Phenotypic Variation in IgG Receptors by Nonclassical FCGR2C Alleles Joris van der Heijden, Willemijn B. Breunis, Judy Geissler, Martin de Boer, Timo K. van den Berg and Taco W. This information is current as Kuijpers of October 2, 2021. J Immunol 2012; 188:1318-1324; Prepublished online 23 December 2011; doi: 10.4049/jimmunol.1003945 http://www.jimmunol.org/content/188/3/1318 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Phenotypic Variation in IgG Receptors by Nonclassical FCGR2C Alleles

Joris van der Heijden,* Willemijn B. Breunis,*,† Judy Geissler,* Martin de Boer,* Timo K. van den Berg,* and Taco W. Kuijpers*,†

The balance between activating and inhibitory signals from the different FcgRs for IgG ensures homeostasis of many inflammatory responses. FCGR2C is the product of an unequal crossover of the FCGR2A and FCGR2B genes encoding the activating FcgRIIa (CD32a) and inhibitory FcgRIIb (CD32b), respectively. A single nucleotide polymorphism (SNP) in exon 3 of FCGR2C results in either expression of the activating FcgRIIc (CD32c) (FCGR2C-open reading frame [ORF]) or its absence because of a stop codon (FCGR2C-Stop). Two additional variations in FcgRIIb/c expression on leukocytes have now been identified. In case of “nonclas- sical” FCGR2C-ORF alleles, FcgRIIc expression was unexpectedly absent, because of novel splice site mutations near exon 7 leading to another stop codon. In some individuals with FCGR2C-Stop alleles FcgRIIb was detected on NK cells, which normally Downloaded from are devoid of this protein. Individuals with these nonclassical FCGR2C-Stop alleles carried a deletion of FCGR2C-FCGR3B that extends into the promoter region of the adjacent FCGR2B gene and probably deletes a negative regulatory element in the FCGR2B promoter in NK cells. FcgRIIb expression on NK cells effectively inhibited killing mediated by FcgRIIIa (CD16a) in Ab-dependent cytotoxicity tests. Our findings demonstrate a more extensive and previously unnoticed variation in FcgR expres- sion with relevance to immunity and inflammation. The Journal of Immunology, 2012, 188: 1318–1324. http://www.jimmunol.org/ cgRs are receptors for the Fc region of IgG and are Because of the very high homology between the genes, it is expressed on various cell types, including macrophages, a difficult gene cluster to study. For instance, FCGR2A and F dendritic cells, neutrophils, NK cells, B lymphocytes, and FCGR2B have ∼95% homology in their exons coding for the platelets. The binding of IgG-opsonized pathogens to FcgRs trig- extracellular domains of FcgRIIa and FcgRIIb, respectively (2). gers a variety of cellular responses and contributes to inflamma- The activating FcgRIIa is expressed by neutrophils, monocytes, tion. FcgRs are also involved in Ab-mediated tumor cell elimi- macrophages, dendritic cells, and platelets, in which it mediates nation. The balance between activating and inhibitory signals various functions such as phagocytosis and Ab-dependent cellular from the different receptor subtypes ensures control over immune cytotoxicity (ADCC). The inhibitory FcgRIIb exists in two splice complex-driven inflammatory responses (1). Depending on their variants, namely, FcgRIIb1 (which is exclusively expressed by by guest on October 2, 2021 affinity for IgG, the receptors can be divided into low-affinity B cells) and FcgRIIb2 (expressed by B cells, macrophages, and FcgRs, which can be subdivided into FcgRIIa (CD32a), FcgRIIb dendritic cells). FcgRIIb inhibits signaling of ITAM-associated (CD32b), FcgRIIc (CD32c), FcgRIIIa (CD16a), and FcgRIIIb receptors by recruitment of phosphotyrosine phosphatases that (CD16b), and the high-affinity FcgRI (CD64). In humans, FcgRIIb dephosphorylate the tyrosine residues in several ITAM-induced is the only inhibitory FcgR, because it contains an ITIM in its activation pathway effectors (3). cytoplasmic tail. All other FcgRs signal through an ITAM, either Single nucleotide polymorphisms (SNPs) have been described encoded within their cytoplasmic tail or within associated adaptor in both FCGR2A and FCGR2B. A functionally relevant SNP in proteins such as the FcRg. FCGR2A leads to either a histidine or an arginine in the ligand- The genes encoding the FcgRs are located on chromosome 1, of binding domain of FcgRIIa (H/R131) (4). This results in differ- which the genes FCGR2A, FCGR2B, and FCGR2C are located in ences in binding affinity for human IgG2 and murine IgG1 (4, 5). one gene cluster with the FCGR3A and FCGR3B genes. In FCGR2B, one SNP leads to either an isoleucine or a threonine in the transmembrane region of FcgRIIb (I/T232). The T232 al- *Sanquin Research and Landsteiner Laboratory, Department of Blood Cell Research, lotype has been suggested to result in functional impairment Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam, The through exclusion from lipid rafts (6, 7). Additional SNPs in the † Netherlands; and Department of Pediatric Hematology, Immunology and Infectious FCGR2B Disease, Emma Children’s Hospital, Academic Medical Center, 1105 AZ Amster- promoter region influence transcriptional activity of dam, The Netherlands (and FCGR2C) (8, 9). FCGR2C, encoding FcgRIIc, appears to be Received for publication December 7, 2010. Accepted for publication November 22, the product of an unequal crossover event between FCGR2A and 2011. FCGR2B (10). FCGR2C contains eight exons, which are highly This work was partially supported by a grant from ZorgOnderzoek Nederland–Medi- homologous to exons 1–4 from FCGR2B and exons 5–8 from sche Wetenschapp (920-03-391) (to W.B.B.). FCGR2A. An SNP in exon 3 of FCGR2C determines the func- Address correspondence and reprint requests to Joris van der Heijden, Sanquin Re- tional expression of this gene. This SNP results in either an open search, Department of Blood Cell Research, Room U202, Plesmanlaan 125, 1066CX Amsterdam, The Netherlands. E-mail address: [email protected] reading frame (FCGR2C-ORF) or the more common stop codon Abbreviations used in this article: ADCC, Ab-dependent cellular cytotoxicity; CNV, (FCGR2C-Stop), in which case FCGR2C represents a pseudogene copy number variation; GUS, b-glucuronidase; IP, immunoprecipitation; MLPA, (11). FcgRIIc is expressed on NK cells from FCGR2C-ORF multiplex ligation-dependent probe amplification; ORF, open reading frame; rADCC, donors, acting as an activating IgG receptor capable of inducing redirected Ab-dependent cellular cytotoxicity; SNP, single nucleotide polymorphism. ADCC and a rise in Ca2+ concentration after receptor cross- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 linking (12, 13). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003945 The Journal of Immunology 1319

Differences in receptor expression levels on the cell surface can PCR and sequence analysis alter the balance between activating and inhibitory signals and Quantitative PCR analysis and sequencing of FcgRIIc transcripts, as well as therefore change the cellular response toward IgG. Apart from the gene-specific, long-range PCR analysis and sequencing of FCGR2C, SNPs, we and others have shown that several FCGR genes are were performed as described previously (12). subject to copy number variation (CNV). CNV in FCGR2C, NK cell purification FCGR3A,andFCGR3B has been shown to correlate with protein expression levels. For instance, deletion of one FCGR3A allele PBMCs were isolated from whole blood by Percoll density gradient cen- results in reduced target cell killing in an FcgRIIIa-specific trifugation. NK cells were isolated from PBMCs by MACS with CD56 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), following the ADCC (14). These SNPs and CNVs in FCGR genes have been manufacturer’s instructions. NK cell purity was verified by flow cytometry. linked with (chronic) inflammatory and autoimmune diseases (12, 15). It is therefore essential to further investigate genetic variation mRNA isolation and reverse transcription in this gene cluster, because that may be highly relevant for the mRNA was isolated from 1 3 106 purified NK cells by use of the QiaAmp discovery of disease risk factors, therapeutic efficacy of bio- RNA blood mini kit (Qiagen), according to the manufacturer’s instruc- logicals, and the development of new treatment strategies. In this tions. Subsequently, first-strand cDNA was synthesized with the Super- script III first-strand synthesis system for RT-PCR (Invitrogen, Breda, The study, we identify two additional genetic variations in the FCGR Netherlands). In short, RNA was primed with 2.5 mM oligo(dT) for 5 min gene cluster that modulate the expression of FcgRIIb and at 65˚C. Reverse transcription was performed with 10 U/ml Superscript III FcgRIIc. in the presence of 5 mM MgCl2, 20 mM Tris-HCl, 50 mM KCl (pH 8.4, reverse transcriptase buffer), 0.5 mM 29-deoxynucleoside 59-triphosphate, and 2 U/ml RNaseOUT [without DTT, for reasons described by Lekanne

Materials and Methods Deprez et al. (18)] for 50 min at 50˚C. Thereafter, Superscript III was Downloaded from Subjects inactivated by incubation for 5 min at 85˚C, followed by chilling on ice. Immediately thereafter, 2 U RNase H were added, and the mixture was Healthy adult white volunteers (n = 146) served as controls. These donors incubated at 37˚C for 20 min. Subsequently, cDNA was stored at 20˚C until were not known to have hematological disorders of any kind in the past or further use. at present. The study was approved by the Medical Ethics Committee of the Academic Medical Center and Sanquin (Amsterdam, The Netherlands) Detection and relative quantification of FcgRII and was performed in accordance with the Declaration of Helsinki. isoform-specific mRNA http://www.jimmunol.org/ Abs and reagents The detection and relative quantification of FcgRII mRNA have been described in detail elsewhere (19). In short, intron-spanning primers were The following mAbs were used in flow cytometry and for immunopre- designed to specifically amplify cDNA and exclude amplification of ge- cipitation (IP): anti-CD3 clone SK7, anti-CD14 clone M5E2 and anti- b CD56 clone B159 (BD Pharmingen, San Diego, CA), anti-FcgRII clone nomic DNA, yielding products of 100 bp for -glucuronidase (GUS), 243 g g AT10 (AbD Serotec, Oxford, U.K.), and anti-FcgRIIb/c clone 2B6 bp for Fc RIIb2, and 162 bp for Fc RIIc1 as the most abundant productive g (Macrogenics, Rockville, MD). Western blots were stained with polyclonal Fc RIIc transcript in immune cells (12). The following primers were used: b 9 9 IgG against either FcgRIIa/c [rabbit antiserum CT10; a gift from Dr. -glucuronidase (GUS), 5 -GAAAATATGTGGTTGGAGAGCTCATT-3 (forward primer) and 59-CCGAGTGAAGATCCCCTTTTTA-39 (reverse A. Verhoeven, Department of Medical Biochemistry, Academic Medical primer); FcgRIIb2, 59-GGAAAAAGCGCATTTGAGCCAATC-39 (for- Centre, University of Amsterdam (16)] or FcgRIIb (Epitomics, Burlin- ward primer) and 59-GGAAATACGAGATCTTCCCTCTCTG-39 (reverse

game, CA). Abs used in cytotoxicity assays were anti-FcgRII clone AT10 by guest on October 2, 2021 g 9 9 (azide-free; AbD Serotec) and anti-FcgRIII clone 3G8 (low-endotoxin primer); and Fc RIIc1, 5 -ATCATTGTGGCTGTGGTCACTGG-3 (for- 9 9 azide-free; BioLegend). ward primer) and 5 -CTTTCTGATGGCAATCATTTGACG-3 (reverse IP lysis buffer is PBS containing 10% glycerol (w/v), 1% Nonidet P-40 primer). Quantification of cDNA was performed with the Lightcycler In- strument (Roche Applied Science). Serial 10-fold dilutions of the cDNA (w/v), and 13 Complete, EDTA-free Protease Inhibitor Mixture (Roche were made and quantified with the method described in Technical Note Applied Science, Almere, The Netherlands). Laemmli sample buffer is No. LC 13/2001 (Roche Applied Science). 50 mM Tris-HCl (pH 6.8), 10% glycerol (v/v), 5 mM DTT (DL-DTT; Sigma-Aldrich), 1% 2-ME, 1% SDS (w/v), and 100 mg/ml bromephenol IP and Western blotting of FcgRII blue. Complete medium is IMDM (PAA, Pasching, Austria) supple- mented with 10% FCS(v/v) (Bodinco, Alkmaar, The Netherlands), IP was performed at 4˚C. Cells were pretreated with 10 mM diisopropyl penicillin (100 U/ml), streptomycin (100 mg/ml;PAA),and2mML- fluorophosphate for 10 min and lysed with IP lysis buffer for 1 h. Lysates glutamine (PAA). were centrifuged at 21,000 3 g for 15 min. Supernatants were incubated with pan-FcgRII Abs (10 mg/ml) overnight. Subsequently, they were in- Genotyping cubated with 5% protein G-Sepharose 4 Fast Flow (w/v) (GE Healthcare Genomic DNA was isolated from whole blood with the Gentra Puregene Life Sciences) for 1 h. Protein G-Sepharose was washed three times with IP lysis buffer by centrifugation at 21,000 3 g for 15 s and incubated in kit (Qiagen, Hilden, Germany), according to the manufacturer’s instruc- Laemmli sample buffer for 30 min at 95˚C. tions. Samples were run on a 12.5% Bis-Tris gel (w/v) and subsequently blotted CNV and SNPs in the low-affinity FCGR genes FCGR2A, FCGR2B, g FCGR2C, FCGR3A,andFCGR3B were determined with an FCGR-specific on a nitrocellulose membrane. Fc RIIb and -c were detected by polyclonal multiplex ligation-dependent probe amplification (MLPA) assay. The MLPA Abs against their cytoplasmic tails. Bands were visualized by ECL sub- assay was performed essentially as described previously (12, 17). The strate (Pierce, Rockford, IL). FCGR MLPA includes gene-specific probes to determine the CNV of the Cytotoxicity assays genes. It also includes probes to detect the following SNPs: FCGR2A 27Q . W (rs9427397 rs9427398), FCGR2A 131H . R (rs1801274), FCGR2B Cytotoxicity of NK cells was determined by Ab-dependent cellular cyto- 232I . T (rs1050501), FCGR2B/C –386C . G (rs3219018), –120A . T, toxicity (ADCC) and redirected ADCC (rADCC). NK cells were isolated FCGR3A 158V . F (rs396991), and FCGR3B (HNA1a/HNA1b/HNA1c). from FCGR2C-ORF, FCGR2C-Stop, and nonclassical FCGR2C-Stop The assay also contains a probe specific for the open reading frame in exon donors and kept in complete medium. Target cells were labeled with 51Cr 3 of the FCGR2C gene and a nonspecific FCGR2B/C probe to detect the for 1 h and subsequently washed three times. Cells were plated in a 96-well stop codon in exon 3. For selected donors, we designed and used probes to plate at 5 3 103 cells/well. NK cells were added at varying E:T cell ratios determine the extent of the FCGR2C/FCGR3B deletion. Probe sequences and incubated in triplicate. In case of ADCC, chimeric therapeutic mAbs are 59-GGGTTCCCTAAGGGTTGGAGAAGCATGACTCTTCTAAAAC- against CD20 (rituximab) were added at 10 mg/ml. In case of rADCC, AAGTTAATAAT-39 and 59-AATGACATCTTTTCATGAACAAGCAATTG- mouse mAbs against either human FcgRII or human FcgRIII were added AGTCTAGATTGGATCTTGCTGGCAC-39 for the region between FCGR3A at 10 mg/ml. As a control, target and NK cells were plated without Abs. and FCGR2C and 59-GGGTTCCCTAAGGGTTGGACACCATCTCAGTA- Target cells were also plated without NK cells to determine the sponta- GTAATTTCTGACACATCACTTT-39 and 59-CAGAAGGCTCATAAGCC- neous release or with saponine to determine the maximum release of 51Cr. AACTCAGATACAACCGTCTAGATTGGATCTTGCTGGCGC-39 for the The total volume per well was 100 ml. Cells were centrifuged at 300 3 g region between FCGR3B and FCGR2B. for 30 s and subsequently incubated at 37˚C and 5% CO2 for 4 h. Cells 1320 VARIATION IN IgG RECEPTORS were centrifuged again at 300 3 g for 30 s, and supernatants were col- Table II. FCGR2C allele combinations in 146 healthy white lected and counted in a gamma counter. individuals, determined by MLPA Specific killing was determined by the formula: percentage specific 3 2 killing = 100 (experimental release spontaneous release)/(maximum FCGR2C Exon 3 No. of Donors release 2 spontaneous release). Stop 8 Statistics ORF 2 Flow cytometry data were analyzed with Student t test; p , 0.05 was Stop/Stop 94 considered statistically significant. Data are expressed as mean 6 SEM. Stop/ORF 21 ORF/ORF 1 Stop/Stop/Stop 15 Results Stop/Stop/ORF 3 Mismatches between FCGR2C genotype and phenotype Stop/ORF/ORF 2 ORF/ORF/ORF 0 We have analyzed the FcgR expression by genotyping CNV and SNPs within the FCGR2A, FCGR2B, FCGR2C, FCGR3A, and FCGR3B genes by MLPA in 146 healthy adult white individuals FcgRII in which exon 6 is not spliced out is the B cell-specific (Tables I, II; data not shown) and phenotyping a selection of 47 of FcgRIIb1. Besides these most commonly found transcripts in the these individuals with different mAbs against FcgRII (pan-CD32), three nonclassical FCGR2C-ORF donors, additional noncoding FcgRIIb/c (CD32b/c), and FcgRIIIa/b (CD16). transcripts were detected as well. Premature stop codons can result There is no mAb available that can differentiate between surface- in various nonsense mRNAs. It is likely that these less frequently expressed FcgRIIb and FcgRIIc because of the completely iden- found transcripts fall in this category. In normal FCGR2C-ORF Downloaded from tical extracellular domains. NK cells express FcgRIIc but not donors, these noncoding transcripts were not detected (data not FcgRIIb (12), and these cells can therefore be used to distinguish shown). FCGR2C-Stop from FCGR2C-ORF donors at the level of surface A gene-specific, long-range PCR was performed to amplify expression with a FcgRIIb/c-specific mAb. FCGR2C from genomic DNA. Sequence analysis of the PCR When we related the genotype for FCGR2C with the FcgRIIc product revealed that all three nonclassical FCGR2C-ORF donors expression on NK cells as measured by flow cytometry (Fig. 1), → studied thus far carried a G A point mutation at the donor splice http://www.jimmunol.org/ we observed a strong and positive correlation between genotype site of intron 7, which explains a splicing defect that results in the and average protein surface expression levels. However, we also loss of exon 7. In addition to this mutation, two donors also had detected two exclusive sets of individuals that showed definite aG→C point mutation at the acceptor splice site of intron 7. In genotype–phenotype mismatches. First, we identified individuals these donors, a cryptic acceptor splice site 62 bp upstream of the who repeatedly showed no FcgRIIb/c surface expression on NK normal exon–intron boundary was used, which explains the in- cells in the presence of a classical FCGR2C-ORF genotype (group sertion of part of intron 7 in these two donors (Fig. 2C). A). Second, we identified some individuals whose NK cells were stained for FcgRIIb/c while carrying classical FCGR2C-Stop Nonclassical FCGR2C-Stop because of gene deletions in the FCGR cluster (group B) alleles, considered to be true pseudogenes (group B). by guest on October 2, 2021 Nonclassical FCGR2C-ORF by novel splice site mutations Because FCGR2C-Stop donors are expected to be unable to ex- (group A) press any isoform of FcgRII on NK cells, we were surprised to find that the NK cells from some of these healthy individuals did Expression of FcgRIIc is known to be determined by the p.Q13X bind mAb 2B6, which is specific for FcgRIIb and FcgRIIc. Al- (ORF versus Stop) mutation in exon 3 of the FCGR2C gene. In- though genotyping errors in FCGR2C were considered, there was terestingly, NK cells of some of the FCGR2C-ORF donors did not an important phenotypic difference with the binding pattern with express FcgRIIc. The lack of expression was not limited to NK the same mAb on immune cells from individuals carrying classical cells but was also apparent on myeloid cells, as indicated for FCGR2C-ORF (Fig. 3A). Neutrophils and monocytes from these monocytes and neutrophils (Fig. 2A). To investigate whether RNA four individuals from group B showed a similar lack of surface transcripts for FcgRIIc were detected in the PBMC fraction of nonclassical FCGR2C-ORF donors, PCR analysis was performed on the RNA from three donors. Sequence analysis of the PCR products revealed various FcgRIIc transcripts. Most abundantly, we found transcripts from which exon 7 was spliced out. In two of these donors, the transcripts contained in addition a 62-bp inser- tion, which was found to be part of intron 7 (Fig. 2B). Both un- usual transcripts result in a frameshift and premature stop codon. Exon 6 was spliced out from all transcripts but that is normal for myeloid cells from all FCGR2C-ORF donors. The only isoform of

Table I. Copy numbers of FCGR2B, FCGR2C, and FCGR3B

CNV (No. of Donors) FIGURE 1. Binding of FcgRIIb/c-specific mAb 2B6 to NK cells from Copies FCGR2B FCGR2C FCGR3B healthy individuals genotyped as FCGR2C-Stop or FCGR2C-ORF donors. 0 000Boxes A and B indicate subgroups with an unexpected mAb binding 1 0 10 9 pattern when considering the individuals’ genotypes. The difference in 2 146 116 121 2B6 binding between donor groups was statistically significant (***p = 3 0 20 16 0.0005). The Journal of Immunology 1321

tion. The deletion of FCGR2C and FCGR3B was found to include part of the sequence between the FCGR3B gene and the presumed promoter region of the FCGR2B gene (Fig. 3B). We hypothesized that a regulatory element in this region could be involved in repression of FcgRIIb expression and that deletion of this element results in FcgRIIb expression on NK cells. Therefore, RNA was isolated from NK cells from individuals genotyped and phenotyped as FCGR2C-ORF, FCGR2C-Stop, and nonclassical FCGR2C-Stop (group B) donors. FcgRIIb1 mRNA was not de- tectable in NK cells, but some FcgRIIb2 mRNA was found to be present in NK cells of all individuals. In subsequent quantitative PCR experiments, the relative amounts of FcgRIIb2 and the major FcgRIIc (FcgRIIc1) mRNA were determined, compared with a household gene. Upon comparison of FCGR2C-ORF and -Stop donors, the nonclassical FCGR2C-Stop donors showed strongly increased transcriptional levels of FcgRIIb2 mRNA. FcgRIIc1 mRNA was comparable between classical and nonclassical FCGR2C-Stop and was—as expected—strongly elevated in the FCGR2C-ORF donors (Fig. 3C). This led to the hypothesis that FcgRIIb and not FcgRIIc is Downloaded from expressed on the NK cells of these nonclassical FCGR2C-Stop donors. The assumption was further verified at the protein level. IPs of FcgRII with pan-FcgRII mAb AT10 were performed from lysates of highly purified NK cell fractions obtained from indi- viduals with the FCGR2C-ORF, -Stop, and nonclassical Stop signature after combined geno-/phenotyping. These immunopre- http://www.jimmunol.org/ cipitates were used for Western blotting and stained with Abs specific for the intracellular tail of either FcgRIIa/FcgRIIc (data not shown) or FcgRIIb (Fig. 3D). The experiments confirmed that NK cells from nonclassical FCGR2C-Stop donors indeed express FcgRIIb as the single FcgRII. To test whether expression of FcgRIIb on NK cells had func- tional consequences, cytotoxicity was tested in rADCC assays (Fig. 4). Killing of target cells in the presence of mAbs for by guest on October 2, 2021 FIGURE 2. Nonclassical ORF donors do not express FcgRIIc. A, FcgRIII was comparable between NK cells from all donor types. 2 Binding of FcgRIIb/c mAb 2B6 to NK cells (CD3 CD56+), monocytes As we had also previously published (11, 12), NK cells from + + (CD14 ), neutrophils, and B cells (CD19 ) compared between classical FCGR2C-ORF donors were able to kill target cells in the presence (n = 9) and nonclassical (n =3)FCGR2C-ORF donors. The differences of mAbs for FcgRII, whereas NK cells from FCGR2C-Stop in 2B6 binding between donor groups in NK cells, monocytes, and neu- donors, not able to bind these mAbs, were unable to kill under trophils were statistically significant (p = 0.004, 0.009, and , 0.001, re- these rADCC conditions (Fig. 4A). As expected, NK cells from spectively), in contrast to B cells, where this difference was not statistically significant (p = 0.27). Data are expressed as mean 6 SEM. B, Repre- nonclassical FCGR2C-Stop donors, expressing the inhibitory sentation of FcgRIIc transcripts found in PBMCs from classical and FcgRIIb, were not able to kill target cells in the presence of mAbs nonclassical FCGR2C-ORF donors. White boxes, Exons that are spliced for FcgRII (Fig. 4B). Rare individuals carrying both an FCGR2C- out. *, The location of stop codons; +62, the insertion of a 62-bp sequence ORF allele and a nonclassical FCGR2C-Stop allele will express from intron 7 (see C). C, Splice site mutations in FCGR2C intron 7 in both FcgRIIb and FcgRIIc on NK cells. We had identified nonclassical FCGR2C-ORF donors. The intronic splice sites are under- a healthy donor with this genotype. The NK cells from this in- lined. Mutated splice sites are in italics, with the altered nucleotides in dividual were unable to kill the target cells in the rADCC, despite bold. Gray boxes represent exons that are retained, and white boxes rep- expression of FcgRIIc. Thus, FcgRIIb on NK cells is unable resent exons that are being spliced out as a consequence of these muta- to mediate cytotoxicity, but effectively inhibits killing mediated tions. Cx, cytoplasmic domain; ECx, extracellular domain, MFI, mean through FcgRIIc when coexpressed and ligated in the rADCC test fluorescence intensity; Sx, signal sequence; TM, transmembrane domain. system (Fig. 4B). Because NK cells also express FcgRIIIa, the effect of FcgRIIb binding of mAb 2B6 as did normal FCGR2C-Stop donors in and FcgRIIc expression on cytotoxicity was tested in a classical contrast to the usual myeloid expression observed in classical ADCC test in which the clinically applied chimeric CD20 mAb FCGR2C-ORF donors. This difference excluded genotyping rituximab was used to opsonize the Raji B cell lymphoma cell line errors as a possible explanation of our findings. as target cell. In these experiments, we observed that the expres- Further studies of these donors in which the NK cells unex- sion of FcgRIIb on the NK cells of nonclassical FCGR2C-Stop pectedly showed CD32b/c surface expression were performed at donors resulted in decreased NK cell-mediated killing of the Raji the genomic level. MLPA analysis of DNA from these so-called target cells when preincubated with the CD20 mAb (Fig. 4C). nonclassical FCGR2C-Stop donors showed that these individuals all carried a large deletion in the FCGR locus, resulting in the Discussion combined loss of both FCGR2C and FCGR3B on one of their Expression of FcgR on NK cells is normally limited to the acti- alleles (14). MLPA probes were designed to analyze the regions vating FcgRIIIa (CD16a). We and others (12, 13) have previously located around these genes to investigate the extent of this dele- shown that NK cells from ∼20% of healthy individuals express 1322 VARIATION IN IgG RECEPTORS Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. Nonclassical FCGR2C-Stop donors express FcgRIIb on NK cells. A, Surface binding of FcgRIIb/c mAb 2B6 to NK cells (CD32CD56+), monocytes (CD14+), neutrophils, and B cells (CD19+), as determined by flow cytometry, compared among classical FCGR2C-Stop (n = 29), classical FCGR2C-ORF (n = 9), and nonclassical FCGR2C-Stop (n = 4) donors. The differences in 2B6 binding between classical FCGR2C-Stop and -ORF donors in NK cells, monocytes, and neutrophils are statistically significant (p , 0.0001), in contrast to binding to B cells (p = 0.39). The differences in 2B6 binding between classical and nonclassical FCGR2C-Stop donors are statistically significant in NK cells (p , 0.0001) but not in monocytes, neutrophils, and B cells (p = 0.97, 0.18, and 0.82, respectively). Binding of 2B6 to classical FCGR2C-ORF and nonclassical FCGR2C-Stop donors is statistically different in monocytes and neutrophils (p = 0.01 and 0.003, respectively) but not in NK cells and B cells (p = 0.17 and 0.40, respectively). Data are expressed as mean 6 SEM. All values are corrected by subtracting values measured with isotype mAb controls. B, Representation of the FCGR gene locus. Arrows indicate the direction of transcription. The distance between the FCGR genes is mentioned between the arrows. The dashed bar shows the extent of the deletion found in nonclassical FCGR2C-Stop donors. C, Relative amounts of FcgRIIb and FcgRIIc transcripts in NK cells from classical FCGR2C-Stop, classical FCGR2C-ORF, and nonclassical FCGR2C-Stop donors. Values are relative to mRNA levels for the household gene GUS. Values were normalized, with the relative amounts of transcripts found in FCGR2C-Stop donors set to 1. D, Western blot analysis of FcgRIIb expression. Lanes 1 and 2, Whole-cell lysates of PMN and the human B cell line Daudi. Lanes 3, 4, and 5, NK cells from different donor types subjected to IP with a mAb binding all isoforms of FcgRII. Nonclassical FCGR2C-Stop donors are abbreviated as n.c.FCGR2C-Stop. a second activating FcgR (i.e., FcgRIIc). Individuals expressing mutations at the splice sites of intron 7 in their FCGR2C-ORF FcgRIIc may have an increased response toward IgG-mediated alleles were shown to exist, resulting in another stop codon. Al- signals on several cell types of the immune system. We have though our previous study on FCGR2C-ORF in ITP (12) has be- previously shown that surface expression of FcgRIIc in such come even more significant because of the lower frequency of individuals is not limited to NK cells but is also present on nonclassical FCGR2C-ORF patients with ITP compared with monocytes (W.B. Breunis, J. van der Heijden, J. Geissler, M. de the healthy control population (data not shown), nonclassical Boer, N. Laddach, M. Tanck, D. Burgner, I.M. Kuipers, D. Roos, FCGR2C-ORF alleles may lead to the misinterpretation of cohort and T.W. Kuijpers, submitted for publication) and, as shown in studies when only analyzed for the typical FCGR2C-ORF exon 3 this study, on neutrophils as well. alleles. Future experiments should include analysis of the intron 7 Classically, an SNP in exon 3 of the gene encoding for FcgRIIc splice sites to determine the number of classical FCGR2C-ORF determines whether a stop codon in exon 3 or an ORF is present. donors. We have found that ∼20% of the FCGR2C-ORF donors However, in a number of healthy individuals, genotyping of from our cohort of healthy white adults carry nonclassical FCGR2C by the SNP in exon 3 alone is insufficient. Additional FCGR2C-ORF alleles and are not expressing any FcgRIIc. From The Journal of Immunology 1323 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. Cytotoxicity of NK cells from different types of donors. A and B, Killing of P815 mouse mastocytoma cells through FcgRII and FcgRIII measured in an rADCC assay, comparing NK cells from FCGR2C-ORF (n = 4) and FCGR2C-Stop (n = 3) donors (A), as well as NK cells from nonclassical FCGR2C-Stop donors (n = 3) and cells from a rare donor carrying an FCGR2C-ORF in combination with a nonclassical FCGR2C-Stop allele (B). C, Killing of Raji cell lines measured in classical ADCC using clinical-grade chimeric CD20 mAb rituximab, comparing the same types of donor as in A and B (n = 3 for all donor types). Values are corrected for killing in the absence of mAbs. Data are expressed as mean 6 SEM. Nonclassical FCGR2C-Stop is abbreviated as n.c.FCGR2C-Stop. *p , 0.05. the phenotypic point of view, nonclassical FCGR2C-ORF donors non have been reported before, this was not further explored (13, should be included in the FCGR2C-Stop group because FcgRIIc is 20). We have now identified that such donors have a deletion at the not expressed in these donors. FCGR locus that includes FCGR2C, FCGR3B, and part of the We have also shown in this study that some individuals express region between FCGR3B and FCGR2B. The prevalence of such FcgRIIb on all circulating NK cells, which normally do not show deletions among healthy white individuals is ∼3%, but another any FcgRIIb expression. Although indications for this phenome- racial background may result in a different prevalence. 1324 VARIATION IN IgG RECEPTORS

Our data suggest that deletion of one or more inhibitory elements 5. Parren, P. W., P. A. Warmerdam, L. C. Boeije, J. Arts, N. A. Westerdaal, A. Vlug, P. J. Capel, L. A. Aarden, and J. G. van de Winkel. 1992. On the interaction of in the 30-kb DNA sequence between the FCGR3B and the IgG subclasses with the low affinity FcgRIIa (CD32) on human monocytes, FCGR2B genes results in the unusual expression of FcgRIIb on neutrophils, and platelets: analysis of a functional polymorphism to human IgG2. NK cells. In these individuals, the B cell expression of FcgRIIb is J. Clin. Invest. 90: 1537–1546. 6. Li, X., J. Wu, R. H. Carter, J. C. Edberg, K. Su, G. S. Cooper, and R. P. Kimberly. unaltered, and the circulating neutrophils and monocytes do not 2003. A novel polymorphism in the Fcg receptor IIB (CD32B) transmembrane show the FcgRIIb expression, as we demonstrated by flow region alters receptor signaling. Arthritis Rheum. 48: 3242–3252. cytometry and IP (Fig. 3). The fact that these donors do not show 7. Floto, R. A., M. R. Clatworthy, K. R. Heilbronn, D. R. Rosner, P. A. MacAry, A. Rankin, P. J. Lehner, W. H. Ouwehand, J. M. Allen, N. A. Watkins, and (increased) expression of FcgRIIb on other tested immune cells K. G. Smith. 2005. Loss of function of a lupus-associated FcgRIIb polymor- may indicate that this regulatory element normally acts to selec- phism through exclusion from lipid rafts. Nat. Med. 11: 1056–1058. tively suppress the FcgRIIb expression in NK cells. 8. Su, K., J. Wu, J. C. Edberg, X. Li, P. Ferguson, G. S. Cooper, C. D. Langefeld, and R. P. Kimberly. 2004. A promoter haplotype of the immunoreceptor Because of the inhibitory nature of FcgRIIb (13, 21), the NK cell tyrosine-based inhibitory motif-bearing FcgRIIb alters receptor expression and expression of FcgRIIb is presumed to result in a reduction in Ab- associates with autoimmunity. I. Regulatory FCGR2B polymorphisms and their association with systemic lupus erythematosus. J. Immunol. 172: 7186–7191. dependent cytotoxicity by NK cells. Cross-linking FcgRIIb alone 9. Su, K., X. Li, J. C. Edberg, J. Wu, P. Ferguson, and R. P. Kimberly. 2004. A in an rADCC assay did not induce killing of target cells. How- promoter haplotype of the immunoreceptor tyrosine-based inhibitory motif- ever, in case of NK cells expressing both FcgRIIb and FcgRIIc, bearing FcgRIIb alters receptor expression and associates with autoimmunity. II. Differential binding of GATA4 and Yin-Yang1 transcription factors and FcgRIIb completely inhibited killing induced by FcgRIIc cross- correlated receptor expression and function. J. Immunol. 172: 7192–7199. linking. In a classical ADCC, all FcgR on the NK cell can bind 10. Warmerdam, P. A., N. M. Nabben, S. A. van de Graaf, J. G. van de Winkel, and mAbs on target cells. FcgRIIIa is the main FcgR on NK cells, P. J. Capel. 1993. The human low affinity immunoglobulin G Fc receptor IIC gene is a result of an unequal crossover event. J. Biol. Chem. 268: 7346–7349. expressed by all donors and at much higher levels than FcgRIIb or 11. Metes, D., L. K. Ernst, W. H. Chambers, A. Sulica, R. B. Herberman, and Downloaded from FcgRIIc. The series of experiments performed in classical ADCC P. A. Morel. 1998. Expression of functional CD32 molecules on human NK cells was too small to identify any effect of additional FcgRIIc ex- is determined by an allelic polymorphism of the FcgRIIC gene. Blood 91: 2369– 2380. pression on the NK cells of FCGR2C-ORF donors when com- 12. Breunis, W. B., E. van Mirre, M. Bruin, J. Geissler, M. de Boer, M. Peters, pared with FCGR2C-Stop donors. Nonetheless, expression of D. Roos, M. de Haas, H. R. Koene, and T. W. Kuijpers. 2008. Copy number variation of the activating FCGR2C gene predisposes to idiopathic thrombocy- FcgRIIb on NK cells resulted in decreased killing of Raji cells in topenic purpura. Blood 111: 1029–1038. the presence of therapeutic mAbs, indicating that the expression of 13. Ernst, L. K., D. Metes, R. B. Herberman, and P. A. Morel. 2002. Allelic poly- the inhibitory FcgRIIb in nonclassical FCGR2C-Stop donors is morphisms in the FcgRIIC gene can influence its function on normal human http://www.jimmunol.org/ natural killer cells. J. Mol. Med. 80: 248–257. functionally relevant in Ab-dependent cytotoxicity. 14. Breunis, W. B., E. van Mirre, J. Geissler, N. Laddach, G. Wolbink, E. van der Taken together, we have identified several novel genetic var- Schoot, M. de Haas, M. de Boer, D. Roos, and T. W. Kuijpers. 2009. Copy iations in the FcgRII/III locus, thereby demonstrating that the number variation at the FCGR locus includes FCGR3A, FCGR2C and FCGR3B but not FCGR2A and FCGR2B. Hum. Mutat. 30: E640–E650. degree of genetic variation within this locus is considerably larger 15. Bournazos, S., J. M. Woof, S. P. Hart, and I. Dransfield. 2009. Functional and than determined previously. Apart from its natural cytotoxic ac- clinical consequences of Fc receptor polymorphic and copy number variants. Clin. Exp. Immunol. 157: 244–254. tivity toward tumors and viral infections, evidence for a regulatory 16. Ibarrola, I., P. J. Vossebeld, C. H. Homburg, M. Thelen, D. Roos, and role of NK cells in inflammation and infectious disease has ac- A. J. Verhoeven. 1997. Influence of tyrosine phosphorylation on protein inter- cumulated during the past decade (22–27). It will be of interest to action with FcgRIIa. Biochim. Biophys. Acta 1357: 348–358. 17. Schouten, J. P., C. J. McElgunn, R. Waaijer, D. Zwijnenburg, F. Diepvens, and by guest on October 2, 2021 see whether the presence of FcgRIIb is linked to the susceptibility G. Pals. 2002. Relative quantification of 40 nucleic acid sequences by multiplex for cancer and inflammatory diseases. ligation-dependent probe amplification. Nucleic Acids Res. 30: e57. Clearly, a more accurate FcgR genotyping, including the cur- 18. Lekanne Deprez, R. H., A. C. Fijnvandraat, J. M. Ruijter, and A. F. Moorman. 2002. Sensitivity and accuracy of quantitative real-time polymerase chain re- rently described variants, will be useful for improving our un- action using SYBR green I depends on cDNA synthesis conditions. Anal. Bio- derstanding of the role of FcgR in disease. chem. 307: 63–69. 19. van Mirre, E., W. B. Breunis, J. Geissler, C. E. Hack, M. de Boer, D. Roos, and T. W. Kuijpers. 2006. Neutrophil responsiveness to IgG, as determined by fixed Acknowledgments ratios of mRNA levels for activating and inhibitory FcgRII (CD32), is stable We thank Prof. Dirk Roos for helpful discussions and critical reading of the over time and unaffected by cytokines. Blood 108: 584–590. 20. Stewart-Akers, A. M., A. Cunningham, M. C. Wasko, and P. A. Morel. 2004. manuscript and Prof. Arthur Verhoeven for providing the FcgRIIa/c-specific FcgR expression on NK cells influences disease severity in rheumatoid arthritis. 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