in a FcγRIIB-dependent inhibition of IgE-induced responses of both human and mouse basophils; (5) that FcγRIIB has a similar dominant inhibitory effect in basophils from virtually all normal donors; and (6) that IL-3 upregulates the expression of both activating and inhibitory FcγR on human basophils from normal donors, but further enhances FcγRIIB-dependent inhibition. FcγR therefore function as a regulatory module, made of two subunits with antagonistic properties, that prevents IgG-induced and controls IgE-induced basophil activation in both mice and humans.