The function of Fcγ receptors in dendritic cells and macrophages.

REVIEWS

The function of Fcγ receptors in dendritic cells and macrophages Martin Guilliams1,2, Pierre Bruhns3,4, Yvan Saeys1,2, Hamida Hammad1,2 and Bart N. Lambrecht1,2,5

Abstract | Dendritic cells (DCs) and macrophages use various receptors to recognize foreign antigens and to receive feedback control from adaptive immune cells. Although it was long believed that all immunoglobulin Fc receptors are universally expressed by phagocytes, recent findings indicate that only monocyte-derived DCs and macrophages express high levels of activating Fc receptors for IgG (FcγRs), whereas conventional and plasmacytoid DCs express the inhibitory FcγR. In this Review, we discuss how the uptake, processing and presentation of antigens by DCs and macrophages is influenced by FcγR recognition of immunoglobulins and immune complexes in the steady state and during inflammation.

Laboratory of Immunoregulation, VIB Inflammation Research Center, 9052 Ghent, Belgium. 2 Department of Respiratory Medicine, Ghent University, 9000 Ghent, Belgium. 3 Institut Pasteur, Département d’Immunologie, Laboratoire Anticorps en Thérapie et Pathologie, 75015 Paris, France. 4 Institut National de la Santé et de la Recherche Médicale, U760, 75015 Paris, France. 5 Department of Pulmonary Medicine, Erasmus University Medical Center, 3015 Rotterdam, The Netherlands. Correspondence to M.G. and B.N.L. e-mails: martin.guilliams@ irc.vib-ugent.be; bart.lambrecht@ irc.vib-ugent.be doi:10.1038/nri3582 Published online 21 January 2014 Corrected online 7 April 2014 1

Dendritic cells (DCs) and macrophages bridge innate and adaptive immunity by recognizing and internalizing foreign antigens and by subsequently processing the antigens for presentation to cells of the adaptive immune system. Once the adaptive immune response has been initiated, innate immune cells receive important feedback signals from adaptive immune cells; for example, T cell-derived cytokines increase the innate effector functions of macrophages and neutrophils. Importantly, B cell-derived immunoglobulins that develop a few days after antigen encounter also regulate the function of innate immune cells, as most innate immune cells express various Fc receptors (FcRs) for IgG (FcγRs), IgM, IgA and IgE. The killing of infected cells by neutrophils and natural killer (NK) cells is facilitated by opsonization by IgGs — a process that is known as antibody-dependent cell-mediated cytotoxicity (ADCC). Similarly, the degranulation of mast cells and basophils is induced by crosslinking of IgE that is bound to the high-affinity FcR for IgE (FcεRI)1,2. In this Review we address the feedback control of DC and macrophage function by immunoglobulins and by antigen–antibody complexes, which are known as immune complexes, focusing mainly on the functions of FcγRs. Targeting antigens to phagocytes via FcRs markedly affects antigen uptake, endosomal maturation, antigen processing and cellular activation. Most papers that address the function of FcRs on phagocytes have conceptually grouped DCs, macrophages and monocytes together as cells of the common mononuclear phagocyte system (MPS), which has led to the dogma that all FcRs are expressed by all cells of the

MPS. The concept of the MPS has undergone considerable changes in the past 10 years, and now different subsets of DCs and macrophages that differ in their FcR expression can be clearly delineated (BOX 1). Given these recent developments, we summarize in this Review what is currently known about FcγR triggering on DC and macrophage subsets in the steady state and in inflammatory disease states, and we identify areas for future research.

A primer on FcγRs Myeloid cells express various FcγRs that facilitate their interaction with monomeric or aggregated IgGs, immune complexes and opsonized (antibodycoated) particles or cells (TABLES 1,2). Most receptors bind extracellular IgGs, with the exception of the neonatal FcR (FcRn)3 and the intracellular FcR tripartite motif-containing protein 21 (TRIM21)4,5, which bind to immunoglobulins following their internalization. The various FcγRs are functionally divided into activating and inhibitory receptors. Activating FcγRs have an immunoreceptor tyrosine-based activation motif (ITAM) in their intracytoplasmic domain or, in the case of the high-affinity FcR for IgG (FcγRI; also known as CD64) and FcγRIIIA, associate with the ITAM-containing signalling subunit FcR common γ‑chain (encoded by FCER1G) (TABLES 1,2). Following receptor activation by immune complexes, the ITAMs activate signalling cascades via SRC family kinases and spleen tyrosine kinase (SYK)2,6,7. The inhibitory FcγR, FcγRIIB, has an immunoreceptor tyrosine-based inhibition motif (ITIM) in its intracytoplasmic domain8.

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REVIEWS Box 1 | Use of FcRs to distinguish moDCs and macrophages from cDCs The high-affinity Fc receptor I for IgG (FcγRI; also known as CD64) and the high-affinity Fc receptor for IgE (FcεRI) have recently been suggested to be the best markers to separate monocyte-derived cells (that is, macrophages and monocyte-derived dendritic cells (moDCs)) from conventional DCs (cDCs). The idea that Fc receptor (FcR) expression can be used to distinguish between myeloid cell subpopulations is not new. In fact, in one of the two original papers152 in which Nobel prize laureate Ralph Steinman described the discovery of splenic DCs, he noticed that splenic DCs are very different from macrophages in that they poorly bind immune complexes and antibody-coated sheep red blood cells. The Malissen team36,37 and the Randolph team25 have recently independently found that an antibody against FcγRI could be used to discriminate moDCs and macrophages from cDCs. The Malissen team36,37 tested antibodies that are specific for known monocyteand/or macrophage-specific markers (that is, F4/80, CD115 (which is the macrophage colony-stimulating factor (M‑CSF) receptor), CD68, CX3C-chemokine receptor 1 (CX3CR1), LY6C, CD43 and FcγRI) and DC‑specific markers (that is, CD11c, 33D1 and high expression levels of MHC class II molecules) in mixed bone-marrow chimeric mice that had been reconstituted with 50% bone marrow from wild-type mice and 50% bone marrow from CC‑chemokine receptor 2 (Ccr2)–/– mice. Bone marrow from Ccr2−/− mice was used because monocytes require CCR2 for their egress from the bone marrow142. As a result, in these mixed chimeric mice monocyte-derived cells can be identified because these cells will almost all be derived from the bone marrow from wild-type mice and not from the bone marrow from Ccr2−/− mice, whereas all the other non-monocytic cells will have a mixed chimerism because they will be derived from both the wild-type and the Ccr2−/− bone marrow. They found that only FcγRI expression facilitated the correct separation of moDCs and macrophages from cDCs36,37. The Randolph laboratory25, through their participation in the Immunological Genome Consortium (see Further information), found that the expression of FcγRI was one of the best markers to discriminate macrophages from cDCs, together with the expression of the tyrosine protein kinase MER (MERTK). In addition, the Lambrecht laboratory23,144 found that FcεRI is highly expressed by moDCs and combining an FcγRI-specific antibody (clone X54–5/7.1) and an FcεRI-specific antibody (clone MAR‑1) is the most specific and sensitive way to distinguish moDCs from cDCs compared with other commonly used discriminating markers. Interestingly, the Amigorena laboratory43 found that human inflammatory moDCs but not macrophages express high levels of FcεRI, which identifies FcεRI as a good moDC marker in mice and humans.

Antibody-dependent cell-mediated cytotoxicity (ADCC). A mechanism by which cytotoxic effector cells, including natural killer (NK) cells, kill other cells, for example, virus-infected target cells that are coated with antibodies. The Fc portions of the coating antibodies interact with the Fc receptor that is expressed by the cytotoxic effector cell, thereby initiating a signalling cascade that leads to cellular activation and target cell killing. The precise killing mechanism depends on the type of cytotoxic effector cell.

This ITIM recruits SH2 domain-containing inositol 5ʹ‑phosphatase 1 (SHIP1; encoded by INPP5D)9 and thus counteracts the signals that are mediated by activating FcγRs10,11. Another classification of FcγRs is based on the affinity of the receptor for IgG: FcγRs with different affinities for different IgG isotypes can bind to multiple classes of immunoglobulin12 (TABLES 1,2). A few receptors — such as FcγRI, FcγRIV and FcRn — can bind to monomeric IgG (which is the definition of highaffinity receptors), whereas the other receptors mainly bind to aggregated IgGs. Although for some researchers the definition of FcγRIV as a high-affinity receptor is debatable2,13, we think that the main factor for consideration when dividing FcγRs into high-affinity or low-affinity receptors should be their capacity to bind monomeric IgGs; on the basis of this criterion, we consider FcγRIV to be a high-affinity receptor 14,15. It was initially thought that the high-affinity FcγRs were unavailable for immediate immunoglobulin-dependent responses in vivo because they were occupied or saturated by endogenous immunoglobulins; however, this viewpoint is no longer supported14–17. Adding to the complexity of FcγR nomenclature and biology, polymorphisms have been described in FcγRs of mice and

humans; for example, polymorphisms in FCGR2A (the gene encoding FcγRIIA) and FCGR3A (the gene encoding FcγRIIIA) modulate the affinity of the receptors they encode for some human IgG subclasses12, and some of these polymorphisms have been linked to disease18. The binding characteristics of IgG subclasses to particular FcγRs can be modified by altering critical amino acid residues, or their glycosylation status, in the amino acid backbone of the Fc fragment of the antibody, in or near the site of interaction with the FcγR. In particular, the nature and the presence of N‑linked glycan structures at residue Asn297 in IgG can modulate or even abrogate FcγR binding, which thus affects the immune response that is induced. These modifications are now being exploited to alter the effector functions of therapeutic antibodies that are used in cancer treatment and autoimmune disease (reviewed in REF. 19).

Expression of FcγRs by DCs and macrophages There is a consensus that different subsets of DCs carry out different functions20 (BOX 2). There are two main developmentally distinct subsets of conventional DCs (cDCs): CD172α (also known as SIRPα)+ cDCs are functionally specialized to present exogenous antigens to CD4+ T cells and to help humoral immunity 21–23, whereas XC-chemokine receptor 1 (XCR1)+ cDCs are specialized for the cross-presentation of exogenous antigens to CD8+ T cells. Plasmacytoid DCs (pDCs) provide an important and early source of type I interferon (IFN) during viral infections. Monocytes are separated in classical monocytes (LY6Chi in mice and CD14hi in humans) and patrolling monocytes (CX3C-chemokine receptor 1 (CX3CR1)hiLY6Clow in mice and CD14lowCD16hi in humans). In tissues, classical monocytes can give rise to monocyte-derived DCs (moDCs), the function of which is to control local effector CD4+ and CD8+ T cell responses. Macrophages have been separated into tissue-resident macrophages (such as microglial cells in the brain, Kupffer cells in the liver and alveolar macrophages in the lungs) and recruited macrophages. Recruited macro phages and moDCs are absent from most tissues in the steady state but rapidly accumulate from newly recruited monocytes following the induction of inflammation. As in vitro-generated moDCs have long been considered to represent in vivo DCs, and as their maturation could be enhanced through the stimulation of activating FcγRs and suppressed through the inhibitory FcγR, it was thought that all FcγRs were broadly expressed by all DC subsets2,11,24. However, by compiling publicly available gene expression data (from the Immunological Genome Consortium 25,26 and from published research articles) from freshly isolated DC and macrophage subsets, it is evident that the expression of FcγRs is highly selective (FIG. 1; see Supplementary information S1 (figure)). Activating FcγR mRNAs (Fcgr1, Fcgr3 and Fcgr4 in mice and FCGR1, FCGR2A, FCGR2C and FCGR3A in humans) are predominantly found in monocytes, macrophages and moDCs. Inhibitory Fcgr2b mRNA is broadly expressed by mouse cDCs and pDCs, as well as by moDCs and macrophages. Human cDCs and pDCs

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VOLUME 14 | FEBRUARY 2014 | 95 © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS Table 1 | Human Fc receptors for IgG Structure

Name

CD

Gene

Alleles* IgG1

IgG2

IgG3

IgG4

Major function

FcγRI

CD64

FCGR1A

–

6x10

No binding

6x10

3x10

Activation

FcγRIIA

CD32A

FCGR2A

His131 Arg131

5x106 3x106

4x105 1x105

9x105 9x105

2x105 2x105

Activation

FcγRIIB

CD32B

FCGR2B

Ile232 Thr232

1x105 1x105

2x104 2x104

2x105 2x105

2x105 2x105

Inhibition

FcγRIIC

CD32C

FCGR2C

Gln13 Stop13

1x105

2x104

2x105

2x105

Activation

FcγRIIIA

CD16A

FCGR3A

Val158 Phe158

2x105 1x105

7x104 3x104

10x106¶ 2x105 8x106¶ 2x105

Activation

FcγRIIIB‡ CD16B

FCGR3B

NA1, NA2 or SH

2x105

No binding

1x106

No binding

Decoy; activation?

FcRn§

None assigned

FCGRT

ND||

8x107¶

5x107¶

3x107¶

2x107¶

IgG recycling and transport

TRIM21§

None assigned

TRIM21

ND

5x106¶

5x106¶

2x106¶

5x106¶

Activation and proteasome targeting

ITAM

γ2

7¶

7¶

7¶

α

α Immune complexes Complexes of antigens that are bound to antibodies and, sometimes, components of the complement system. The concentration of immune complexes is increased in many autoimmune disorders, in which the immune complexes become deposited in tissues and cause tissue damage.

ITIM

α

α

Mononuclear phagocyte system (MPS). Bone marrow-derived cells with different morphologies (that is, monocytes, macrophages and dendritic cells) that are mainly responsible for phagocytosis, cytokine secretion and antigen presentation.

γ2

α

GPI anchor

Neonatal FcR (FcRn). Unrelated to classical Fc receptors (FcRs) and binds to a different region in the antibody Fc fragment. It is structurally related to the family of MHC class I molecules and is responsible for regulating IgG half-life.

Cross-presentation The initiation of a CD8+ T cell response to an antigen that is not present within antigenpresenting cells (APCs). This exogenous antigen must be taken up by APCs and then re‑routed to the MHC class I pathway of antigen presentation.

Monocyte-derived DCs (moDCs). In vitro-generated monocyte-derived DCs are the most studied DC subset and can be obtained in large quantities by culturing mouse bone marrow cells in granulocyte–macrophage colony-stimulating factor (GM‑CSF), or by culturing human peripheral blood monocytes in GM‑CSF and interleukin‑4 (IL‑4).

β2m α

β2m, β2-microglobulin; FcγR, Fc receptor for IgG; FcRn, neonatal FcR; GPI, glycosyl phosphotidylinositol; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; TRIM21, tripartite motif-containing protein 21. *Gene polymorphisms identified either by the position in the protein and the amino acid substitutions (for example, His131 or Arg131), or by the name of the allele (NA1, NA2 or SH). ‡Associates with integrins140. §Intracellular receptor50,52. ||No alleles have been described to date that affect binding affinity or that are linked with disease. ¶Affinity value corresponding to a high-affinity interaction. The binding affinity values of the FcγRs for the various immunoglobulin subclasses are depicted in M-1 unit.

also express FCGR2B mRNA, as well as that for the activating FcγR FCGR2A. Both mouse and human + CD172α cDCs |express low levels of FcγRI, as deterNature Reviews Immunology mined by flow cytometry 21,27–29. These data suggest that macrophages and moDCs express mRNA for most activating and inhibitory FcγRs, whereas cDCs and pDCs mainly express mRNA for the inhibitory FcγRIIB. Although mRNA expression does not always predict whether a protein is expressed or not, these mRNA expression data are supported by recent human and mouse flow cytometry data28,30–34. Of note, these data were compiled from representative DC and tissue-resident macrophage subsets in a limited number of tissues under steady-state and disease conditions, and it remains to be determined whether they are applicable to all situations.

However, Kupffer cells of the liver and osteoclasts also express all activating FcγRs (REF. 35; M.G., unpublished observations). Furthermore, it remains to be shown whether particular cytokines or inflammatory mediators can increase the expression of activating FcγR on cDCs. There are some important similarities with respect to FcγR expression between mice and humans (FIG. 1); however, there are also some subtle but important differences in both species. Although mouse moDCs and macrophages constitutively express high levels of Fcgr1 mRNA in the steady state23,27,36,37, human cultured moDCs and macrophages express very low levels of FCGR1 (REFS 28,38,39). This could be due to the use of interleukin‑4 (IL‑4) in the human cultures, which is known to downregulate FcγRI expression39–42. FcγRI



REVIEWS Table 2 | Mouse Fc receptors for IgG* Structure

Name

Gene

IgG1

IgG2a

IgG2b

IgG3

Major function

FcγRI

Fcgr1

NB

1x10

1x10

+

Activation

FcγRIIB

Fcgr2b

3x106

4x105

2x106

No binding

Inhibition

FcγRIII

Fcgr3

3x105

7x105

6x105

No binding

Activation

FcγRIV

Fcgr4

NB

3x107¶

2x107¶

No binding

Activation

FcRn§

Fcgrt

8x106

+

+

+

IgG recycling and transport

TRIM21§

Trim21

2x106

+

+

+

Activation and proteasome targeting

ITAM

γ2

5

‡

α

ITIM

8¶

α

α

γ2

α

β2m α

+, binds receptor but the binding affinity is unknown; β2m, β2-microglobulin; FcγR, Fc receptor for IgG; FcRn, neonatal FcR; Nature Reviews | Immunology ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; TRIM21, tripartite motif-containing protein 21. *The binding affinity values of the FcγRs for the various immunoglobulin subclasses are depicted in M-1 unit. ‡Under debate151. §Intracellular receptor50,52. ¶Affinity value corresponding to a high-affinity interaction.

is highly expressed on human macrophages and moDCs that have been directly isolated from inflamed tissues (such as from tumour ascites from patients with cancer 43 or from the inflamed colon of patients with inflammatory bowel disease27). In addition, mouse pDCs do not express activating FcγRs on their cell surface as determined by flow cytometry 34, whereas human pDCs express the activating FcγRIIA33,44, albeit at low levels compared with monocytes and macrophages (for protein expression of FcγRIIA see REFS 33,44; for mRNA expression see FIG. 1). FcγRIV is only present in mice, and FcγRIIA, FcγRIIC and FcγRIIIB are only present in humans. However, mouse FcγRIV has been suggested to be the homologue of human FcγRIIIA40, and mouse FcγRIII has been suggested to be the homologue of human FcγRIIA (REF. 12; J. Lejeune and H. Watier, personal communication). Furthermore, as mouse FcγRIV can also bind IgE, it was thought to be functionally equivalent to the human IgE receptor FcεRI when it is expressed on monocytes and macrophages15. The difference in expression of activating FcγRs between cDCs and moDCs is so striking that three research groups have independently hypothesized that expression of FcγRI, along with FcεRI, can be used as an effective discriminative marker to separate moDCs and macrophages from cDCs in mice and humans (BOX 2).

Throughout this Review, it is important to make a conceptual distinction between phagocytes found in the steady state and those found in conditions of inflammation. The cDCs that populate the peripheral tissues in homeostasis mainly express the inhibitory FcγR and express low levels of activating FcγRs. Many pathogen encounters and tissue ‘insults’ lead to neutrophil and monocyte recruitment into tissues. Monocytes can rapidly differentiate into macrophages and moDCs in situ, and these cells express almost all types of FcγRs. moDCs do not migrate well, therefore it is difficult to envisage how they could function as antigen-presenting cells (APCs) for the naive T cells that recirculate through the lymph nodes. However, immunoglobulins only come into play a few days into the primary immune response or during a memory response, when primed T cells are poised to migrate to peripheral tissues. Thus, we hypothesize that the main function of activating FcγRs is to modify the encounter of moDCs and T cells at sites of inflammation, and to promote the clearance of pathogens in the periphery, as well as from filtering areas in central lymphoid organs, by macrophages. However, we do not exclude the possibility that particular activation states may induce higher expression of activating FcγRs on cDCs and that this might induce cDCs to respond to immune complexes in such environments. This is an area of research that requires more attention.



REVIEWS Box 2 | DC and macrophage subsets XCR1+ conventional DCs • Ontogeny: conventional dendritic cells (cDCs) that are derived from pre-cDCs and that depend on the transcription factor basic leucine zipper transcriptional factor ATF-like 3 (BATF3) • Mouse surface markers: these cells express XC-chemokine receptor 1 (XCR1) and DC natural killer lectin group receptor 1 (DNGR1; also known as CLEC9A) in all tissues, and they differentially express CD8a, CD103 or CD207 depending on the tissue • Human surface markers: these cells express XCR1, DNGR1 and BDCA3 (also known as CD141) • Main function: the cross-presentation of antigen for the activation of effector CD8+ T cells CD172α+ conventional DCs • Ontogeny: cDCs that are derived from pre-cDCs and that depend on the transcription factor interferon-regulatory factor 4 (IRF4) • Mouse surface markers: these cells express CD172α (also known as SIRPα) in all tissues, and they express CD11b or CD4 depending on the tissue • Human surface markers: these cells express CD172α and BDCA1 (also known as CD1c) • Main functions: the induction of T helper 2 (TH2) or TH17 cells, and the promotion of humoral immune responses Plasmacytoid DCs • Ontogeny: derived from pre-plasmacytoid DCs and depend on the transcription factor E2.2 • Mouse surface markers: these cells express Siglec‑H, bone marrow stromal antigen 2 (BST2) and LY6C • Human surface markers: these cells express BDCA2 and BDCA4 • Main function: the production of type I interferon (IFN) during viral infections Monocyte-derived DCs • Ontogeny: derived from monocytes • Mouse surface markers: these cells express the high-affinity Fc receptor I for IgG (FcγRI) and the high-affinity Fc receptor for IgE (FcεRI); LY6C expression is lost with time • Human surface markers: these cells express FcεRI; FcγRI expression is upregulated on activation • Main functions: the promotion of local T cell responses, enhancement of inflammation and production of chemokines Macrophages • Ontogeny: mostly of primitive origin but can be derived from monocytes during inflammation • Mouse surface markers: these cells express F4/80, FcγRI and tyrosine protein kinase MER (MERTK) • Human surface markers: these cells express CD68; expression of FcγRI is upregulated on activation • Main functions: sentinel immune function, the elimination of pathogens and tissue homeostasis

Antigen internalization and degradation by FcγRs Internalization of opsonized material or immune complexes represents the only function shared by all FcγRs that are expressed at the cell surface, irrespective of whether they have an ITAM or an ITIM. However, the molecular mechanisms that underlie this internalization are different. The internalization of immune complexes via ITAM-bearing FcγRs relies on the tyrosines of the ITAM present in the FcγR complex 45, whereas the internalization of immune complexes via ITIM-bearing FcγRIIB relies on the presence of a di‑leucine motif in its intracellular domain46. Importantly, although both receptor types rapidly endocytose the receptor complex and its bound ligands47, it is thought that the type of FcγR that mediates the internalization influences the degradative pathway in which the antigens will subsequently be routed. The model suggests that internalization by activating FcγRs favours a degradative route for antigen processing and presentation that results in T cell activation, whereas internalization by FcγRIIB favours a retention pathway that preserves the intact antigen for subsequent transfer to B cells48. It was recently shown that IgG opsonization enhances antigen presentation to CD4+ T cells only when antigen and IgG are present within the same phagosome; indeed, cells that

contain phagosomes with either antigen or IgG alone failed to efficiently present antigens49. Therefore, a specific mechanism may be responsible for the efficient routing of internalized antigen when it is bound to an antibody and internalized by an FcγR. FcRn has been suggested to facilitate the transport of IgG-bound antigens through particular intracellular routes to favour antigen presentation and subsequent immune responses50,51. FcRn is expressed by macrophages and DCs in humans and mice and enables immune complex uptake and antigen processing by DCs3,52. FcRn is also required for efficient phagocytosis of IgG-opsonized bacteria by FcγRs53. Importantly, FcRn does not bind to IgG at the physiological pH (that is, 7.4) of the extra cellular milieu, and only binds when histidine residues in the Fc portion of IgG become protonated in the acidic environment of endocytic vacuoles (that is, pH≤6.5)3. Immune complexes bind to FcγRs on the surface of DCs or macrophages, they are internalized and they subsequently bind to FcRn, which controls the intracellular routing to antigen-processing endosomes 48,49 (FIG. 2) and/or recycling endosomes. It is also possible that the ubiquitously expressed intracellular receptor TRIM21 binds to IgG-opsonized (or IgM-opsonized) particles4



REVIEWS Human FcγR receptor expression Classical Patrolling monocyte monocyte Macrophage moDC Activating FCGR1

pDC

XCR1+ cDC

CD172α+ cDC

Activating FCGR2A Inhibitory FCGR2B Activating FCGR2C Activating FCGR3A

XCR1 + cDC

Spleen

Skin

Blood Skin

pDC

Blood

Blood

moDC

In vitro generated Inflammation (ascites)

4 6 8 10 12 14

In vitro generated Inflammation

Log2 expression

Alveolar In vitro generated Inflammation (ascites)

Blood

Blood

Decoy FCGR3B

Mouse FcγR receptor expression Classical Patrolling monocyte monocyte Macrophage

CD172α+ cDC

Activating Fcgr1 Inhibitory Fcgr2b Activating Fcgr3

Spleen Skin-draining lymph node Lung

4 6 8 10 12 14

Spleen Skin-draining lymph node

Log2 expression

Alveolar Spleen In vitro generated Inflammation

Blood

Blood

Activating Fcgr4

Figure 1 | Compilation of microarray data of human and mouse FcγR expression by DCs and macrophages. Expression values were extracted from published, publicly Nature Reviews | Immunology available microarray data sets and represent log2 expression levels that were obtained after quantile normalization of the data using the Robust Multi-array Average (RMA) procedure (see Supplementary information S1 (figure)). Expression values were subsequently colour-coded, varying from white (showing low expression), to orange (showing medium expression) and to red (showing high expression). Note that low mRNA expression levels do not necessarily correspond to no Fc receptor for IgG (FcγR) expression; for example, the low mRNA levels of that encoding Fcγ receptor IIB (Fcgr2b) in mouse plasmacytoid dendritic cells (pDCs) are sufficient for protein expression, as shown by flow cytometry34. When merging microarray data from different platforms, data were integrated at the gene level, keeping the probe sets that had the highest expression levels when multiple probe sets were available. A final quantile normalization was then carried out across all platforms and samples were aggregated and the median expression value for each cell type was calculated. All mouse microarray data were obtained from the publicly available Immunological Genome Consortium (REF. 145; NCBI gene expression omnibus (GEO) data repository GSE15907), except for the monocyte-derived DC (moDC) samples, which were obtained from REF. 146 (NCBI GEO data repository GSE2197) and REF. 147 (NCBI GEO data repository GSE42101). Human microarray data were obtained from REF. 43 (NCBI GEO data repository GSE40484) for monocytes, inflammatory macrophages and inflammatory DCs; from REF. 30 (NCBI GEO data repository GSE35459) for pDCs and conventional DCs (cDCs); from REF. 148 (NCBI GEO data repository GSE18816) for alveolar macrophages; from REF. 149 (NCBI GEO data repository GSE45466) for moDCs and from REF. 150 (NCBI GEO data repository GSE35433) for monocyte-derived macrophages that were generated in vitro. Both of the methods used for the array compilation, as well as the Fc receptor (FcR) expression data for additional groups cells, including B cell, T cells, natural killer cells and neutrophils are included in Supplementary information S1 (figure). XCR1, XC-chemokine receptor 1.

following internalization by FcRs that are expressed on the cell surface (FIG. 2). The recognition of intracellular antibodies by TRIM21 activates signalling pathways that lead to cell activation and production of pro-inflammatory molecules54, and routes antibody-bound viruses to the proteasome through its E3 ubiquitin ligase activity 5,55. It is so far unclear whether TRIM21‑dependent signalling pathways also affect the sorting of FcγR-internalized antigens (that is, not only of opsonized viruses) to particular endosomal compartment routes. The uptake through distinct FcγRs will influence not only whether an antigen is presented or not but also through which degradative pathway it is processed and the repertoire of epitopes that is presented. In mice, FcγRIIB expression was found to result in the presentation of a restricted set of T cell epitopes compared with FcγRIII expression. This difference relies on the ability of FcγRIII to trigger the SYK signalling pathway and promote FcR targeting to lysosomes56,57. In addition, the short intracytoplasmic domains of the human activating receptors FcγRI and FcγRIIIA contain serine or threonine phosphorylation motifs that have been reported to regulate internalization (and phagocytosis) efficiency 58,59. There are several isoforms of the inhibitory FcγRIIB in humans and mice that have different antigen internalization and presentation properties6. A systematic analysis of the degradative pathways and T cell repertoire generation following antigen internalization by each FcγR that is expressed by macrophages and DCs remains to be carried out.

Role of FcγRs in phagocyte activation In addition to facilitating the capture and the internalization of antibody-bound antigens or pathogens, most FcγRs induce ITAM- or ITIM-mediated intracellular signalling. This signalling strongly influences core functions of both macrophages and DCs, including their functional polarization, their capacity to kill pathogens and their regulation of T cell responses. Through concomitant expression of both activating FcγRs and the inhibiting FcγRIIB, the immune system can set strict thresholds for phagocyte activation. Modulation of macrophage polarization. Macrophages have been conceptually separated into classically activated macrophages (M1 macrophages, which are activated by IFNγ and are specialized for pathogen killing) and alternatively activated macrophages (M2 macrophages, which are activated by IL‑4 and/or IL‑13 and are specialized for tissue remodelling). Although crosslinking of activating FcγRs on monocytes and macrophages induces the production of several pro-inflammatory cytokines and chemokines60,61, immune complex‑mediated signalling via activating FcγRs together with Toll-like receptor (TLR) triggering induces a specific M2 activation state in macrophages — macrophages in this state were termed ‘M2b’ or ‘regulatory’ macrophages. These cells produce low levels of IL‑12 and high levels of IL‑10, tumour necrosis factor (TNF), IL‑1 and IL‑6 (REFS 62–64). Importantly, such combined signalling of FcγR and TLR triggering leads to lower IL‑12 production than TLR triggering alone in mouse macrophages62.



REVIEWS

Virus Immune complex

IgG

FcγR

Antigen Common γ-chain ITAM SYK

Internalization and sorting

Increased endosomal maturation

FcRn β2m

Processed antigen

Late acidic endosome

moDCs

?

Protection from degradation

TRIM21

Proteasome

Early endosome Peptides MHC class II loading in the MIIC

MHC class I loading in the ER

Signal 1: Presentation to CD4+ T cells

Signal 1: Cross-presentation to CD8+ T cells

Figure 2 | Efficient processing of antibody-coating antigens by moDCs. Triggering of Fc receptors for IgG (FcγRs) on monocyte-derived DCs (moDCs) induces a more efficient immunoreceptor tyrosine-based activation Nature Reviewsmotif | Immunology (ITAM)-dependent uptake of the antigen. Moreover, signalling through the activating FcγRs via spleen tyrosine kinase (SYK) activates moDCs and facilitates endosomal maturation, increased lysosomal fusion and efficiently facilitates the delivery of processed antigens to the MHC class II compartment (MIIC) for enhanced MHC class II presentation to CD4+ T cells. In addition, antigens coupled to antibodies are more efficiently cross-presented than unbound antigens. This is thought to be the result of two independent mechanisms: first, neonatal FcRn (FcRn)-mediated protection from degradation and efficient delivery of the antigen to the cytosol; and second, tripartite motif-containing protein 21 (TRIM21)-mediated increased delivery to the proteasome. Note that TRIM21‑mediated delivery to the proteasome has been shown to occur for opsonized particles (including viruses), but not directly for antigen-containing immune complexes (question mark). TRIM21 also functions in the absence of activating FcγRs54 (dashed arrows). However, uptake of antibody-coated viruses via FcγRs may help target them to TRIM21. In addition, all of these experiments were carried out in moDCs and it is currently unknown whether these observations also apply to conventional DCs. β2m, β2-microglobulin; ER, endoplasmic reticulum.

The activation of macrophages by immune complexes is determined by the balance between the triggering of activating ITAM-bearing FcγRs and the triggering of inhibitory ITIM-bearing FcγRIIB. The antigen size, concentration and IgG valence in the immune complex could be additional factors that influence macrophage activation. Macrophages from Fcgr2b–/– mice have a lower activation threshold than macrophages from wildtype mice and these deficient mice are much more sensitive to immune complex‑induced alveolitis65, arthritis66

and sepsis67. However, Fcgr2b–/– mice are more resistant to pneumococcal peritonitis because of the increased ability of their macrophages to clear the bacteria67, and transgenic overexpression of FcγRIIB on macrophages increased mortality after Streptococcus pneumoniae infection68. Taken together, this shows that the increased macrophage activation that is found in the absence of FcγRIIB can be beneficial for the host as it increases the ability of macrophages to clear bacteria, but it can also be detrimental when it increases immunopathology.



REVIEWS Antibody-mediated regulation of macrophage infection. Many pathogens have developed escape mechanisms to inhibit phagolysosomal fusion, and thus degradation, in order to survive in the hostile intracellular environment of macrophages. Legionella pneumophila and Toxoplasma gondii can evade phagolysosomal fusion and can reside within vacuoles that are permissive for replication69,70. However, as mentioned above, the presence of specific antibodies on the surface of the pathogens redirects these pathogens to lysosomes, which inhibits intracellular replication and facilitates efficient elimination by macrophages71. This process requires the expression of activating FcγRs, which target the bacteria to the lysosomes following uptake. Similarly, the absence of FcγRIIB on Mycobacterium tuberculosis-infected macrophages induces increased IL‑12 production and increased resistance to infection, whereas the absence of the FcR common γ‑chain is associated with increased susceptibility to infection72. The delivery of pathogens to macrophages and the concomitant activation of these cells could represent one of the major mechanisms that underlies the protective function of antibodies against intracellular pathogens73. However, delivering pathogens to macrophages is not always favourable for the host. The long-lived nature of macrophages74–77 may be an explanation for why these cells represent an attractive niche for pathogens that induced chronic infections. Increased uptake of such opsonized macrophage-tropic microorganisms through FcγRs may therefore result in antibody-enhanced infection. This can occur via increased uptake of the pathogen or by subversion of macrophage activation78; for example, antibodies against dengue virus facilitate its uptake by macrophages79. When the level of maternal dengue virus-specific antibodies in young infants is below the protective level for neutralization but still high enough to mediate antibody-enhanced infection, these antibodies increase the infectivity and the severity of the illness80. Therefore, it has been suggested that antibody-enhanced infection is the main mechanism to explain why, during a dengue virus epidemic in Cuba in 1981, children that had been previously infected presented more severe forms of the infection than children that were too young to have been infected during the epidemic of 1977 (REF. 81). Another mechanism of antibody-enhanced infection involves subversion of macrophage activation. Leishmania major is a macrophage-tropic pathogen that has developed escape mechanisms to ensure its intracellular survival82. A polarized T helper 1 (TH1)‑type immune response has been associated with enhanced parasite clearance through the induction of M1 macrophages, whereas a TH2‑type response has been associated with host susceptibility through the induction of M2 macrophages. Engagement of ITAM-bearing FcRs on macrophages activates the mitogen-activated protein kinase (MAPK) pathway through SYK and induces the downregulation of IL‑12 and the upregulation of IL‑10 production by M2b macrophages. Leishmania amazonensis parasites that are coated with immunoglobulins induce IL‑10 production by macrophages from wildtype mice but not by those from mice that are deficient

for all activating IgE and IgG receptors83. Furthermore, these activating IgE and IgG receptor-deficient mice were more resistant to Leishmania spp. infection84–86. Taken together, these observations show that the expression of FcRs on macrophages influences both the uptake of pathogens by these cells and the concomitant activation of the cells (FIG. 3), which is ultimately an important factor that influences the outcome of infectious diseases.

Role of FcγRs in DC activation Modulation of antigen presentation by FcγRs. Several studies have shown that antibody-bound soluble antigens, particulate antigens or apoptotic tumour cells enable DCs to activate antigen-specific T cells more efficiently than free antigens45,87–92, which implies that FcγRs have a crucial role in augmenting antigen presentation (FIG. 2). In mice, experiments have been carried out in vitro on granulocyte–macrophage colony-stimulating factor (GM‑CSF)-cultured moDCs or in vivo by injecting immune complexes composed of model antigens such as ovalbumin (OVA) complexed with OVA-specific IgG (often IgG raised in rabbits). Although both CD4+ and CD8+ T cell responses can be increased by immune complexes, there seems to be a bias for CD8+ T cell responses, as immune complexes mainly enter cross-presentation pathways87,93–95. Studies using mice in which DCs can be conditionally depleted (Cd11c–DTR (diphtheria toxin receptor) mice) have revealed that antigen presentation in response to immune complex injection depends on a CD11chi cell, which is probably a DC96. Although both inhibitory FcγRIIB and activating FcγRs can mediate the uptake of antigens from immune complexes (see above), it seems that it is mainly activating FcγRs that promote antigen presentation, which is due to their ability to activate DCs and to stimulate the MHC class I cross-presentation machinery 93,97. The precise activating FcγR that is involved in mediating the immunopotentiating effects of immune complexes, as well as the precise subtype of DC that controls the immune response following the in vivo injection of immune complexes, is unknown. However, given the low expression levels of FcγRs on cDCs in the steady state, it is questionable whether these cells are the ones that mediate this effect in vivo98. The presence of immune complexes does not increase the capacity of XCR1+ cDCs to cross-present antigens89 and the probable explanation for this is that XCR1+ cDCs already express receptors that favour cross-presentation, so the presence of a specific antibody does not enhance their already high cross-presentation capacity 89. Early studies suggested that splenic CD172α+ cDCs (identified originally as CD8α− DCs) cross-present immune complex‑associated antigens more efficiently than soluble antigens. It is worth noting that in these early studies there was no clear distinction between CD172α+ cDCs and moDCs (both of which are CD8α−CD11b+CD172α+). However, if these cells were indeed CD172α+ cDCs, then this increased cross-presentation would have to occur through FcγRIIB, as this is the only FcγR that is highly expressed by these cells in mice (FIG. 1). In fact, the increased cross-presentation was shown to depend on



REVIEWS a FcγR function in macrophages Opsonized infected cell • Increased uptake • Lysis of target cell • ADCC

FcγR

Opsonized pathogen • Increased uptake • Phagolysosomal fusion • Pathogen killing

+ TLR ligand

IVIG therapy • Induces increased inhibitory FcγRIIB expression • Induces decreased activating FcγR expression • Increases the activation threshold

b FcγR function in pDCs

Immune complexes • Increased IL-10 production • Decreased IL-12 production • High IL-1, IL-6 and TNF production • M2b (regulatory) activation state

Antibody-enhanced infection • Macrophage-tropic pathogens • Increased uptake • Increased IL-10 production • Pathogen persistence

Particulate antigens • Poor antigen uptake • Poor antigen processing • No antigen presentation

Autoantibody

Self DNA

Chromatin

Immune complexes • Increased antigen uptake • Antigen routing to MHC class II-processing organelles • Antigen presentation to CD4+ T cells • Immune tolerance?

Antimicrobial peptide FcγRIIA

FcγRIIB

HMGB1

Self DNA-containing immune complexes • Phagosomal maturation • Generation of ISC • TLR9 relocalization to the ISC • High type I IFN production • Autoimmunity (SLE)

Figure 3 | FcγR-mediated macrophage and pDC activation. a | Pathogens coated with antibodies (opsonized pathogens) Nature activation Reviews | Immunology are often more efficiently killed by macrophages because of the Fc receptor for IgG (FcγR)-mediated of macrophages, which induces an immunoreceptor tyrosine-based activation motif (ITAM)-dependent increased uptake and increases phagolysosomal fusion, thereby yielding more efficient killing of pathogens. Similarly, opsonized infected cells can be killed through a mechanism called antibody-dependent cell-mediated cytotoxicity (ADCC). However, immune complexes induce a particular macrophage activation status termed the M2b (regulatory) macrophage activation state, which is characterized by increased interleukin‑10 (IL‑10) production and decreased IL‑12 production, but high IL‑1, IL‑6 and tumour necrosis factor (TNF) production. This M2b activation state can facilitate the survival of macrophage-tropic pathogens, such as Leishmania spp., that have developed strategies to subvert macrophage function and to use the macrophage as a preferential cellular niche. Increased uptake of these macrophage-tropic pathogens results in antibody-enhanced infection. Finally, the manipulation of macrophage activation by immune complexes has been suggested to be one of the main mechanisms that underlies intravenous immunoglobulin therapy (IVIG therapy). A high dose of immune complexes is thought to induce higher expression of inhibitory FcγRIIB and lower expression of the activating FcγRs, which yields an increased activation threshold for macrophages. b | Plasmacytoid dendritic cells (pDCs) have poor capacities to capture and present particulate antigens to CD4+ T cells compared with conventional DCs (cDCs) (dashed arrow). Antibody-coated antigens are more efficiently taken up by pDCs and subsequently more efficiently routed to MHC class II‑processing organelles compared with cDCs, which results in better antigen presentation to CD4+ T cells. As pDCs have been shown to be tolerogenic in the steady state, we hypothesize that, in the absence of danger signals, FcγR-mediated uptake and presentation of immune-complexed antigens by pDCs induces the development of immune tolerance. pDCs have also been implicated in the pathogenesis of systemic lupus erythematosus (SLE). In patients with SLE, self DNA-containing immune complexes that are associated with antimicrobial peptides, high-mobility group box 1 protein (HMGB1) and autoantibodies are recognized by FcγRs on pDCs. This triggers phagosomal maturation and the generation of the interferon (IFN) signalling compartment (ISC). Triggering of FcγRs by self DNA-containing immune complexes has been shown to be crucial in the relocalization of Toll-like receptor 9 (TLR9) to the ISC, which then results in high levels of type I IFN production by the pDCs and exacerbates the autoimmune response in patients with SLE.



REVIEWS the expression of the FcR common γ-chain89, which implicates the involvement of an activating FcγR rather than FcγRIIB, and thus the involvement of moDCs rather than cDCs, in this process. Studies in vitro using GM‑CSF-cultured mouse moDCs have shown that both FcγRI and FcγRIII contribute to the enhanced antigen presentation of immune complexes, but the precise role of FcγRIV remains to be determined99. Injection of immune complexes in mice might cause a mild form of inflammation (due to complement activation or to contaminating endotoxin), which would lead to the recruitment and the activation of moDCs. Although direct proof of this scenario is lacking so far, we hypothesize that the injection of immune complexes increases cross-presentation of the complexed antigens, mainly through the recruitment and the activation of moDCs via ITAM-bearing FcγRs, rather than through cDCs. In humans, the presence of antigens in immune complexes also favours cross-presentation by moDCs, and the FcγR that is involved was shown to be activating FcγRIIA, although cross-presentation may be counteracted by the inhibitory FcγRIIB28,100,101. FcγRI was not suggested to be involved. However, as IL‑4 is used to generate human moDCs in vitro, and as IL‑4 induces the rapid downregulation of FcγRI by moDCs40–42, the use of in vitro-generated moDCs may underestimate the importance of FcγRI as an internalization and activating receptor for human moDCs. In human monocytes, FcγRI targets antigens to the MHC class II‑rich late endosomes and leads to enhanced antigen processing and presentation to CD4+ T cells102. Considering the studies of antigen uptake and processing as a whole, we conclude that activating FcγRs on DCs promote antigen presentation to CD4+ and CD8+ T cells. The inhibitory FcγR, possibly in combination with FcRn, on cDCs and pDCs can also promote antigen presentation on MHC class II molecules and preserves some intact antigens for presentation to B cells. The regulated expression of FcγRs by different APC subsets further amplifies the specialized function of DCs to process antigens and of macrophages to degrade antigens.

Group 2 innate lymphoid cells These cells predominantly produce type 2 cytokines and require the transcription factors retinoic acid receptor-related orphan receptor‑α (RORα) and GATA-binding protein 3 (GATA3) for their development and function.

Polarization of adaptive immune responses. T cell polarization is a crucial aspect of immune regulation and is controlled by APCs providing instructive signals to naive T cells in the draining lymph nodes and the spleen. Whether a particular APC instructs naive TH cell differentiation depends on the migratory capacity of the APC and its potential to produce co‑stimulatory molecules and instructive cytokines that influence the T cell differentiation programme. Most experiments that investigate the influence of FcγR triggering on TH cell polarization have been carried out in vitro using mouse or human GM‑CSF-generated moDCs, or in vivo after the artificial introduction of immune complexes in naive mice. There are a few conceptual problems when discussing how FcγR triggering on APCs influences naive TH cell polarization in normal physiology, as high-affinity antibodies and immune complexes only form when adaptive immunity has already been induced. However, natural antibodies are present in unimmunized mice and have a

broader and lower affinity specificity that might trigger FcγRs during a naive T cell response. Moreover, we and others have recently shown that the main DCs that are responsible for the initial induction of T cell responses are migratory cDCs21,23,103, but these cells express very low levels of activating FcγRs. MoDCs express the highest levels of activating FcγRs, but are much more sessile cells that primarily reside within the inflamed tissues23,36. Therefore, FcγR-mediated triggering of DCs would mainly affect the interactions between primed T cells and moDCs in peripheral tissues to maintain TH cell polarization that is initiated by cDCs104,105. Signalling through ITAM-containing activating FcγRs can upregulate the expression of co‑stimulatory receptors (the so‑called signal 2) and the production of TH1‑polarizing cytokines (the so‑called signal 3) (FIG. 4). Indeed, when immune complexes were injected in vivo to promote tumour immunity, or when responses to opsonized Leishmania spp. were studied in naive mice, there was an increase in the number of IFNγ-producing CD4+ TH1 cells, accompanied by an increased production of IL‑12 by DCs95,106,107. The triggering of activating FcγRs on human moDCs can also promote DC activation and can lead to increased antigen uptake, processing and presentation, and to TH1 cell polarization61. This response probably involves the induction of a type I IFN response (FIG. 4), as small interfering RNA (siRNA)-mediated inhibition of the gene encoding signal transducer and activator of transcription 1 (STAT1), which is downstream of the type I IFN receptor, inhibited the upregulation of the co-stimulatory receptors CD80 and CD86, which are markers of DC activation61. However, other groups have found that targeting antigens to activating FcγRs promotes the development of TH2‑type immune responses97,108. In mouse models of asthma, which are driven by type 2 cytokines, triggering of FcγRI or FcγRIII on DCs has been shown to induce the production of IL‑10 and to skew T cell immunity towards the TH2 cell phenotype108,109. In addition, when primed OVA-specific TH2 cells were transferred to mice, OVA-containing immune complexes activated T cells much better than antigen alone110. The triggering of FcγRIII and TLR4 on lung DCs induced the production of IL‑33. IL‑33 signals through its receptor (which consists of ST2 and IL‑1 receptor accessory protein) that is expressed by TH2 cells, group 2 innate lymphoid cells, basophils, natural killer T cells and DCs to promote a type 2 immune response111 — in mice, this leads to the generation of IgG1 and IgE antibodies. FcγRIII can be triggered not only by IgG1‑containing immune complexes but also by IgE. Furthermore, the crosslinking of IgE on moDCs was shown to suppress IL‑12 production and to increase IL‑10 production in an FcγRIII-dependent manner 112. However, how FcγR triggering on DCs affects TH cell polarization still needs further study. Role of the inhibitory FcγR on DCs. Our review of published studies showed that inhibitory FcγRIIB is expressed by all macrophages and DC subsets (FIG. 1) and is the predominant FcγR that is expressed by cDCs and pDCs. As in many cell types, triggering of FcγRIIB



REVIEWS on DCs has the potential to suppress the effects that are mediated by activating FcγRs. Mice that lack FcγRIIB generally mount an exaggerated T cell response following the injection of immune complexes, and gene expression profiling of moDCs showed exaggerated DC activation when this receptor was absent 96,113. Furthermore, mice that selectively lack FcγRIIB on DCs showed enhanced T cell responses to injection of immune complexes in vivo93. Targeting of antigens to FcγRIIB might also be necessary for maintaining tolerance to self antigens that are derived from apoptotic cells114, but it remains to be shown whether mice that specifically lack FcγRIIB on DCs develop signs of autoimmunity. The induction of mucosal tolerance leads to the generation of IgG-containing immune complexes in the nasal draining lymph nodes, which was shown to be suppressed in FcγRIIB-deficient mice because of a failure to induce the development of CD4+CD25hi regulatory T (TReg) cells115. The maturation of human moDCs is accompanied by the downregulation of FcγRIIB expression, which hence lowers their immunoglobulin-mediated activation threshold38,101. When this receptor was blocked, moDCs were shown to produce more IL‑12p70 and to induce more T cell proliferation in response to immune complex‑mediated stimulation61. Triggering of FcγRIIB also subverted the normal activation of DCs by the TLR4 agonist lipopolysaccharide116. In addition, triggering of FcγRIIB by immune complexes might affect the differentiation of moDCs. When moDCs develop from monocytes in vitro in the presence of immune complexes, their differentiation is hampered and they no longer produce IL‑12 in response to TLR4 agonists117. Furthermore, the important role that FcγRIIB has in regulating DC responsiveness to immune complexes is supported by the fact that its expression relative to that of activating FcγRs is tightly regulated. Type 2 cytokines (including IL‑4, IL‑10 and transforming growth factor-β (TGFβ)) increase FcγRIIB expression by moDCs101,118,119, whereas type 1 cytokines (including IFNγ and TNF) decrease FcγRIIB expression by moDCs120,121. Conversely, IFNγ also increases human FcγRI expression and mouse FcγRIV expression by monocytes, whereas TGFβ and IL‑4 decrease the expression of these FcγRs40,41. Taken together, these observations show that the cytokine milieu can influence the expression of both activating FcγRs and FcγRIIB, and hence can modulate the threshold for moDC maturation. Role of FcγRs on pDCs. pDCs are an important source of early type I IFN and have the capacity to crosspresent exogenous antigens to CD8+ T cells as efficiently as XCR1+ cDCs, despite having a lower uptake of antigens122. However, in humans and mice, pDCs do not present exogenous antigens well to CD4+ T cells. pDCs that have been isolated from patients undergoing clinical DC therapy for melanoma were cultured in vitro and the antigen keyhole limpet haemocyanin (KLH), to which there was no prior exposure, was added to the cultures for the purpose of immunomonitoring the induction of T cell immunity. These pDCs could only present KLH antigen to KLH-specific CD4+ T cells when serum containing

Immune complex FcγR

IgG

Antigen

IFNAR

Type I IFN

ITAM SYK

BTK

LAT

PLCγ

IRF3– IRF7

PI3K

JAK

? AKT PKC MAPK

STAT1

NF-κB

Signal 3: Polarizing cytokines

Signal 2: Co-stimulatory receptors

Figure 4 | Role of FcγRs in moDC maturation. Triggering Nature Reviews | Immunology of Fc receptors for IgG (FcγRs) on monocyte-derived dendritic cells (moDCs) induces immunoreceptor tyrosine-based activation motif (ITAM)-dependent DC maturation and increases T cell responses. On the one hand, ITAM-mediated signalling via spleen tyrosine kinase (SYK) and other signalling intermediary molecules, as depicted, induces the expression of co‑stimulatory molecules, which yields a better signal 2; on the other hand, ITAM-mediated signalling induces the production of polarizing cytokines, which induces an optimal signal 3. Note that it is currently not clear whether FcγR-mediated signalling drives a particular type of T cell response (that is, T helper 1 (TH1), TH2, TH17, T follicular helper (TFH) or regulatory T (TReg) cell response). In addition, all of these experiments were carried out on moDCs and it is currently unknown whether these observations also apply to conventional DCs. Question mark indicates this pathway has been proposed but not formally demonstrated. Dashed line indicates there are additional steps in this pathway. BTK, Bruton’s tyrosine kinase; IFN, interferon; IFNAR, type I IFN receptor; IRF, interferon-regulatory factor; JAK, Janus kinase; LAT, linker for activation of T cell; MAPK, mitogen-activated protein kinase; NK-κB, nuclear factor-κB; PI3K, phosphoinositide 3‑kinase; PKC, protein kinase C; PLCγ, phospholipase Cγ; STAT1, signal transducer and activator of transcription 1.

antigen-specific antibodies was added to the culture. The serum facilitated KLH antigen uptake in endosomes in a process that required FcγRIIA123. KLH uptake was inhibited by TLR9 ligands that accumulate in late endosomes, but not by TLR9 ligands that target early endosomes, suggesting that the immunoglobulin-mediated processing of KLH occurred in late acidic endosomes, which are sites of MHC class II loading 124. Furthermore, transgenic



REVIEWS

Intravenous immunoglobulin therapy (IVIG therapy). Injection of high doses of polyclonal antibodies into patients.

overexpression of the activating human FcγRIIA boosted the uptake of immune complexes by mouse pDCs34. In addition, mouse pDCs only present soluble OVA to CD4+ T cells in the presence of OVA-containing immune complexes. Mouse pDCs mainly express FcγRIIB, and blocking antibodies against this receptor blocked antigen routing to MHC class II‑processing organelles and the subsequent induction of CD4+ T cell proliferation107,125. Why exactly mouse pDCs use inhibitory FcγRIIB whereas human pDCs use activating FcγRIIA remains to be investigated. Both mouse and human pDCs are known to promote the generation of TReg cells that mediate peripheral tolerance and that suppress tumour immunity; however, it remains to be investigated whether targeting immune complexes to pDCs in the steady state promotes the induction of tolerance. Although pDCs control tolerance in the steady state, their activation during immune complex uptake might break tolerance and contribute to autoimmunity. pDCs have been implicated in the pathogenesis of systemic lupus erythematosus (SLE), which is a multisystem autoimmune disorder characterized by autoantibodies that are specific for nuclear components, including chromatin and double-stranded DNA (dsDNA)126. The recognition of bacterial DNA occurs via endosomal TLR9 and in normal conditions self DNA is not recognized by this receptor. However, in patients with SLE, immune complexes that consist of autoantibodies, self DNA, high-mobility group box 1 protein (HMGB1) and neutrophil-derived peptides trigger the production of type I IFN by pDCs in a process that requires FcγRIIA and TLR9 (REFS 44,127). The triggering of TLR9 occurs in a late endosomal compartment termed the IFN signalling compartment (ISC), which contains the signalling adaptor TNF receptor-associated factor 3 (TRAF3), which thus leads to the induction of IFN-regulatory factor 7 (IRF7) and a type I IFN response. For TLR9 to traffic to the ISC, it first needs to traffic from the endoplasmic reticulum to the phagosome — a process that requires UNC93 homolog B (UNC93B). DNA-containing immune complexes stimulate the localization of TLR9 and UNC93B to phagosomes in a process that requires FcγRs. Strikingly, triggering of FcγRs by DNA-containing immune complexes also induces the recruitment of the autophagy protein LC3 and autophagy-related protein 7 (ATG7) to the phagosome, phagosomal maturation and the trafficking of TLR9 to the ISC compartment 128. Therefore, IFNα secretion by pDCs in response to DNA-containing immune complexes depends on a convergence of phagocytic and non-canonical autophagic pathways. Pathogens can also activate pDCs and this response could be influenced by FcγR triggering. The IFNα response to Staphyloccocus aureus in human pDCs has been shown to occur only in the presence of specific antibodies that trigger FcγRIIA, which facilitates the activation of TLR9 by bacterial DNA and thus represents a memory response129. Targeting of CpG oligodeoxynucleotides to FcγRIIA, which is selectively expressed by pDCs), has been suggested to be a valuable pDC activation strategy for human immunotherapy of cancer130. In humans, FcγRIIA seems to be the dominant receptor for enhancing pDC

responsiveness to TLR9 agonists. There are some important differences in mice. In mice, TLR9 is expressed not only by pDCs, but also by other DC subsets and macro phages. Moreover, FcγRIIA is not expressed in mice and many functions of human FcγRIIA are mediated by FcγRIII, which is also expressed by DCs and macrophages. The exact cell type responding to DNA‑containing immune complexes131 has yet to be defined. Therefore, in mice and in humans, pDCs can acquire the capacity to present antigens to CD4+ T cells when these antigens are bound to immune complexes and internalized through FcγRs. Although this pathway is probably involved in mediating tolerance in the steady state, concomitant exposure to TLR ligands or microbial products might promote effector T cell immunity and might cause disease.

Clinical implications The fact that FcγR signalling can influence DC and macrophage activation has important clinical applications. Modulating the ability of a therapeutic antibody to bind to activating versus inhibitory FcγRs could tip the balance in favour of cellular activation or suppression. Cellular activation is desirable for cancer immuno therapy or for vaccination against infectious diseases, whereas suppression is necessary for the induction of immune tolerance in cases of chronic inflammation and autoimmunity. Adoptive DC therapy using autologous moDCs or pDCs might be greatly facilitated by targeting antigens to activating FcγRs, particularly when the inhibitory FcγR is also blocked. The feasibility of this concept has been shown in preclinical mouse models93,132 and in human ex vivo studies28,61,123. Intravenous immunoglobulin therapy (IVIG therapy) has been used to treat various autoimmune diseases, although the precise mechanism that underlies its protective effect is still under debate 133. It has been suggested that injection of a high dose of IgGs would simply compete with the immune complexes that are present in many autoimmune diseases for binding to individual FcγRs. However, IVIG does not function in FcγRIIB-deficient mice134–136, which suggests that IVIG does not simply compete for binding to activating ITAM-bearing FcRs. IVIG was shown to increase the expression of FcγRIIB and to decrease the expression of FcγRIV on effector macrophages within arthritic lesions and inflamed kidneys134,137. This may be one of the crucial immunomodulatory mechanisms that is induced by IVIG, as IVIG increases the threshold for macrophage activation. Importantly, the effect of IVIG seems to be independent of FcγRIIB expression at the initiation of the immune response, but requires FcγRIIB expression on macrophages within the inflamed tissues138. Indeed, the in vitro treatment of spleen cells from both wild-type and FcγRIIB-deficient mice with immunoglobulins followed by the transfer of these cells to wild-type mice could reproduce the beneficial effects of IVIG138. CD11c+ cells but not CD11c– cells were found to be the main cells responsible for this beneficial effect, which suggests that there is a role for DCs in the initiation of IVIGinduced immunosuppression. As inhibitory FcγRIIB



REVIEWS does not seem to be involved in the initiation of this immunosuppression, and as splenic mouse cDCs do not express marked levels of the activating FcγRs (FIG. 1), it is probable that the moDCs that are induced by the immunoglobulin treatment are the main CD11c+ cells responsible for this IVIG effect. Indeed, the transfer of in vitro immunoglobulin-treated moDCs was sufficient to protect mice from immunothrombocytopenia138. Thus, IVIG seems to function through distinct FcγRs on several cell types in different locations and at different time points; through activating FcγRs on moDCs in the spleen during the initiation phase of the immune response, thereby imprinting a tolerogenic phenotype on these cells (possibly by inducing high IL‑10 production by these cells); and by increasing FcγRIIB expression on macrophages within the inflamed tissue, thereby increasing the threshold of macrophage activation during the effector phase of the immune response. Human moDCs that had been treated with IVIG in vitro also showed increased IL‑10 production, decreased IL‑12 production and impaired maturation139. The mechanism by which IVIG-triggered moDCs may influence FcγRIIB expression on inflammatory macrophages may involve the induction of a TH2‑type response140. Indeed, moDCs that have been stimulated with immunoglobulins produce IL‑33 (REF. 112), which in turn could induce the production of IL‑4, leading to an increase in the expression of FcγRIIB on macrophages. Although the conversion of moDCs into TH2‑type response-inducing cells in some mouse studies was suggested to occur through FcγRIII108, in humans this may occur through the C‑type lectin DC‑specific ICAM3‑grabbing non-integrin (DC-SIGN; also known as CD209), which also functions as a receptor for sialic acid-rich IgG glycoforms. Indeed, IVIG treatment of transgenic mice that express human DC-SIGN results in the IL‑33‑mediated induction of IL‑4 production by basophils, which in turn increases the expression of FcγRIIB by macrophages in arthritic lesions140. FcRn Jönsson, F. & Daëron, M. Mast cells and company. Front. Immunol. 3, 16 (2012). 2. Nimmerjahn, F. & Ravetch, J. V. Fcγ receptors as regulators of immune responses. Nature Rev. Immunol. 8, 34–47 (2008). 3. Roopenian, D. C. & Akilesh, S. FcRn: the neonatal Fc receptor comes of age. Nature Rev. Immunol. 7, 715–725 (2007). 4. James, L. C., Keeble, A. H., Khan, Z., Rhodes, D. A. & Trowsdale, J. Structural basis for PRYSPRY-mediated tripartite motif (TRIM) protein function. Proc. Natl Acad. Sci. USA 104, 6200–6205 (2007). 5. Mallery, D. L. et al. Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21). Proc. Natl Acad. Sci. USA 107, 19985–19990 (2010). 6. Daëron, M. Fc receptor biology. Annu. Rev. Immunol. 15, 203–234 (1997). 7. Blank, U., Launay, P., Benhamou, M. & Monteiro, R. C. Inhibitory ITAMs as novel regulators of immunity. Immunol. Rev. 232, 59–71 (2009). 8. Amigorena, S. et al. Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes. Science 256, 1808–1812 (1992). 9. Ono, M., Bolland, S., Tempst, P. & Ravetch, J. V. Role of the inositol phosphatase SHIP in negative regulation of the immune system by the receptor FcγRIIB. Nature 383, 263–266 (1996). 10. Daëron, M., Jaeger, S., Du Pasquier, L. & Vivier, E. Immunoreceptor tyrosine-based inhibition motifs: a quest in the past and future. Immunol. Rev. 224, 11–43 (2008). 1.

has also been suggested to be important in one of the mechanisms that underlie the protective action of IVIG in K/BxN mice, which are a model for autoimmune arthritis141.

Concluding remarks During an adaptive immune response, the direct binding of immunoglobulins to FcγRs or the formation of immune complexes containing specific antigens provide an important source of feedback that controls the function of APCs. The outcome of this feedback can be the enhanced phagocytic function of macrophages or it can involve the increased targeting of antigens to DCs that have become more proficient in antigen uptake and processing, and in polarizing TH cell responses to the pathogen. In addition, during repeated antigen encounter, the presence of antigen-specific immunoglobulins that are derived from memory B cells or from plasma cells could greatly facilitate the recognition and clearance of pathogens by DCs and macrophages. Whereas the expression of activating FcγRs was once thought to be ubiquitous on macrophages and DCs, we now realize that cDCs and pDCs in the steady state express only low levels of activating FcγRs, but express the inhibitory FcγR that is involved in maintaining tolerance. It remains to be studied whether cDCs upregulate FcγRs under conditions of inflammation and how this affects their function. Following an encounter with pathogens, monocytes are recruited that rapidly develop into macrophages and DCs in situ, and these cells express almost all types of activating FcγRs. We hypothesize that the main function of activating FcγRs on moDCs is the modification of DC and T cell encounters at sites of inflammation, whereas the function of FcγRs on macrophages is to promote the clearance of pathogens in the periphery. These pathways could be exploited to boost immune responses to tumours, and the dysregulation of FcγR function might contribute to the development of autoimmunity.

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FURTHER INFORMATION Immunological Genome Consortium: http://www.immgen.org/

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The function of Fcγ receptors in dendritic cells and macrophages Martin Guilliams, Pierre Bruhns, Yvan Saeys, Hamida Hammad and Bart N. Lambrecht Nature Reviews Immunology 14, 94–108 (2014)

In the version of this Review that was initially published, the images in Table 2 showing the structure of FcγRIIB and FcγRIII were in the wrong order. This error has been corrected in the online HTML and PDF versions of the article. Nature Reviews Immunology apologizes for this error.

© 2014 Macmillan Publishers Limited. All rights reserved

The immunobiology of human Fc gamma receptors on hematopoietic cells and tissue macrophages.

Binding of monomeric immunoglobulins to Fc receptors of mouse macrophages.

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Regulation of IL-10 and IL-12 production and function in macrophages and dendritic cells.

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Macrophages and dendritic cells in the post-testicular environment.

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Fc gamma receptors: gene structure and receptor function.

Structure and function of renal macrophages and dendritic cells from lupus-prone mice.

The effect of cytokines on the expression and function of Fc receptors for IgG on human myeloid cells.

Intestinal macrophages and dendritic cells: what's the difference?

Metabolic reprogramming in macrophages and dendritic cells in innate immunity.

Identification and structural characterization of Fc gamma-receptors on pulmonary alveolar macrophages.

Fc receptors on mouse placenta and yaol sac cells.

Properties of the Fc receptor on macrophages.

Role of scavenger receptors in dendritic cell function.

Epidermal Langerhans cells bear Fc and C3 receptors.

gamma 2 receptors on macrophages, polymorphonuclear cells, and lymphocytes.

Phenotype and function of nasal dendritic cells.

Herpesviral Fc receptors and their relationship to the human Fc receptors.