Diversification of IgG effector functions

Fc effector functions

Abstract

IgG is the major immunoglobulin class produced during an immune response against foreign antigens and efficiently provides protection through its bifunctional nature. While the Fab domains confer highly specific recognition of the antigen, the Fc domain mediates a wide range of effector functions that modulate several aspects of innate and adaptive immunity. Engagement of the various types of Fcγ receptors (FcγRs) by an IgG Fc domain can activate distinct immunomodulatory pathways with pleiotropic functional consequences for several leukocyte types. Fc effector functions are not limited to phagocytosis and cytotoxicity of IgG-opsonized targets but exhibit remarkable diversity and include modulation of leukocyte activity and survival, cytokine and chemokine expression, maturation of antigen-presenting cells, antigen processing and presentation, B-cell selection and IgG affinity maturation, as well as regulation of IgG production. These functions are initiated upon specific interactions of the Fc domain with the various types of FcγRs—a process that is largely determined by the structural heterogeneity of the IgG Fc domain. Modulation of the Fc-associated glycan structure and composition along with differences in the primary amino acid sequence among the IgG subclasses represent the two main diversification mechanisms of the Fc domain that generate a spectrum of Fc domain phenotypes with distinct affinity for the various FcγR types and differential capacity to activate immunomodulatory pathways.

Introduction

IgG represents the main immunoglobulin class produced during an immune response against foreign antigens and is abundantly present in circulation, constituting >75% of total circulating immunoglobulins. The protective functions of IgG are the result of the activity of its two functional domains: the Fab domain that mediates highly specific antigenic recognition and the Fc domain that contributes to the IgG effector activity. Fc effector activity is mediated through specific interactions of the Fc domain with Fcγ receptors (FcγRs), specialized receptors widely expressed by several effector leukocyte types.

Although the classical definition of Fc effector function has historically been the induction of phagocytosis by phagocytic leukocytes and antibody-dependent cellular cytotoxicity by NK cells, substantial evidence strongly suggests that the Fc domain of IgG has the capacity to mediate a wide spectrum of effector activities through specific interactions with the different types of FcγRs. These effector activities span several aspects of innate and adaptive immunity and can potently influence the functional activity of several leukocyte types through the specific activation of distinct immunomodulatory pathways. Here, we discuss the diversity of Fc effector activities and the biological consequences on the development and regulation of an immune response. We begin by describing the structural properties and the functional characteristics of the different types of FcγRs, discussing the mechanisms that mediate diversification of the Fc domain that determine its capacity to engage the various FcγR types and activate specific signaling pathways.

Type I and Type II

FcγRs: structural properties and functional characteristicsFcγRs are divided into two main types (Type I and II), with each type having unique functional and structural features (Table 1) (1). All members of the Type I FcγR family belong to the immunoglobulin receptor superfamily because their extracellular region, which binds IgG, contains two or three immunoglobulin domains (2). In contrast, Type II FcγRs are C-type lectin receptors, which exhibit diverse ligand specificity (3). Indeed, in addition to interacting with the Fc domain of IgG, Type II FcγRs have the capacity to engage diverse ligands, including carbohydrate structures, heavily glycosylated proteins and IgE (4–6).

Table 1.

Properties and characteristics of Type I and Type II FcγRs

Property/characteristic	Type I	Type II
Structural domain family	Immunoglobulin	C-type lectin
IgG Fc binding site	Hinge-proximal CH2	CH2–CH3 interface
Binding stoichiometry (receptor:IgG)	1:1	2:1
Ligand specificity	High (exclusive for IgG)	Low (IgG, IgE, carbohydrates, other glycoproteins)

Apart from the differences in the structure and ligand specificity among different FcγR types, Type I and Type II FcγRs interact with the Fc domain at distinct regions. Whereas the CH2-proximal hinge and the CH2 domain of the IgG Fc serve as the binding site for Type I FcγRs, Type II FcγRs interact with the Fc domain at the CH2–CH3 interface at a 2:1 stoichiometric complex (receptor:IgG) (2, 3). The differences between Type I and Type II FcγRs are not limited to their structural characteristics, but also extend to their functional properties, as receptor engagement by IgG Fc induces the activation of distinct immunomodulatory pathways (discussed in detail in the next sections) (7).

Upon receptor cross-linking by the Fc domains of IgG immune complexes, Type I FcγRs trigger signaling events through their intracellular signaling motifs, inducing either pro-inflammatory or anti-inflammatory processes. On the basis of the functional consequences initiated upon receptor engagement, Type I FcγRs are classified into either activating or inhibitory (8). Activating FcγRs contain ITAMs at their cytoplasmic domain, whereas ITIMs are present in inhibitory FcγRs (9–11).

Humans express six different type I FcγRs: FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and FcγRIIIb, each with distinct functional properties (1). FcγRI represents the sole FcγR capable of interacting with the Fc domain of IgG with high affinity (K_D: 10⁻¹⁰ M), whereas the rest of the FcγRs represent low-affinity IgG Fc receptors that are normally engaged through multivalent, low affinity, high avidity interactions with multimeric IgG immune complexes. This ensures that while monomeric IgGs cannot induce receptor engagement and thus inappropriate signaling under steady-state conditions, low-affinity FcγRs can be engaged only by IgG immune complexes generated during an immune response. Indeed, low-affinity Type I FcγRs initiate a number of signaling cascades upon receptor cross-linking that mediate diverse immunomodulatory events with the capacity to influence several aspects of innate and adaptive immunity (9, 10, 12, 13).

Several studies have elucidated the precise signaling events that follow Type I FcγR cross-linking by IgG immune complexes (11, 13–25). In particular, activating Type I FcγR interactions with multivalent IgG immune complexes induce receptor clustering and aggregation that subsequently lead to the recruitment of Syk and Src family kinases at their intracellular domains (17, 19, 22, 26, 27). Interactions of these kinases with the ITAMs of activating Type I FcγRs trigger their phosphorylation and the activation of several pro-inflammatory signaling pathways that involve the phosphorylation of diverse families of kinases, actin remodeling, Ca²⁺ influx and the up-regulation of genes with pro-inflammatory and pro-survival properties (18, 24, 28–31). These processes readily influence the functional activity of innate leukocytes and determine the effector functions during IgG-mediated inflammation.

Since engagement of activating Type I FcγRs initiates a number of pro-inflammatory pathways with profound consequences on innate leukocyte activity, their signaling function is tightly regulated by an inhibitory Type I FcγR. For the majority of innate leukocytes, activating Type I FcγR expression is coupled with the expression of FcγRIIb, the sole inhibitory FcγR (21). The main function of FcγRIIb is to antagonize the signaling activity of ITAM-containing Type I FcγRs on innate leukocytes and of the BCR on B cells, which is accomplished by the phosphorylation of FcγRIIb ITIMs upon receptor cross-linking. ITIMs induce the recruitment of SHIP family phosphatases, which promote the hydrolysis of phosphatidylinositol 3,4,5-triphosphate to phosphatidylinositol 4,5-biphosphate, inhibiting thereby the recruitment and activation of Src kinases and PLCγ (11, 21, 32, 33). These events efficiently over-ride any signals initiated upon cross-linking of activating Type I FcγRs or the BCR.

Given the role of FcγRIIb to regulate the activity of key immunoreceptors of innate and adaptive immunity, FcγRIIb represents the main mechanism for the control of IgG-mediated inflammation, B-cell activation and selection, as well as tolerance. Indeed, modulation of FcγRIIb expression on innate leukocytes and B cells largely determines the in vivo Fc effector activity of IgG antibodies and influences the affinity and durability of IgG humoral responses (32–35).

In contrast to Type I FcγRs, the precise signaling events that are initiated upon receptor cross-linking by IgG immune complexes are poorly defined for Type II FcγRs. The Type II FcγRs are CD23 and DC-SIGN, which are both members of the C-type lectin receptor family and share a characteristic oligomeric structure that is stabilized by an α-helical coiled-coil stalk domain at the extracellular, ligand-binding region (3). Previous studies on CD23 signaling focused primarily on the events that are initiated upon IgE–CD23 interactions, as CD23 was originally described as the low-affinity FcR for IgE. CD23 exists as two isoforms (CD23a and CD23b), which exhibit distinct cellular expression patterns and signaling activity. CD23a is constitutively expressed by B cells, whereas CD23b expression is induced by IL-4 in several leukocyte types, including monocytes and T cells (36). A 6-amino-acid difference in the cytoplasmic region among the two isoforms also determines the receptor signaling activity. Whereas both isoforms induce an increase in cAMP levels upon cross-linking, CD23a also has the capacity to activate the PLC pathway and mediate pleiotropic signaling activities, including activation of MAP kinases and NF-κB (37–40).

DC-SIGN, the other Type II FcγR, associates with the adaptor protein LSP1 at its cytoplasmic domain and signals through the guanine-nucleotide exchange factor protein LARG to activate Rho- and Ras-GTPases, which consequently leads to the induction of diverse immunostimulatory pathways upon receptor cross-linking (41–43). Although a number of previous studies have provided insights into the signaling activity of Type II FcγRs, it is unknown which exact signaling pathways are modulated following receptor engagement by the IgG Fc domain. Indeed, given the diversity of ligands that have the capacity to interact with Type II FcγRs, it is anticipated that the affinity as well as the nature of the interaction between Type II FcγRs and their respective ligands represent the major determinants for the precise downstream signaling pathways activated upon receptor cross-linking.

Although the precise signaling pathways that are activated upon receptor engagement by the IgG Fc domain are unknown, several recent studies have defined the biological consequences induced upon Type II FcγR–Fc interactions. For example, engagement of DC-SIGN on regulatory macrophages by the IgG Fc triggers the expression and release of IL-33, a potent T_h2-polarizing cytokine with profound effects on innate leukocyte responsiveness to IgG-mediated inflammation (44–48). Likewise, IgG Fc–CD23 interactions on B cells increase the expression of the inhibitory Type I FcγR, FcγRIIb in an autocrine manner, regulating thereby B-cell selection and IgG affinity maturation (3, 34). All these effects have a major impact on several aspects of innate and adaptive immune responses and reflect the astonishing diversity of effector activities mediated through interactions of the Fc domain of IgG with the different types of FcγRs.

IgG Fc domain heterogeneity

Given the capacity of Type I and Type II FcγRs to activate diverse immunomodulatory pathways upon engagement, several regulatory mechanisms exist to control their activity and prevent inappropriate or excessive activation of FcγR-mediated pathways. Type I and Type II FcγRs follow a characteristic expression pattern among the various leukocyte populations, with several different cell types often co-expressing more than one FcγR type at a given time throughout their development or differentiation (Table 2) (7). In addition, the expression of Type I and Type II FcγRs is subject to regulation by chemokines and cytokines, which influence the FcγR expression profile of effector leukocytes, affecting thereby their responsiveness to IgG-mediated inflammation. For example, IFN-γ induces the expression of FcγRI on myeloid cells and FcγRIIIb on eosinophils, whereas IL-4 up-regulates FcγRIIb expression on myeloid cells and induces the expression of CD23 on T cells, monocytes, granulocytes and macrophages (36, 49–52). Such alterations in the FcγR expression pattern readily affect the outcome of FcγR-mediated signaling activities, as well as the immunomodulatory functions initiated upon FcγR cross-linking by IgG immune complexes.

Table 2.

Type I and Type II FcγR expression profile in leukocyte populations

Receptor		Myeloid cells					Lymphoid cells
		Monocytes	Neutrophils	Eosinophils	DCs	Mφ	B cells	T cells	NK cells
Type I	FcγRI	+	#	#	#	+/–	–	–	–
	FcγRIIa	+	+	+	+	+	–	–	–
	FcγRIIb	+	+	+	+	+	+	–	–/^
	FcγRIIc	–	–	–	–	–	–	–	^
	FcγRIIIa	+/–	–	–	#	+/–	–	–	+
	FcγRIIIb	–	+	#	–	–	–	–	–
Type II	DC-SIGN	+/–	–	–	+	+/–	–	–	–
	CD23	#	#	#	#	#	+	#	–

(+), expressed; (–), not expressed; (+/–), subsets only; (#), inducible only; (^), allele-dependent expression; DCs, dendritic cells; Mφ, macrophages.

Although the levels of FcγR expression on the surface of effector leukocytes influence the functional consequences of FcγR-mediated signaling by IgG immune complexes, the primary structure of the IgG Fc domain represents the main determinant for the capacity of Type I and Type II FcγRs to engage IgG immune complexes and initiate downstream effector activities (1, 3). Despite being part of the constant region of the IgG molecule, the Fc domain exhibits substantial heterogeneity that determines its affinity for the various FcγR types.

Such heterogeneity originates from differences in the primary amino acid sequence among the various subclasses and Gm allotypes (i.e. allotypes within the IgG constant regions), as well as in the structure and composition of the Fc-associated N-linked glycan (53). Indeed, IgG subclasses exhibit differential affinity for the various FcγR types; for example, in humans, IgG1 and IgG3 have the capacity to interact with most FcγRs, whereas IgG2 and IgG4 demonstrate minimal affinity for FcγRs.

Likewise, another source of structural heterogeneity of the Fc domain is the composition of the Fc-associated glycan. This glycan structure is conjugated at a highly conserved site (Asn297) that is present among all IgG subclasses even in diverse mammalian species and is essential for the maintenance of the Fc domain at a conformation permissive for interactions with FcγRs (53). The Fc-associated glycan is present at a hydrophobic cleft between the two CH2 domains of the Fc domain and its presence ensures that the Fc domain adopts its characteristic horseshoe-like conformation, in which the two CH3 domains are tightly associated, whereas the CH2 domains remain spatially separated (3). This conformation is essential for the hinge-proximal region of the CH2 domains to interact with Type I FcγRs, as destabilization of the Fc domain conformation through removal of the glycan structure readily results in the collapse of the CH2 domains and subsequently in the loss of Fc–FcγR interactions (54, 55).

Although the presence of the Fc-associated glycan is a critical determinant for the regulation of the Fc domain structure and Fc–FcγR interactions, the precise composition of the glycan structure determines the affinity of the Fc domain for Type I and Type II FcγRs. In particular, the core glycan structure comprises a heptasaccharide moiety of mannose and N-acetylglucosamine residues, which can be modified through the selective addition of other saccharide units, including fucose, galactose, N-acetylglucosamine and sialic acid (53). The presence of specific saccharide units at the core glycan structure regulates the flexibility of the Fc domain and influences its capacity to interact with Type I and Type II FcγRs.

Analyses of the Fc-associated glycan structure and composition from IgG purified from human serum revealed that under steady-state conditions, there is substantial heterogeneity in the Fc-associated glycoforms, which exhibit differential FcγR affinity (34). For example, the presence of a branching fucose residue at the core glycan structure is associated with differences in the affinity for the activating Type I FcγR, FcγRIIIa. IgG glycovariants lacking this fucose residue exhibit higher affinity for FcγRIIIa and demonstrate increased effector activity through an enhanced capacity to interact with and activate FcγRIIIa-expressing effector leukocytes, such as NK cells and macrophages (56–59). Likewise, the presence of terminal sialic acid residues at the Fc glycan confers increased flexibility of the Fc domain through destabilization of the Fc domain quaternary structure, which in turn exposes the Type II FcγR-binding site at the CH2–CH3 interface (3). Indeed, sialylated Fc glycoforms have the capacity to interact with the Type II FcγRs, DC-SIGN and CD23, mediating pleiotropic immunomodulatory effects on macrophages and B cells, respectively (34, 44, 46, 47).

Given the critical role of the Fc glycan structure and composition in the control of Fc domain affinity for Type I and Type II FcγRs, regulatory mechanisms exist that dynamically control Fc glycan modifications during an immune response. Indeed, recent analyses of the Fc glycan composition revealed distinct fluctuations at the levels of specific glycoforms upon vaccination, during pregnancy, in chronic inflammatory and neoplastic diseases, in response to systemic metabolic changes and during infection (34, 60–65). Examples include inflammatory diseases, such as rheumatoid arthritis and Wegener’s granulomatosis, which are characterized by reduced galactosylation and sialylation, as well as infectious diseases, such as Dengue infection, in which increased levels of afucosylated glycoforms are associated with clinical disease severity and thrombocytopenia (60, 61, 63–65).

A number of recent mechanistic studies in human vaccination cohorts and in mice with specific deletions of the enzymes that catalyze the addition of saccharide units to the core IgG Fc glycan structure provided novel insights into the dynamic regulation of Fc domain glycosylation and its importance in the control of downstream effector functions (34, 65). For example, analysis of the Fc glycan profile of antigen-specific IgGs elicited upon influenza vaccination in humans revealed specific enrichment in certain Fc glycoforms at different time points following vaccination (34). These effects correlated with characteristic changes in the expression of enzymes that catalyze the addition of saccharide moieties to the core glycan structure, including the sialyltransferase St6Gal1 and the fucosyltransferase, Fut8 in plasmablasts and memory B cells at different time points (34).

Likewise, specific modulation of Fc glycosylation was also observed in mouse models of immune complex-mediated nephrotoxic nephritis and collagen-induced arthritis (CIA), in which immunization induced a significant reduction of Fc sialylation of antigen-specific IgGs (65). These asialylated IgGs were a key determinant for IgG-mediated inflammation and were required for disease development. These observations were further validated in mouse strains encompassing conditional genetic deletion of St6gal1 in B cells (AID-Cre, St6gal1^fl/fl) (65). In these mice, complete loss of sialylation of anti-collagen II IgGs elicited upon immunization further exacerbated joint inflammation in the CIA model, whereas ex vivo sialylation of arthritogenic IgGs diminished their pathogenic potency in mouse models of arthritis (65). These studies highlight the existence of regulatory mechanisms that modulate Fc glycosylation during an immune response—a critical process that determines downstream effector responses and greatly influences innate and adaptive immunity.

Diversity of Fc effector functions

Specific modulation of the Fc glycan structure and composition coupled with differences in the primary amino acid sequence of IgG subclass and Gm allotype variants represent the two main diversification mechanisms of the Fc domain that generate a spectrum of Fc domain phenotypes with distinct affinity for the various FcγR types and differential capacity to activate immunomodulatory pathways. Indeed, engagement of the different types of FcγRs initiates distinct signaling pathways with pleiotropic biological consequences for several aspects of innate and adaptive immunity (7). These effects are largely dependent on the types of FcγR-expressing effector leukocytes involved and the balancing activity between activating and inhibitory Type I FcγRs, as well as Type II FcγRs. For example, activating-FcγR cross-linking on effector leukocytes is accompanied by specific signaling events that involve the activation of several families of kinases and pro-inflammatory pathways that lead rapidly to cellular activation (15, 16, 19, 24, 28, 66–68).

For granulocytes, such as neutrophils, eosinophils and mast cells, cross-linking FcγRs triggers rapid degranulation, and the generation and release of reactive oxygen and nitrogen intermediates with potent bactericidal activity (20, 30, 31, 67, 69–73). Likewise, FcγRIIIa-cross-linking on NK cells triggers cellular activation and the release of enzymes, such as perforin and granzymes, from intracellular granules that induces the formation of pores at the cell membrane of IgG-coated cellular targets, leading ultimately to the activation of pro-apoptotic pathways and cell death (22, 74). Analogous cellular activation events are initiated upon FcγR cross-linking in other FcγR-expressing cell types, such as platelets. Indeed, engagement of platelet FcγRIIa by IgG immune complexes triggers platelet activation and degranulation, leading to platelet aggregation and the activation of pro-thrombotic pathways (27, 75, 76).

For phagocytes and antigen-presenting cells, such as macrophages and dendritic cells, FcγR-mediated cellular activation is accompanied by the efficient internalization of the IgG-opsonized particles and their shuffling to endosomal compartments for subsequent degradation in lysosomes (13, 77–81). Indeed, phagocytosis of IgG-coated targets through activating FcγR pathways induces enhanced endosomal maturation and efficient degradation of lysosomal contents, which translates to improved antigen processing and presentation on MHC-II molecules, inducing thereby potent T-cell responses (13, 77, 80–82). Additionally, dendritic cell maturation is associated with the balanced signaling activity of activating and inhibitory Type I FcγRs. Without stimulation, dendritic cells express FcγRIIa and FcγRIIb and cell maturation by IgG immune complexes is restricted through the inhibitory activity of FcγRIIb (49, 77, 80, 82, 83). Genetic deletion of Fcgr2b, mAb-mediated FcγRIIb blockade or specific engagement of FcγRIIa on dendritic cells stimulates robust dendritic cell maturation and up-regulation of co-stimulatory molecule expression, leading to potent induction of T-cell responses (49, 50, 82, 84, 85).

Apart from the immediate effects observed shortly after FcγR cross-linking by IgG immune complexes, FcγR-mediated signaling also induces the up-regulation of pro-inflammatory gene expression, which leads to the production and release of several cytokines and chemokines with potent immunomodulatory activity. These mediators readily affect cellular differentiation and FcγR expression levels, as well as cell survival. For example, Type I FcγR-cross-linking on macrophages influences macrophage polarization, leading to the induction of a polarization state that resembles the M2 phenotype and is characterized by enhanced migratory and phagocytic activity, as well as increased IL-1, IL-6 and IL-10 expression (32, 50, 86, 87). As mentioned earlier, engagement of the Type II FcγR, DC-SIGN on regulatory macrophages by sialylated Fc IgG molecules up-regulates IL-33 expression, which in turn triggers the production and release of IL-4 by basophils (46). This potent T_h2-polarizing response increases the expression of FcγRIIb on innate effector leukocytes, altering thereby the balance between activating and inhibitory Type I FcγRs and limiting IgG-mediated inflammation (44, 46).

A similar example of orchestrated Type I and Type II FcγR activity with potent immunomodulatory function is also evident in B cells. B cells co-express the inhibitory Type I FcγR, FcγRIIb and the Type II FcγR, CD23, and engagement of these FcγRs readily influences B-cell selection and survival, as well as IgG affinity maturation (34). Since B cells do not express any activating Type I FcγR, the main role of FcγRIIb signaling activity is to regulate the function of the BCR. In particular, in cases of weak or absent BCR signaling, B cells undergo apoptosis through FcγRIIb engagement, eliminating thereby B cells with low or no affinity for the BCR (11, 21, 33). In contrast, FcγRIIb–BCR co-engagement attenuates pro-apoptotic signals, leading to B-cell survival and the selection of B cells with high-affinity BCRs.

This critical role for FcγRIIb in B-cell selection is reflected in studies using Fcgr2b-deficient strains, which are characterized by high titer and low-affinity IgG responses, indicative of inefficient B-cell selection (88–90). Likewise, genetic variants of FCGR2B that influence receptor expression or activity constitute risk factors for the development of autoimmune pathologies in humans. Since FcγRIIb expression on B cells represents a critical factor for B-cell selection, fluctuations in the FcγRIIb expression levels could readily impact IgG responses (91–95). Indeed, engagement of CD23 on B cells by sialylated IgG immune complexes up-regulates B-cell FcγRIIb expression, which subsequently raises the threshold for B-cell selection, leading to the generation of high-affinity IgG responses (34). This cross-talk between CD23 and FcγRIIb regulates precisely the signaling activity of the BCR and is a critical component that controls B-cell selection and IgG responses. Similar to B cells, FcγRIIb cross-linking by IgG immune complexes activates pro-apoptotic pathways in plasma cells, regulating thereby plasma cell survival and IgG production during an immune response (21, 96). This negative feedback mechanism prevents inappropriate IgG production and constitutes an efficient approach to efficiently terminate humoral immune responses.

Conclusions

IgG antibodies, a major component of adaptive immunity, not only confer protective activity against foreign antigens, but, through diverse Fc effector functions, have the capacity to influence the outcome of IgG-mediated inflammation and immunity. Through distinct mechanisms of diversification, the IgG Fc domain exhibits substantial heterogeneity that regulates its capacity to interact and activate FcγR pathways with potent immunomodulatory activity. A substantial body of evidence supports that Fc effector functions are not limited to IgG-mediated phagocytosis or cytotoxicity, but also extend to several critical functions that modulate the functional activity of leukocytes and influence several components of the innate and adaptive immunity. Understanding the complexity and diversity of Fc effector functions could result in the development and optimization of therapeutic strategies against infection and in autoimmune and chronic inflammatory pathologies through specific activation of FcγR-mediated immunomodulatory pathways.

Funding

Research reported in this publication was supported in part by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (P01AI100148, U19AI111825, U19AI109946), the National Cancer Institute (R35CA196620, P01CA190174) and the Bill & Melinda Gates Foundation (OPP1124068). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. S.B. is an amfAR Mathilde Krim Fellow in Basic Biomedical Research (109519-60-RKVA).

Conflict of Interest statement: The authors have no conflicting financial interests.