Download Type i and type ii Fc receptors regulate innate and adaptive immunity

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Type I and type II Fc receptors regulate
innate and adaptive immunity
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© 2014 Nature America, Inc. All rights reserved.
Andrew Pincetic1,2, Stylianos Bournazos1,2, David J DiLillo1,2, Jad Maamary1, Taia T Wang1, Rony Dahan1,
Benjamin-Maximillian Fiebiger1 & Jeffrey V Ravetch1
Antibodies produced in response to a foreign antigen are characterized by polyclonality, not only in the diverse epitopes to which
their variable domains bind but also in the various effector molecules to which their constant regions (Fc domains) engage. Thus,
the antibody’s Fc domain mediates diverse effector activities by engaging two distinct classes of Fc receptors (type I and type II)
on the basis of the two dominant conformational states that the Fc domain may adopt. These conformational states are regulated by
the differences among antibody subclasses in their amino acid sequence and by the complex, biantennary Fc-associated N-linked
glycan. Here we discuss the diverse downstream proinflammatory, anti-inflammatory and immunomodulatory consequences of the
engagement of type I and type II Fc receptors in the context of infectious, autoimmune, and neoplastic disorders.
Antibodies are the key components that link the innate and adaptive
branches of immunity and are normally produced in response to a
foreign antigen to mediate host protection. A defining characteristic
of the antibody response is its polyclonality; antibodies have the
capacity to target a seemingly limitless array of antigens through
almost unlimited diversification of their antigen-binding Fab domains
by somatic recombination and mutation. However, it is becoming
increasingly clear that polyclonality of the antibody response applies
as well to the effector molecules that are engaged by the antibodyantigen complex. Thus, while Fab-antigen interactions are crucial to
the specificity of the antibody response, there is a crucial role for the
Fc domain in mediating the diverse effector properties triggered by
antigen recognition, even for processes attributed solely to recognition by the Fab, such as neutralization of toxins and viruses1,2. Specific
interactions of the immunoglobulin G (IgG) Fc domain with distinct
receptors expressed by diverse leukocyte cell types result in pleiotropic
effector functions for IgG, including the clearance of pathogens and
toxins, lysis and removal of infected or malignant cells, modulation
of the innate and adaptive branches of immunity to shape an immune
response, and initiation of anti-inflammatory pathways that actively
suppress immunity3,4.
As with every aspect of the immune system, multiple layers of regulation exist to finely tune the interaction of an IgG Fc domain with
its cognate receptors to eliminate any potential for self-destructive
autoimmunity and uncontrolled inflammation. More specifically, the
ability of an IgG molecule to engage the various Fc receptors (FcRs)
is a dynamic and tightly regulated process that is controlled mainly
by the intrinsic structure and heterogeneity of the Fc domain of IgG.
1The
Laboratory of Molecular Genetics and Immunology, The Rockefeller
University, New York, New York, USA. 2These authors contributed equally to this
work. Correspondence should be addressed to J.V.R. ([email protected]).
Received 11 April; accepted 9 June; published online 21 July 2014;
doi:10.1038/ni.2939
nature immunology VOLUME 15 NUMBER 8 AUGUST 2014
While it is traditionally considered the invariant region of an IgG
molecule, the Fc domain displays considerable heterogeneity that
arises from the differences among the four subclasses of human IgG
(IgG1, IgG2, IgG3 and IgG4) in their amino acid sequences and in their
Gm (genetic marker) allotypes and from the complex, biantennary
N-linked glycan attached at Asn297, a site that is conserved in all the
IgG subclasses5 and species examined so far. The net result of this Fc
heterogeneity translates into hundreds of different Fc structures that
can associate with any one variable region of a receptor. Ultimately,
this considerable structural heterogeneity of the Fc domain allows
extrinsic modulation of its conformation, which results in selective engagement of particular classes of FcRs with distinct effector
activities. Thus, for any single Fab, a diversity of Fc structures is
possible, which results in distinct effector responses for any given
antigen-binding activity.
On the basis of the two dominant conformational states that the
Fc domain can adopt, two structurally distinct sets of FcRs for IgG
are now recognized, with selective abilities to engage each of these
conformational states. Type I FcRs belong to the immunoglobulin
receptor superfamily and are represented by the canonical Fcγ receptors, including the activating receptors FcγRI, FcγRIIa, FcγRIIc,
FcγRIIIa and FcγRIIIb and the inhibitory receptor FcγRIIb. Each of
these receptors binds Fc domains in the open conformation near the
hinge-proximal region6 (Fig. 1) in a complex with a stoichiometry
of 1:1 (receptor/antibody). On the other hand, type II FcRs, represented by the family of C-type lectin receptors, which includes CD209
(DC-SIGN; homologous to SIGN-R1 in mice) and CD23, specifically
bind Fc domains in the closed conformation at the interface of the
constant domains of the immunoglobulin heavy chains (CH2-CH3)7
in a complex with a stoichiometry of 2:1 (receptor/antibody). Given
the ability of each receptor family to initiate distinct effector and
immuno­modulatory pathways, the conformational diversity of the
IgG Fc domain serves as a general strategy for shifting receptor specificity to actively effect different immunological outcomes.
707
review
Type I FcRs
b
a
Type I FcR binding
FcγRI
Type I FcR binding
CH2
FcγRIIa
FcγRIIb
Type II FcRs
FcγRIIc
FcγRIIIa
FcγRIIIb
CD23
DC-SIGN
CH2
Type II
FcR
binding
Type II
FcR
binding
C H3
Type II
FcR
binding
Type II
FcR
binding
CH 3
Gene
FCGR1A
FCGR2A FCGR2B FCGR2C
FCER2
CD209
Locus
1q21
1q23
1q23
19q13
19q13
72
40
50–80
47
MW (kDa)
Expression Monocytes,
MΦs,
PMNs
(induced)
Myeloid
cells,
DCs,
platelets
B cells,
myeloid
cells,
DCs
NK cells
FCGR3A
MΦs
NK cells,
monocytes
(subset)
FCGR3B
PMNs
B cells,
IL-4-induced:
T cells,
monocytes,
FDCs,
PMNs, MΦs
46
DCs, MΦ
subset,
monocytes,
endothelial
cells
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© 2014 Nature America, Inc. All rights reserved.
Figure 1 Reciprocal engagement of type I and type II FcRs by the IgG Fc domain. (a) The Fc domain alternates between the open conformation (left)
and closed conformation (right), depending on the sialylation status of the Fc glycan. Nonsialylated Fc (Protein Data Bank accession code, 3AVE) adopts
an open conformation able to bind type I FcRs near the hinge-proximal surface, whereas the binding site for type II FcRs remains inaccessible. Upon
conjugation of sialic acid, the Fc acquires a closed conformation that occludes the type I FcR–binding site and reveals a binding site for type II FcRs
(A. Ahmed (California Institute of Technology), J. Giddens (University of Maryland), L.X. Wang (University of Maryland) and P. Bjorkman (California
Institute of Technology), personal communication). (b) Characteristics and expression profiles of the human type I and type II receptors (diagrams,
above) known to bind the Fc domain of IgG. MΦ, macrophage; PMN, polymorphonuclear leukocyte; NK, natural killer.
The well-known roles of type I FcRs in mediating antibodytriggered inflammation, as seen, for example, in autoimmune diseases, have been extensively reviewed8–13. In this Review, we discuss
new observations about the structural heterogeneity of the IgG Fc
domain that highlight the intrinsic flexibility of this domain in adopting distinct conformational states (open or closed) with preferential
binding to either type I FcRs or type II FcRs. We also discuss the
functional consequences of the Fc domain’s interaction with these two
types of FcRs, as well as the molecular events that are initiated upon
engagement of the receptor, in the context of studies of infectious,
auto­immune and neoplastic disorders. Defining the molecular and
structural determinants that govern the selection of Fc-mediated effector pathways will provide greater understanding of how antibodies
regulate an immune response and will also inform the development
of therapeutic antibodies with specific effector properties.
Structural determinants of IgG
IgG molecules consist of two identical pairs of polypeptide chains;
each chain is organized into modular units of approximately 110
amino acids characterized by genetically variable or constant domain
sequences. These domains adopt a common tertiary structural motif,
the immunoglobulin fold, which is defined by antiparallel β-sheets
stabilized by a disulfide bond and hydrophobic core interactions14.
Within the Fab domain, the immunoglobulin fold serves as a robust
protein scaffold that accommodates highly variable loop regions that
link the strands of the β-sheets. The recognition of antigen, as determined by crystal structures of antigen-antibody complexes, involves
contacts with the complementarity-determining regions formed by
these hypervariable loops in the variable domains of the heavy and light
chains15. Variation in the amino acid sequences of complementaritydetermining regions functions as the structural basis for the broad
repertoire of antigen-specific antibodies by providing a heterogeneous
protein-binding surface able to interact with diverse ligands.
In contrast to the observed sequence variability of the Fab fragment,
the carboxy-terminal constant domains of both heavy chains (CH2 and
CH3) form the Fc fragment, which assumes a familiar horseshoe-like
topology in which both CH3 domains remain tightly associated and the
CH2 domains remain further apart. In the cleft between the CH2 domains
lies an Fc-conjugated carbohydrate structure that shields the hydrophobic core of the Fc from solvent (Fig. 2a). Despite a nomenclature
708
that traditionally emphasizes the invariant nature of the Fc, careful analysis
of structural and functional data has revealed that the Fc domain also
displays considerable heterogeneity that arises in part from differences among various IgG subclasses in amino acid sequences16 and
heterogeneity in glycan composition, as well as from highly dynamic
regions that encompass the hinge-proximal surface and the flexible
loops in the immunoglobulin fold of the CH2 domains17,18.
The hinge-proximal region of the Fc serves as a consensus binding site for type I FcRs19,20. Crystallographic analysis of several IgG1
Fc structures has consistently shown the hinge-proximal region in a
disordered state, albeit with relatively minor variability in the overall
orientation of the CH2 domain. However, rather than lacking defined
structure, the hinge-proximal region and the flexible loops involved
in receptor binding probably alternate among multiple conformational states, with certain conformers preferentially interacting with
individual ligands. In support of this model, two published high­resolution structures of human IgG4 Fc have revealed conformational
folds of the flexible loops of the CH2 domain that differ from those of
other published high-resolution structures of human IgG1 Fc21. More
specifically, the FG loop of IgG4 flips away from the CH2 domain to
prevent Pro329 from intercalating between tryptophan residues in
type I FcRs in the so-called ‘proline sandwich’ configuration necessary
for receptor binding20. Such conformational differences correspond
to the specialized effector functions attributed to both IgG subclasses,
with IgG1 demonstrating greater antibody-dependent cell-mediated
cytotoxicity (ADCC) than that of IgG4 as a result of enhanced binding
affinity for activating type I FcRs22.
Fc glycosylation
Several studies have demonstrated that differences in the glycosylation
of the Fc fragment modulates the effector function of IgG23–25; this has
established the Fc-associated glycan as a critical regulatory determinant for antibody activity. The complex, biantennary Fc glycan, which
consists of a core heptasaccharide structure conjugated to a highly
conserved glycosylation site at Asn297, shows considerable heterogeneity in sugar composition due to the variable addition of fucose,
galactose, bisecting N-acetylglucosamine, or sialic acid (Fig. 2a,d).
Interestingly, the glycoform profile of serum IgG varies predictably
among health and disease states, with autoantibodies produced during
autoimmune or inflammatory reactions frequently lacking galactose
VOLUME 15 NUMBER 8 AUGUST 2014 nature immunology
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a
P329
b
c
N297
D265
Y296
α1,3 arms
F241
W87
α1,6 arm
P329
W110
F243
E258
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Figure 2 Glycan-dependent modulation of Fc structure. (a) Crystal structure
α1,3 arm
of the Fc fragment of human IgG1 (Protein Data Bank accession code, 1H3Y)
Core glycan
depicting the orientation of the glycan arms relative to that of the C H2 domains
GlcNAc
Man
Gal
Sial
of the two heavy chains. The interaction between the α1,3 arms maintains the
Fc in the appropriate conformation for FcγR binding. (b) The ‘proline sandwich’
GlcNAc
GlcNAc
Man
configuration represents a key contact point between Pro329 (P329) on the Fc
(red) and two tryptophan residues (W87 and W110) on FcγRIIb (blue; Protein
GlcNAc
Man
Gal
Sial
Data Bank accession code, 1E4K). (c) Side-chain interactions of glycan residues
with their respective amino acids (red, individual glycan–amino acid interactions).
α1,6 arm
The ring structure of F241 rotates ~90° in the structure of a sialylated Fc (Protein
Fuc
GlcNAc
Data Bank accession code, 4Q6Y; (A. Ahmed (California Institute of Technology),
P. Bjorkman (California Institute of Technology), J. Giddens (University of Maryland) and
L.X. Wang (University of Maryland) personal communication). (d) Principal glycosylation structures attached to Asn297 of the Fc. The core glycan
structure (inside dashed rectangular outline) shows the invariable heptasaccharide group conjugated to the Fc. Sugars beyond the core are attached
with varying frequencies. GlcNAc, N-acetylglucosamine; Fuc, fucose; Man, mannose; Gal, galactose; Sial, N-acetylneuraminic acid (sialic acid).
d
N297
© 2014 Nature America, Inc. All rights reserved.
K246
and sialic acid residues compared with bulk antibodies present in the
steady state23,26,27. These differences are observed in not only mouse
models of autoimmunity, such as the K/BxN and MRL/lpr strains that
develop rheumatoid arthritis–like disease, but also cohorts of patients
with rheumatoid arthritis, Crohn’s disease28, systemic lupus erythematosus29 and other conditions. Differences in Fc glycosylation result
in altered binding affinity for FcγRs and complement factors, which
ultimately influences the effector pathways elicited by the Fc domain.
For example, fucosylation and sialylation represent two extensively
investigated glycan modifications of Fc that reduce affinity specifically
for FcγRIIIa30 and all type I FcRs23, respectively. Sialylation results
in increased conformational flexibility of the CH2 domain such that
the conformational states that preferentially engage type II receptors
are more frequently sampled in the population by Fc (A. Ahmed,
(California Institute of Technology), P. Bjorkman (California Institute
of Technology), J. Giddens (University of Maryland) and L.X. Wang
(University of Maryland), personal communication).
Binding to type I or type II FcRs requires, at a minimum, the presence of the core glycan group on the Fc, despite crystallographic
analysis demonstrating that the glycan does not directly interact
with receptors20. This suggests that the glycan modulates the affinity with which the antibody binds to the receptor by determining
the conformational state of the Fc. In support of this view, crystal
structures of aglycosylated Fc fragments typically resolve the Fc in
a ‘closed’ conformation in which the CH2 domains collapse together
to occlude the type I FcR–binding site31. Consequently, aglycosylated
nature immunology VOLUME 15 NUMBER 8 AUGUST 2014
antibodies fail to bind type I FcRs and lack any effector function
in vivo32. Thus, a key role for the Fc glycan is to stabilize the Fc in
an ‘open’ conformation that is accessible for interactions with type I
FcRs. Due to the biantennary construction of the glycan, one branch
(the α1,6 arm) folds along the protein backbone of the CH2 domain
and makes several noncovalent contacts with amino acid side chains,
whereas the second branch (the α1,3 arm) extends into the cavity
between the CH2 domains, where it interacts with the other α1,3
arm conjugated to the opposite heavy chain (Fig. 2b). By occupying
this space, the α1,3 arms limit the interdomain flexibility of the CH2
domains to maintain the Fc in the appropriate ‘open’ conformation
for binding type I FcRs.
Crystal structures of Fc fragments resolve carbohydrate branches
with well-defined electron density, which suggests that protein-sugar
interactions restrain mobility of the Fc glycan. However, nuclear magnetic resonance analysis demonstrates that the glycan arms exhibit
greater motion than initially assumed33. This suggests that modifying
the Fc glycan with various sugar moieties may not only alter proteinglycan interactions but also influence glycan mobility as a means of
fine-tuning Fc conformation. For example, the addition of a terminal
galactose to the α1,6 arm more firmly anchors this branch to the Fc
protein backbone34, although an unbound state also exists33. In contrast, the α1,3 arm remains highly dynamic, with the addition of sialic
acid further increasing branch mobility. Such glycan dynamics may be
a critical parameter, since the position of the glycan arms determines
the spacing between the CH2 domains and receptor specificity.
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Intrinsic flexibility of the Fc domain
Several groups have demonstrated that sialylation of the Fc glycan
switches the in vivo activity of IgG23,35–37. Whereas nonsialylated
IgG stimulates proinflammatory pathways upon the engagement of
activating type I FcRs, sialylated IgG suppresses inflammation associated with autoimmunity. This change in antibody effector properties coincides with a change in receptor-binding affinity whereby
sialylated Fc preferentially binds type II FcRs, such as CD209 (DCSIGN) and CD23, but not type I FcRs7,36. Although this finding was
initially surprising, it now seems that reciprocal engagement of disparate classes of receptors is a general property of antibodies. For
example, like IgG, IgE also binds to either type I FcRs (FcεRI) or type
II FcRs (CD23), which results in either inflammatory responses or
immunosuppressive responses, respectively. This has been attributed
to the intrinsic flexibility of the Cε3 domain on the Fc fragment
of IgE38; crystal structures of IgE in complex with CD23 or FcεRI
have revealed two mutually exclusive Fc conformations: a closed
(CD23-bound) one or an open (FcεRI-bound) one25,39. The CH2
domain of IgG lacks the intrinsic flexibility of the Cε3 domain of IgE
in the absence of sialylation. However, upon attachment of sialic acid,
structural perturbations are observed, consistent with the acquisition of greater flexibility by the Fc fragment7. Molecular models
that simulate the binding of sialylated Fc to CD209 (DC-SIGN) indicate that the Fc adopts a closed conformation as a result of movement of the sialylated α1,3 arms out of the internal cavity. Consistent
with this possibility, a crystal structure of a fully sialylated Fc preparation has been solved in which the Fc in the crystals is found in
both ‘open’ and ‘closed’ states (A. Ahmed, J. Giddens, L.X. Wang and
P. Bjorkman, personal communication). Although the α1,3 arms
are poorly resolved in this structure, an intriguing finding is that
the aromatic ring of Phe241, which normally forms a hydrophobic
stacking interaction with a mannose residue at the base of the
α1, 3 arm, rotates away from the glycan (Fig. 2c). The rotation of
Phe241 might sever its interaction with the glycan and provide
a structural link to the greater motion of α1,3 arm that has
been measured by nuclear magnetic resonance and is indicated by
molecular modeling.
Fc glycosylation in disease
The regulatory mechanisms that control the glycan composition of Fc
are not well understood. In terms of the sialylation of Fc glycan, many
studies of humans have demonstrated findings consistent with strict
control of ST6Gal1, the glycosyltransferase responsible for terminal
sialylation of Fc glycan. For example, modulation in the abundance of
sialylated Fc of antigen-specific IgG following immunization suggests
active regulation of ST6Gal1 over the course of a vaccine response40.
Further, patients with rheumatoid arthritis or Wegener’s granulomatosis are observed to have specific modulations in the abundance
of sialylation of antigen-specific IgG Fc domains that correspond
with the severity of clinical disease; antibodies to citrullinated protein in rheumatoid arthritis and antibodies specific for proteinase 3
in Wegener’s granulomatosis have been observed to decrease their
abundance of sialylated Fc during disease flares, whereas sialylation is elevated during periods of clinical remission27,41. Whether
diminished sialylation on the Fc glycans of antibodies to citrullinated
protein or proteinase 3 has a role in the pathogenesis of either disease remains to be determined. These and other observations in the
literature suggest that precise regulation of the activity of ST6Gal1
(and probably that of other glycosyltransferases that act on the Fc
glycan) is a fundamental homeostatic process; this remains an open
area for investigation.
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Immunomodulatory functions of type I and type II FcRs
Upon exposure to antigens, specific IgG antibodies in the peripheral repertoire or generated early in the antibody response result in
the formation of immune complexes; in turn, depending on their Fc
conformations, these interact either with type I FcRs or type II FcRs on
effector cells and on B cells to modulate both humoral immune processes and innate immune processes. Balanced positive and negative
signaling through type I and type II FcRs is essential for the development of appropriate immune responses to soluble protein antigens
or microorganisms.
As described above, the specific composition of IgG subclasses
and Fc glycans in immune complexes dictates the degree to which
type I or type II FcRs are engaged. The distribution of IgG subclasses is regulated by both the cytokine milieu and the biochemical
nature of the antigen. The determinants of the composition of Fc
glycans on IgG elicited during an active immune response are not
well understood, but data suggest that antigen exposure can induce
glycan modifications on antigen-specific IgG antibodies and that
such modifications may have a role in shaping an ongoing antibody
response37,40,42. Profound immunomodulation through signaling
via immune complexes seen in the regulation of vaccine responses,
in antigen presentation by dendritic cells (DCs), in B cell selection,
and in the exacerbation of infection by some pathogens; we will consider each of these processes separately.
Vaccine responses
Type I FcR–dependent immunomodulation through vaccination with
immune complexes can affect the resulting IgG titer and/or its avidity
for antigen. Activating type I FcRs on DCs, follicular DCs (FDCs)
and macrophages are most relevant in adaptive immune responses,
and bone marrow–chimera experiments have shown that DCs and
macrophages provide the greatest contribution to priming antibody
responses43. The expression of type I FcRs critically regulates the
responses of T cells generated by immune complex-primed DCs
by influencing the maturation and antigen presentation of DCs44.
Signaling through the immunoreceptor tyrosine-based activation
motifs of FcR γ-chains matures DCs and upregulates the expression
of major histocompatibility complex (MHC) and costimulatory molecules, a process required for their function as antigen-presenting
cells. Immune complexes have also been demonstrated to polarize
macrophages toward the M2b phenotype; these cells have enhanced
antigen-presentation activity through increased expression of costimulatory molecules45 (Fig. 3a). Signaling through the pathway of
the inhibitory FcγRIIb immunoreceptor tyrosine-based inhibitory
motif regulates the IgG-dependent maturation of both DCs and macrophages; FcγRIIb-mediated inhibition can be attenuated by the presence of signaling via Toll-like receptors in conjunction with signaling
via activating type I FcRs. Notably, DCs that do not receive inhibitory signaling via FcγRIIb undergo spontaneous maturation, which
demonstrates the critical function of this receptor in regulating immunological activation44,46,47. A level of control over activation of cells
of the immune system may also occur through inhibitory signaling
via immunoreceptor tyrosine-based activation motifs, induced by
low-valence targeting of activating type I FcRs48.
Antigen presentation
A well-defined consequence of the engagement of activating type I FcRs
is uptake of immune complexes by endocytosis or ­phagocytosis49,50.
DCs internalize immune complexes through type I FcR–mediated
pathways and efficiently process and present the antigen on both MHC
class I molecules and MHC class II molecules in a process central
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a
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© 2014 Nature America, Inc. All rights reserved.
b
Katie Vicari/Nature Publishing Group
+
Figure 3 Immunomodulatory functions of type I and type II FcRs.
CD8
Activating
Inhibitory
T cell
FcγR
FcγR
(a) For antigen presentation, antibodies bind to soluble antigens to
CD23
form immune complexes, then the recognition of immune complexes
CD4+
by activating type I FcγRs expressed on DCs results in the engulfment
Immune
DC
T cell
B cell
complex
of the complexes by endocytosis and the maturation of DCs, which
?
includes upregulation of the expression of MHC costimulatory molecules.
↑ MHC-II
↑ Ag presentation
The internalized antigens are processed and presented on MHC
↑ Co-stimulation
B cell
class I and class II molecules to CD4+ or CD8+ T cells, which results
activation
in activation of the T cells so they can mediate specific effector functions.
(b) The selection of B cells with high-affinity B cell receptors occurs
in the germinal center. FDCs express the inhibitory receptor FcγRIIb,
GC
Memory/PC
which binds immune complexes and presents them to germinal center
B cell
differentiation
B cells. The type I Fc receptor FcγRIIb and the type II Fc receptor CD23
are also expressed on the surface of B cells in the germinal center,
Apoptosis
which facilitates the binding of immune complexes. Preferential
binding of antigen by the B cell receptor results in the positive
selection of the respective B cells; this allows them to further
differentiate into antibody-producing plasma cells or memory B cells.
FDC
In contrast, exclusive binding of the presented immune complex via
the inhibitory receptor FcγRIIb leads to the induction of apoptosis,
thereby setting a threshold for the selection of B cells with high-affinity
B cell receptors. Furthermore, since sialylated immune complexes may
also interact with CD23 on B cells in the germinal center, regulation of Fc glycosylation may provide a means by which to modulate cell activation
and/or affinity maturation in an as-yet-uncharacterized pathway.
to the induction of adaptive cellular immune responses (Fig. 3a).
The activation of DCs and subsequent priming of immune responses
mediated by either CD4+ T cells or CD8+ T cells are substantially
enhanced when antigen is internalized as an immune complex
through activating type I FcRs51–53. Signaling in cis through the inhibitory FcγRIIb on DCs negatively regulates antigen presentation, as
DCs derived from FcγRIIb-deficient mice are more potent inducers
of T cell activation both in vitro and in vivo44,54. Other innate effector cell types also demonstrate increased uptake of antigens that are
present as immune complexes. The activation of type I FcγR pathways
on granulocytes, monocytes and macrophages triggers degradation of
antigens in lysosomal compartments and production of proinflammatory chemokines and cytokines45,55. This activity, as with DCs, is
moderated by FcγRIIb signaling. It is important to note that macrophage and DC subsets can differ in FcR expression patterns, a topic
that has been reviewed56.
FcγRIIb expression, mice generate antibodies of lower avidity, presumably due to an absence of B cell selection based on the specificity
or avidity of the receptor62. Since sialylated immune complexes may
also interact with CD23 (ref. 7) on B cells in the germinal center,
regulation of Fc glycosylation probably provides a means with which
to modulate cell activation and/or affinity maturation in an as-yetuncharacterized pathway that remains to be elucidated (Fig. 3b).
Plasma cells have little or no expression of the B cell receptor
and display elevated amounts of FcγRIIb; they are therefore subject
to unopposed proapoptotic FcγRIIb signaling, which is probably
involved in the homeostasis of long-lived plasma cells. During an
ongoing immune response, immune complexes can induce FcγRIIbdependent apoptosis in existing long-lived plasma cells, which suggests a possible mechanism for maintaining a reservoir of relatively
consistent size that allows the addition of new cells with different
antibody specificities63,64.
B cell selection
B cells express a single type I FcR, FcγRIIb, throughout development,
while the type II FcR, CD23, also has variable expression during
B cell maturation57,58. FcγRIIb on B cells is a key regulator of
affinity maturation and the B cell repertoire. Exclusive engagement of FcγRIIb on B cells is proapoptotic; however, coligation of
FcγRIIb and the B cell receptor by immune complexes results in
attenuation of apoptotic signaling in a process mediated by the
inositol polyphosphate phosphatase SHIP59. In this way, B cells with
higher affinity for antigen are selected for survival, while those with
receptors of irrelevant specificity or low affinity are more likely to
undergo apoptosis.
In the germinal center, somatically mutated B cells with receptors of higher affinity for antigen are selected through interactions
with immune complexes retained on a specialized stromal cell type,
the FDC (Fig. 3b). As with B cells, FDCs express FcγRIIb and complement receptors, both of which are involved in the retention of
immune complexes. This has been shown in studies of mice with
selective deficiency in FcγRIIb on either FDCs or B cells; mice with
selective FcγRIIb deficiency on FDCs generate antibody responses of
higher avidity, which probably results from the lack of competition for
binding of FcγRIIb by B cells60,61. In contrast, when only B cells lack
Antibody-dependent enhancement of infection and/or disease
A long-appreciated yet not fully understood immunomodulatory
property of type I FcRs is their occasional ability to mediate enhanced
infection and/or clinical disease. Perhaps the best studied example of
such antibody-dependent enhancement is secondary infection with
dengue virus, which represents a case of an increase in both viral
infection and the associated sequelae: while primary infection with
dengue virus in humans is often asymptomatic or mild in presentation, subsequent infection with a distinct strain of dengue virus can
cause exponentially higher viral titers, along with severe and even
fatal disease65,66. This enhanced disease phenotype during secondary
infection is thought to be caused by low-avidity, cross-reactive, nonneutralizing antibodies generated during the primary infection that
mediate increased infection of monocytes and macrophages through
type I FcRs (Fig. 4a). Elevated replication of virus in these cells results
in a massive release of cytokines that can cause gastrointestinal hemorrhage and vascular leakage that results in the clinical phenotype of
dengue hemorrhagic fever or shock syndrome67.
An example of antibody-dependent enhancement of infection
may have been present in the STEP trial of a vaccine against human
immunodeficiency virus (HIV), in which preexisting antibodies to
the adeno­virus vector used to deliver HIV antigens correlated with
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increased risk of infection with HIV68. Antibody-dependent enhancements of infectious disease that are not secondary to increased infection or replication have occurred during outbreaks of infection with
respiratory syncytial virus or measles in humans previously vaccinated with formalin-inactivated viral proteins and in some severe
pandemic infections with influenza virus. Disease enhancement
in these situations was thought to occur when immune complexes
formed from non-neutralizing IgG deposited in tissues or formed
with viral antigens expressed on host cells, which results in cytotoxicity and/or complement deposition and inflammation69–71.
Why some microbes cause enhanced infection and/or clinical disease through antibody opsonization is not well understood.
Adaptation to productive replication in monocytes or macrophages
may be one determinant of cytokine-associated disease enhancement.
A second determinant might be antigenic variability due to multiple
serotypes, as with dengue viruses, or to continuous selection of novel
variants, as with influenza viruses; such antigenic variability predisposes the host to the generation of cross-reactive, non-neutralizing
antibodies that may mediate enhanced uptake and infection through
FcRs or, in rare circumstances, may form insoluble immune complexes
that cause disease secondary to engagement of type I FcRs and
inflammation or direct cytotoxicity.
Role of FcRs in the activity of therapeutic antibodies
Type I FcRs serve crucial roles during humoral immune responses
to foreign pathogens or to epitopes expressed by malignant cells
during tumor immunity. IgG antibodies serve to bridge the target, recognized by the antibody’s Fab region, and the activating
type I FcRs that are expressed by natural killer cells, monocytes,
macrophages and other innate immune effector cells that mediate
the antibody’s activity. After crosslinking of activating type I FcRs
and phosphorylation of immuno­receptor tyrosine-based activation
motifs, effector cells mediate cytotoxicity through the release of
cytotoxic mediators or are activated to induce phagocytosis of the
opsonized target3. Here we discuss the roles of type I FcRs during
the effects carried out by antiviral, antitumor and immunomodulatory antibodies in vivo.
Type I FcRs during immunity to infectious disease
The importance of activating type I FcRs during immune responses is
exemplified by antibodies that engage antigens expressed by various
infectious agents. While some results of in vitro viral neutralization
712
a
Monocyte/
macrophage
Activating
FcγR
↑ of virus uptake
Inhibitory
FcγR
b
Virus
particle
ADCC
Monocyte/
macrophage
NK cell
Ab-coated tumor,
infected cell,
Treg cell
c
Phagocytosis
Anti-CD40
+
CD40
APC
FcγRIIb+
cell
CD40
FcγRIIbmediated scaffold
Activation
↑ TNFR (CD40)
signaling
↑ Ag presentation
↑ Costimulation
↑ T cell population
expansion
Katie Vicari/Nature Publishing Group
© 2014 Nature America, Inc. All rights reserved.
Figure 4 Type I FcR–mediated effector functions. (a) In antibodydependent enhancement of infection and/or disease, preexisting
antibodies present from a primary infection (for example, infection with
dengue virus) that are at too low a concentration to neutralize the target
bind to viral particles during a secondary infection with virus of a different
serotype. These immune complexes are bound by activating type I FcγRs
expressed on monocytes and macrophages, which mediates increased
virus uptake and replication that results in the enhancement of infection.
(b) Antibody-opsonized pathogens interact with activating type I FcγRs
on monocytes and/or macrophages, which leads to phagocytosis and
clearance. Antibody (Ab)-coated malignant, virus-infected or Treg cells
also engage monocytes and/or macrophages or natural killer cells, which
results in phagocytosis or cell-mediated cytotoxicity (ADCC) of the target
cells. Removal of infected or tumor cells leads to clearance of disease,
while removal of Treg cells leads to enhanced cellular immunity. (c) The
agonistic effect of antibodies to the TNFR family requires the expression
of type I FcγRIIb. For example, anti-CD40 binds CD40 on antigen (Ag)presenting cells (APC). FcγRIIb+ cells bind CD40-bound antibody in trans
and thereby act as a scaffold for the clustering of TNFR molecules on the
membrane to mimic the effect of multimeric ligand engagement and to
activate TNFR-related pathways (CD40 signaling and cellular activation).
assays correlate with in vivo protection by a neutralizing antibody, in
other studies the in vivo protective abilities of an antibody are much
greater than their in vitro effects. This disparity can be explained by
the protective contributions FcR-expressing effector cells. Thus, the
involvement of interactions between activating type I FcRs and antibodies targeting antigens, including the viral M2 protein or hemagglutinin, has been indicated during protection against influenza
virus infection in vivo2,72,73. While they are dispensable for in vitro
neutralization, Fc-FcR interactions are required for in vivo protection mediated by broadly neutralizing antibodies to influenza virus
directed at the stalk domain of hemagglutinin2. In vivo protection
requires Fc-FcR interactions after entry of the virus into target cells,
which suggests that effector cells mediate ADCC of infected cells
expressing hemagglutinin on their surface. Similarly, neutralizing
antibodies to HIV mutated to abrogate engagement of type I FcRs
have shown impaired ability to protect macaques from infection with
simian-human immunodeficiency virus74; this suggests that antibodies
to HIV also require FcR-mediated effector function to mediate their
activity in vivo. Furthermore, in vivo protection from infection
with Bacillus anthracis mediated by antibodies to protective antigen
absolutely requires the engagement of activating type I FcRs1,75.
The Fc domains of antibodies to pathogens can even be engineered
to enhance interactions with activating type I FcRs to augment the
protective abilities of the antibodies in vivo1,2. In each of these models,
FcR-expressing effector cells may function at one or both of the following two stages: the phagocytosis or cytotoxicity of the antibodycoated pathogen itself, or the phagocytosis or cytotoxicity of infected
cells (Fig. 4b). Thus, various classes of pathogen-specific antibodies
interact with activating type I FcRs on innate effector cells to mediate
their in vivo protective effects.
Type I FcRs recruited by antitumor antibodies
Various therapeutic antibodies to tumor-specific antigens are now
being investigated or used in the clinical setting, including those that
directly induce cell death, block survival signals, neutralize growthsupporting ligands or directly engage type I FcR–expressing effector
cells. Rituximab, the first therapeutic monoclonal antibody approved
for the treatment of cancer, is widely used to treat various classes
VOLUME 15 NUMBER 8 AUGUST 2014 nature immunology
npg
© 2014 Nature America, Inc. All rights reserved.
review
of lymphomas and leukemias expressing the B cell–specific surface
antigen CD20. The mechanism of rituximab has been investigated
intensively, and while the induction of apoptosis and activation of
the complement cascade have been proposed as possible modes of
action, the recruitment of FcR+ effector cells dominates in vivo76
(Fig. 4b). Thus, functional polymorphisms of human FcγRIIa and
FcγRIIIa correlate with the efficacy of the depletion of CD20+ cells
in lymphoma and autoimmune disease77,78. Studies of various mouse
models have clearly demonstrated that antibodies to CD20 absolutely
require interactions with activating type I FcRs expressed by monocytes or macrophages for the depletion of CD20+ cells79,80. A similar
mechanism governs the function of trastuzumab, an antibody to the
epidermal growth factor receptor HER2/neu, since the V158F polymorphism in human FcγRIIIa that affects the affinity of the receptor
for IgG1 correlates with antibody efficacy81,82, and in vivo animal
models require expression of activating FcγRs to prevent the growth
of HER2+ tumors79. Variants of alleles encoding FcγRIIa and FcγRIIIa
are also predictive of efficacy in the treatment of colorectal cancer
with cetuximab, an antibody to the epidermal growth factor receptor83. Further, modulating the ability of antibodies to engage activating versus inhibitory type I FcRs on effector cells, either through
genetic deletion of the inhibitory FcγRIIb or engineering of the IgG
Fc to selectively engage activating type I FcRs, modulates the cytotoxic
potential of antitumor antibodies for enhanced ADCC and tumor
clearance79,84. A striking example of this importance of the engagement of type I FcRs by antitumor antibodies is provided by obinutuzumab, a monoclonal antibody to CD20 (approved by the US Food
and Drug Administration) with enhanced binding of FcγRIIIa that
extends by a year the survival of patients with chronic lymphocytic
leukemia relative to the survival afforded by rituximab, an unmodified CD20-specific antibody85. Thus, numerous antitumor antibodies
require interactions with activating type I FcRs on innate effector cells
to activate ADCC and mediate their therapeutic effects on malignant
cells. Optimization of this pathway is thus critical to the clinical efficacy of this broad class of therapeutics.
Antitumor activity of immunomodulatory antibodies
Antibodies that target immunoregulatory receptors, such as T cell
checkpoints or signals for the maturation of antigen-presenting cells,
have received intense interest from the cancer immunotherapy field. To
enhance an antitumor immune response, immunomodulatory antibodies that target immunoreceptors on the surface of the cell can act as
agonists or antagonists to either stimulate or block, respectively, a target
antigen; this results in enhanced T cell–mediated responses. Since these
antibodies modulate the signaling pathways triggered by their targets, it
was generally accepted that they would act without the need of effector
function or to engage FcR-expressing cells. However, the unexpected
contribution of various members of the type I FcR family to the activities of immunomodulatory antibodies has been described.
Interactions with the inhibitory receptor FcγRIIb are required for
the agonistic activity of antibodies that target various members of
the tumor-necrosis receptor (TNFR) superfamily. For example, an
absolute requirement for FcγRIIb has been described for the in vivo
immunostimulatory and antitumor activities of agonistic antibodies
to the costimulatory receptor CD40 (refs. 86,87). Population expansion and activation of T cells induced by CD40-specific antibodies
has been observed in mice lacking all activating type I FcRs (Fcer1g−/−
mice) but not in mice deficient in FcγRIIb (Fcgr2b−/− mice). Moreover,
dramatically enhanced antitumor responses mediated by CD8+ T cells
have been observed through the use of IgG antibodies to CD40 in
which the Fc domain of IgG was engineered to augment interactions
nature immunology VOLUME 15 NUMBER 8 AUGUST 2014
with FcγRIIb86,87. Studies of antibodies targeting other members of
the TNFR superfamily have reported a general requirement for the
engagement of FcγRIIb by this class of agonistic antibodies. Animal
models of FcR-deficient mice or comparison of Fc variants with
distinct FcR-binding abilities have been used to demonstrate that toxicity triggered by agonistic antibodies to the TNFR superfamily member CD95 (Fas) or antibodies to the death receptors DR4 and DR5
has a common requirement for coengagement of FcγRIIb for optimal
therapeutic effects of these antibodies88–91. The general mechanistic
basis for these anti-TNFR therapeutics has been described92. First,
the agonistic antibodies function in trans (i.e., they engage FcγRIIb
on a distinct cell other than the antibody-bound target cell). Second,
this trans coengagement is independent of downstream signaling
via FcγRIIb. Third, distinct FcγRIIb-expressing cell populations are
required for the activity of the various antibodies to members of the
TNFR superfamily92. Thus, the type I inhibitory receptor FcγRIIb
acts as a scaffold to mediate the clustering of TNFR molecules on the
membrane and thereby mimic the effect of the engagement of these
receptors by multimeric ligands (Fig. 4c).
Therapeutic immunomodulatory antibodies that target cell-surface
immunoregulatory molecules, such as the immunological checkpoint
receptor CTLA-4, were initially thought to function solely through
antagonizing signals mediated by their targets. However, reports have
demonstrated a requirement for activating type I FcRs in the antitumor
therapeutic effects of immunomodulatory antibodies to CTLA-4 and
to the immunomodulatory receptor GITR93,94. Antibodies to CTLA-4
substantially lose their antitumor activity in mice that lack activating
FcRs in colon and in B16 melanoma tumor models. The antitumor
efficacy of antibodies to CTLA-4 was found to be associated with FcRmediated depletion of intratumoral regulatory T cells (Treg cells) but
not such depletion of peripheral Treg cells94 (Fig. 4b), which called into
question the common mechanistic paradigm that antagonist antibodies function by blocking inhibitory signaling. Similarly, depletion of
intratumoral Treg cells has been observed after in vivo administration
of antibodies to GITR in tumor models93. Both CTLA-4 and GITR
are also expressed on effector T cells (Teff cells), which are essential
for the antitumor immune response. However, the higher expression
of these immunoreceptors on the surface of Treg cells than on Teff cells
and the presence of activated FcR-expressing effector cells in the tumor
microenvironment contributes to the increased ratio of Teff cells to
Treg cells after depletion of Treg cells in the tumor microenvironment;
this leads to effective antitumor immunity. Ipilimumab, an anti-CTLA-4
therapeutic now in use in the clinic, has an IgG1 Fc isotype and therefore has the potential to engage effector cells to mediate the depletion
of Treg cells, but it remains unknown whether depletion of Treg cells
also occurs in patients treated with antibodies to CTLA-4. Given the
revised paradigms for the requirements for type I FcRs in the optimal
activity of immunmodulatory antibodies, the development of future
antibody-based therapeutics should incorporate Fc domains that
interact specifically with the appropriate FcRs.
Immunosuppressive activity of type I and type II FcRs
Intravenous immunoglobulin (IVIG) consists of polyclonal IgG molecules purified from thousands of blood donors. IVIG therapy was initially developed as a replacement therapy for immuno­deficient patients
with impaired antibody production. However, in the 1980s, administration of high doses of IVIG was reported to restore platelet numbers
in patients suffering from idiopathic thrombocytopenic purpura, an
antibody-mediated autoimmune disease in which the immune system
depletes platelets from the blood95. Since then, IVIG administration has
been included as an approved therapy for many chronic autoimmune
713
Autoantibody
?
immune complex
Unidentified
mediators
Inhibitory
Effector
FcγR
macrophage
Activating
Upregulation
FcγR
of inhibitory
FcγRIIb
IL-4
Unidentified
immune cell types
DC-SIGN+
regulatory
macrophage
or DC
IL-33
Basophil
DC-SIGN
+
© 2014 Nature America, Inc. All rights reserved.
Sialylated
IgG
npg
Unidentified
type II FcR
and CD23
Asialylated
IgG
Katie Vicari/Nature Publishing Group
review
Figure 5 Both type I FcRs and type II FcRs mediate the anti-inflammatory
effects of IVIG or sialylated IgG. Immune complexes of autoantibodies
and autoantigens crosslink activating type I FcγRs, which promotes
the activation of macrophages and inflammatory autoimmune disease.
Sialylated Fcs engage DC-SIGN- or SIGN-RI-expressing macrophages or
DCs, which promotes expression of IL-33. This IL-33 signals activated
FcεRI+ innate leukocytes (basophils) to produce IL-4. IL-4 in turn
promotes upregulation of FcγRIIb expression on effector macrophages and
thereby increases the activation threshold needed to trigger inflammation.
Alternatively, as-yet-unidentified type II FcRs expressed by various cell
types may also mediate the anti-inflammatory effects of IVIG or sialylated
IgG through unidentified pathways, depending on the type and location of
the inflammation.
diseases, such as Guillain-Barrè syndrome, Kawasaki disease, and
chronic inflammatory demyelinating polyneuropathy.
The mode of action of IVIG has been the subject of numerous studies and reviews4,96,97. The following three key components of the pathway triggered by IVIG infusion have been discovered to be integral for
its immunomodulatory activity in various systems: the sialylated Fc
fraction of the immunoglobulins present in the IVIG preparation35,98;
the type II FcRs, such as CD209 (DC-SIGN in humans; SIGN-R1 in
mice)36; and expression of the inhibitory type I receptor FcγRIIb99.
Thus, the immunoinhibitory effect of IVIG has been recapitulated in a
mouse model of rheumatoid arthritis through the use of a recombinant
IgG Fc fragment galactosylated and sialylated in vitro35. Since sialylation of the Fc glycan results in diminished affinity for type I FcRs, an
additional receptor triggered by this ligand must be responsible for
the anti-inflammatory activity. Mouse studies with IVIG have demonstrated that the sialylated IgG fraction binds to mouse SIGN-R1
expressed on regulatory macrophages, an interaction necessary for the
inhibitory activity of the sialylated IgG fraction in vivo36. Various studies have also demonstrated that FcγRIIb is required for the immunoinhibitory activity of IVIG in models of autoimmune disease35,99–101,
since FcγRIIb-deficient mice fail to respond to treatment with IVIG or
sialyalted IgG Fc in K/BxN arthritis, immunothrombocytopenia, nephrotoxic nephritis and epidermolysis bullosa acquisita. In addition, IVIG
has been shown to upregulate FcγRIIb expression on monocytes and
B cells among clinically responding patients with chronic inflammatory demyelinating polyneuropathy102, and functional polymorphisms
of both CD209 (which encodes CD209 (DC-SIGN)) and FCGR2B
(which encodes FcγRIIb) are associated with the clinical response to
IVIG therapy among patients with Kawasaki disease103.
Experiments elucidating the mechanism by which sialylated Fc,
the type II FcR CD209 (DC-SIGN; SIGN-R1) and the inhibitory
type I Fc receptor FcγRIIb function in tandem to regulate inflammation have been carried out with the K/BxN model of serum-induced
joint inflammation. The engagement of the type II FcR SIGN-R1 on
714
r­ egulatory macrophages by sialylated IgG induces the secretion of
interleukin 33 (IL-33), a potent T helper type 2–polarizing cytokine
known for its pleiotropic effects on various effector cells of the
immune system. The secretion of IL-33 subsequently signals basophils
to release IL-4 at sites of inflammation, which upregulates expression
of the type I inhibitory receptor FcγRIIb on effector inflammatory
monocytes7 (Fig. 5). This upregulation of FcγRIIb expression raises
the threshold of activation for inflammatory effector macrophages
and thereby attenuates IgG-mediated inflammation.
New data also suggest that other sialylated IgG–mediated mechanisms may be involved in regulating inflammation during auto­
immunity (Fig. 5). For example, IL-4 and IL-33 are not involved in
the IVIG-dependent suppression of idiopathic thrombocytopenic
purpura104,105. That observation is consistent with the finding that
SIGN-R1 is necessary for the activity of IVIG in prophylactic models of disease but is dispensable in therapeutic models of idiopathic
thrombocytopenic purpura and during the late phase of epidermolysis
bullosa acquisita and models of inflammatory arthritis99. Thus, it is
likely that additional receptors for sialylated IgG may exist that act
during the later phases of inflammation. Sialylated IgG also engages
CD23, a type II FcR7. CD23 exists in two isoforms: CD23a, which is
present on mature B cells; and CD23b, which requires induction by
IL-4 for expression on T cells, monocytes, Langerhans cells, eosinophils and macrophages. While early studies indicated that B cells
are dispensable for IVIG activity, only the cytokine production aspect
of B cells was assessed. Thus, the effect of the ligation of CD23 by
sialylated IgG on the production of antibodies and autoantibodies
by B cells, as well as the engagement of CD23 on other innate cellular
populations during autoimmunity, remains to be determined.
Conclusions
The structural diversity and conformational flexibility of IgG Fc, controlled by the amino acid sequence of the Fc isotypes and Fc glycan
composition, are essential for regulating antibody effector functions
through differences in the engagement of particular members of the
type I and type II FcR families. Thus, for any individual antibody’s
Fab, a diversity of Fc effector functions are possible. It is clear that the
isotype and glycosylation of the Fc domain, and thus the effector function of the antibody, are tightly regulated during immune responses
and are potentially dysregulated during autoimmunity. However,
the signals that dictate the Fc structure and effector function of IgG
in vivo during the development of an immune response remain poorly
understood. Antibody therapeutics for the treatment of autoimmune
disorders, infectious disease or tumors require multiple respective
effector functions and the development of such therapeutics must
consider not only target specificity but also which downstream effector functions will be required for optimal therapeutic efficacy. Thus,
optimal interactions with either activating or inhibitory type I or type
II FcRs must be manipulated during new vaccination strategies and
must be engineered into next-generation antibody therapeutics.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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