Download Alleles FCGR2C Nonclassical Phenotypic Variation in IgG

Phenotypic Variation in IgG Receptors by
Nonclassical FCGR2C Alleles
This information is current as
of August 3, 2017.
Joris van der Heijden, Willemijn B. Breunis, Judy Geissler,
Martin de Boer, Timo K. van den Berg and Taco W.
Kuijpers
J Immunol 2012; 188:1318-1324; Prepublished online 23
December 2011;
doi: 10.4049/jimmunol.1003945
http://www.jimmunol.org/content/188/3/1318
Subscription
Permissions
Email Alerts
This article cites 27 articles, 9 of which you can access for free at:
http://www.jimmunol.org/content/188/3/1318.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
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.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
References
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*,†
F
cgRs are receptors for the Fc region of IgG and are
expressed on various cell types, including macrophages,
dendritic cells, neutrophils, NK cells, B lymphocytes, and
platelets. The binding of IgG-opsonized pathogens to FcgRs triggers a variety of cellular responses and contributes to inflammation. FcgRs are also involved in Ab-mediated tumor cell elimination. The balance between activating and inhibitory signals
from the different receptor subtypes ensures control over immune
complex-driven inflammatory responses (1). Depending on their
affinity for IgG, the receptors can be divided into low-affinity
FcgRs, which can be subdivided into FcgRIIa (CD32a), FcgRIIb
(CD32b), FcgRIIc (CD32c), FcgRIIIa (CD16a), and FcgRIIIb
(CD16b), and the high-affinity FcgRI (CD64). In humans, FcgRIIb
is the only inhibitory FcgR, because it contains an ITIM in its
cytoplasmic tail. All other FcgRs signal through an ITAM, either
encoded within their cytoplasmic tail or within associated adaptor
proteins such as the FcRg.
The genes encoding the FcgRs are located on chromosome 1, of
which the genes FCGR2A, FCGR2B, and FCGR2C are located in
one gene cluster with the FCGR3A and FCGR3B genes.
*Sanquin Research and Landsteiner Laboratory, Department of Blood Cell Research,
Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam, The
Netherlands; and †Department of Pediatric Hematology, Immunology and Infectious
Disease, Emma Children’s Hospital, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands
Received for publication December 7, 2010. Accepted for publication November 22,
2011.
This work was partially supported by a grant from ZorgOnderzoek Nederland–Medische Wetenschapp (920-03-391) (to W.B.B.).
Address correspondence and reprint requests to Joris van der Heijden, Sanquin Research, Department of Blood Cell Research, Room U202, Plesmanlaan 125, 1066CX
Amsterdam, The Netherlands. E-mail address: [email protected]
Abbreviations used in this article: ADCC, Ab-dependent cellular cytotoxicity; CNV,
copy number variation; GUS, b-glucuronidase; IP, immunoprecipitation; MLPA,
multiplex ligation-dependent probe amplification; ORF, open reading frame; rADCC,
redirected Ab-dependent cellular cytotoxicity; SNP, single nucleotide polymorphism.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003945
Because of the very high homology between the genes, it is
a difficult gene cluster to study. For instance, FCGR2A and
FCGR2B have ∼95% homology in their exons coding for the
extracellular domains of FcgRIIa and FcgRIIb, respectively (2).
The activating FcgRIIa is expressed by neutrophils, monocytes,
macrophages, dendritic cells, and platelets, in which it mediates
various functions such as phagocytosis and Ab-dependent cellular
cytotoxicity (ADCC). The inhibitory FcgRIIb exists in two splice
variants, namely, FcgRIIb1 (which is exclusively expressed by
B cells) and FcgRIIb2 (expressed by B cells, macrophages, and
dendritic cells). FcgRIIb inhibits signaling of ITAM-associated
receptors by recruitment of phosphotyrosine phosphatases that
dephosphorylate the tyrosine residues in several ITAM-induced
activation pathway effectors (3).
Single nucleotide polymorphisms (SNPs) have been described
in both FCGR2A and FCGR2B. A functionally relevant SNP in
FCGR2A leads to either a histidine or an arginine in the ligandbinding domain of FcgRIIa (H/R131) (4). This results in differences in binding affinity for human IgG2 and murine IgG1 (4, 5).
In FCGR2B, one SNP leads to either an isoleucine or a threonine
in the transmembrane region of FcgRIIb (I/T232). The T232 allotype has been suggested to result in functional impairment
through exclusion from lipid rafts (6, 7). Additional SNPs in the
promoter region influence transcriptional activity of FCGR2B
(and FCGR2C) (8, 9). FCGR2C, encoding FcgRIIc, appears to be
the product of an unequal crossover event between FCGR2A and
FCGR2B (10). FCGR2C contains eight exons, which are highly
homologous to exons 1–4 from FCGR2B and exons 5–8 from
FCGR2A. An SNP in exon 3 of FCGR2C determines the functional expression of this gene. This SNP results in either an open
reading frame (FCGR2C-ORF) or the more common stop codon
(FCGR2C-Stop), in which case FCGR2C represents a pseudogene
(11). FcgRIIc is expressed on NK cells from FCGR2C-ORF
donors, acting as an activating IgG receptor capable of inducing
ADCC and a rise in Ca2+ concentration after receptor crosslinking (12, 13).
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
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 “nonclassical” 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
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 expression with relevance to immunity and inflammation. The Journal of Immunology, 2012, 188: 1318–1324.
The Journal of Immunology
Differences in receptor expression levels on the cell surface can
alter the balance between activating and inhibitory signals and
therefore change the cellular response toward IgG. Apart from
SNPs, we and others have shown that several FCGR genes are
subject to copy number variation (CNV). CNV in FCGR2C,
FCGR3A, and FCGR3B has been shown to correlate with protein
expression levels. For instance, deletion of one FCGR3A allele
results in reduced target cell killing in an FcgRIIIa-specific
ADCC (14). These SNPs and CNVs in FCGR genes have been
linked with (chronic) inflammatory and autoimmune diseases (12,
15). It is therefore essential to further investigate genetic variation
in this gene cluster, because that may be highly relevant for the
discovery of disease risk factors, therapeutic efficacy of biologicals, and the development of new treatment strategies. In this
study, we identify two additional genetic variations in the FCGR
gene cluster that modulate the expression of FcgRIIb and
FcgRIIc.
Materials and Methods
Healthy adult white volunteers (n = 146) served as controls. These donors
were not known to have hematological disorders of any kind in the past or
at present. The study was approved by the Medical Ethics Committee of
the Academic Medical Center and Sanquin (Amsterdam, The Netherlands)
and was performed in accordance with the Declaration of Helsinki.
Abs and reagents
The following mAbs were used in flow cytometry and for immunoprecipitation (IP): anti-CD3 clone SK7, anti-CD14 clone M5E2 and antiCD56 clone B159 (BD Pharmingen, San Diego, CA), anti-FcgRII clone
AT10 (AbD Serotec, Oxford, U.K.), and anti-FcgRIIb/c clone 2B6
(Macrogenics, Rockville, MD). Western blots were stained with polyclonal
IgG against either FcgRIIa/c [rabbit antiserum CT10; a gift from Dr.
A. Verhoeven, Department of Medical Biochemistry, Academic Medical
Centre, University of Amsterdam (16)] or FcgRIIb (Epitomics, Burlingame, CA). Abs used in cytotoxicity assays were anti-FcgRII clone AT10
(azide-free; AbD Serotec) and anti-FcgRIII clone 3G8 (low-endotoxin
azide-free; BioLegend).
IP lysis buffer is PBS containing 10% glycerol (w/v), 1% Nonidet P-40
(w/v), and 13 Complete, EDTA-free Protease Inhibitor Mixture (Roche
Applied Science, Almere, The Netherlands). Laemmli sample buffer is
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
blue. Complete medium is IMDM (PAA, Pasching, Austria) supplemented with 10% FCS(v/v) (Bodinco, Alkmaar, The Netherlands),
penicillin (100 U/ml), streptomycin (100 mg/ml; PAA), and 2 mM Lglutamine (PAA).
Genotyping
Genomic DNA was isolated from whole blood with the Gentra Puregene
kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions.
CNV and SNPs in the low-affinity FCGR genes FCGR2A, FCGR2B,
FCGR2C, FCGR3A, and FCGR3B were determined with an FCGR-specific
multiplex ligation-dependent probe amplification (MLPA) assay. The MLPA
assay was performed essentially as described previously (12, 17). The
FCGR MLPA includes gene-specific probes to determine the CNV of the
genes. It also includes probes to detect the following SNPs: FCGR2A 27Q .
W (rs9427397 rs9427398), FCGR2A 131H . R (rs1801274), FCGR2B
232I . T (rs1050501), FCGR2B/C –386C . G (rs3219018), –120A . T,
FCGR3A 158V . F (rs396991), and FCGR3B (HNA1a/HNA1b/HNA1c).
The assay also contains a probe specific for the open reading frame in exon
3 of the FCGR2C gene and a nonspecific FCGR2B/C probe to detect the
stop codon in exon 3. For selected donors, we designed and used probes to
determine the extent of the FCGR2C/FCGR3B deletion. Probe sequences
are 59-GGGTTCCCTAAGGGTTGGAGAAGCATGACTCTTCTAAAACAAGTTAATAAT-39 and 59-AATGACATCTTTTCATGAACAAGCAATTGAGTCTAGATTGGATCTTGCTGGCAC-39 for the region between FCGR3A
and FCGR2C and 59-GGGTTCCCTAAGGGTTGGACACCATCTCAGTAGTAATTTCTGACACATCACTTT-39 and 59-CAGAAGGCTCATAAGCCAACTCAGATACAACCGTCTAGATTGGATCTTGCTGGCGC-39 for the
region between FCGR3B and FCGR2B.
PCR and sequence analysis
Quantitative PCR analysis and sequencing of FcgRIIc transcripts, as well as
the gene-specific, long-range PCR analysis and sequencing of FCGR2C,
were performed as described previously (12).
NK cell purification
PBMCs were isolated from whole blood by Percoll density gradient centrifugation. NK cells were isolated from PBMCs by MACS with CD56
MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), following the
manufacturer’s instructions. NK cell purity was verified by flow cytometry.
mRNA isolation and reverse transcription
mRNA was isolated from 1 3 106 purified NK cells by use of the QiaAmp
RNA blood mini kit (Qiagen), according to the manufacturer’s instructions. Subsequently, first-strand cDNA was synthesized with the Superscript III first-strand synthesis system for RT-PCR (Invitrogen, Breda, The
Netherlands). In short, RNA was primed with 2.5 mM oligo(dT) for 5 min
at 65˚C. Reverse transcription was performed with 10 U/ml Superscript III
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
Deprez et al. (18)] for 50 min at 50˚C. Thereafter, Superscript III was
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
incubated at 37˚C for 20 min. Subsequently, cDNA was stored at 20˚C until
further use.
Detection and relative quantification of FcgRII
isoform-specific mRNA
The detection and relative quantification of FcgRII mRNA have been
described in detail elsewhere (19). In short, intron-spanning primers were
designed to specifically amplify cDNA and exclude amplification of genomic DNA, yielding products of 100 bp for b-glucuronidase (GUS), 243
bp for FcgRIIb2, and 162 bp for FcgRIIc1 as the most abundant productive
FcgRIIc transcript in immune cells (12). The following primers were used:
b-glucuronidase (GUS), 59-GAAAATATGTGGTTGGAGAGCTCATT-39
(forward primer) and 59-CCGAGTGAAGATCCCCTTTTTA-39 (reverse
primer); FcgRIIb2, 59-GGAAAAAGCGCATTTGAGCCAATC-39 (forward primer) and 59-GGAAATACGAGATCTTCCCTCTCTG-39 (reverse
primer); and FcgRIIc1, 59-ATCATTGTGGCTGTGGTCACTGG-39 (forward primer) and 59-CTTTCTGATGGCAATCATTTGACG-39 (reverse
primer). Quantification of cDNA was performed with the Lightcycler Instrument (Roche Applied Science). Serial 10-fold dilutions of the cDNA
were made and quantified with the method described in Technical Note
No. LC 13/2001 (Roche Applied Science).
IP and Western blotting of FcgRII
IP was performed at 4˚C. Cells were pretreated with 10 mM diisopropyl
fluorophosphate for 10 min and lysed with IP lysis buffer for 1 h. Lysates
were centrifuged at 21,000 3 g for 15 min. Supernatants were incubated
with pan-FcgRII Abs (10 mg/ml) overnight. Subsequently, they were incubated with 5% protein G-Sepharose 4 Fast Flow (w/v) (GE Healthcare
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
Laemmli sample buffer for 30 min at 95˚C.
Samples were run on a 12.5% Bis-Tris gel (w/v) and subsequently blotted
on a nitrocellulose membrane. FcgRIIb and -c were detected by polyclonal
Abs against their cytoplasmic tails. Bands were visualized by ECL substrate (Pierce, Rockford, IL).
Cytotoxicity assays
Cytotoxicity of NK cells was determined by Ab-dependent cellular cytotoxicity (ADCC) and redirected ADCC (rADCC). NK cells were isolated
from FCGR2C-ORF, FCGR2C-Stop, and nonclassical FCGR2C-Stop
donors and kept in complete medium. Target cells were labeled with 51Cr
for 1 h and subsequently washed three times. Cells were plated in a 96-well
plate at 5 3 103 cells/well. NK cells were added at varying E:T cell ratios
and incubated in triplicate. In case of ADCC, chimeric therapeutic mAbs
against CD20 (rituximab) were added at 10 mg/ml. In case of rADCC,
mouse mAbs against either human FcgRII or human FcgRIII were added
at 10 mg/ml. As a control, target and NK cells were plated without Abs.
Target cells were also plated without NK cells to determine the spontaneous release or with saponine to determine the maximum release of 51Cr.
The total volume per well was 100 ml. Cells were centrifuged at 300 3 g
for 30 s and subsequently incubated at 37˚C and 5% CO2 for 4 h. Cells
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
Subjects
1319
1320
VARIATION IN IgG RECEPTORS
were centrifuged again at 300 3 g for 30 s, and supernatants were collected and counted in a gamma counter.
Specific killing was determined by the formula: percentage specific
killing = 100 3 (experimental release 2 spontaneous release)/(maximum
release 2 spontaneous release).
Statistics
Flow cytometry data were analyzed with Student t test; p , 0.05 was
considered statistically significant. Data are expressed as mean 6 SEM.
Results
Mismatches between FCGR2C genotype and phenotype
Nonclassical FCGR2C-ORF by novel splice site mutations
(group A)
Expression of FcgRIIc is known to be determined by the p.Q13X
(ORF versus Stop) mutation in exon 3 of the FCGR2C gene. Interestingly, NK cells of some of the FCGR2C-ORF donors did not
express FcgRIIc. The lack of expression was not limited to NK
cells but was also apparent on myeloid cells, as indicated for
monocytes and neutrophils (Fig. 2A). To investigate whether RNA
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 insertion, which was found to be part of intron 7 (Fig. 2B). Both unusual 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
FCGR2C Exon 3
No. of Donors
Stop
ORF
Stop/Stop
Stop/ORF
ORF/ORF
Stop/Stop/Stop
Stop/Stop/ORF
Stop/ORF/ORF
ORF/ORF/ORF
8
2
94
21
1
15
3
2
0
FcgRII in which exon 6 is not spliced out is the B cell-specific
FcgRIIb1. Besides these most commonly found transcripts in the
three nonclassical FCGR2C-ORF donors, additional noncoding
transcripts were detected as well. Premature stop codons can result
in various nonsense mRNAs. It is likely that these less frequently
found transcripts fall in this category. In normal FCGR2C-ORF
donors, these noncoding transcripts were not detected (data not
shown).
A gene-specific, long-range PCR was performed to amplify
FCGR2C from genomic DNA. Sequence analysis of the PCR
product revealed that all three nonclassical FCGR2C-ORF donors
studied thus far carried a G→A point mutation at the donor splice
site of intron 7, which explains a splicing defect that results in the
loss of exon 7. In addition to this mutation, two donors also had
a G→C point mutation at the acceptor splice site of intron 7. In
these donors, a cryptic acceptor splice site 62 bp upstream of the
normal exon–intron boundary was used, which explains the insertion of part of intron 7 in these two donors (Fig. 2C).
Nonclassical FCGR2C-Stop because of gene deletions in the
FCGR cluster (group B)
Because FCGR2C-Stop donors are expected to be unable to express any isoform of FcgRII on NK cells, we were surprised to
find that the NK cells from some of these healthy individuals did
bind mAb 2B6, which is specific for FcgRIIb and FcgRIIc. Although genotyping errors in FCGR2C were considered, there was
an important phenotypic difference with the binding pattern with
the same mAb on immune cells from individuals carrying classical
FCGR2C-ORF (Fig. 3A). Neutrophils and monocytes from these
four individuals from group B showed a similar lack of surface
Table I. Copy numbers of FCGR2B, FCGR2C, and FCGR3B
CNV (No. of Donors)
Copies
0
1
2
3
FCGR2B
FCGR2C
FCGR3B
0
0
146
0
0
10
116
20
0
9
121
16
FIGURE 1. Binding of FcgRIIb/c-specific mAb 2B6 to NK cells from
healthy individuals genotyped as FCGR2C-Stop or FCGR2C-ORF donors.
Boxes A and B indicate subgroups with an unexpected mAb binding
pattern when considering the individuals’ genotypes. The difference in
2B6 binding between donor groups was statistically significant (***p =
0.0005).
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
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
(Tables I, II; data not shown) and phenotyping a selection of 47 of
these individuals with different mAbs against FcgRII (pan-CD32),
FcgRIIb/c (CD32b/c), and FcgRIIIa/b (CD16).
There is no mAb available that can differentiate between surfaceexpressed FcgRIIb and FcgRIIc because of the completely identical extracellular domains. NK cells express FcgRIIc but not
FcgRIIb (12), and these cells can therefore be used to distinguish
FCGR2C-Stop from FCGR2C-ORF donors at the level of surface
expression with a FcgRIIb/c-specific mAb.
When we related the genotype for FCGR2C with the FcgRIIc
expression on NK cells as measured by flow cytometry (Fig. 1),
we observed a strong and positive correlation between genotype
and average protein surface expression levels. However, we also
detected two exclusive sets of individuals that showed definite
genotype–phenotype mismatches. First, we identified individuals
who repeatedly showed no FcgRIIb/c surface expression on NK
cells in the presence of a classical FCGR2C-ORF genotype (group
A). Second, we identified some individuals whose NK cells were
stained for FcgRIIb/c while carrying classical FCGR2C-Stop
alleles, considered to be true pseudogenes (group B).
Table II. FCGR2C allele combinations in 146 healthy white
individuals, determined by MLPA
The Journal of Immunology
binding of mAb 2B6 as did normal FCGR2C-Stop donors in
contrast to the usual myeloid expression observed in classical
FCGR2C-ORF donors. This difference excluded genotyping
errors as a possible explanation of our findings.
Further studies of these donors in which the NK cells unexpectedly showed CD32b/c surface expression were performed at
the genomic level. MLPA analysis of DNA from these so-called
nonclassical FCGR2C-Stop donors showed that these individuals
all carried a large deletion in the FCGR locus, resulting in the
combined loss of both FCGR2C and FCGR3B on one of their
alleles (14). MLPA probes were designed to analyze the regions
located around these genes to investigate the extent of this dele-
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 detectable 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
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 individuals with the FCGR2C-ORF, -Stop, and nonclassical Stop
signature after combined geno-/phenotyping. These immunoprecipitates 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 functional consequences, cytotoxicity was tested in rADCC assays
(Fig. 4). Killing of target cells in the presence of mAbs for
FcgRIII was comparable between NK cells from all donor types.
As we had also previously published (11, 12), NK cells from
FCGR2C-ORF donors were able to kill target cells in the presence
of mAbs for FcgRII, whereas NK cells from FCGR2C-Stop
donors, not able to bind these mAbs, were unable to kill under
these rADCC conditions (Fig. 4A). As expected, NK cells from
nonclassical FCGR2C-Stop donors, expressing the inhibitory
FcgRIIb, were not able to kill target cells in the presence of mAbs
for FcgRII (Fig. 4B). Rare individuals carrying both an FCGR2CORF allele and a nonclassical FCGR2C-Stop allele will express
both FcgRIIb and FcgRIIc on NK cells. We had identified
a healthy donor with this genotype. The NK cells from this individual were unable to kill the target cells in the rADCC, despite
expression of FcgRIIc. Thus, FcgRIIb on NK cells is unable
to mediate cytotoxicity, but effectively inhibits killing mediated
through FcgRIIc when coexpressed and ligated in the rADCC test
system (Fig. 4B).
Because NK cells also express FcgRIIIa, the effect of FcgRIIb
and FcgRIIc expression on cytotoxicity was tested in a classical
ADCC test in which the clinically applied chimeric CD20 mAb
rituximab was used to opsonize the Raji B cell lymphoma cell line
as target cell. In these experiments, we observed that the expression of FcgRIIb on the NK cells of nonclassical FCGR2C-Stop
donors resulted in decreased NK cell-mediated killing of the Raji
target cells when preincubated with the CD20 mAb (Fig. 4C).
Discussion
Expression of FcgR on NK cells is normally limited to the activating FcgRIIIa (CD16a). We and others (12, 13) have previously
shown that NK cells from ∼20% of healthy individuals express
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
FIGURE 2. Nonclassical ORF donors do not express FcgRIIc. A,
Binding of FcgRIIb/c mAb 2B6 to NK cells (CD32CD56+), monocytes
(CD14+), neutrophils, and B cells (CD19+) compared between classical
(n = 9) and nonclassical (n = 3) FCGR2C-ORF donors. The differences
in 2B6 binding between donor groups in NK cells, monocytes, and neutrophils were statistically significant (p = 0.004, 0.009, and , 0.001, respectively), in contrast to B cells, where this difference was not statistically
significant (p = 0.27). Data are expressed as mean 6 SEM. B, Representation of FcgRIIc transcripts found in PBMCs from classical and
nonclassical FCGR2C-ORF donors. White boxes, Exons that are spliced
out. *, The location of stop codons; +62, the insertion of a 62-bp sequence
from intron 7 (see C). C, Splice site mutations in FCGR2C intron 7 in
nonclassical FCGR2C-ORF donors. The intronic splice sites are underlined. Mutated splice sites are in italics, with the altered nucleotides in
bold. Gray boxes represent exons that are retained, and white boxes represent exons that are being spliced out as a consequence of these mutations. Cx, cytoplasmic domain; ECx, extracellular domain, MFI, mean
fluorescence intensity; Sx, signal sequence; TM, transmembrane domain.
1321
1322
VARIATION IN IgG RECEPTORS
a second activating FcgR (i.e., FcgRIIc). Individuals expressing
FcgRIIc may have an increased response toward IgG-mediated
signals on several cell types of the immune system. We have
previously shown that surface expression of FcgRIIc in such
individuals is not limited to NK cells but is also present on
monocytes (W.B. Breunis, J. van der Heijden, J. Geissler, M. de
Boer, N. Laddach, M. Tanck, D. Burgner, I.M. Kuipers, D. Roos,
and T.W. Kuijpers, submitted for publication) and, as shown in
this study, on neutrophils as well.
Classically, an SNP in exon 3 of the gene encoding for FcgRIIc
determines whether a stop codon in exon 3 or an ORF is present.
However, in a number of healthy individuals, genotyping of
FCGR2C by the SNP in exon 3 alone is insufficient. Additional
mutations at the splice sites of intron 7 in their FCGR2C-ORF
alleles were shown to exist, resulting in another stop codon. Although our previous study on FCGR2C-ORF in ITP (12) has become even more significant because of the lower frequency of
nonclassical FCGR2C-ORF patients with ITP compared with
the healthy control population (data not shown), nonclassical
FCGR2C-ORF alleles may lead to the misinterpretation of cohort
studies when only analyzed for the typical FCGR2C-ORF exon 3
alleles. Future experiments should include analysis of the intron 7
splice sites to determine the number of classical FCGR2C-ORF
donors. We have found that ∼20% of the FCGR2C-ORF donors
from our cohort of healthy white adults carry nonclassical
FCGR2C-ORF alleles and are not expressing any FcgRIIc. From
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
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.
The Journal of Immunology
1323
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
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
should be included in the FCGR2C-Stop group because FcgRIIc is
not expressed in these donors.
We have also shown in this study that some individuals express
FcgRIIb on all circulating NK cells, which normally do not show
any FcgRIIb expression. Although indications for this phenome-
non have been reported before, this was not further explored (13,
20). We have now identified that such donors have a deletion at the
FCGR locus that includes FCGR2C, FCGR3B, and part of the
region between FCGR3B and FCGR2B. The prevalence of such
deletions among healthy white individuals is ∼3%, but another
racial background may result in a different prevalence.
1324
Acknowledgments
We thank Prof. Dirk Roos for helpful discussions and critical reading of the
manuscript and Prof. Arthur Verhoeven for providing the FcgRIIa/c-specific
Ab for Western blot analysis.
Disclosures
The authors have no financial conflicts of interest.
References
1. Nimmerjahn, F., and J. V. Ravetch. 2008. Fcg receptors as regulators of immune
responses. Nat. Rev. Immunol. 8: 34–47.
2. Brooks, D. G., W. Q. Qiu, A. D. Luster, and J. V. Ravetch. 1989. Structure and
expression of human IgG FcRII(CD32): functional heterogeneity is encoded by
the alternatively spliced products of multiple genes. J. Exp. Med. 170: 1369–1385.
3. Ravetch, J. V., and L. L. Lanier. 2000. Immune inhibitory receptors. Science 290:
84–89.
4. Warmerdam, P. A., J. G. van de Winkel, E. J. Gosselin, and P. J. Capel. 1990.
Molecular basis for a polymorphism of human Fcg receptor II (CD32). J. Exp.
Med. 172: 19–25.
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
IgG subclasses with the low affinity FcgRIIa (CD32) on human monocytes,
neutrophils, and platelets: analysis of a functional polymorphism to human IgG2.
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.
2003. A novel polymorphism in the Fcg receptor IIB (CD32B) transmembrane
region alters receptor signaling. Arthritis Rheum. 48: 3242–3252.
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
K. G. Smith. 2005. Loss of function of a lupus-associated FcgRIIb polymorphism through exclusion from lipid rafts. Nat. Med. 11: 1056–1058.
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
tyrosine-based inhibitory motif-bearing FcgRIIb alters receptor expression and
associates with autoimmunity. I. Regulatory FCGR2B polymorphisms and their
association with systemic lupus erythematosus. J. Immunol. 172: 7186–7191.
9. Su, K., X. Li, J. C. Edberg, J. Wu, P. Ferguson, and R. P. Kimberly. 2004. A
promoter haplotype of the immunoreceptor tyrosine-based inhibitory motifbearing FcgRIIb alters receptor expression and associates with autoimmunity.
II. Differential binding of GATA4 and Yin-Yang1 transcription factors and
correlated receptor expression and function. J. Immunol. 172: 7192–7199.
10. Warmerdam, P. A., N. M. Nabben, S. A. van de Graaf, J. G. van de Winkel, and
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.
11. Metes, D., L. K. Ernst, W. H. Chambers, A. Sulica, R. B. Herberman, and
P. A. Morel. 1998. Expression of functional CD32 molecules on human NK cells
is determined by an allelic polymorphism of the FcgRIIC gene. Blood 91: 2369–
2380.
12. Breunis, W. B., E. van Mirre, M. Bruin, J. Geissler, M. de Boer, M. Peters,
D. Roos, M. de Haas, H. R. Koene, and T. W. Kuijpers. 2008. Copy number
variation of the activating FCGR2C gene predisposes to idiopathic thrombocytopenic purpura. Blood 111: 1029–1038.
13. Ernst, L. K., D. Metes, R. B. Herberman, and P. A. Morel. 2002. Allelic polymorphisms in the FcgRIIC gene can influence its function on normal human
natural killer cells. J. Mol. Med. 80: 248–257.
14. Breunis, W. B., E. van Mirre, J. Geissler, N. Laddach, G. Wolbink, E. van der
Schoot, M. de Haas, M. de Boer, D. Roos, and T. W. Kuijpers. 2009. Copy
number variation at the FCGR locus includes FCGR3A, FCGR2C and FCGR3B
but not FCGR2A and FCGR2B. Hum. Mutat. 30: E640–E650.
15. Bournazos, S., J. M. Woof, S. P. Hart, and I. Dransfield. 2009. Functional and
clinical consequences of Fc receptor polymorphic and copy number variants.
Clin. Exp. Immunol. 157: 244–254.
16. Ibarrola, I., P. J. Vossebeld, C. H. Homburg, M. Thelen, D. Roos, and
A. J. Verhoeven. 1997. Influence of tyrosine phosphorylation on protein interaction with FcgRIIa. Biochim. Biophys. Acta 1357: 348–358.
17. Schouten, J. P., C. J. McElgunn, R. Waaijer, D. Zwijnenburg, F. Diepvens, and
G. Pals. 2002. Relative quantification of 40 nucleic acid sequences by multiplex
ligation-dependent probe amplification. Nucleic Acids Res. 30: e57.
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 reaction using SYBR green I depends on cDNA synthesis conditions. Anal. Biochem. 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
ratios of mRNA levels for activating and inhibitory FcgRII (CD32), is stable
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.
FcgR expression on NK cells influences disease severity in rheumatoid arthritis.
Genes Immun. 5: 521–529.
21. Clynes, R. A., T. L. Towers, L. G. Presta, and J. V. Ravetch. 2000. Inhibitory Fc
receptors modulate in vivo cytotoxicity against tumor targets. Nat. Med. 6: 443–
446.
22. Lünemann, A., J. D. Lünemann, and C. Münz. 2009. Regulatory NK-cell
functions in inflammation and autoimmunity. Mol. Med. 15: 352–358.
23. Gao, N., P. Jennings, Y. Guo, and D. Yuan. 2011. Regulatory role of natural killer
(NK) cells on antibody responses to Brucella abortus. Innate Immun. 17: 152–
163.
24. Empson, V. G., F. M. McQueen, and N. Dalbeth. 2010. The natural killer cell:
a further innate mediator of gouty inflammation? Immunol. Cell Biol. 88: 24–31.
25. Culley, F. J. 2009. Natural killer cells in infection and inflammation of the lung.
Immunology 128: 151–163.
26. Erten, G., E. Aktas, and G. Deniz. 2008. Natural killer cells in allergic inflammation. Chem. Immunol. Allergy 94: 48–57.
27. Harrington, L., C. V. Srikanth, R. Antony, H. N. Shi, and B. J. Cherayil. 2007. A
role for natural killer cells in intestinal inflammation caused by infection with
Salmonella enterica serovar Typhimurium. FEMS Immunol. Med. Microbiol. 51:
372–380.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
Our data suggest that deletion of one or more inhibitory elements
in the 30-kb DNA sequence between the FCGR3B and the
FCGR2B genes results in the unusual expression of FcgRIIb on
NK cells. In these individuals, the B cell expression of FcgRIIb is
unaltered, and the circulating neutrophils and monocytes do not
show the FcgRIIb expression, as we demonstrated by flow
cytometry and IP (Fig. 3). The fact that these donors do not show
(increased) expression of FcgRIIb on other tested immune cells
may indicate that this regulatory element normally acts to selectively suppress the FcgRIIb expression in NK cells.
Because of the inhibitory nature of FcgRIIb (13, 21), the NK cell
expression of FcgRIIb is presumed to result in a reduction in Abdependent cytotoxicity by NK cells. Cross-linking FcgRIIb alone
in an rADCC assay did not induce killing of target cells. However, in case of NK cells expressing both FcgRIIb and FcgRIIc,
FcgRIIb completely inhibited killing induced by FcgRIIc crosslinking. In a classical ADCC, all FcgR on the NK cell can bind
mAbs on target cells. FcgRIIIa is the main FcgR on NK cells,
expressed by all donors and at much higher levels than FcgRIIb or
FcgRIIc. The series of experiments performed in classical ADCC
was too small to identify any effect of additional FcgRIIc expression on the NK cells of FCGR2C-ORF donors when compared with FCGR2C-Stop donors. Nonetheless, expression of
FcgRIIb on NK cells resulted in decreased killing of Raji cells in
the presence of therapeutic mAbs, indicating that the expression of
the inhibitory FcgRIIb in nonclassical FCGR2C-Stop donors is
functionally relevant in Ab-dependent cytotoxicity.
Taken together, we have identified several novel genetic variations in the FcgRII/III locus, thereby demonstrating that the
degree of genetic variation within this locus is considerably larger
than determined previously. Apart from its natural cytotoxic activity toward tumors and viral infections, evidence for a regulatory
role of NK cells in inflammation and infectious disease has accumulated during the past decade (22–27). It will be of interest to
see whether the presence of FcgRIIb is linked to the susceptibility
for cancer and inflammatory diseases.
Clearly, a more accurate FcgR genotyping, including the currently described variants, will be useful for improving our understanding of the role of FcgR in disease.
VARIATION IN IgG RECEPTORS