Dynamics of intracellular neonatal Fc receptor-ligand interactions in primary macrophages using biophysical fluorescence techniques.

The neonatal Fc receptor (FcRn) is responsible for the recycling of endocytosed albumin and IgG, and contributes to their long plasma half-life. We recently identified an FcRn-dependent recycling pathway from macropinosomes in macrophages; however, little is known about the dynamics of intracellular FcRn-ligand interactions to promote recycling. Here we demonstrate a multiplexed biophysical fluorescent microscopy approach to resolve the spatiotemporal dynamics of albumin-FcRn interactions in living bone marrow-derived macrophages (BMDMs). We used the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) to detect the interaction of a FcRn-mCherry fusion protein with endocytosed Alexa Fluor 488-labeled human serum albumin (HSA-AF488) in BMDMs, and raster image correlation spectroscopy (RICS) analysis of single fluorescent-labeled albumin molecules to monitor the diffusion kinetics of internalized albumin. Our data identified a major fraction of immobile HSA-AF488 molecules in endosomal structures of human FcRn-positive mouse macrophages and an increase in FLIM-FRET following endocytosis, including detection of FRET in tubular-like structures. A nonbinding mutant of albumin showed minimum FLIM-FRET and high mobility. These data reveal the kinetics of FcRn-ligand binding within endosomal structures for recruitment into transport carriers for recycling. These approaches have wide applicability for analyses of intracellular ligand-receptor interactions.

Therapeutic exploitation of neutrophils to fight cancer.

Antibody-based immunotherapy is a promising strategy in cancer treatment. Antibodies can directly inhibit tumor growth, induce complement-dependent cytotoxicity and induce Fc receptor-mediated elimination of tumor cells by macrophages and natural killer cells. Until now, however, neutrophils have been largely overlooked as potential effector cells, even though they are the most abundant type of immune cells in the circulation. Neutrophils display heterogeneity, especially in the context of cancer. Therefore, their role in cancer is debated. Nevertheless, neutrophils possess natural anti-tumor properties and appropriate stimulation, i.e. specific targeting via antibody therapy, induces potent tumor cell killing, especially via targeting of the immunoglobulin A Fc receptor (FcαRI, CD89). In this review we address the mechanisms of tumor cell killing by neutrophils and the role of neutrophils in induction of anti-tumor immunity. Moreover, possibilities for therapeutic targeting are discussed.

Fc Receptor Variants and Disease: A Crucial Factor to Consider in the Antibody Therapeutics in Clinic.

The fragment crystallizable (Fc) domain of antibodies is responsible for their protective function and long-lasting serum half-life via Fc-mediated effector function, transcytosis, and recycling through its interaction with Fc receptors (FcRs) expressed on various immune leukocytes, epithelial, and endothelial cells. Therefore, the Fc-FcRs interaction is a control point of both endogenous and therapeutic antibody function. There are a number of reported genetic variants of FcRs, which include polymorphisms in (i) extracellular domain of FcRs, which change their affinities to Fc domain of antibodies; (ii) both cytoplasmic and intracellular domain, which alters the extent of signal transduction; and (iii) the promoter region of the FcRs gene, which affects the expression level of FcRs, thus being associated with the pathogenesis of disease indications. In this review, we firstly describe the correlation between the genetic variants of FcRs and immunological disorders by individual differences in the extent of FcRs-mediated regulations. Secondly, we discuss the influence of the genetic variants of FcRs on the susceptibility to infectious diseases or cancer in the perspective of FcRs-induced effector functions. Overall, we concluded that the genetic variants of FcRs are one of the key elements in the design of antibody therapeutics due to their variety of clinical outcomes among individuals.

The neonatal Fc receptor in cancer FcRn in cancer.

Since the neonatal IgG Fc receptor (FcRn) was discovered, it was found to be involved in immunoglobulin recycling and biodistribution, immune complexes routing, antigen presentation, humoral immune response, and cancer immunosurveillance. The latest data show that FcRn plays a part in cancer pathophysiology. In various types of cancers, such as lung and colorectal cancer, FcRn has been described as an early marker for prognosis. Dysregulation of FcRn expression by cancer cells allows them to increase their metabolism, and this process could be exploited for passive targeting of cytotoxic drugs. However, the roles of this receptor depend on whether the studied cell population is the tumor tissue or the infiltrating cells, bringing forward the need for further studies.

Serum IgA Fc effector functions in infectious disease and cancer.

Immunoglobulin (Ig) A is the most abundant antibody isotype present at mucosal surfaces and the second most abundant in human serum. In addition to preventing pathogen entry at mucosal surfaces, IgA can control and eradicate bacterial and viral infections through a variety of antibody-mediated innate effector cell mechanisms. The role of mucosal IgA in infection (e.g. neutralization) and in inflammatory homeostasis (e.g. allergy and autoimmunity) has been extensively investigated; by contrast, serum IgA is comparatively understudied. IgA binding to fragment crystallizable alpha receptor plays a dual role in the activation and inhibition of innate effector cell functions. Mounting evidence suggests that serum IgA induces potent effector functions against various bacterial and some viral infections including Neisseria meningitidis and rotavirus. Furthermore, in the era of immunotherapy, serum IgA provides an interesting alternative to classical IgG monoclonal antibodies to treat cancer and infectious pathogens. Here we discuss the role of serum IgA in infectious diseases with reference to bacterial and viral infections and the potential for IgA as a monoclonal antibody therapy.

Integration of Ontogeny Into a Physiologically Based Pharmacokinetic Model for Monoclonal Antibodies in Premature Infants.

An understanding of pediatric pharmacokinetics (PK) is essential for first-in-pediatric dose selection and clinical trial design. At present, there is no reliable way to scale the PK of monoclonal antibodies and immunoglobulin G drug products from adults to young children or to premature infants-a vulnerable population with a rapidly growing drug development pipeline. In this work, pediatric physiologically based PK models are constructed in PK-Sim and Mobi to explore the PK of pagibaximab, palivizumab, MEDI8897, and intravenous immunoglobulin in preterm infants. In addition to considering ontogeny in pediatric organ volumes, organ composition, blood flow rates, and hematocrit, advanced ontogeny is applied for 3 key parameters: capillary surface area, hematopoietic cell concentration, and lymph flow rate. The role and importance of each parameter for determining pediatric clearance (CL) and volume of distribution at steady state (VSS ) are quantitatively assessed with a local sensitivity analysis. In addition, the uncertainty around parameters with limited information in pediatrics is addressed (eg, free neonatal Fc receptor concentration). The full ontogeny parameterization yields pediatric PK predictions that are within 1.5-fold prediction error >90% of the time for preterm infants, with an absolute average fold error of 1.05. This result suggests that many of the key factors related to ontogeny are appropriately addressed. Overall, this study makes a first step toward developing a platform pediatric physiologically based PK model for monoclonal antibodies and immunoglobulin G drug products by solidifying existing parameterizations, integrating new concepts, and drawing attention to unmet needs for physiologic knowledge in children.

FcRn mediates fast recycling of endocytosed albumin and IgG from early macropinosomes in primary macrophages.

The neonatal Fc receptor (FcRn) rescues albumin and IgG from degradation following endocytosis and thereby extends the half-life of these plasma proteins. However, the pathways for the uptake of these soluble FcRn ligands, and the recycling itinerary of the FcRn-ligand complexes, have not been identified in primary cells. Here, we have defined the recycling of human albumin and IgG in primary mouse macrophages selectively expressing the human FcRn. Albumin is internalised by macropinocytosis; in the absence of FcRn, internalised albumin is rapidly degraded, while in the presence of FcRn albumin colocalises to SNX5-positive membrane domains and is partitioned into tubules emanating from early macropinosomes for delivery in transport carriers to the plasma membrane. Soluble monomeric IgG was also internalised by macropinocytosis and rapidly recycled by the same pathway. In contrast, the fate of IgG bound to surface Fcγ receptors differed from monomeric IgG endocytosed by macropinocytosis. Overall, our findings identify a rapid recycling pathway for FcRn ligands from early macropinosomes to the cell surface of primary cells.

Immunoglobulin A: magic bullet or Trojan horse?

BACKGROUND: Neutrophils participate in the first line of defense by executing several killing mechanisms, including phagocytosis, degranulation and the release of neutrophil extracellular traps. Additionally, they can orchestrate the adaptive immune system by secreting cytokines and chemokines. Opsonization with antibodies aids in the recognition of pathogens, via binding to Fc receptors on the neutrophil surface. Immunoglobulin A (IgA) is the most abundant antibody at mucosal sites and has multiple functions in homeostasis and immunity. Neutrophils and IgA can interact via the IgA Fc receptor Fc?RI (CD89), leading to pro- or anti-inflammatory responses. AIMS: The aim of this review is to give a concise overview of the interplay between IgA, Fc?RI and neutrophils and to explore potential therapies for autoimmune diseases and cancer. RESULTS: Crosslinking of FcαRI by IgA-immune complexes yields potent neutrophil activation and pro-inflammatory effector functions, including the recruitment of neutrophils. This can lead to neutrophil accumulation and tissue destruction during IgA-autoantibody mediated diseases. Conversely, for cancer treatment, the myriad of powerful neutrophil effector functions after targeting FcαRI may contribute to effective immunotherapy. CONCLUSION: By interfering with or actively promoting the interaction between IgA and FcαRI, therapies for multiple maladies could be developed.

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.

The Implication and Significance of Beta 2 Microglobulin: A Conservative Multifunctional Regulator.

OBJECTIVE: This review focuses on the current knowledge on the implication and significance of beta 2 microglobulin (ß2M), a conservative immune molecule in vertebrate. DATA SOURCES: The data used in this review were obtained from PubMed up to October 2015. Terms of ß2M, immune response, and infection were used in the search. STUDY SELECTIONS: Articles related to ß2M were retrieved and reviewed. Articles focusing on the characteristic and function of ß2M were selected. The exclusion criteria of articles were that the studies on ß2M-related molecules. RESULTS: ß2M is critical for the immune surveillance and modulation in vertebrate animals. The dysregulation of ß2M is associated with multiple diseases, including endogenous and infectious diseases. ß2M could directly participate in the development of cancer cells, and the level of ß2M is deemed as a prognostic marker for several malignancies. It also involves in forming major histocompatibility complex (MHC class I or MHC I) or like heterodimers, covering from antigen presentation to immune homeostasis. CONCLUSIONS: Based on the characteristic of ß2M, it or its signaling pathway has been targeted as biomedical or therapeutic tools. Moreover, ß2M is highly conserved among different species, and overall structures are virtually identical, implying the versatility of ß2M on applications.

The role of Fc-receptors in the uptake and transport of therapeutic antibodies in the retinal pigment epithelium.

In the ophthalmological clinic, intravitreally applied antibodies or Fc-containing fusion proteins are frequently used, but the biology and pharmacokinetics of these therapeutics in the retina are not well understood. We have previously shown intracellular uptake of Fc-containing molecules in RPE cells. In this study, we investigated the involvement of Fc-receptors, both Fcγ-receptors and the neonatal Fc-receptor (FcRn) in the uptake and intracellular trafficking of the VEGF-antagonists bevacizumab, aflibercept and the anti-CD20 antibody rituximab in three different model systems, primary porcine RPE cells, ARPE-19 cells and porcine RPE/choroid explants. The expression of Fcγ-receptors was tested in primary porcine RPE cells, and the expression of Fcγ-receptors I and II could be shown in RT-PCR and qRT-PCR, while the expression of FcRn was additionally confirmed in Western blot and immunocytochemistry. All three compounds, bevacizumab, rituximab and aflibercept, were taken up into the cells and displayed a characteristic time-dependent pattern, as shown in Western blot and immunohistochemistry. The uptake was not altered by the inhibition of Fcγ-receptors using different inhibitors (TruStain FcX, genistein, R406). However, the inhibition of FcRn with an antagonistic antibody reduced intracellular IgG in porcine RPE cells (rituximab) and ARPE-19 cells (bevacizumab, rituximab). Colocalisations between the tested compounds and myosin7a could be found. In addition, limited colocalization with FcRn and the tested compounds, as well as triple localization between compound, FcRn and myosin7a could be detected, indicating a role of myosin7a in FcRn mediated transport. However, the colocalizations are restricted to small fractions of the Fc-containing compounds. Furthermore, the FcRn is mainly found in the membrane section, where only minute amounts of the Fc-containing compounds are seen, suggesting a limited interaction. An apical to choroidal transport of IgG through the RPE/choroid can be found in RPE/choroid explants. Inhibition of FcRn increases the amount of bevacizumab found on the choroidal side, suggesting a role of FcRn in the recycling of bevacizumab. In conclusion, our data indicate a role for FcRn, but not Fcγ-receptors, in the uptake and transport of Fc-containing molecules in the RPE and indicate a recycling function of FcRn in the retina.

FCRL regulation in innate-like B cells.

Coelomic cavity-derived B-1 and splenic marginal zone (MZ) B lymphocytes play principal roles in frontline host protection at homeostasis and during primary humoral immune responses. Although they share many features that enable rapid and broad-based defense against pathogens, these innate-like subsets have disparate B cell receptor (BCR) signaling features. Members of the Fc receptor-like (FCRL) family are preferentially expressed by B cells and possess tyrosine-based immunoregulatory function. An unusual characteristic of many of these cell surface proteins is the presence of both inhibitory (ITIM) and activating (ITAM-like) motifs in their cytoplasmic tails. In mice, FCRL5 is a discrete marker of splenic MZ and peritoneal B-1 B cells and has both ITIM and ITAM-like sequences. Recent work explored its signaling properties and identified that FCRL5 differentially influences innate-like BCR function. Closer scrutiny of these differences disclosed the ability of FCRL5 to counter-regulate BCR activation by recruiting SHP-1 and Lyn to its cytoplasmic motifs. Furthermore, the disparity in FCRL5 regulation between MZ and B-1 B cells correlated with relative intracellular concentrations of SHP-1. These findings validate and extend our understanding of the unique signaling features in innate-like B cells and provide new insight into the complexity of FCRL modulation.

Reversal of Arthritis by Human Monomeric IgA Through the Receptor-Mediated SH2 Domain-Containing Phosphatase 1 Inhibitory Pathway.

OBJECTIVE: Rheumatoid arthritis (RA), one of the most frequent chronic inflammatory rheumatic disorders, is characterized by the presence of autoantibodies and joint infiltration by activated immune cells, leading to cartilage and bone destruction. IgA occurs predominantly as monomers (mIgA) in plasma and regulates many cell responses through interaction with the Fcα receptor type I (FcαRI). FcαRI targeting by anti-FcαRI Fab inhibits activating receptors by inducing an inhibitory immunoreceptor tyrosine-based activation motif (ITAMi) configuration through SH2 domain-containing phosphatase 1 (SHP-1) recruitment. The aim of this study was to investigate the potential utility of mIgA for the treatment of arthritis by acting as an inducer of ITAMi signaling. METHODS: The effect of plasma-derived human mIgA on inhibition of multiple heterologous receptors was evaluated on FcαRI+ cell transfectants, blood phagocytes from healthy individuals, and synovial cells from RA patients. FcαRI-transgenic mice and wild-type mice treated with mIgA were studied in models of collagen antibody-induced arthritis (CAIA) and collagen-induced arthritis (CIA). The mice were assessed for development of arthritis using an arthritis score, and joint tissue samples were evaluated for the extent of leukocyte infiltration and expression of phosphatase. RESULTS: Treatment with mIgA impaired cell activation in an FcαRI-FcRγ-dependent manner, involving ITAMi signaling. Human mIgA or anti-FcαRI Fab were strongly effective in either preventing or attenuating CAIA or CIA in FcαRI-transgenic mice. Administration of mIgA markedly inhibited the recruitment of leukocytes to the inflamed joints of mice, which was associated with induction of SHP-1 phosphorylation in joint tissue cells. Moreover, mIgA reversed the state of inflammation in the synovial fluid of RA patients by inducing an ITAMi configuration. CONCLUSION: These results demonstrate a therapeutic potential of human mIgA in experimental arthritis. The findings support future clinical exploration of mIgA for the treatment of RA.

FcRn Rescues Recombinant Factor VIII Fc Fusion Protein from a VWF Independent FVIII Clearance Pathway in Mouse Hepatocytes.

We recently developed a longer lasting recombinant factor VIII-Fc fusion protein, rFVIIIFc, to extend the half-life of replacement FVIII for the treatment of people with hemophilia A. In order to elucidate the biological mechanism for the elongated half-life of rFVIIIFc at a cellular level we delineated the roles of VWF and the tissue-specific expression of the neonatal Fc receptor (FcRn) in the biodistribution, clearance and cycling of rFVIIIFc. We find the tissue biodistribution is similar for rFVIIIFc and rFVIII and that liver is the major clearance organ for both molecules. VWF reduces the clearance and the initial liver uptake of rFVIIIFc. Pharmacokinetic studies in FcRn chimeric mice show that FcRn expressed in somatic cells (hepatocytes or liver sinusoidal endothelial cells) mediates the decreased clearance of rFVIIIFc, but FcRn in hematopoietic cells (Kupffer cells) does not affect clearance. Immunohistochemical studies show that when rFVIII or rFVIIIFc is in dynamic equilibrium binding with VWF, they mostly co localize with VWF in Kupffer cells and macrophages, confirming a major role for liver macrophages in the internalization and clearance of the VWF-FVIII complex. In the absence of VWF a clear difference in cellular localization of VWF-free rFVIII and rFVIIIFc is observed and neither molecule is detected in Kupffer cells. Instead, rFVIII is observed in hepatocytes, indicating that free rFVIII is cleared by hepatocytes, while rFVIIIFc is observed as a diffuse liver sinusoidal staining, suggesting recycling of free-rFVIIIFc out of hepatocytes. These studies reveal two parallel linked clearance pathways, with a dominant pathway in which both rFVIIIFc and rFVIII complexed with VWF are cleared mainly by Kupffer cells without FcRn cycling. In contrast, the free fraction of rFVIII or rFVIIIFc unbound by VWF enters hepatocytes, where FcRn reduces the degradation and clearance of rFVIIIFc relative to rFVIII by cycling rFVIIIFc back to the liver sinusoid and into circulation, enabling the elongated half-life of rFVIIIFc.

Fc receptors as adaptive immunoreceptors.

Most biological activities of antibodies depend on their ability to engage Receptors for the Fc portion of immunoglobulins (FcRs) on a variety of cell types. As FcRs can trigger positive and negative signals, as these signals control several biological activities in individual cells, as FcRs are expressed by many cells of hematopoietic origin, mostly of the myeloid lineage, as these cells express various combinations of FcRs, and as FcR-expressing cells have different functional repertoires, antibodies can exert a wide spectrum of biological activities. Like B and T Cell Receptors (BCRs and TCRs), FcRs are bona fide immunoreceptors. Unlike BCRs and TCRs, however, FcRs are immunoreceptors with an adaptive specificity for antigen, with an adaptive affinity for antibodies, with an adaptive structure and with an adaptive signaling. They induce adaptive biological responses that depend on their tissue distribution and on FcR-expressing cells that are selected locally by antibodies. They critically determine health and disease. They are thus exquisitely adaptive therapeutic tools.

FcRn: from molecular interactions to regulation of IgG pharmacokinetics and functions.

The neonatal Fc receptor, FcRn, is related to MHC class I with respect to its structure and association with ß2microglobulin (ß2m). However, by contrast with MHC class I molecules, FcRn does not bind to peptides, but interacts with the Fc portion of IgGs and belongs to the Fc receptor family. Unlike the 'classical' Fc receptors, however, the primary functions of FcRn include salvage of IgG (and albumin) from lysosomal degradation through the recycling and transcytosis of IgG within cells. The characteristic feature of FcRn is pH-dependent binding to IgG, with relatively strong binding at acidic pH (<6.5) and negligible binding at physiological pH (7.3-7.4). FcRn is expressed in many different cell types, and endothelial and hematopoietic cells are the dominant cell types involved in IgG homeostasis in vivo. FcRn also delivers IgG across cellular barriers to sites of pathogen encounter and consequently plays a role in protection against infections, in addition to regulating renal filtration and immune complex-mediated antigen presentation. Further, FcRn has been targeted to develop both IgGs with extended half-lives and FcRn inhibitors that can lower endogenous antibody levels. These approaches have implications for the development of longer lived therapeutics and the removal of pathogenic or deleterious antibodies.

Fc receptor-dependent mechanisms of monoclonal antibody therapy of cancer.

Targeted therapies like treatment with monoclonal antibodies (mAbs) have entered the arsenal of modern anticancer drugs. mAbs combine specificity with multiple effector functions that can lead to reduction of tumour burden. Direct mechanisms of action, including induction of apoptosis or growth inhibition, depend on the biology of the target antigen. Fc tails of mAbs have furthermore the potential to initiate complement-dependent lysis as well as immune effector cell-mediated tumour cell killing via binding to Fc receptors. Natural killer cells can induce apoptosis via antibody-dependent cellular cytotoxicity (ADCC), whereas macrophages are able to phagocytose mAb-opsonized tumour cells (antibody-dependent cellular phagocytosis; ADCP). Finally, neutrophils can induce non-apoptotic tumour cell death, especially in the presence of immunoglobulin A (IgA) antitumour mAbs. In spite of promising clinical successes in some malignancies, improvement of mAb immunotherapy is required to achieve overall complete remission in cancer patients. New strategies to enhance Fc receptor-mediated mechanisms of action or to overcome the immunosuppressive microenvironment of the tumour in mAb therapy of cancer are therefore currently being explored and will be addressed in this chapter.