Binding of IgG Abs to FcγRs on immune cells induces FcγR cross-linking that leads to cellular effector functions, such as phagocytosis, Ab-dependent cellular cytotoxicity, and cytokine release. However, polymorphisms in low affinity FcγRs have been associated with altered avidity toward IgG, thereby substantially impacting clinical outcomes of multimodular therapy when targeting cancer or autoimmune diseases with mAbs as well as the frequency and severity of autoimmune diseases. In this context, we investigated the consequences of three nonsynonymous single nucleotide polymorphisms (SNPs) for the high affinity receptor for IgG, FcγRI. Only SNP V39I, located in the extracellular domain of FcγRI, reduces immune-complex binding of FcγRI whereas monomeric IgG binding is unaffected. This leads to reduced FcγRI effector functions, including Fc receptor γ-chain signaling and intracellular calcium mobilization. SNPs I301M and I338T, located in the transmembrane or intracellular domain, respectively, have no influence on monomeric IgG or immune complex binding, but FcRγ signaling is decreased for both SNPs, especially for I338T. We also found that the frequency of these SNPs in a cohort of healthy Dutch individuals is very low within the population. To our knowledge, this study addresses for the first time the biological consequences of SNPs in the high affinity FcγR, and reveals reduction in several FcγRI functions, which have the potential to alter efficacy of therapeutic Abs.

The Fc receptors bind the constant domain (Fc) of Abs. Ab-opsonized pathogens or cells can cross-link Fc receptors on leukocytes, leading to effector functions like phagocytosis, Ab-dependent cellular cytotoxicity, and cytokine release. In this way, Fc receptors are essential bridges between the adaptive and innate immune system. Genetic polymorphisms of IgG receptors (FcγR) have been described and extensively characterized for several members of this family, including FcγRIIA, FcγRIIIA, and FcγRIIIB (14). These single nucleotide polymorphisms (SNPs) can alter ligand binding and thereby influence the outcome of immune responses.

For both FcγRIIA and FcγRIIIA, one functional SNP has been described: 131H/R (histidine/arginine) for FcγRIIA and 158F/V (phenylalanine/valine) for FcγRIIIA. FcγRIIA-131HH has a higher binding capacity for IgG2 than FcγRIIA-131RR (5). Functionally, individuals homozygous for H131 have increased responses against opsonized bacteria, including increased phagocytosis, degranulation, and cytokine release, compared with individuals homozygous for R131 (68). Furthermore, antitumor responses mediated by the therapeutic mAb rituximab were higher in patients with FcγRIIA-131HH (9, 10). For FcγRIIIA, 158VV has a higher avidity for IgG1, IgG3, and IgG4 compared with 158FF (2). Consequently, IgG-stimulated NK cells from individuals with FcγRIIIA-158VV had higher intracellular calcium mobilization, NK cell activation, and apoptosis induction in target cells compared with FcγRIIIA-158FF (11). The FcγRIIIA 158F/V SNP is also associated with the clinical outcome of mAb treatment against cancer. Patients with FcγRIIIA-158VV have better responses and progression-free survival after treatment with rituximab, trastuzumab, or cetuximab (9, 1214). In contrast, the FcγRIIIA-158FF genotype is associated with increased incidence of autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus (11, 15). Thus, these FcγR SNPs appear to directly impact the effectiveness of immune responses in humans.

FcγRI is the high affinity IgG receptor and is expressed on monocytes, macrophages, eosinophils, and dendritic cells, as well as on neutrophils after activation. Expression of FcγRI is upregulated within hours on neutrophils during sepsis and can very efficiently be used as a biomarker for sepsis or systemic infection (16). In the plasma membrane, FcγRI associates with FcRγ, which contains the ITAMs required for intracellular signaling after receptor cross-linking (17). For many years, FcγRI has been far less studied than the low affinity IgG receptors. In part, this is caused by the dogma that FcγRI is always saturated with monomeric IgG due to its high affinity, and thereby not able to participate in mAb therapy or other immune responses. However, FcγRI can very efficiently bind to immune complexes (IC) even when preoccupied with monomeric IgG (18). Cytokines can increase this IC binding greatly, leading to a specific activation of FcγRI at the site of infection or inflammation (18). Furthermore, several studies have implicated an important role for FcγRI during inflammation, autoimmune responses, and mAb immunotherapy in tumor models (1922). In addition, FcγRI can efficiently induce MHC class II Ag presentation and cross-presentation (23, 24).

The gene encoding human FcγRI (FCGRIA) is located on human chromosome 1q21. Two very similar genes, FCGR1B and FCGR1C, are considered pseudogenes of FCGRIA and most likely originate from gene duplication. Only transcription of FCGR1A leads to the human FcγRI protein because the two pseudogenes contain stop codons in the extracellular domain (EC) 3 and are barely transcribed (25). FCGR1A encodes for six exons: the signal peptide is encoded by the first two exons; EC1, 2, and 3 are encoded by exon 3, 4, and 5 respectively; and exon 6 encodes the transmembrane region and intracellular domain. Several polymorphisms have also been identified for FcγRI: three nonsynonymous SNPs (rs7531523, V39I; rs12078005, I301M; and rs142350980, I338T), and two SNPs resulting in stop codons (rs74315310, R92*; and rs1338887, Q224*) (26, 27). Until now, the functional consequences of these three nonsynonymous SNPs have not been studied (26), although they might have important consequences as was shown for the low affinity FcγR single nucleotide changes. Therefore, we explored the frequency of these SNPs in a cohort of healthy Dutch volunteers and the possible differences in receptor function.

Blood was obtained from healthy donors at the University Medical Center Utrecht and mononuclear cells were isolated using Ficoll density centrifugation (GE Healthcare). Frozen mononuclear cells were used for DNA isolation via the MagnaPure Compact System (Roche Diagnostics). Cell samples were thawed at 37°C, dissolved in 9 ml RPMI 1640 (Life Technologies) supplemented with 20% heat-inactivated FCS (Bodinco), and centrifuged for 10 min at 380 × g. Prior to DNA extraction, cells were dissolved in PBS (Sigma-Aldrich) at a concentration of 5 × 106 cells/ml.

PCR reactions were designed for genotyping FcγRI SNPs using genomic DNA as template. Two regions of the FCGR1A gene were sequenced: exon 3 and exon 6, encoding for the EC1 and transmembrane/cytosolic domain, respectively. For exon 3, the forward primer 5′-CCCAACACCCAGATGAGGAT-3′ and reverse primer 5′-GGAGAGAACGCAGAGGAAGA-3′ were used. For exon 6, the forward primer 5′-CCAAACATAACTCAGCTAGACC-3′ and reverse primer 5′-GGACGGTCCAGATCGATG-3′ were used. Both PCR reactions were performed with 100 ng DNA, 10 pmol of each primer, 10 nmol 2'-deoxynucleoside 5'-triphosphates, 10 pmol of MgCl2, 1× Phusion HF buffer, and 2 U of Phusion HF Polymerase in a 25 μl reaction volume (New England Biolabs). PCR started with 98°C for 3 min, 30 cycles of denaturing at 98°C for 15 s, annealing at 65°C for 30 s, and extension at 72°C for 1 min with a final extension at 72°C for 10 min. PCR products were analyzed on a 1% agarose gel for amplification of the correct product size (538 bp for exon 3; 389 bp for exon 6). One microliter of the PCR reaction and 10 pmol primer were combined in 10 μl final volume and the sequencing reactions were performed by EZ-Seq (Macrogen).

The retroviral vector pMX human FcγRI was described previously (28). Using site-directed mutagenesis, the FcγRI SNPs were introduced into this plasmid. The following mutagenesis primers were used (nucleotide changes are underlined): for V39I (nucleotide G115A), 5′-GTTCCAAGAGGAAACCATAACCTTGCACTGTGAG-3′; for I301M (nucleotide A903G), 5′-CTATCTGGCAGTGGGAATGATGTTTTTAGTGAACAC-3′; and for I338T (nucleotide T1013C), 5′-GGTCATGAGAAGAAGGTAACTTCCAGCCTTCAAGAAG-3′. Given here are the forward primers, for each reaction the reverse complement primers were also used.

Ba/F3 (murine pro-B cell line) and HEK293T cells were cultured in RPMI 1640 medium (RPMI 1640 Glutamax; Life Technologies) supplemented with 10% FCS, penicillin/streptomycin, and 0.1 ng/ml murine IL-3 (ImmunoTools) for Ba/F3 cells. P388D1 (murine macrophage-like cell line) cells were cultured in DMEM medium (DMEM Glutamax; Life Technologies) supplemented with 10% FCS and penicillin/streptomycin. Amphotropic viral particles produced in HEK293T cells were used to transduce both Ba/F3 and P388D1 cells. Puromycin selection generated stable Ba/F3-FcγRI and P388D1-FcγRI cell populations. Expression of FcγRI was evaluated using an Alexa Fluor 647–labeled anti-FcγRI Ab (clone 10.1; BioLegend) measured on a FACSCanto II (BD Biosciences).

To evaluate the expression of murine FcγR on Ba/F3 cells, cells were incubated with RPE-labeled anti-mFcγRI (clone X54-4/7.1.1; BioLegend), Alexa Fluor 647–labeled anti-mFcγRII Ab (clone K9.361, Ly17.2 specific, own production), or allophycocyanin-labeled anti-mFcγRIV Ab (clone 9E9; BioLegend) for 45 min at 4°C. For staining of mFcγRIII, cells were first incubated with unlabeled anti-mFcγRII Ab (clone K9.361, Ly17.2 specific, own production) for 45 min at 4°C. After washing the cells, FITC-labeled anti-mFcγRII/III Ab (clone 2.4G2; BD Biosciences) was added for 45 min at 4°C. As a positive control, P388D1 cells were stained with these Abs. After washing, the cells were fixed with 1% paraformaldehyde (PFA) (Sigma) and fluorescence intensity was measured on a FACSCanto II.

To measure monomeric IgG binding, 1 × 105 Ba/F3-FcγRI cells were incubated with different concentrations of human IgG1 (hIgG1) [hIgG1 anti–respiratory syncytial virus F-protein, no F(ab) binding; Abbott] or mouse IgG2c [mouse IgG2c anti-human CD20, no F(ab) binding; own production] for 1 h at 4°C followed by two washes with PBS. The cells were then incubated with saturating concentrations of PE-conjugated goat–anti-human IgG (Southern Biotech) or allophycocyanin-conjugated goat–anti-mouse IgG (H and L chains) (Southern Biotech) for 45 min at 4°C. After washing, the cells were fixed with 1% PFA and fluorescence intensity was measured on a FACSCanto II.

Soluble IC were generated by combining 10 μg/ml hIgG1 with 5 μg/ml allophycocyanin-conjugated goat F(ab)2 anti-human IgG F(ab)2 (Jackson ImmunoResearch) in PBS, followed by a 2× dilution series. The Abs were incubated at 37°C for 30 min, followed by 10 min incubation at 4°C. A total of 1 × 105 Ba/F3-FcγRI cells were combined with the soluble IC, mixed well, and incubated for 1 h at 4°C followed by two washes with PBS. The cells were fixed with 1% PFA and fluorescence intensity was measured on a FACSCanto II.

The rosette assay using Dynabeads was adapted from a previously described protocol (29). Epoxy-Dynabeads (4.5 μm) were coupled to DNP24-BSA following the manufacturer’s instructions (Thermo Fisher Scientific). DNP24-BSA Dynabeads were opsonized with rabbit IgG anti-DNP (polyclonal Ab; Vector Laboratories) using the indicated concentrations. A total of 1 × 105 Ba/F3-FcγRI cells were combined with 3.5 × 105 beads and incubated for 1 h at 4°C on a shaker. Afterwards, cells were fixed with 3% PFA. Rosette formation was evaluated using bright-field microscopy at 20× magnification; cells bound to five or more beads were defined as rosettes. For each condition, triplicates were measured and on average 400–600 cells were counted per well.

Mouse FcγRI/III−/− mice on C57BL/6 background were kindly provided by Dr. J. S. Verbeek and maintained in the Animal Facility of University Medical Center Utrecht. Experiments were approved by the Animal Ethical Committee of the University Medical Center Utrecht. Bone marrow (BM) cells were harvested from mFcγRI/III−/− mice and the RBCs were lysed. A total of 3 × 106 BM cells were seeded in a six-well plate. BM cells were cultured in RPMI 1640 medium supplemented with 10% FCS, penicillin/streptomycin, and 10 ng/ml GM-CSF (Immunex). Amphotropic viral particles produced in HEK293T cells were used to transduce the adherent BM cells on days 3 and 4 with human FcγRI (wild type [WT] or SNPs). Medium was refreshed on these days and again on day 7. At day 8, adherent cells were harvested with 50 mM EDTA. The BM-derived macrophages (BMDMs) were used in the IgG-coated beads assay, described above. The IC binding was corrected for the percentage FcγRI-positive BMDMs, as measured by flow cytometry.

Microtiter plates were coated with 10 μg/ml DNP24-BSA (Thermo Fisher Scientific) overnight and incubated with 0, 1, or 10 μg/ml of rabbit IgG anti-DNP for 1 h to generate IC. A total of 7.5 × 105 Ba/F3-FcγRI cells or 2.5 × 105 P388D1-FcγRI were placed onto the IC for 5 min at 37°C and afterward lysed in 20 μl of hot reducing Laemmli sample buffer. Total lysates from 1.5 × 106 Ba/F3-FcγRI cells or 0.5 × 106 P388D1-FcγRI cells were subjected to SDS-PAGE and Western blotting. The following Abs were used: p-ERK (clone E10; Cell Signaling), total ERK (clone 3A7; Cell Signaling), and α-tubulin (clone YL1/2; Merck Millipore). For detection of the p-ERK and total ERK Abs, goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) was used; for detection of α-tubulin, goat anti-rat IgG-HRP (Santa Cruz Biotechnology) was used. After detection of p-ERK, the blot was washed and reprobed for α-tubulin detection. For quantification of p-ERK levels, nonsaturated images were used. The p-ERK signal was divided by the α-tubulin signal of the same lane and p-ERK levels of FcγRI-WT were set to one.

For calcium assays, 5 × 105 Ba/F3-FcγRI cells were incubated for 20 min with 8 μM Fluo-3 and 10 μM Fura Red (Invitrogen) at 37°C. Next, anti-FcγRI Ab (clone m22, own production) was added and cells were incubated for 10 min at 37°C. Cells were washed twice and resuspended in HBSS supplemented with 10% FCS. Cytosolic calcium levels were measured on a FACSCanto II as the ratio of Fluo-3/Fura Red (cytosolic Ca2+ level). After 30 s of baseline measurement, a goat anti-mouse IgG Ab (Southern Biotech) was added and calcium flux was measured for 3.5 min. Subsequently, 2 μg/ml ionomycin (Calbiochem) was added as a loading control. Area under the curve (AUC) was calculated using FlowJo and GraphPad Prism 6 software and AUC of FcγRI-WT was set to one.

Statistical analysis was performed using GraphPad Prism 6 software. Multiple comparison ANOVA was used to compare each FcγRI SNP to FcγRI-WT. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All graphs represent mean ± SD of triplicate measurements, unless indicated otherwise.

Data from the 1000 Genomes Project Phase 3 suggest SNP frequency data for V39I, R92*, and I338T (30). However, in this project ∼400 genomes from European citizens were sequenced, and none of them were of Dutch or German individuals (30). To validate the findings in an independent Dutch cohort for SNPs located within FcγRI, we designed two PCR reactions to amplify specific regions from genomic DNA of 158 healthy donors. V39I and R92* are located in exon 3 of FCGR1A, and I301M and I338T are located in exon 6 of FCGR1A (approximate locations in the FcγRI protein are depicted in Fig. 1A). Exon 3 and exon 6 were fully sequenced and the frequency of the FcγRI SNPs is listed in Table I. In total, we found two donors that were heterozygous for the V39I SNP (GA genotype), whereas the rest of the donors were all homozygous for the major alleles. In this cohort we did not observe any of the other SNPs. The minor allele frequency we found for V39I (0.63%) corresponds to the frequency found by the 1000 Genomes Project (0.5%) (Table I). However, R92* and I338T have minor allele frequencies very similar to V39I, although we did not observe these SNPs in the Dutch cohort. Together, these data indicate, in line with data from the 1000 Genomes Project, that the frequency of FcγRI SNPs is very low within the population. Therefore, we focused on the further functional analysis of rs7531523, rs12078005, and rs142350980.

FIGURE 1.

FcγRI SNPs location and expression on Ba/F3 cells. (A) Schematic representation of the location of FcγRI SNPs in the FcγRI protein. V39I is located in EC1 of FcγRI, I301M is located in the transmembrane region that associates with the FcRγ, and I338T is located in the intracellular domain of FcγRI. Downstream signaling after FcγRI cross-linking involves phosphorylation of the FcRγ and part of the further signaling machinery is depicted. (B) Ba/F3 cells were stably transduced with FcγRI, either WT or the SNP variants. FcγRI expression was measured using flow cytometry and untransduced Ba/F3 (ctrl) cells were used as control.

FIGURE 1.

FcγRI SNPs location and expression on Ba/F3 cells. (A) Schematic representation of the location of FcγRI SNPs in the FcγRI protein. V39I is located in EC1 of FcγRI, I301M is located in the transmembrane region that associates with the FcRγ, and I338T is located in the intracellular domain of FcγRI. Downstream signaling after FcγRI cross-linking involves phosphorylation of the FcRγ and part of the further signaling machinery is depicted. (B) Ba/F3 cells were stably transduced with FcγRI, either WT or the SNP variants. FcγRI expression was measured using flow cytometry and untransduced Ba/F3 (ctrl) cells were used as control.

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Table I.
FcγRI SNP allele frequency in Dutch individuals
SNPGenotypeaPhenotypebFrequency in Dutch Individualsc
Frequency in 1000 Genomes Project Phase 3
Major AlleleMinor AlleleMajor Allele (%)Minor Allele (%)
rs7531523 G115A V39I 314 (99.37%) 2 (0.63%) 99.5 0.5 
rs74315310 C274T R92*d 316 (100%) ― 99.6 0.4 
rs12078005 A903G I301M 316 (100%) ― No data No data 
rs142350980 T1013C I338T 316 (100%) ― 99.7 0.3 
SNPGenotypeaPhenotypebFrequency in Dutch Individualsc
Frequency in 1000 Genomes Project Phase 3
Major AlleleMinor AlleleMajor Allele (%)Minor Allele (%)
rs7531523 G115A V39I 314 (99.37%) 2 (0.63%) 99.5 0.5 
rs74315310 C274T R92*d 316 (100%) ― 99.6 0.4 
rs12078005 A903G I301M 316 (100%) ― No data No data 
rs142350980 T1013C I338T 316 (100%) ― 99.7 0.3 

A dash (―) denotes that in this study none of the tested donors had this allele.

a

Genotype indicates major allele (left), the nucleotide position in FCGR1A (middle), and the minor allele (right).

b

Phenotype indicates the major amino acid (left), the amino acid position in the FcγRI protein (middle), and the minor amino acid (right).

c

Frequencies are based on sequencing genomic DNA of n = 158 Dutch individuals.

d

Stop codon is indicated by an asterisk (*).

To study the functional consequences of the nonsynonymous FcγRI SNPs, we generated plasmids for the different FcγRI SNPs using site-directed mutagenesis. We did not include the R92* SNP because this stop codon in EC1 does not lead to a functional protein (27). Stable surface expression of FcγRI was obtained after transduction of Ba/F3 cells (Fig. 1B, Supplemental Fig. 1A), a murine pro-B cell line used before to study FcγRI function which endogenously expresses the FcRγ (18) but does not express any murine FcγRs (Supplemental Fig. 1A). However, small differences in FcγRI expression level were observed between FcγRI-WT and FcγRI-I301M or FcγRI-I338T (Supplemental Fig. 1B). We isolated two single cell clones from the FcγRI-WT bulk population with different FcγRI expression (Supplemental Fig. 1C) to investigate the influence of these different FcγRI expression levels. As explained in Supplemental Fig. 2, we corrected data for FcγRI expression when applicable throughout this study.

Because FcγRI is a high affinity IgG receptor, monomeric IgG can efficiently bind to the receptor. We performed monomeric IgG binding assays comparing FcγRI-WT with the FcγRI SNP variants. Two different IgG isotypes, hIgG1 and mouse IgG2c, were used, of which mouse IgG2c binds with a slightly higher affinity to FcγRI than hIgG1. For both isotypes we observed a dose-dependent increase in IgG binding for all FcγRI variants. However, there were no differences in monomeric IgG binding between FcγRI-WT and any of the FcγRI SNPs (Fig. 2). This indicates that the FcγRI SNPs do not affect monomeric IgG binding of FcγRI.

FIGURE 2.

Monomeric IgG binding of FcγRI SNPs. Binding of monomeric IgG to untransduced Ba/F3 (ctrl) or Ba/F3-FcγRI cells was measured using flow cytometry. (A and B) hIgG1 or (C and D) mouse IgG2c (mIgG2c) was added in the indicated concentrations. Median fluorescence intensity (MFI) is depicted. (B and D) Monomeric ligand binding at 2.5 μg/ml is shown relative to FcγRI-WT, with WT set to 100% binding. The data represented here are corrected for the FcγRI expression on the Ba/F3 cells and one representative of n = 4 independent experiments is shown.

FIGURE 2.

Monomeric IgG binding of FcγRI SNPs. Binding of monomeric IgG to untransduced Ba/F3 (ctrl) or Ba/F3-FcγRI cells was measured using flow cytometry. (A and B) hIgG1 or (C and D) mouse IgG2c (mIgG2c) was added in the indicated concentrations. Median fluorescence intensity (MFI) is depicted. (B and D) Monomeric ligand binding at 2.5 μg/ml is shown relative to FcγRI-WT, with WT set to 100% binding. The data represented here are corrected for the FcγRI expression on the Ba/F3 cells and one representative of n = 4 independent experiments is shown.

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As monomeric IgG binding is known not to trigger downstream signaling of FcγRI or effector functions, we next investigated IC binding to the SNP variants of FcγRI. First, we generated soluble IC by incubating hIgG1 with an anti-hIgG F(ab)2-binding Ab and these soluble IC were then added to Ba/F3-FcγRI cells. Binding of FcγRI-I301M and FcγRI-I338T to soluble IC was comparable to FcγRI-WT. However, the binding of FcγRI-V39I, the only SNP located extracellularly, was significantly reduced compared with FcγRI-WT (Fig. 3A). Next, we measured the binding of FcγRI to IgG-coated beads, which are much larger IC compared with the soluble IC and better resemble opsonized bacteria or tumor cells. Ba/F3-FcγRI cells that bound five or more beads were scored as rosettes (Supplemental Fig. 3). Again, Ba/F3 cells expressing FcγRI-V39I had reduced IC binding compared with FcγRI-WT, as indicated by reduced rosette formation (Fig. 3B). As expected due to their transmembrane and intracellular location, the other two SNPs, I301M and I338T, had no effect on IC binding.

FIGURE 3.

IC binding of FcγRI SNPs. (A) Fluorescently labeled soluble IC were comprised of hIgG1 and anti-hIgG F(ab)2-binding Abs and added to untransduced Ba/F3 (ctrl) or Ba/F3-FcγRI cells at 0.5 μg/ml. Binding of soluble IC is depicted as median fluorescence intensity (MFI). The data represented here are corrected for the FcγRI expression on the Ba/F3 cells. (B) Dynabeads (4.5 μm) were coupled to DNP24-BSA and opsonized with the indicated concentrations of rabbit IgG anti-DNP, resulting in IgG-coated beads that serve as large IC. Binding of these IgG-coated beads to Ba/F3-FcγRI cells was evaluated using microscopy, and rosettes were defined as cells bound with ≥5 beads. (C) BMDMs from mFcγRI/III−/− mice were transduced with FcγRI, either WT or the SNP variants, on days 3 and 4 of culture. On day 8, BMDMs were harvested and IC binding was measured using IgG-coated beads. Mock transduced BMDMs (ctrl) show no IC binding. One representative of n = 3 independent experiments is shown. ***p < 0.001, ****p < 0.0001.

FIGURE 3.

IC binding of FcγRI SNPs. (A) Fluorescently labeled soluble IC were comprised of hIgG1 and anti-hIgG F(ab)2-binding Abs and added to untransduced Ba/F3 (ctrl) or Ba/F3-FcγRI cells at 0.5 μg/ml. Binding of soluble IC is depicted as median fluorescence intensity (MFI). The data represented here are corrected for the FcγRI expression on the Ba/F3 cells. (B) Dynabeads (4.5 μm) were coupled to DNP24-BSA and opsonized with the indicated concentrations of rabbit IgG anti-DNP, resulting in IgG-coated beads that serve as large IC. Binding of these IgG-coated beads to Ba/F3-FcγRI cells was evaluated using microscopy, and rosettes were defined as cells bound with ≥5 beads. (C) BMDMs from mFcγRI/III−/− mice were transduced with FcγRI, either WT or the SNP variants, on days 3 and 4 of culture. On day 8, BMDMs were harvested and IC binding was measured using IgG-coated beads. Mock transduced BMDMs (ctrl) show no IC binding. One representative of n = 3 independent experiments is shown. ***p < 0.001, ****p < 0.0001.

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To test the function of these FcγRI SNPs in primary myeloid-derived cells, mouse BMDM from mFcγRI/III−/− mice were transduced with FcγRI-WT or the FcγRI SNP variants. Expression of FcγRI was confirmed by flow cytometry (data not shown) and BMDMs were incubated with IgG-coated beads to measure IC binding. Whereas mock transduced BMDMs did not bind IC, FcγRI-WT BMDMs efficiently bound IC in a concentration-dependent manner (Fig. 3C). BMDMs expressing FcγRI-V39I had significantly lower IC binding compared with FcγRI-WT, whereas BMDMs expressing FcγRI-I301M or FcγRI-I338T had similar IC binding as FcγRI-WT (Fig. 3C). Together, these data indicate that the only the FcγRI SNP located in the EC of FcγRI, V39I, reduces IC binding.

Cross-linking of FcγRI induces phosphorylation of the ITAMs in FcRγ by kinases of the Src family. This leads to recruitment of SYK to the ITAMs and initiation of downstream signaling, including a rapid increase in intracellular Ca2+ concentrations. This rise in Ca2+ levels is considered relevant for FcγR-mediated phagocytosis (31). We hypothesized that reduced IC binding of FcγRI-V39I would also lead to reduced downstream receptor signaling. In addition, SNPs located in the transmembrane and intracellular domain of FcγRI might also impact signaling. To study this, Ba/F3-FcγRI cells were loaded with calcium dyes and intracellular calcium levels were measured using flow cytometry. Within seconds after inducing cross-linking in FcγRI, the intracellular Ca2+ levels increased and this effect lasts for ∼2 min (Fig. 4A). Quantification of multiple experiments based on the AUC indeed showed a decreased intracellular Ca2+ flux of FcγRI-V39I compared with FcγRI-WT, indicating reduced FcRγ signaling. Interestingly, whereas the intracellular Ca2+ flux of FcγRI-I301M was also less compared with FcγRI-WT (similar to FcγRI-V39I), cross-linking of FcγRI-I338T resulted in an even stronger reduction (∼50%) in intracellular Ca2+ flux compared with FcγRI-WT (Fig. 4B), most likely due to its close proximity with the further signaling machinery (Fig. 1A). As a control, all Ba/F3-FcγRI–expressing cells showed an equally strong increase in intracellular Ca2+ levels after addition of ionomycin (Supplemental Fig. 4A).

FIGURE 4.

FcγRI cross-linking induced intracellular calcium mobilization and ERK phosphorylation. (A) Intracellular calcium mobilization kinetics in Ba/F3-FcγRI cells. Cells were loaded with calcium dyes and FcγRI-dependent calcium flux was induced using an anti-FcγRI Ab (m22), cross-linked with a goat anti-mouse Ab (addition of this Ab is indicated by the arrow). (B) Summary of four to six independent experiments (mean ± SEM) comparing AUC of the calcium flux. FcγRI-WT was set to one. (C) Western blot analysis of Ba/F3-FcγRI after 5 min cross-linking by plate-bound IC of the indicated concentrations. Cell lysates were analyzed for total ERK 1/2 (p42/p44), p-ERK 1/2, and α-tubulin. (D) Quantification of p-ERK 1/2 levels. The p-ERK signal was divided by the α-tubulin signal of the same lane and p-ERK levels of FcγRI-WT were set to one. Data from n = 3 independent experiments were combined. (E) Western blot analysis of P338D1-FcγRI after 5 min cross-linking by 10 μg/ml plate-bound IC. (F) Quantification of P388D1-FcγRI p-ERK 1/2 levels. Data from n = 4 independent experiments were combined. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4.

FcγRI cross-linking induced intracellular calcium mobilization and ERK phosphorylation. (A) Intracellular calcium mobilization kinetics in Ba/F3-FcγRI cells. Cells were loaded with calcium dyes and FcγRI-dependent calcium flux was induced using an anti-FcγRI Ab (m22), cross-linked with a goat anti-mouse Ab (addition of this Ab is indicated by the arrow). (B) Summary of four to six independent experiments (mean ± SEM) comparing AUC of the calcium flux. FcγRI-WT was set to one. (C) Western blot analysis of Ba/F3-FcγRI after 5 min cross-linking by plate-bound IC of the indicated concentrations. Cell lysates were analyzed for total ERK 1/2 (p42/p44), p-ERK 1/2, and α-tubulin. (D) Quantification of p-ERK 1/2 levels. The p-ERK signal was divided by the α-tubulin signal of the same lane and p-ERK levels of FcγRI-WT were set to one. Data from n = 3 independent experiments were combined. (E) Western blot analysis of P338D1-FcγRI after 5 min cross-linking by 10 μg/ml plate-bound IC. (F) Quantification of P388D1-FcγRI p-ERK 1/2 levels. Data from n = 4 independent experiments were combined. *p < 0.05, **p < 0.01, ***p < 0.001.

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Besides a rise in intracellular Ca2+ levels, Fc receptor–mediated triggering of ITAM signaling leads to activation of the MAPK–ERK pathway (31). To determine the activation of this pathway in Ba/F3-FcγRI cells, plate-bound IC were used to induce FcRγ signaling through FcγRI cross-linking on Ba/F3 cells. After 5 min of incubation, ERK phosphorylation was strongly induced in a concentration-dependent manner (Fig. 4C). Cross-linking of all three FcγRI SNPs resulted in decreased p-ERK levels compared with FcγRI-WT (Fig. 4D). Similar to the intracellular Ca2+ flux, this decrease was most pronounced for FcγRI-I338T.

To confirm these findings in a myeloid cell line, we transduced P388D1 cells, a macrophage-like cell line, with the human FcγRI constructs and measured downstream signaling. Because these cells, like mouse BMDMs, do not induce a measurable intracellular Ca2+ flux upon Fc receptor cross-linking (data not shown), we studied ERK phosphorylation. Stable and equal surface expression of FcγRI-WT or SNP variants was obtained after transduction of P388D1 cells (Supplemental Fig. 4B). Basal p-ERK levels were higher in P388D1-FcγRI cells compared with Ba/F3-FcγRI cells. As expected, stimulation with IC increased the ERK phosphorylation of P388D1-FcγRI WT cells (Fig. 4E). However, this increased p-ERK signal was significantly lower in all three FcγRI SNPs after receptor cross-linking by IC (Fig. 4E, 4F). Together, these data indicate that downstream signaling of FcγRI is negatively influenced by the FcγRI SNPs, both for intracellular Ca2+ mobilization and induction of the MAPK-ERK pathway.

Abs of the IgG isotype bind to FcγR on immune cells, which is essential for the induction of their cellular effector functions and efficient immune responses. Polymorphisms in these FcγRs have been associated with altered avidity toward IgG, leading to changes in immune-mediated responses of Abs. Most of the work done on FcγR SNPs focuses on the low affinity FcγRs, including FcγRIIA, FcγRIIIA, and FcγRIIIB. To our knowledge, we describe in this study for the first time functional nonsynonymous SNPs for the high affinity receptor for IgG, FcγRI. The only SNP located in the EC of FcγRI, V39I, reduces IC binding of FcγRI and this leads to reduced FcRγ signaling and intracellular calcium mobilization, suggesting that this area of FcγRI is involved in binding of IC. In line with the location of SNPs I301M and I338T, the transmembrane or intracellular domain, respectively, they have no influence on IC binding. However, these SNPs substantially reduced FcγRI signaling. This effect is most pronounced for FcγRI-I338T, suggesting that this intracellular part of FcγRI plays a key role in interacting with the intracellular signaling machinery and therefore might be interesting as a potential target to further enhance or inhibit FcγRI-mediated effects. Of note, there was no difference in p-ERK levels between the three SNPs in P388D1-FcγRI cells, although they were all decreased compared with FcγRI-WT. The basal p-ERK levels were also higher in these cells compared with Ba/F3-FcγRI cells, indicating that these signaling pathways might be differentially regulated in these myeloid cells. However, both cell lines show that all three SNPs negatively influence FcγRI downstream signaling.

The V39I SNP is located in EC1 of FcγRI and is largely buried within this structure (26). Furthermore, the IgG binding site is located in EC2 of FcγRI (32, 33), although EC1 is positioned very close to the IgG binding site. Our data indicate that monomeric IgG binding is not affected by the change of a valine to isoleucine at position 39, which is in line with how monomeric IgG binds to FcγRI. However, IC binding is reduced in the V39I variant, possibly indicating an important role for this amino acid, specifically in IC binding. It is tempting to speculate that cross-linking of multiple FcγRI by the IC requires a slightly altered FcγRI structure and that a valine at position 39 is necessary to obtain this. Interestingly, V39 appears highly conserved within the FcγR family (26), possibly implying an important role of this amino acid in structure and/or function of FcγR. Currently, it is unknown which amino acids of FcγRI interact with FcRγ. However, the reduced FcRγ signaling of the I301M variant might indicate that this isoleucine in the transmembrane region of FcγRI is involved in this interaction. It is unclear how the introduction of a threonine at position 338 in the intracellular domain of FcγRI leads to a strong reduction in FcRγ signaling. If this threonine is constitutively phosphorylated in cells, this might interfere with efficient recruitment of signaling molecules like SYK to FcRγ (Fig. 1A). Computational prediction of the phosphorylation of FcγRI-I338T indicates that several serine/threonine kinases, including MAP kinase-activated protein kinase 5, nuclear dbf2-related kinase, and tyrosine kinase-like, might phosphorylate threonine 338 (data not shown).

The frequency of these FcγRI SNPs is quite low in the population. In Dutch individuals, we observed two V39I heterozygotes in 158 healthy donors (0.63%). The frequency of V39I reported by the 1000 Genomes Project Phase 3 (0.5%) corresponds with the frequency we observed. However, in this data set the V39I SNP is only observed within the African population (30), which might indicate that the two heterozygous donors in our cohort are of African ancestry. The frequency of I301M is 0% in our cohort and no frequency data are available in the 1000 Genomes Project. The frequency of the I338T SNP is comparable to the V39I SNP according to the 1000 Genomes Project (0.3%), with the highest prevalence in the European population (30). We did not observe the I338T SNP in our cohort, which might be explained by the smaller size (158 donors) compared with the 1000 Genomes Project (2504 donors) or the fact that no Dutch/German individuals were part of this larger project. The very low frequency of the FcγRI SNPs might indicate that having these less-functional variants of FcγRI is evolutionary unfavorable. However, four individuals from one family lacking expression of FcγRI, caused by the R92* premature stop codon, do not appear to have increased susceptibility for infections or autoimmune diseases (27). Nonetheless, in these individuals it is likely that redundancy within the FcγR family compensates for the FcγRI deficiency.

Our data indicate that naturally occurring functional SNPs of FcγRI exist, albeit with very low prevalence. Although the impact of these individual SNPs is low due to the low frequency, this study allowed to get major insights into the functional role of different parts of FcγRI. The area around V39 in EC1 is most likely an important binding site for IC or it affects the folding of the extracellular parts of FcγRI, whereas the intracellular region containing I338 has a major role in downstream signaling of FcγRI. Both mutations are therefore most likely avoided in the general population as they are associated with a functional disadvantage. Together, these insights in FcγRI biology will allow the development of more targeted therapies to either enhance or decrease activity of FcγRI in the context of patients receiving Abs as treatment.

We thank Dr. Eric Spierings for helpful discussions on SNP analysis and sequencing data, and Dr. Sjef Verbeek for providing mFcγRI/III−/− mice. In addition, we thank Jessica Anania and Prof. Mark Hogarth for input on IC binding, and Willemijn Janssen and Karin van Veghel for assistance with the intracellular calcium measurements.

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • AUC

    area under the curve

  •  
  • BM

    bone marrow

  •  
  • BMDM

    BM-derived macrophage

  •  
  • EC

    extracellular domain

  •  
  • hIgG1

    human IgG1

  •  
  • IC

    immune complex

  •  
  • PFA

    paraformaldehyde

  •  
  • SNP

    single nucleotide polymorphism

  •  
  • WT

    wild type.

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The authors have no financial conflicts of interest.

Supplementary data