FcγRIV: A Novel FcR with Distinct IgG Subclass Specificity

Immunity, Vol. 23, 41–51, July, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.immuni.2005.05.010

Fc␥RIV: A Novel FcR with Distinct IgG Subclass Specificity Falk Nimmerjahn,1,2 Pierre Bruhns,1,2,3 Ken Horiuchi,1 and Jeffrey V. Ravetch1,* 1 Laboratory of Molecular Genetics and Immunology The Rockefeller University 1230 York Avenue New York, New York 10021

Summary Mouse IgG subclasses display a hierarchy of in vivo activities, with IgG2a and IgG2b showing the greatest protective and pathogenic properties. These enhanced activities result, in part, from their ability to bind to a novel, ␥ chain-dependent, activating IgG Fc receptor, Fc␥RIV. Fc␥RIV maps in the 75 kb genomic interval between Fc␥RII and Fc␥RIII; its expression is restricted to myeloid lineage cells, and it binds to IgG2a and IgG2b with intermediate affinity. No binding to IgG1 or IgG3 was observed. Blocking Fc␥RIV binding to pathogenic anti-platelet antibodies is sufficient to protect mice from antibody-induced thrombocytopenia. Thus, the Fc␥R system has evolved distinct activation receptors displaying selectivity for IgG subclasses, with IgG1 antibodies exclusively dependent on Fc␥RIII, whereas IgG2a and IgG2b show preferential dependence on Fc␥RIV activation. These distinct binding affinities for the IgG subclasses to Fc␥Rs account for their differential protective and pathogenic activities in vivo. Introduction Fc receptors for IgG couple innate and adaptive immunity through their ability to activate effector cells by antibody-antigen complexes and opsonized target cells. Two broad classes of these receptors have been described: those that activate effector cell responses and those that inhibit those responses (Ravetch, 2003). Activation receptors such as FcγRI and FcγRIII upon crosslinking by IgG transduce signals through immunoreceptor tyrosine-based activation motif (ITAM) sequences, usually found on the common γ chain subunit. Activation responses are dependent on the sequential activation of members of the src and syk kinase families, resulting in the recruitment of potent signaling molecules such as PI3kinase and protein kinase C, for example. Inhibitory signals are transduced upon cocrosslinking of the inhibitory FcγRIIB receptor to an ITAM-containing receptor. Phosphorylation of an immunoreceptor tyrosine-based inhibitory motif (ITIM) sequence found in the cytoplasmic domain results in the recruitment of the SH2-containing inositol polyphosphate phosphatase (SHIP) and the hydrolysis of PI3K *Correspondence: [email protected] 2 These authors contributed equally to this work. 3 Present address: The Pasteur Institute, 25 rue Dr. Roux, 75015 Paris, France.

products such as PIP3, resulting in the termination of ITAM-initiated activation (Bolland and Ravetch, 1999). Coexpression of activation and inhibitory FcRs is a general property of effector cells such as macrophages, neutrophils, and mast cells and will determine whether an IgG-initiated response results in downstream effector cell coupling (Clynes et al., 1999). Similarly, the ability of immune complexes (ICs) to convert immature dendritic cells into cells capable of stimulating naive T cells is also determined by the balance between activation and inhibitory FcγRs (Kalergis and Ravetch, 2002; Dhodapkar et al., 2005). Thus, the threshold for cellular activation by ICs is determined by the relative ratios of these opposing Fc receptor molecules. In the mouse and in man, low-affinity activation and inhibitory receptors have been studied most extensively for the IgG1 subclass. Mice express a single activation FcR for IgG1, FcγRIII, and a single inhibitory receptor for this subclass, FcγRIIB. In vivo disruption of each of these genes results in attenuation or enhancement, respectively, of IgG1-mediated inflammatory responses (Takai et al., 1994, 1996; Hazenbos et al., 1996, 1998; Meyer et al., 1998; Wernersson et al., 1999). As was found for IgG1, IgG2a and IgG2b are also dependent on FcR expression for their ability to mediate effector responses such as ADCC, clearance of opsonized particles, or triggering release of inflammatory mediators (Clynes et al., 1998, 2000; Fossati-Jimack et al., 2000; Uchida et al., 2004). In common with IgG1, mice deficient in the FcR-common γ chain were equally unable to mediate effector responses to IgG2a or IgG2b antibodies in models of immune thrombocytopenia, IgG-mediated RBC or B cell depletion, tumor clearance, and the reverse passive Arthus reactions (Takai et al., 1994; Sylvestre and Ravetch, 1994; Clynes and Ravetch, 1995; Clynes et al., 2000; Samuelsson et al., 2001). Surprisingly, however, when IgG2a- and IgG2bmediated responses were studied in mice deficient for the α subunit, responsible for IgG binding, for the known activation IgG FcγRs, i.e., FcγRI or FcγRIII, only partial or no reduction was observed in the ability of IgG2a and IgG2b antibodies to trigger effector cell responses (Meyer et al., 1998; Hazenbos et al., 1998; Fossati-Jimack et al., 2000; Barnes et al., 2002; Ioan-Facsinay et al., 2002; Uchida et al., 2004). These results suggested that other γ chain-dependent FcγRs α subunits might be involved in the in vivo activity of these subclasses of antibodies. To identify a mouse Fc receptor α subunit with specificity for IgG2a and IgG2b, we turned to the recently described family of Fc receptor homologs (FcRHs) identified through homology searching of the mouse and human genomes (Davis et al., 2002). These genes share a genomic structure and sequence similarity to the classical FcR genes and are tightly linked to the FcR genes on chromosome 1 in man and 1 and 3 in the mouse. The biological function of these molecules, such as their ligand specificity is, as yet, unknown. One such gene, called FcRL3 or CD16-2, showed 63% sequence identity to the human FcγRIII α chain gene, with

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Figure 1. Genomic Organization of FcγR Genes on Mouse Chromosome 1 (A) Schematic representation of the mouse FcγRIV gene in its genomic context. Filled boxes represent genes, and arrows indicate the direction of transcription. Representative restriction sites are indicated. The scale indicates the position of the genes on chromosome 1 according to the ensembl database in mega base pairs (Mbp). Bac clones corresponding to the locus are shown underneath the schematic drawing of chromosome 1 and were derived from C57BL/6 or 129 genomic DNA. Abbreviations: Cen, centromere and Chr1-H2, chromosome 1 band H2. (B) Exon-intron structure of the FcγRIV gene. Exons are shown as black boxes, and representative restriction sites are indicated. Abbreviations: S, signal sequence; EC, extracellular domain; TM, transmembrane region; and IC, intracellular tail.

a two-domain structure similar to those found for the low-affinity FcγRII and FcγRIII genes (Davis et al., 2002; Mechetina et al., 2002). Northern blot analysis demonstrated transcripts for this gene in hematopoeitic organs such as the spleen, liver, and bone marrow. The mouse genome consortium mapping placed the gene on mouse chromosome 1, adjacent to the FcγRII and FcγRIII genes (Waterston et al., 2002). However, evidence for IgG binding, protein expression, or biological function was lacking. As described below, this gene has now been identified the α subunit of a novel IgG2a, IgG2b FcγR, whose biological properties define it as an authentic activation IgG FcR accounting for the in vivo activities of IgG2a and IgG2b antibodies. We have designated this mouse IgG Fc receptor mFcγRIV, in accordance with the convention established in 1991 (Ravetch and Kinet, 1991) and in conformity with the World Health Organization nomenclature standards (World Health Organization, 1994). Results Gene Mapping and Genomic Organization of Fc␥RIV Previous reports have linked FcγRIV (FcγRL3, CD16-2) to the mouse low-affinity FcγRIIB and FcγRIII genes based on the alignments generated by the genome consortium. These linkages placed the FcγRIV gene either centromeric to FcγRIIB or telomeric to FcγRIII (Davis et al., 2002; Mechetina et al., 2002). To determine the position of the FcγRIV gene, we isolated a series of overlapping bacterial artificial chromosome (BAC) clones from both the 129 and C57BL/6 strains that span the FcγRII–FcγRIII locus, as shown in Figure 1A. Restriction mapping and sequence analysis of these clones established that FcγRIV mapped in the intergenic interval between FcγRII and FcγRIII. Fine structure mapping of the FcγRIV gene, shown in Figure 1B,

revealed the canonical split signal exon of classical FcR genes, two extracellular domains encoded on separate exons, and a single exon encoding the transmembrane and cytoplasmic domains, features conserved in the mouse and human FcγRIII α chain genes. In addition, FcγRIV contains a charged amino acid in the putative transmembrane domain, a feature shared with FcRs that associate with the common γ chain (Bolland and Ravetch, 1999). The chromosomal linkage, genomic organization, and sequence characteristics of FcγRIV point to a common ancestor for the FcγRIII and FcγRIV genes and suggest that FcγRIV may share biological properties with the FcγRIII gene. Subunit Composition To determine the cellular localization and subunit requirements for FcγRIV protein expression, cDNAs were isolated for FcγRIV from a spleen library by using DNA primers based on the published sequence encoding this gene. The FcγRIV cDNA was modified with a FLAG tag at the amino terminus and was transfected into 293T or Chinese hamster ovary (CHO) cells to determine if surface expression of tagged FcγRIV could be detected. As shown in Figure 2A, expression of FLAGFcγRIV was not detected when the isolated chain was transfected into 293T cells. In contrast, cotransfection of FLAG-FcγRIV with a cDNA encoding the common γ chain resulted in efficient surface expression of FLAGFcγRIV, detected either by anti-FLAG staining (Figure 2A) or by staining with an FcγRIV-specific mAb, 9G8.1, (Figure 2B), thus establishing the requirement for γ chain coexpression to result in surface expression for FcγRIV, as has been demonstrated for FcγRI and FcγRIII. Coprecipitation studies demonstrated a physical association of γ chain and FcγRIV (Figure S1 available in the Supplemental Data with this article online). γ chain expression was also required for FcγRIV expression in vivo, as demonstrated in Figure 2C. FACS analy-

FcγRIV: A Novel IgG Fc Receptor 43

Figure 2. Cell Surface Expression of FcγRIV Is γ Chain Dependent (A) 293T cells were transiently transfected either with FLAG-tagged mouse FcγRIV (mFcγRIV) alone (left) or additionally cotransfected with an expression plasmid coding for the common γ chain (mFcγRIV + γ chain). Cells were analyzed 24 hr later by flow cytometric analysis for FcγRIV cell surface expression with a FLAG epitope-specific antibody (shaded histograms) or an isotype control antibody (open histograms). (B) Characterization of the FcγRIV-specific monoclonal antibody 9G8.1. Chinese hamster ovary (CHO) cells stably transfected with FLAGtagged murine versions of FcγRI, FcγRIIB, FcγRIII, and FcγRIV (mFcγRI, mFcγRIIB, mFcγRIII, mFcγRIV, respectively) were either stained with the 9G8.1 antibody (top) or a FLAG epitope-specific antibody (anti-FLAG; bottom). Shaded histograms represent the FLAG or FcγRIV antibody staining, and open histograms indicate the staining with isotype-matched control antibodies. mFcγRI, mFcγRIII, and mFcγRIV were cotransfected with an expression plasmid coding for the common γ chain. (C) Peripheral blood mononuclear cells were isolated from C57BL/6 wild-type (B6) or C57BL/6 γ chain knockout mice (B6.γ −/−) and stained with antibodies specific for Mac-1 and FcγRIV (9G8.1). Representative density blots from three independent experiments are shown. Numbers indicate the percentage of cells in the respective quadrants.

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Figure 3. Cell-Type-Specific Expression of FcγRIV and Cytokine-Mediated Regulation (A) Peripheral blood mononuclear cells were stained with antibodies specific for Mac-1, Gr-1, and FcγRIV. The density plot shows the Mac-1/Gr-1 staining pattern, and gates identify Mac-1+/Gr-1− monocytes and Mac-1+/ Gr-1high neutrophils that were analyzed for FcγRIV expression. Shaded histograms represent FcγRIV staining, and open histograms represent staining with an isotype-matched control antibody. One out of three representative experiments is shown. (B) FcγRIV expression on bone marrowderived dendritic cells (BM-DCs). Bone marrow cells of C57BL/6 mice were cultured in the presence of GM-CSF for 6 days and either left untreated (top) or stimulated with LPS for 24 hr (bottom). Mature dendritic cells were identified by staining for MHC II (I-Ab), CD80, CD86, and FcγRIIB/FcγRIII (2.4G2) as described (Banchereau and Steinman, 1998). Gates in density plots identify cell populations that were analyzed for FcγRIV expression. Shaded histograms represent FcγRIV staining, and open histograms represent staining with an isotype-matched control antibody. One out of four representative experiments is shown. (C) FcγRIV expression on monocytes, macrophages, and neutrophils. Bone marrowderived monocytes (BM-Monocytes) were generated by culturing bone marrow cells for 7 days in the presence of M-CSF. F4/80+ cells were gated and analyzed for FcγRIV expression. Neutrophils (Mac-1high/Gr-1high) or macrophages (Mac-1+/F4/80+) were elicited by intraperitoneal injection of thioglycollate, collected by peritoneal lavage after 6 hr (neutrophils) or 4 days (macrophages), and analyzed for FcγRIV expression (shaded histograms). One out of three representative experiments is shown. (D) Cytokine-mediated regulation of FcγRIV expression on BM-Monocytes. Flow cytometric analysis of FcγRIV expression on M-CSF elicited BM-Monocytes (F4/80+) that were either left untreated (open histograms) or stimulated with the indicated cytokines for 24 hr (shaded histograms). One out of three representative experiments is shown.

sis of peripheral blood cells isolated from either wildtype (wt) or γ chain-deficient mice (γ−/−) stained with Mac-1, and an anti-FcγRIV-specific monoclonal antibody (9G8.1) indicated that the FcγRIV+Mac-1+ population is not detected in γ−/− mice. This γ chain dependence in vivo was also observed for FcγRI and FcγRIII, as reported (Takai et al., 1994). Fc␥RIV Expression on Cellular Populations mAb 9G8.1 was used to determine the expression pattern of FcγRIV on selective cellular subsets, as shown in Figures 3A–3D. FcγRIV expression was detected on myeloid cells and absent from lymphoid populations. Peripheral blood monocytes, splenic and bone marrow dendritic cells, bone marrow, and thioglycollate-elicited macrophages and neutrophils all expressed FcγRIV. No

expression was observed on T cells, B cells, NK cells, or other granulocytes (Table 1). Expression of the heterooligomeric complex of FcγRIV and the γ chain could be significantly upregulated on bone marrow-derived monocytes by interferon-γ (IFN-γ) and LPS and downregulated by TGF-β and IL-4 as has been observed for other Fc receptors before (Okayama et al., 2000; Pricop et al., 2001; Tridandapani et al., 2003). The expression pattern of FcγRIV is distinctive among the γ chainexpressing FcRs and suggests a more specialized role for FcγRIV in mediating cellular activation on myeloid cells. Binding Affinity and Specificity for IgG Subclasses Binding studies on FcγRIV were performed by using surface plasmon resonance (SPR) with soluble FcγRIV

FcγRIV: A Novel IgG Fc Receptor 45

Table 1. Cell-Type-Specific Expression of FcγRIV Cell Type

Marker/Antibody

Origin

Expression Levela

B cells

CD19

T cells

TCR-β

γδ-T cells NK cells Monocytes/macrophages

TCR-γδ NK1.1 Mac-1high/Gr-1− Mac-1high/Gr-1− Mac-1high/Gr-1− F4/80 F4/80/Mac1+ CD11chigh/CD8+ or CD8− CD11c+/CD80low/CD86low CD11c+/CD80high/CD86high Mac-1high/Gr-1high Mac-1high/Gr-1high Mac-1high/Gr-1high Gr-1+/ Mac-1− Gr-1+/ Mac-1− 6A6 antibody

Blood Spleen Bone marrow Blood Spleen Spleen Spleen Blood Spleen Bone marrow M-CSF Thioglycollate Spleen Bone marrow (DCi) Bone marrow (DCm) Blood Spleen Thioglycollate Blood Spleen Blood

− − − − − − − ++ ++ + ++ +++ +/−b + +/− ++ ++ ++ − − −

Dendritic cells

Neutrophils

Other granulocytes Platelets

Expression of FcγRIV on the indicated cell populations from different organs of C57BL/6 mice was analyzed by flow cytometric analysis with the indicated cell-type-specific markers in combination with the FcγRIV-specific monoclonal antibody 9G8.1. Abbreviations: DCi, immature dendritic cells and DCm, mature dendritic cells. a The relative level of expression was compared between different cell types on the basis of the mean fluorescence intensity obtained with the directly labeled 9G8.1 antibody at different dilutions in three independent experiments. b Expression was only slightly above background.

and a panel of mouse immunoglobulins that were engineered to express the same VH and VL regions and either the IgG1, IgG2a, IgG2b, or IgG3 constant regions. These Fc regions were all derived from the C57BL/6 strain and expressed in 293T cells to minimize variation in allelic difference, antibody glycosylation, or H + L structure. SPR analysis has been demonstrated to yield comparable results to classical approaches like analytical ultracentrifugation or scatchard analysis (Figure S2) (Ghirlando et al., 1995; Miller et al., 1996; Sondermann et al., 1999; Mimura et al., 2001). FcγRIV binding to this series of immunoglobulins was compared to FcγRIIB and FcγRIII expressed as soluble receptors as described for human FcRs (Wines et al., 2000; Maenaka et al., 2001). FcγRIV bound to IgG2a and IgG2b with an affinity intermediate between that observed for the lowaffinity receptors FcγRII and FcγRIII and the high-affinity receptor FcγRI. As seen in Figure 4A and Table 2, FcγRIV binds with an affinity to IgG2a of 3 × 107 (M−1), an affinity five to ten times greater than IgG2a binding to FcγRIII or FcγRIIB, respectively. No binding was detected to IgG1 or IgG3. The binding specificity of FcγRIV was similar when ICs were used to probe CHO cells expressing FcγRIV + γ chain (Figure 4B). No binding to FcγRIV was observed when IgM ICs were used (Figure 4B). This unique binding specificity of FcγRIV was further studied in DT40 cells transfected with FcγRIV + γ chain. Crosslinking of FcγRIV on these cells with IgG2a ICs resulted in a calcium flux characteristic of FcR signaling through γ chain (Kurosaki et al., 1992) (Figure 4C). In contrast, IgG1 complexes were unable to trigger DT40 calcium mobilization, consistent with the undetectable binding affinity for FcγRIV to IgG1.

Fc␥RIV Binding Accounts for the Pathogenic Activity of IgG2a and IgG2b Antibodies To determine the in vivo role of FcγRIV in mediating the biological activity of IgG2a and IgG2b antibodies, we investigated the ability of a panel of anti-platelet antibodies derived from the 6A6 V region and bearing IgG1, IgG2a, or IgG2b constant domains to mediate platelet clearance in intact and FcR-deficient mice. 6A6-IgG2b induces a rapid thrombocytopenia in wt mice that is abrogated in γ chain-deficient mice, as shown in Figure 5A, indicating that this antibody is FcR dependent in its in vivo activity. FcγRI- or FcγRIII-deficient animals, however, do not exhibit evidence of reduced platelet clearance in response to 6A6-2b treatment (Figures 5B and 5C). However, pretreatment of wt mice with a hamster mAb 2C6 specific for FcγRIV and able to block binding of IgG2a or IgG2b resulted in significant (p < .001) protection in mice injected with 6A6-2b (Figures 5D and 5E). Moreover, in vivo blocking of FcγRIV activity resulted in impaired platelet depletion by a 6A6IgG2a antibody (Figure 5E). In contrast, 6A6-G1-induced thrombocytopenia in wt mice was unaffected by pretreatment with FcγRIV-blocking antibodies but was abolished in FcγRIII-deficient mice (Figure 5F). FcγRIV thus is necessary for the in vivo biological activity of IgG2a and IgG2b mAbs in their ability to mediate platelet clearance, whereas the same platelet specificity when associated with an IgG1-constant region is FcγRIV independent and completely dependent on FcγRIII.

Discussion The immune response has evolved selective pathways to engage and eliminate the constant threat posed by

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Figure 4. Antibody-Isotype Specificity of FcγRIV (A) Kinetic analysis of FcγRIV binding to C57BL/6 IgG antibody isotypes by surface plasmon resonance. A soluble variant of FcγRIV lacking the transmembrane and intracellular portions was injected at the indicated concentrations through flow cells containing immobilized antibody isotypes at a flow rate of 30 ␮l/min. Background binding to flow cells with immobilized BSA was automatically subtracted in all experiments. (B) Immune complex (IC) binding to FcγRIV. CHO cells stably transfected with mouse FcγRIV (mFcγRIV) and the common γ chain were incubated with TNP-BSA-biotin/anti-TNP ICs (TNP-ICs) followed by detection of bound TNP-ICs with Pe-Avidin and flow cytometric analysis. Open histograms represent binding of TNP-BSA-biotin alone, and open shaded histograms represent binding of TNP-ICs. (C) FcγRIV-mediated IC-triggered calcium release. DT40 cells expressing mFcγRIV and the common γ chain were analyzed for FcγRIV expression (left). To determine calcium influx mediated by FcγRIV crosslinking, DT40-FcγRIV cells were loaded with Fluo-3 and either left untreated (broken line) or incubated with IgG1 (thin line) or IgG2a TNP-ICs (thick line). Calcium influx was monitored by flow cytometric analysis.

microbial pathogens while preventing these same pathways from triggering unnecessary tissue damage and systemic disease. This polarization is seen at many levels of the immune response, from the mechanisms by which dendritic cells are capable of inducing both tolerogenic and immunogenic response, to the pathways that give rise to TH1 or TH2 responses, to the selective recruitment of specific effector cells elicited in response to specific pathogens (Sher and Coffman, 1992; Steinman et al., 2003). Immunoglobulin isotypes represent another such example of polarization of the

immune function in response to specific challenges and are linked to the polarization of TH1 and TH2 cells. TH1 responses are optimized to facilitate the elimination of intracellular pathogens and through the release of cytokines such as IFN-γ result in the activation of macrophages and enhance the production of specific immunoglobulin subclasses, such as IgG2a and IgG3. TH2 cytokines such as IL-4 and IL-5, on the other hand, are generally produced in response to extracellular microbial challenge and lead to enhanced B cell activation and the production of IgG1 and IgE and the secretion

FcγRIV: A Novel IgG Fc Receptor 47

Table 2. Affinities of Fcγ Receptors for Antibody Isotypes sFcγRIIB

sFcγRIII

KA (1/M)c IgG1 IgG2a IgG2b IgG3 IgG1-D265A Hu-IgG1 Hu-IgG1-N297A

6

3.33 x 10 0.42 x 106 2.23 x 106 −/−a −/−a n.d.b n.d.b

KD (M)c

sFcγRIV

KA (1/M)c −7

3.01 x 10 2.39 x 10−6 4.48 x 10−7 −/−a −/−a n.d.b n.d.b

6

0.31 x 10 0.68 x 106 0.64 x 106 −/−a −/−a n.d.b n.d.b

KD (M)c

KA (1/M)c −6

3.2 x 10 1.46 x 10−6 1.55 x 10−6 −/−a −/−a n.d.b n.d.b

a

-/2.9 x 107 1.7 x 107 −/−a −/−a 9.6 x 106 −/−a

KD (M)c -/-a 3.45 x 10−8 5.9 x 10−8 −/−a −/−a 1.04 x 10−7 −/−a

Affinity constants were measured by surface plasmon resonance. Soluble versions of murine Fcγ receptors IIb, III, and IV were injected through flow cells containing the indicated immobilized antibodies at five different concentrations (4, 2, 1, 0.5, and 0.25 ␮g/ml). Background binding to a reference flow cell containing immobilized BSA was subtracted. IgG1-D265A and IgG1-N297A are mutant IgG1 molecules with alanin (A) instead of aspartate (D) or asparagin (N) at the indicated position in the Fc portion. Abbreviations: hu, human and sFcγR, soluble Fcγ receptor. a −/− indicates no detectable binding. b n.d. indicates not done. c The standard deviation for KA and KD was below 5%.

of IgA (Mosmann and Coffman, 1989; Finkelman et al., 1990). Differential roles for IgG subclasses in host defense have been studied most extensively for IgG1 and IgG2a in their ability to confer protection in models of viral and fungal infection (Coutelier et al., 1987; Schlageter and Kozel, 1990; Baldridge and Buchmeier, 1992; MarkineGoriaynoff and Coutelier, 2002; Taborda et al., 2003). These studies have suggested that IgG2a is the most potent subclass in mediating protection. Similarly, in

models of passive antibody-induced cellular depletion, such as immune thrombocytopenia, hemolytic anemia, and B cell depletion induced by anti-CD20 antibodies, IgG2a is the most potent subclass in inducing these activities (Kipps et al., 1985; Kaminski et al., 1986; Fossati-Jimack et al., 2000; Azeredo da Silveira et al., 2002; Uchida et al., 2004). Previous studies have shown that mice deficient in the common FcR γ chain, required for assembly and surface expression of FcγRI, FcγRIII, and FcγRIV, fail to demonstrate IgG2a-mediated protection Figure 5. Analysis of FcγRIV Function In Vivo (A–C) For analysis of FcγRIV function in vivo, experimental immune thrombocytopenia was induced in wt (A), FcγRI−/− (B), and FcγRIII−/− (C) mice by intravenous injection of 3.5 ␮g of an IgG2b variant of the 6A6 anti-platelet antibody (red lines). As a control, platelet depletion in γ−/− mice (g−/−) was included to demonstrate FcR dependence (blue lines). (D) To block IC binding to FcγRIV in vivo, mice were pretreated with 200 ␮g of an FcγRIV-specific monoclonal antibody (2C6) that interferes with IC binding to this Fc receptor or an isotype-matched control antibody, followed by injection with the platelet depleting 6A6-IgG2b antibody. *p < 0.001; **p < 0.01. (E) The percentage of platelet depletion 4 hr after injection of a 6A6-IgG1, 6A6-IgG2a, or 6A6-IgG2b isotype variant with or without (mock) previous injection of the 2C6- or 9E9blocking antibodies or isotype-matched control antibodies (Iso) is shown. (F) Platelet depletion mediated by the 6A6IgG1 antibody is FcγRIII dependent. C57BL/6 wt, common γ−/−, and FcγRIII−/− mice were injected with the 6A6-IgG1 isotype variant, and platelet counts were followed over time. Additionally, mice were injected with 200 ␮g of the FcγRIV-blocking antibody 2C6 or an isotype control antibody before injection with 6A6-IgG1. One out of three independent experiments with five mice per group is shown. For each group, the mean ± SD was calculated, and Student’s t test was used for statistical analysis.

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or pathology, whereas mice deficient in complement C3 display no diminution of IgG2a-induced activity (Clynes and Ravetch, 1995; Sylvestre et al., 1996; Ravetch and Clynes, 1998). IgG2a, despite its ability to activate complement through the classical pathway in vitro, mediates its in vivo activity through Fc receptor activation. However, because mice deficient either in FcγRI or FcγRIII retained IgG2a activity, the precise Fc receptor responsible and the basis for the enhanced activity were not clear. With the identification of FcγRIV, a γ chain-dependent activation FcγR expressed on myeloid cells with intermediate binding affinity for IgG2a and IgG2b, a mechanism can be proposed for the enhanced activity of IgG2a over that observed for IgG1. IgG1 binds exclusively with low affinity (0.3 × 106 M−1) to the activation receptor FcγRIII, whereas IgG2a binds with low affinity (0.7 × 106 M−1) to FcγRIII and with 40-fold higher affinity to FcγRIV (2.9 × 107 M−1). It will also bind to FcγRI with high affinity (w1 × 109 M−1). The enhanced activity of IgG2a is thus most easily explained by its higher-affinity binding to FcγRIV with additional contributions from FcγRIII. The induction of FcγRI and FcγRIV expression on macrophages by IFN-γ suggests that TH1 activation induces both IgG2a expression and its activation receptors, thereby amplifying the role of this subclass in mediating effector responses in vivo. IgG2b binds with 10-fold higher affinity to FcγRIV as compared to FcγRIII, thereby accounting for the preferential role of FcγRIV to the in vivo activity of IgG2b. We have previously demonstrated that the activity of IgG1 antibodies in vivo is dependent on the ratio of activation to inhibitory receptor expression (FcγRIII: FcγRIIB) (Clynes et al., 2000). Because IgG1 demonstrates a 10-fold higher affinity for the inhibitory receptor than its activation counterpart, small changes in expression of the inhibitory receptor result in pronounced changes in the activation threshold and effector cell responses. In contrast, IgG2a has a 70-fold higher affinity for the activation receptor FcγRIV than the inhibitory receptor FcγRIIB, suggesting that IgG2a activity will be less sensitive to changes in FcγRIIB expression than IgG1. Thus, the higher binding affinity of IgG2a to FcγRIV and the reduced affinity to FcγRIIB insure that IgG2a functions as a highly effective opsonin to induce clearance of pathogens through effector cell activation. In contrast, the preferential binding of IgG1 to the inhibitory receptor may provide a protective mechanism for this most abundant isotype of serum antibody to prevent incidental activation of circulating myeloid cells by ICs and the systemic inflammation that could follow. The diversity of cellular receptors for immunoglobulins mirrors the complexity of responses that are required to effectively deal with microbial pathogens. Through the segregation of specific isotypes and subclasses to receptors with distinct cellular distributions, affinities, and signaling pathways, the selectivity of responses needed can be provided. Differential regulation of these receptors provides yet another level of control, reflecting the polarization of responses to specific pathogens. A more complete picture has begun to emerge with the addition of the FcγRIV to the specifi-

cities required to effectively account for the different roles of immunoglobulin in effective immunity. Experimental Procedures Mice C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME). γ−/−, FcγRII−/−, and FcγRIII−/− mice were generated in our laboratory and backcrossed for 12 generations to the C57BL/6 background. FcγRI−/− 129/B6 mice were generously provided by Dr. Hogarth (The Austin Research Institute, Victoria, Australia). Female mice at 2–4 months of age were used for all experiments and maintained at the Rockefeller University animal facility. All experiments were done in compliance with federal laws and institutional guidelines and have been approved by the Rockefeller University (New York, New York). Cell Culture and Cell Isolation CHO-K1(CHO), DT40, and 293T cells were cultured according to ATCC guidelines. CHO-FcγRI, CHO-FcγRIII, CHO-FcγRIV, and DT40-FcγRIV stable transfectants were obtained by cotransfection of the pNTneo-FcγRI, FcγRIII, or FcγRIV plasmids (neomycin resistant) and pNTzeo-γ chain (zeocin resistant) followed by subsequent selection in 1 mg/mL geneticin (Invitrogen) and 0.5 mg/mL zeocin (Invitrogen). The CHO-FcγRII cell line was generated by transfection of CHO cells with pNT-neo-FcγRII and subsequent selection in Geneticin. Transient expression of FcγRIV alone or in combination with the γ chain in 293T cells was achieved by calcium phosphate transfection. C57BL/6 bone marrow-derived monocytes (BM-Mo) were generated by culturing nonadherent bone marrow cells for 6–8 days in DMEM medium supplemented with 10% FBS and 500 ng/ml M-CSF (Peprotech). For some experiments, these BM-Mo were cultured in medium supplemented with INF-γ (50 ng/ml), LPS (100 ng/ml), TGF-β (100 ng/ml), or IL-4 (100 ng/ml) for 24–36 hr. Bone marrow-derived dendritic cells were generated as described (Lutz et al., 1999) and matured by culturing the cells in the presence of 1 ␮g/ml LPS (Sigma) for 24–48 hr. For flow cytometric analysis, peripheral blood, bone marrow, and spleen cells were isolated followed by red blood cell lysis. Thioglycollate-elicited neutrophils and macrophages were generated by intraperitoneal injection of 2 ml of 3% thioglycollate (Sigma). After 6 hr (neutrophils) or 4 days (macrophages), cells were collected by peritoneal lavage and used for FACS analysis. Neutrophils were identified by their high expression levels of Mac-1 and GR-1, as described (Ramalingam et al., 2003). Antibodies Antibodies 2.4G2, Mac-1, GR-1, CD19, TCR-β, TCR-γδ, CD11c, Nk1.1, CD80, CD86, and I-Ab were purchased from Pharmingen (San Diego, CA). The F4/80 antibody was from Serotec (Oxford, UK). The TNP-specific mouse antibodies IgG1,λ (clone 107.3), IgG1,κ (clone A111-3), and IgM (clone G155-228) were from Pharmingen. The TNP-specific mouse IgG2a (clone 7B4) and IgG2b (clone 1B4) were kind gifts from Dr. B. Heyman (Uppsala University, Uppsala, Sweden). Fluorescein isothiocyanate (FITC)-labeled F(ab#)2 goat anti-hamster IgG and DNP-specific IgG3 (clone MADNP-4) were purchased from Serotec, FITC anti-FLAG from Sigma, phycoerythrin (PE)-F(ab#)2 anti-mouse Fab-specific, and FITC goat anti-armenian hamster IgG from Jackson Immunoresearch (West Grove, PA). Neutravidin-PE was from Molecular Probes (Eugene, OR). For Biacore analysis and in vivo platelet depletion experiments, C57BL/6 IgG isotype variants of 6A6 were produced by transient transfection of 293T cells with Fc-engineered heavy chains and the native light chain, as described (Wardemann et al., 2003), and subsequent purification of recombinant antibodies from serum-free culture supernatants via protein A or protein G affinity columns (Amersham) as suggested by the manufacturer. The human IgG1 and IgG1-N297A (nonglycosylated) antibodies were kindly provided by Dr. Stavenhagen (Macrogenics, Inc.). FcγRIV-specific antibodies were produced at the monoclonal antibody facility of the Memorial Sloan Kettering Cancer Center. Briefly, Armenian hamsters were injected with an FcγRIV-IgG1 fusion pro-

FcγRIV: A Novel IgG Fc Receptor 49

tein consisting of the extracellular domain of FcγRIV fused to a mouse IgG1 Fc portion (D265A-variant deficient in Fc-receptor binding) kindly provided by S. Burke (Macrogenics, Inc.). After immunizations and boosting, hamster splenic B cells were fused to a mouse fusion partner, and hybridoma clones were selected for secreting antibodies that bound to CHO-K1-FcγRIV cells expressing mFcγRIV in a flow cytometric assay. In this study, three of these clones, 9G8.1, 2C6, and 9E9, were used. In contrast to 9G8.1, the 2C6 and 9E9 antibodies interfere with IC binding to FcγRIV. The 2C6 antibody shows crossreactivity to FcγRI but does not interfere with IC or monomeric antibody binding to this receptor (data not shown). Both of these antibodies were purified from hybridoma supernatants by protein G affinity chromatography. For FACS analysis, the 9G8.1 antibody was directly labeled with the Alexa 488 or Alexa 647 dyes (Molecular Probes) as suggested by the manufacturer. IC Binding Assay ICs were preformed by incubating biotinylated BSA-(5)-TNP at 10 ␮g/ml with anti-TNP/DNP monoclonal antibodies at 15 ␮g/ml for 1 hr at 37°C in PBS. 2 × 105 cells were incubated with these ICs for 2 hr at 4°C. After washing, bound ICs were detected by using Neutravidin-PE at 2 ␮g/ml for 1 hr at 4°C, followed by washing and flow cytometric analysis using a FACSCalibur cytometer (Becton Dickinson). cDNA and Cloning The FcγRIV cDNA was amplified from C57BL/6 spleen cDNA by PCR using the primers FcγRIV-forward (5#-ATCTGCTCTAGAAGC ATGTGGCAGCTACTA-3#) and FcγRIV-reverse (5#-CATGCGATAAGA GCTCACTTGTCCTGAGGT-3#). DNA sequencing revealed a complete match to the Ensembl database sequence (http://www.ensembl. org). cDNA coding for mFcγRIIB1 (Ly17.2 haplotype), mFcγRIII, and γ chain was previously cloned in the laboratory, mFcγRI cDNA was a kind gift from B. Seed. To enable cell surface detection, a FLAG epitope tag was inserted immediately 3# of the signal sequence cleavage site in mFcγRI, mFcγRII, mFcγRIII, and mFcγRIV by PCR using overlapping primers. Resulting constructs were cloned into pNT NEO (a gift from M. Daëron). For the generation of soluble variants of FcγRII, FcγRIII, and FcγRIV, the extracellular domains of FcγRII (amino acids 1–211), FcγRIII (amino acids 1–216), and FcγRIV (amino acids 1–201) were amplified by PCR and cloned in frame with a C-terminal hexahistidine or FLAG epitope tag at the C terminus into pcDNA3.1. The 6A6-antibody heavy and light chains were cloned out of the parental hybridoma by using a 5#RACE approach (Invitrogen). After sequencing individual clones, the most abundant sequences similar to known antibody heavy and light chain sequences were fused to the Fc portions of IgG1, IgG2a, and IgG2b derived from C57BL/6 mice (kindly provided by Dr. Fukuyama, Rockefeller University, New York, NY), expressed by transient transfection in 293T cells, and tested for their ability to deplete platelets (see below). Production of Soluble Mouse Fc␥ Receptors Soluble Fcγ receptors and recombinant antibodies were generated by transient transfection of 293T cells with calcium phosphate transfection. For protein production, cells were cultured in DMEM medium supplemented with 1% Nutridoma SP (Roche). Cell culture supernatants were harvested 6 days after transfection, and recombinant proteins were purified with Ni-NTA (Qiagen) or anti-FLAG agarose (Sigma) by affinity chromatography. All proteins were dialyzed against PBS and further purified by gel filtration (Amicon Ultra, Millipore). Purity was assessed by SDS-PAGE followed by Coomassie blue staining and was estimated to be greater than 90%.

using restriction enzyme digestion, followed by southern blot analysis using additional probes corresponding to the 5# and the 3# regions of each FcγR murine gene to determine gene orientation. Results found were crosschecked by multiple alignments (MegAlign: DNAstar, Lasergene) using our cDNA and BAC clone sequencing results and the mouse Ensembl database sequences for mouse chromosome 1: the FcγRIV gene mapping and orientation corresponds to the Ensembl gene NM_144559 but differs from the results Mechetina et al. (2002) for positioning and orientation. Flow Cytometric Analysis of Ca2+ Mobilization The intracellular-free calcium concentration was determined by preloading 1 × 106 DT40 cells with 5 mM Fluo-3 AM (Molecular Probes, Eugene, OR) in the presence of 0.2% Pluronic F-127 (Sigma, St. Louis, MO) for 30 min at room temperature. Cells were washed three times in RPMI 1640 and resuspended at 1 × 106 cells/ ml in complete medium, followed by incubation with IgG1 or IgG2a ICs as described above; the intracellular-free calcium concentration was monitored with a flow cytometer. The mean [Ca2+]i was evaluated with FCS assistant 1.2.9 b software (Becton Dickinson). SPR Analysis A Biacore 3000 biosensor system was used to assay the interaction of soluble Fcγ receptors II, III, and IV with the indicated antibody isotypes. Antibodies or BSA as a control protein were immobilized at high and low densities to flow cells of CM5 sensor chips (Biacore) by standard amine coupling as suggested by the manufacturer. Soluble Fcγ receptors were injected at five different concentrations through flow cells at room temperature in HBS-EP running buffer (10 mM Hepes, [pH 7.4], 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20) at a flow rate of 30 ␮l/min. Soluble Fc receptors were injected for 3 min, and dissociation of bound molecules was observed for 15 min. Background binding to control flow cells was subtracted automatically. Control experiments were performed to exclude mass transport limitations (data not shown). Affinity constants were derived from sensorgram data by using simultaneous fitting to the association and dissociation phases and global fitting to all curves in the set. As described for soluble human Fc receptors, a 1:1 Langmuir binding model closely fitted the observed sensorgram data and was used in all experiments (Maenaka et al., 2001). Alternatively, soluble Fc receptors were immobilized to sensor chips with the same result described above. Experimental Immune Thrombocytopenia Mice (2–4 months) were injected intravenously with 3.5 ␮g of the recombinant 6A6-IgG2b variant diluted in 200 ␮l of PBS. Alternatively, mice were injected with 5.5 ␮g of the 6A6-IgG1 antibody variant. As described before, for other antibody switch variants (Azeredo da Silveira et al., 2002) the IgG1 isotype proved to be less efficient in mediating platelet depletion and had to be injected at a slightly higher dose. Platelet counts before injection and at indicated time points after injection were determined by blood collection (40 ␮l) from the retro-orbital plexus and measuring platelet counts of a 1:10 dilution in PBS/5%BSA in an Advia 120 hematology system (Bayer). Alternatively, mice were injected 30 min before administration of the 6A6 antibody variants with 200 ␮g of the blocking FcγRIV antibodies 2C6 or 9E9 or with 200 ␮g of hamster isotype control antibodies (Pharmingen).

Supplemental Data Supplemental Data include three figures and are available with this article online at http://www.immunity.com/cgi/content/full/23/1/41/ DC1/. Acknowledgments

Gene Mapping C57BL/6 genomic DNA BAC clone libraries (IncyteGeomics) were screened by using the 32P-labeled cDNA encoding the first extracellular domain of mFcγRIV as a probe. Positive clones were found to be the same as the ones positive for murine FcγRIIB and/or murine FcγRIII genes. Precise gene mapping for FcγRIV was done by

We are grateful to S. Burke, L. Huang, and S. Johnson (Macrogenics Inc, Rockville, MD) for purified mFcγRIV fusion proteins and cDNA constructs; B. Heyman and A. Cazenave for monoclonal antibodies; H. Fukuyama for mouse Fc constant regions; O. Malbec, M. Daëron, Betty Diamond, and Matthew Scharff for helpful discus-

Immunity 50

sions and reagents; Dr. B. Coller for providing access to the Advia hematology system; and the members of the Ravetch laboratory for technical assistance, helpful discussions, and suggestions. F.N. is supported by the Deutsche Forschungsgemeinschaft (Grant NI711/2-1). P.B. is the Margaret Dammann Eisner-Irvington Institute Postdoctoral fellow. This work was supported in part by grants from the National Institutes of Health to J.V.R. Received: February 24, 2005 Revised: May 4, 2005 Accepted: May 11, 2005 Published: July 26, 2005 References Azeredo da Silveira, S., Kikuchi, S., Fossati-Jimack, L., Moll, T., Saito, T., Verbeek, J.S., Botto, M., Walport, M.J., Carroll, M., and Izui, S. (2002). Complement activation selectively potentiates the pathogenicity of the IgG2b and IgG3 isotypes of a high affinity antierythrocyte autoantibody. J. Exp. Med. 195, 665–672.

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