Tissue niche occupancy determines the contribution of fetal- versus bone-marrow-derived macrophages to IgG effector functions.

Understanding the mechanisms underlying cytotoxic immunoglobulin G (IgG) activity is critical for improving therapeutic antibody activity and inhibiting autoantibody-mediated tissue pathology. While prior research highlights the important role of the mononuclear phagocytic system for removing opsonized target cells, it remains unclear which monocyte or macrophage subsets stemming from fetal or post-natal bone-marrow (BM)-associated definitive hematopoiesis are involved in target cell depletion. By using a titrated irradiation approach as well as Kupffer-cell-specific deletion of activated Fcγ receptor signaling, we establish conditions under which the contribution of BM-derived monocytes versus yolk-sac-derived liver-resident macrophages to cytotoxic IgG activity can be studied. Our results demonstrate that liver-resident macrophages originating from either fetal or adult hematopoiesis play a central role in IgG-mediated depletion of opsonized target cells from the peripheral blood under steady-state conditions, highlighting the impact of the tissue niche and not macrophage origin for cytotoxic antibody activity.

My Name Is Legion, for We Are Many-The Complex Community of Antibody Receptors.

Antibodies act as the central mediators of immunological defense mechanisms, therapeutic agents within clinics, and the mediators of various immune-mediated disorders [...].

Mixed IgG Fc immune complexes exhibit blended binding profiles and refine FcR affinity estimates.

Immunoglobulin G (IgG) antibodies coordinate immune effector responses by interacting with effector cells via fragment crystallizable γ (Fcγ) receptors. The IgG Fc domain directs effector responses through subclass and glycosylation variation. Although each Fc variant has been extensively characterized in isolation, during immune responses, IgG is almost always produced in Fc mixtures. How this influences effector responses has not been examined. Here, we measure Fcγ receptor binding to mixed Fc immune complexes. Binding of these mixtures falls along a continuum between pure cases and quantitatively matches a mechanistic model, except for several low-affinity interactions mostly involving IgG2. We find that the binding model provides refined estimates of their affinities. Finally, we demonstrate that the model predicts effector cell-elicited platelet depletion in humanized mice. Contrary to previous views, IgG2 exhibits appreciable binding through avidity, though it is insufficient to induce effector responses. Overall, this work demonstrates a quantitative framework for modeling mixed IgG Fc-effector cell regulation.

Immunoglobulin G-dependent inhibition of inflammatory bone remodeling requires pattern recognition receptor Dectin-1.

Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases. In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution of inflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.

Mixed IgG Fc immune complexes exhibit blended binding profiles and refine FcR affinity estimates.

Immunoglobulin (Ig)G antibodies coordinate immune effector responses by selectively binding to target antigens and then interacting with various effector cells via the Fcγ receptors. The Fc domain of IgG can promote or inhibit distinct effector responses across several different immune cell types through variation based on subclass and Fc domain glycosylation. Extensive characterization of these interactions has revealed how the inclusion of certain Fc subclasses or glycans results in distinct immune responses. During an immune response, however, IgG is produced with mixtures of Fc domain properties, so antigen-IgG immune complexes are likely to almost always be comprised of a combination of Fc forms. Whether and how this mixed composition influences immune effector responses has not been examined. Here, we measured Fcγ receptor binding to immune complexes of mixed Fc domain composition. We found that the binding properties of the mixed-composition immune complexes fell along a continuum between those of the corresponding pure cases. Binding quantitatively matched a mechanistic binding model, except for several low-affinity interactions mostly involving IgG2. We found that the affinities of these interactions are different than previously reported, and that the binding model could be used to provide refined estimates of these affinities. Finally, we demonstrated that the binding model can predict effector-cell elicited platelet depletion in humanized mice, with the model inferring the relevant effector cell populations. Contrary to the previous view in which IgG2 poorly engages with effector populations, we observe appreciable binding through avidity, but insufficient amounts to observe immune effector responses. Overall, this work demonstrates a quantitative framework for reasoning about effector response regulation arising from IgG of mixed Fc composition. Summary points: The binding behavior of mixed Fc immune complexes is a blend of the binding properties for each constituent IgG species.An equilibrium, multivalent binding model can be generalized to incorporate immune complexes of mixed Fc composition.Particularly for low-affinity IgG-Fcγ receptor interactions, immune complexes provide better estimates of affinities.The FcγR binding model predicts effector-elicited cell clearance in humanized mice.

There Is Strength in Numbers: Quantitation of Fc Gamma Receptors on Murine Tissue-Resident Macrophages.

Many of the effector functions of antibodies rely on the binding of antibodies/immune complexes to cellular Fcγ receptors (FcγRs). Since the majority of innate immune effector cells express both activating and inhibitory Fc receptors, the outcome of the binding of immune complexes to cells of a given population is influenced by the relative affinities of the respective IgG subclasses to these receptors, as well as by the numbers of activating and inhibitory FcγRs on the cell surface. A group of immune cells that has come into focus more recently is the various subsets of tissue-resident macrophages. The central functions of FcγRs on tissue macrophages include the clearance of opsonized pathogens, the removal of small immune complexes from the circulation and the depletion of antibody-opsonized cells in the therapy of autoimmunity and cancer. Despite these essential functions of FcγRs on tissue-resident macrophages, an in-depth quantification of FcγRs is lacking. Thus, the aim of our current study was to quantify the various Fcγ receptors on macrophages in murine liver, lung, kidney, brain, skin and spleen. Our study identified a pronounced heterogeneity between FcγR expression patterns of the different tissue macrophages, which may reflect their specialized functions within their unique niches in different organ environments.

Corrigendum: There Is (Scientific) Strength in Numbers: A Comprehensive Quantitation of Fc Gamma Receptor Numbers on Human and Murine Peripheral Blood Leukocytes.

[This corrects the article DOI: 10.3389/fimmu.2020.00118.].

There Is (Scientific) Strength in Numbers: A Comprehensive Quantitation of Fc Gamma Receptor Numbers on Human and Murine Peripheral Blood Leukocytes.

Antibodies are essential mediators of immunological defense mechanisms, are clinically used as therapeutic agents, but are also functionally involved in various immune-mediated disorders. Whereas IgG antibodies accomplish some of their biological tasks autonomously, many functions depend on their binding to activating and inhibitory Fcγ receptors (FcγR). From a qualitative point of view expression patterns of FcγR on immunologically relevant cell types are well-characterized both for mice and humans. Surprisingly, however, there is only quite limited information available on actual quantities of FcγR expressed by the different leukocyte populations. In this study we provide a comprehensive data set assessing quantitatively how many individual human and mouse FcγRs are expressed on B cells, NK cells, eosinophils, neutrophils, basophils and both classical, and non-classical monocytes under steady state conditions. Moreover, among human donors we found two groups with different expression levels of the inhibitory FcγRIIb on monocytes which appears to correlate with haplotypes of the activating FcγRIIIa.

The Immunological Organ Environment Dictates the Molecular and Cellular Pathways of Cytotoxic Antibody Activity.

Cytotoxic immunoglobulin G antibodies are an essential component of therapeutic approaches aimed at depleting self-reactive or malignant cells. More recent evidence suggests that the tissue in which the target cell resides influences the underlying molecular and cellular pathways responsible for cytotoxic antibody activity. By studying cytotoxic IgG activity directed against natural killer cells in primary and secondary immunological organs, we show that distinct organ-specific effector pathways are responsible for target cell depletion. While in the bone marrow, the classical complement pathway and the high-affinity Fcγ-receptor I expressed on organ-resident macrophages were both involved in removing opsonized target cells; in the spleen and blood, all activating FcγRs but not the classical complement pathway were critical for target cell killing. Our study suggests that future strategies aimed at optimizing overall cytotoxic antibody activity may need to consider organ-specific pathways to achieve a maximal therapeutic effect.

Dissecting FcγR Regulation through a Multivalent Binding Model.

Many immune receptors transduce activation across the plasma membrane through their clustering. With Fcγ receptors (FcγRs), this clustering is driven by binding to antibodies of differing affinities that are in turn bound to multivalent antigen. As a consequence of this activation mechanism, accounting for and rationally manipulating immunoglobulin (Ig)G effector function is complicated by, among other factors, differing affinities between FcγR species and changes in the valency of antigen binding. In this study, we show that a model of multivalent receptor-ligand binding can effectively account for the contribution of IgG-FcγR affinity and immune complex valency. This model in turn enables us to make specific predictions about the effect of immune complexes of defined composition. In total, these results enable both rational immune complex design for a desired IgG effector function and the deconvolution of effector function by immune complexes.

Tumor location determines tissue-specific recruitment of tumor-associated macrophages and antibody-dependent immunotherapy response.

Despite recent advances in activating immune cells to target tumors, the presence of some immune cells, such as tumor-associated macrophages (TAMs) or tumor-associated neutrophils (TANs), may promote rather than inhibit tumor growth. However, it remains unclear how antibody-dependent tumor immunotherapies, such as cytotoxic or checkpoint control antibodies, affect different TAM or TAN populations, which abundantly express activating Fcγ receptors. In this study, we show that the tissue environment determines which cellular effector pathways are responsible for antibody-dependent tumor immunotherapy. Although TAMs derived from Ly6Chigh monocytes recruited by the CCL2-CCR2 axis were critical for tumor immunotherapy of skin tumors, the destruction of lung tumors was CCL2-independent and required the presence of colony-stimulating factor 2-dependent tissue-resident macrophages. Our findings suggest that TAMs may have a dual role not only in promoting tumor growth in certain tissue environments on the one hand but also in contributing to tumor cell destruction during antibody-mediated immunotherapy on the other hand.

Enzymatic lipid oxidation by eosinophils propagates coagulation, hemostasis, and thrombotic disease.

Blood coagulation is essential for physiological hemostasis but simultaneously contributes to thrombotic disease. However, molecular and cellular events controlling initiation and propagation of coagulation are still incompletely understood. In this study, we demonstrate an unexpected role of eosinophils during plasmatic coagulation, hemostasis, and thrombosis. Using a large-scale epidemiological approach, we identified eosinophil cationic protein as an independent and predictive risk factor for thrombotic events in humans. Concurrent experiments showed that eosinophils contributed to intravascular thrombosis by exhibiting a strong endogenous thrombin-generation capacity that relied on the enzymatic generation and active provision of a procoagulant phospholipid surface enriched in 12/15-lipoxygenase-derived hydroxyeicosatetraenoic acid-phosphatidylethanolamines. Our findings reveal a previously unrecognized role of eosinophils and enzymatic lipid oxidation as regulatory elements that facilitate both hemostasis and thrombosis in response to vascular injury, thus identifying promising new targets for the treatment of thrombotic disease.

bIgG time for large eaters: monocytes and macrophages as effector and target cells of antibody-mediated immune activation and repression.

The mononuclear phagocytic system consists of a great variety of cell subsets localized throughout the body in immunological and non-immunological tissues. While one of their prime tasks is to detect, phagocytose, and kill intruding microorganisms, they are also involved in maintaining tissue homeostasis and immune tolerance toward self through removal of dying cells. Furthermore, monocytes and macrophages have been recognized to play a critical role for mediating immunoglobulin G (IgG)-dependent effector functions, including target cell depletion, tissue inflammation, and immunomodulation. For this, monocyte and macrophage populations are equipped with a complex set of Fc-receptors, enabling them to directly interact with pro- or anti-inflammatory IgG preparations. In this review, we will summarize the most recent findings, supporting a central role of monocytes and macrophages for pro- and anti-inflammatory IgG activity.

How immunoglobulin G antibodies kill target cells: revisiting an old paradigm.

The capacity of immunoglobulin G (IgG) antibodies to eliminate virtually any target cell has resulted in the widespread introduction of cytotoxic antibodies into the clinic in settings of cancer therapy, autoimmunity, and transplantation, for example. More recently, it has become apparent that also the protection from viral infection via IgG antibodies may require cytotoxic effector functions, suggesting that antibody-dependent cellular cytotoxicity (ADCC) directed against malignant or virally infected cells is one of the most essential effector mechanisms triggered by IgG antibodies to protect the host. A detailed understanding of the underlying molecular and cellular pathways is critical, therefore, to make full use of this antibody effector function. Several studies over the last years have provided novel insights into the effector pathways and innate immune effector cells responsible for ADCC reactions. One of the most notable outcomes of many of these reports is that cells of the mononuclear phagocytic system rather than natural killer cells are critical for removal of IgG opsonized target cells in vivo.

B cells and CD22 are dispensable for the immediate antiinflammatory activity of intravenous immunoglobulins in vivo.

Intravenous immunoglobulins (IVIgs) efficiently suppress a variety of autoimmune diseases. Over the past few years several potential mechanisms underlying this antiinflammatory activity have become apparent. Among these, terminal sialic acid residues in the sugar moiety of the immunoglobulin G constant fragment have been shown to be critical for the antiinflammatory activity of IVIgs in models of rheumatoid arthritis and immunothrombocytopenia (ITP). More recently, B cells and the sialic acid-binding protein CD22 were suggested to be involved in this IVIg-dependent immunomodulatory pathway. To study whether B cells are directly involved in IVIg-mediated suppression of acute autoimmune diseases, we tested the activity of IVIgs in mice deficient in B cells or CD22. We show that neither B cells nor CD22 are critical for the immediate antiinflammatory activity of IVIgs in mouse models of rheumatoid arthritis and ITP.

FcγRIIB: a modulator of cell activation and humoral tolerance.

An immune response needs to be tightly regulated to prevent excessive inflammation, which may result in the destruction of healthy tissues. At the molecular level, the strength of an immune response is determined by the integration of a multitude of positive and negative signals. This review will focus on IgG-dependent immune responses and discuss how the inhibitory receptor FcγRIIB may be involved in regulating both the afferent and efferent phases of such a response. Furthermore, we will discuss recent evidence suggesting that FcγRIIB may have important functions beyond the negative regulation of signals transduced by the B-cell receptor or activating FcγRs and could be responsible for the activity of agonistic antibodies in vivo.

IVIg-mediated amelioration of ITP in mice is dependent on sialic acid and SIGNR1.

Intravenous immunoglobulin G (IVIg) therapy is widely used to treat autoimmune and inflammatory diseases. Recent evidence suggests that in mice, splenic resident cells might be important for the anti-inflammatory activity of IVIg in a model of serum transfer arthritis. Splenectomized human immunothrombocytopenia (ITP) patients, however, still respond to IVIg therapy. To investigate whether the requirement of the spleen is essential for mouse ITP, we used a passive model of induced ITP and demonstrated that IVIg activity was functional in splenectomized animals. Further analysis showed that the IVIg-mediated amelioration of platelet phagocytosis was fully dependent on terminal sialic acid residues in the IVIg preparation and could be blocked with a specific ICAM3 grabbing nonintegrin-related 1 (SIGNR1) specific antibody. These results suggest that, similar to the human system, a spleen-independent but sialic acid- and SIGNR1-dependent pathway is responsible for IVIg-mediated suppression of autoantibody-dependent platelet depletion in mice.

Low level of FcγRIII expression on murine natural killer cells.

Cellular Fcγ-receptors are crucial for mediating the functions of therapeutic antibodies. Antibody dependent cellular cytotoxicity (ADCC) is an important mechanism by which Fcγ-receptor expressing cells of the innate immune system including natural killer (NK) cells can kill opsonized target cells. During FACS analysis, however, binding of the Fc-fragment of staining antibodies specific for cell type specific receptors can lead to false positive results and wrong interpretation of the data. Current strategies to block such unwanted binding largely target FcγRIIB and FcγRIII but not the recently identified mouse FcγRIV. In this study we demonstrate that Fc-dependent binding of the NK cell specific antibody NK1.1 by FcγRIV on monocytes results in a large overestimation of FcγRIII expression on murine NK cells. These results highlight the importance of blocking unwanted binding of FACS antibodies to FcγRIV and shed new light on the expression level of FcγRIII on NK cells in mice during the steady state.