David A. Mancardi

Specificity and affinity of human Fcγ receptors and their polymorphic variants for human IgG subclasses

Distinct genes encode 6 human receptors for IgG (hFcgammaRs), 3 of which have 2 or 3 polymorphic variants. The specificity and affinity of individual hFcgammaRs for the 4 human IgG subclasses is unknown. This information is critical for antibody-based immunotherapy which has been increasingly used in the clinics. We investigated the binding of polyclonal and monoclonal IgG1, IgG2, IgG3, and IgG4 to FcgammaRI; FcgammaRIIA, IIB, and IIC; FcgammaRIIIA and IIIB; and all known polymorphic variants. Wild-type and low-fucosylated IgG1 anti-CD20 and anti-RhD mAbs were also examined. We found that (1) IgG1 and IgG3 bind to all hFcgammaRs; (2) IgG2 bind not only to FcgammaRIIA(H131), but also, with a lower affinity, to FcgammaRIIA(R131) and FcgammaRIIIA(V158); (3) IgG4 bind to FcgammaRI, FcgammaRIIA, IIB and IIC and FcgammaRIIIA(V158); and (4) the inhibitory receptor FcgammaRIIB has a lower affinity for IgG1, IgG2, and IgG3 than all other hFcgammaRs. We also identified parameters that determine the specificity and affinity of hFcgammaRs for IgG subclasses. These results document how hFcgammaR specificity and affinity may account for the biological activities of antibodies. They therefore highlight the role of specific hFcgammaRs in the therapeutic and pathogenic effects of antibodies in disease.

Mouse and human neutrophils induce anaphylaxis

Anaphylaxis is a life-threatening hyperacute immediate hypersensitivity reaction. Classically, it depends on IgE, FcεRI, mast cells, and histamine. However, anaphylaxis can also be induced by IgG antibodies, and an IgG1-induced passive type of systemic anaphylaxis has been reported to depend on basophils. In addition, it was found that neither mast cells nor basophils were required in mouse models of active systemic anaphylaxis. Therefore, we investigated what antibodies, receptors, and cells are involved in active systemic anaphylaxis in mice. We found that IgG antibodies, FcγRIIIA and FcγRIV, platelet-activating factor, neutrophils, and, to a lesser extent, basophils were involved. Neutrophil activation could be monitored in vivo during anaphylaxis. Neutrophil depletion inhibited active, and also passive, systemic anaphylaxis. Importantly, mouse and human neutrophils each restored anaphylaxis in anaphylaxis-resistant mice, demonstrating that neutrophils are sufficient to induce anaphylaxis in mice and suggesting that neutrophils can contribute to anaphylaxis in humans. Our results therefore reveal an unexpected role for IgG, IgG receptors, and neutrophils in anaphylaxis in mice. These molecules and cells could be potential new targets for the development of anaphylaxis therapeutics if the same mechanism is responsible for anaphylaxis in humans.

FcγRIV is a mouse IgE receptor that resembles macrophage FcεRI in humans and promotes IgE-induced lung inflammation

FcgammaRIV is a recently identified mouse activating receptor for IgG2a and IgG2b that is expressed on monocytes, macrophages, and neutrophils; herein it is referred to as mFcgammaRIV. Although little is known about mFcgammaRIV, it has been proposed to be the mouse homolog of human FcgammaRIIIA (hFcgammaRIIIA) because of high sequence homology. Our work, however, has revealed what we believe to be new properties of mFcgammaRIV that endow this receptor with a previously unsuspected biological significance; we have shown that it is a low-affinity IgE receptor for all IgE allotypes. Although mFcgammaRIV functioned as a high-affinity IgG receptor, mFcgammaRIV-bound monomeric IgGs were readily displaced by IgE immune complexes. Engagement of mFcgammaRIV by IgE immune complexes induced bronchoalveolar and peritoneal macrophages to secrete cytokines, suggesting that mFcgammaRIV may be an equivalent of human FceRI(alphagamma), which is expressed by macrophages and neutrophils and especially in atopic individuals, rather than an equivalent of hFcgammaRIIIA, which has no affinity for IgE. Using mice lacking 3 FcgammaRs and 2 FceRs and expressing mFcgammaRIV only, we further demonstrated that mFcgammaRIV promotes IgE-induced lung inflammation. These data lead us to propose a mouse model of IgE-induced lung inflammation in which cooperation exists between mast cells and mFcgammaRIV-expressing lung cells. We therefore suggest that a similar cooperation may occur between mast cells and hFceRI-expressing lung cells in human allergic asthma.

The high-affinity human IgG receptor FcγRI (CD64) promotes IgG-mediated inflammation, anaphylaxis, and antitumor immunotherapy.

Receptors for the Fc portion of IgG (FcγRs) are mandatory for the induction of various IgG-dependent models of autoimmunity, inflammation, anaphylaxis, and cancer immunotherapy. A few FcγRs have the ability to bind monomeric IgG: high-affinity mouse mFcγRI, mFcγRIV, and human hFcγRI. All others bind IgG only when aggregated in complexes or bound to cells or surfaces: low-affinity mouse mFcγRIIB and mFcγRIII and human hFcγRIIA/B/C and hFcγRIIIA/B. Although it has been proposed that high-affinity FcγRs are occupied by circulating IgG, multiple roles for mFcγRI and mFcγRIV have been reported in vivo. However, the potential roles of hFcγRI that is expressed on monocytes, macrophages, and neutrophils have not been reported. In the present study, we therefore investigated the role of hFcγRI in antibody-mediated models of disease and therapy by generating hFcγRI-transgenic mice deficient for multiple endogenous FcRs. hFcγRI was sufficient to trigger autoimmune arthritis and thrombocytopenia, immune complex-induced airway inflammation, and active and passive systemic anaphylaxis. We found monocyte/macrophages to be responsible for thrombocytopenia, neutrophils to be responsible for systemic anaphylaxis, and both cell types to be responsible for arthritis induction. Finally, hFcγRI was capable of mediating antibody-induced immunotherapy of metastatic melanoma. Our results unravel novel capabilities of human FcγRI that confirm the role of high-affinity IgG receptors in vivo.

Human FcγRIIA induces anaphylactic and allergic reactions

IgE and IgE receptors (FcεRI) are well-known inducers of allergy. We recently found in mice that active systemic anaphylaxis depends on IgG and IgG receptors (FcγRIIIA and FcγRIV) expressed by neutrophils, rather than on IgE and FcεRI expressed by mast cells and basophils. In humans, neutrophils, mast cells, basophils, and eosinophils do not express FcγRIIIA or FcγRIV, but FcγRIIA. We therefore investigated the possible role of FcγRIIA in allergy by generating novel FcγRIIA-transgenic mice, in which various models of allergic reactions induced by IgG could be studied. In mice, FcγRIIA was sufficient to trigger active and passive anaphylaxis, and airway inflammation in vivo. Blocking FcγRIIA in vivo abolished these reactions. We identified mast cells to be responsible for FcγRIIA-dependent passive cutaneous anaphylaxis, and monocytes/macrophages and neutrophils to be responsible for FcγRIIA-dependent passive systemic anaphylaxis. Supporting these findings, human mast cells, monocytes and neutrophils produced anaphylactogenic mediators after FcγRIIA engagement. IgG and FcγRIIA may therefore contribute to allergic and anaphylactic reactions in humans.

Cutting Edge: The Murine High-Affinity IgG Receptor FcγRIV Is Sufficient for Autoantibody-Induced Arthritis

K/BxN serum-induced passive arthritis was reported to depend on the activation of mast cells, triggered by the activating IgG receptor FcγRIIIA, when engaged by IgG1 autoantibodies present in K/BxN serum. This view is challenged by the fact that FcγRIIIA-deficient mice still develop K/BxN arthritis and because FcγRIIIA is the only activating IgG receptor expressed by mast cells. We investigated the contribution of IgG receptors, IgG subclasses, and cells in K/BxN arthritis. We found that the activating IgG2 receptor FcγRIV, expressed only by monocytes/macrophages and neutrophils, was sufficient to induce disease. K/BxN arthritis occurred not only in mast cell-deficient Wsh mice, but also in mice whose mast cells express no activating IgG receptors. We propose that at least two autoantibody isotypes, IgG1 and IgG2, and two activating IgG receptors, FcγRIIIA and FcγRIV, contribute to K/BxN arthritis, which requires at least two cell types other than mast cells, monocytes/macrophages, and neutrophils.

Neutrophils mediate antibody-induced antitumor effects in mice

Tumor engraftment followed by monoclonal antibody (mAb) therapy targeting tumor antigens represents a gold standard for assessing the efficiency of mAbs to eliminate tumor cells. Mouse models have demonstrated that receptors for the Fc portion of immunoglobulin G (FcγRs) are critical determinants of mAb therapeutic efficacy, but the FcγR-expressing cell populations responsible remain elusive. We show that neutrophils are responsible for mAb-induced therapy of both subcutaneous syngeneic melanoma and human breast cancer xenografts. mAb-induced tumor reduction, abolished in neutropenic mice, could be restored in FcγR-deficient hosts upon transfer of FcγR+ neutrophils or upon human FcγRIIA/CD32A transgenic expression. Finally, conditional knockout mice unable to perform FcγR-mediated activation and phagocytosis specifically in neutrophils were resistant to mAb-induced therapy. Our work suggests that neutrophils are necessary and sufficient for mAb-induced therapy of subcutaneous tumors, and represent a new and critical focal point for optimizing mAb-induced immunotherapies that will impact on human cancer treatment.

Neutrophils in local and systemic antibody‐dependent inflammatory and anaphylactic reactions

Neutrophils are notorious for their efficacy in microbial killing. Various mechanisms, such as phagocytosis, production of ROS, cytokines/chemokines and lipid mediators, degranulation of antimicrobials and enzymes, as well as NETosis contribute to this capacity. However, every incidence of neutrophil activation bears a risk to cause damage to the host. Several distinct steps, i.e., adhesion to endothelial cells, transmigration, chemotaxis, cytokine stimulation, and TLR signaling, are thought to control the extent of neutrophil activation. In the absence of a microbial stimulus, other pathways can induce neutrophil activation, among which FcR‐induced activation when neutrophils encounter ICs. In these situations (inflammation, autoimmunity, allergy), neutrophils may act as primary or secondary effectors of immune reactions. In the presence of circulating ICs, neutrophils can indeed get stimulated directly in the bloodstream and trigger an immune response. Upon deposition of antibody complexes inside of tissues, neutrophils are first recruited and primed before being highly activated to amplify the ongoing inflammation. This review focuses on the engagement, activation, and responses of neutrophils to antibody ICs, inside of tissues or in the vasculature.

A hypomorphic mutation in the Gfi1 transcriptional repressor results in a novel form of neutropenia

Using N‐ethyl‐N‐nitrosourea‐induced mutagenesis, we established a mouse model with a novel form of neutropenia resulting from a point mutation in the transcriptional repressor Growth Factor Independence 1 (Gfi1). These mice, called Genista, had normal viability and no weight loss, in contrast to mice expressing null alleles of the Gfi1 gene. Furthermore, the Genista mutation had a very limited impact on lymphopoiesis or on T‐ and B‐cell function. Within the bone marrow (BM), the Genista mutation resulted in a slight increase of monopoiesis and in a block of terminal granulopoiesis. This block occurred just after the metamyelocytic stage and resulted in the generation of small numbers of atypical CD11b+Ly‐6Gint neutrophils, the nuclear morphology of which resembled that of mature WT neutrophils. Unexpectedly, once released from the BM, these atypical neutrophils contributed to induce mild forms of autoantibody‐induced arthritis and of immune complex‐mediated lung alveolitis. They additionally failed to provide resistance to acute bacterial infection. Our study demonstrates that a hypomorphic mutation in the Gfi1 transcriptional repressor results in a novel form of neutropenia characterized by a split pattern of functional responses, reflecting the distinct thresholds required for eliciting neutrophil‐mediated inflammatory and anti‐infectious responses.

Cutting Edge: FcγRIII (CD16) and FcγRI (CD64) Are Responsible for Anti-Glycoprotein 75 Monoclonal Antibody TA99 Therapy for Experimental Metastatic B16 Melanoma

mAb therapy for experimental metastatic melanoma relies on activating receptors for the Fc portion of IgG (FcγR). Opposing results on the respective contribution of mouse FcγRI, FcγRIII, and FcγRIV have been reported using the gp75-expressing B16 melanoma and the protective anti-gp75 mAb TA99. We analyzed the contribution of FcγRs to this therapy model using bioluminescent measurement of lung metastases loads, novel mouse strains, and anti-FcγR blocking mAbs. We found that the TA99 mAb-mediated effects in a combination therapy using cyclophosphamide relied on activating FcγRs. The combination therapy, however, was not more efficient than mAb therapy alone. We demonstrate that FcγRI and, unexpectedly, FcγRIII contributed to TA99 mAb therapeutic effects, whereas FcγRIV did not. Therefore, FcγRIII and FcγRI are, together, responsible for anti-gp75 mAb therapy of B16 lung metastases. Our finding that mouse FcγRIII contributes to Ab-induced tumor reduction correlates with clinical data on its human functional equivalent human FcγRIIIA (CD16A).