Studies on the regulatory mechanisms of Fcγ receptor function

Receptors for immunoglobulins (Fc receptors) play a central role during an immune response, as they mediate the specific recognition of antigens of almost infinite diversity by leukocytes, thereby linking the innate with the adaptive branches of immunity. This thesis undertook a series of studies to further investigate the role of Fc receptors in innate immune responses, using in vitro approaches to examine functional activity and underlying regulatory mechanisms.FcγRIla (CD32a) is a member of the Fcγ receptor family and mediates binding of multivalent IgG. Although CD32a is expressed by a number of myeloid cell types, including neutrophils, macrophages, eosinophils and monocytes, we observed that ligand binding to this receptor was suppressed, as evidenced by the low levels of CD32a-mediated IgG binding to these cells. We therefore aimed to determine the mechanisms that account for the myeloid-specific suppression of CD32a ligand binding. A series of experiments were performed to examine the effects of proteases and sialylation of CD32a in the regulation of IgG binding. In addition, the role of association of CD32a with plasma membrane microdomains rich in cholesterol and sphingolipids in the regulation of IgG binding to CD32a was examined. These membrane microdomains, also termed lipid rafts, have previously been reported to be essential for efficient receptor signalling. Chemical disruption of lipid raft structure by depletion or sequestration of membrane cholesterol greatly inhibited CD32a-mediated IgG binding, strongly implicating lipid rafts in the control of CD32a function. To further investigate this suggestion, specific CD32a mutants were generated, which would be predicted to have altered association with lipid rafts. Both these mutants showed reduced association with lipid rafts (A224S and C241A) and displayed decreased levels of IgG binding compared with wild type CD32a. Additionally, we generated a chimaeric CD32a receptor containing a glycophosphatidylinositol (GPI] lipid anchor consensus sequence, which would constitutively associate with lipid rafts. GPI-anchored CD32a exhibited increased capacity for IgG binding compared with the full-length transmembrane CD32a. Our findings clearly suggest a major role for lipid rafts in the regulation of IgG binding and more specifically, that suppression of CD32a-mediated IgG binding in myeloid cells is achieved by receptor exclusion from lipid raft membrane microdomainsThis thesis also describes an investigation of the association of Fcγ receptor genetic variants with idiopathic pulmonary fibrosis (IPF) susceptibility and progression. In particular, two allelic variants of CD32a (H131/R131) and CD16 (NA1/NA2) were examined that confer altered IgG binding and may therefore contribute to disease pathogenesis. Copy number variation of the FCGR3B gene was also determined using a quantitative PCR-based approach. Susceptibility to IPF was found to be associated with the NA1 allele of FcγRIIIb and increased FCGR3B copy number. In addition, IPF disease severity at disease presentation and progression over a 1 2-month period following diagnosis was found to be linked to the FcγRIIa H131 variant. These results support the involvement of Fcγ-mediated interactions in IPF and reveal a novel role of Fcγ receptors in IPF disease pathogenesis and progression

The mechanistic basis of protection by non-neutralizing anti-alphavirus antibodies

Although neutralizing monoclonal antibodies (mAbs) against epitopes within the alphavirus E2 protein can protect against infection, the functional significance of non-neutralizing mAbs is poorly understood. Here, we evaluate the activity of 13 non-neutralizing mAbs against Mayaro virus (MAYV), an emerging arthritogenic alphavirus. These mAbs bind to the MAYV virion and surface of infected cells but fail to neutralize infection in cell culture. Mapping studies identify six mAb binding groups that localize to discrete epitopes within or adjacent to the A domain of the E2 glycoprotein. Remarkably, passive transfer of non-neutralizing mAbs protects against MAYV infection and disease in mice, and their efficacy requires Fc effector functions. Monocytes mediate the protection of non-neutralizing mAbs in vivo, as Fcγ-receptor-expressing myeloid cells facilitate the binding, uptake, and clearance of MAYV without antibody-dependent enhancement of infection. Humoral protection against alphaviruses likely reflects contributions from non-neutralizing antibodies through Fc-dependent mechanisms that accelerate viral clearance

A mouse model for HIV-1 entry

Passive transfer of neutralizing antibodies against HIV-1 can prevent infection in macaques and seems to delay HIV-1 rebound in humans. Anti-HIV antibodies are therefore of great interest for vaccine design. However, the basis for their in vivo activity has been difficult to evaluate systematically because of a paucity of small animal models for HIV infection. Here we report a genetically humanized mouse model that incorporates a luciferase reporter for rapid quantitation of HIV entry. An antibody’s ability to block viral entry in this in vivo model is a function of its bioavailability, direct neutralizing activity, and effector functions

Cross-reactive coronavirus antibodies with diverse epitope specificities and Fc effector functions

The continual emergence of novel coronavirus (CoV) strains, like SARS-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. From a recovered SARS-CoV donor sample, we identify and characterize a panel of six monoclonal antibodies that cross-react with CoV spike (S) proteins from the highly pathogenic SARS-CoV and SARS-CoV-2, and demonstrate a spectrum of reactivity against other CoV. Epitope mapping reveals that these antibodies recognize multiple epitopes on SARS-CoV-2 S, including the receptor binding domain, N-terminal domain, and S2 subunit. Functional characterization demonstrates that the antibodies mediate phagocytosis - and in some cases trogocytosis - but not neutralization in vitro. When tested in vivo in murine models, two of the antibodies demonstrate a reduction in hemorrhagic pathology in the lungs. The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies

A Combination of Two Human Monoclonal Antibodies Limits Fetal Damage by Zika Virus in Macaques

Human infection by Zika virus (ZIKV) during pregnancy can lead to vertical transmission and fetal aberrations, including microcephaly. Prophylactic administration of antibodies can diminish or prevent ZIKV infection in animal models, but whether passive immunization can protect nonhuman primates and their fetuses during pregnancy has not been determined. Z004 and Z021 are neutralizing monoclonal antibodies to domain III of the envelope (EDIII) of ZIKV. Together the two antibodies protect nonpregnant macaques against infection even after Fc modifications to prevent antibody-dependent enhancement in vitro (ADE) and extend their half-lives. Here we report on prophylactic co-administration of the Fc-modified antibodies to pregnant rhesus macaques challenged 3 times with ZIKV during first and second trimester. The two antibodies did not entirely eliminate maternal viremia but limited vertical transmission protecting the fetus from neurologic damage. Thus, maternal passive immunization with two antibodies to EDIII can shield primate fetuses from the harmful effects of ZIKV

CD32 ligation promotes the activation of CD4+T cells

Low affinity receptors for the Fc portion of IgG (FcγRs) represent a critical link between innate and adaptive immunity. Immune complexes (ICs) are the natural ligands for low affinity FcγRs, and high levels of ICs are usually detected in both, chronic viral infections and autoimmune diseases. The expression and function of FcγRs in myeloid cells, NK cells and B cells have been well characterized. By contrast, there are controversial reports about the expression and function of FcγRs in T cells. Here, we demonstrated that ∼2% of resting CD4+ T cells express cell surface FcγRII (CD32). Analysis of CD32 expression in permeabilized cells revealed an increased proportion of CD4+CD32+ T cells (∼9%), indicating that CD4+ T cells store a CD32 cytoplasmic pool. Activation of CD4+ T cells markedly increased the expression of CD32 either at the cell surface or intracellularly. Analysis of CD32 mRNA transcripts in activated CD4+ T cells revealed the presence of both, the stimulatory FcγRIIa (CD32a) and the inhibitory FcγRIIb (CD32b) isoforms of CD32, being the CD32a:CD32b mRNA ratio ∼5:1. Consistent with this finding, we found not only that CD4+ T cells bind aggregated IgG, used as an IC model, but also that CD32 ligation by specific mAb induced a strong calcium transient in CD4+ T cells. Moreover, we found that pretreatment of CD4+ T cells with immobilized IgG as well as cross-linking of CD32 by specific antibodies increased both, the proliferative response of CD4+ T cells and the release of a wide pattern of cytokines (IL-2, IL-5, IL-10, IL-17, IFN-γ, and TNF-α) triggered by either PHA or anti-CD3 mAb. Collectively, our results indicate that ligation of CD32 promotes the activation of CD4+ T cells. These findings suggest that ICs might contribute to the perpetuation of chronic inflammatory responses by virtue of its ability to directly interact with CD4+ T cells through CD32a, promoting the activation of T cells into different inflammatory profiles.Fil: Holgado, María Pía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Sananez, Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Raiden, Silvina Claudia. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños Pedro Elizalde (ex Casa Cuna); Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Geffner, Jorge Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Arruvito, Maria Lourdes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentin

Maternal Anti-Dengue IgG Fucosylation Predicts Susceptibility to Dengue Disease in Infants

Infant mortality from dengue disease is a devastating global health burden that could be minimized with the ability to identify susceptibility for severe disease prior to infection. Although most primary infant dengue infections are asymptomatic, maternally derived anti-dengue immunoglobulin G (IgGs) present during infection can trigger progression to severe disease through antibody-dependent enhancement mechanisms. Importantly, specific characteristics of maternal IgGs that herald progression to severe infant dengue are unknown. Here, we define \u3e /=10% afucosylation of maternal anti-dengue IgGs as a risk factor for susceptibility of infants to symptomatic dengue infections. Mechanistic experiments show that afucosylation of anti-dengue IgGs promotes FcgammaRIIIa signaling during infection, in turn enhancing dengue virus replication in FcgammaRIIIa(+) monocytes. These studies identify a post-translational modification of anti-dengue IgGs that correlates with risk for symptomatic infant dengue infections and define a mechanism by which afucosylated antibodies and FcgammaRIIIa enhance dengue infections

Understanding the genomic structure of copy number variants of the low-affinity Fcγ receptor region allows confirmation of the association of FCGR3B deletion with rheumatoid arthritis

Fcγ receptors are a family of cell-surface receptors that are expressed by a host of different innate and adaptive immune cells and mediate inflammatory responses by binding the Fc portion of immunoglobulin G (IgG). In humans, five low affinity receptors are encoded by the genes FCGR2A, FCGR2B, FCGR2C, FCGR3A and FCGR3B, which are located in a 82.5kb segmental tandem duplication on chromosome 1q23.3, which shows extensive copy number variation. Deletions of FCGR3B have been suggested to increase the risk of inflammatory diseases such as systemic lupus erythematosus and rheumatoid arthritis. In this study we identify the deletion breakpoints of FCGR3B deletion alleles in the UK population and endogamous Native American populations, and show that some but not all alleles are likely to be identical-by-descent. We also localise a duplication breakpoint, confirming that the mechanism of CNV generation is non-allelic homologous recombination, and identify several alleles with gene conversion events using fosmid sequencing data. We use information on the structure of the deletion alleles to distinguish FCGR3B deletions from FCGR3A deletions in whole genome aCGH data. Reanalysis of published aCGH data supports association of FCGR3B deletion with increased risk rheumatoid arthritis in a large cohort of 1982 cases and 3271 controls (Odds Ratio 1.61, p=2.9x10-3)