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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Metes, D. Articles by Morel, P. A. Search for Related Content PubMed PubMed Citation Articles by Metes, D. Articles by Morel, P. A. Related Collections Immunobiology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 91 No. 7 (April 1), 1998: pp. 2369-2380 Expression of Functional CD32 Molecules on Human NK Cells Is Determined by an Allelic Polymorphism of the FcRIIC Gene By Diana Metes, Linda K. Ernst, William H. Chambers, Andrei Sulica, Ronald B. Herberman, and Penelope A. Morel From the Departments of Pathology, Medicine, and Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine; the University of Pittsburgh Cancer Institute, Pittsburgh, PA; and the Center of Immunology, Institute of Virology, Bucharest, Romania.     ABSTRACT Abstract Introduction Methods Results Discussion References Human natural killer (NK) cells were thought to express only FcRIIIA (CD16), but recent reports have indicated that NK cells also express a second type of FcR, ie, FcRII (CD32). We have isolated, cloned, and sequenced full-length cDNAs of FcRII from NK cells derived from several normal individuals that may represent four different products of the FcRIIC gene. One transcript (IIc1) is identical with the already described FcRIIc form. The other three (IIc2-IIc4) appear to represent unique, alternatively spliced products of the same gene, and include a possible soluble form. Analyses of the full-length clones have revealed an allelic polymorphism in the first extracellular exon, resulting in either a functional open reading frame isoform or a null allele. Stable transfection experiments enabled us to determine a unique binding pattern of anti-CD32 monoclonal antibodies to FcRIIc. Further analyses of NK-cell preparations revealed heterogeneity in CD32 expression, ranging from donors lacking CD32 expression to donors expressing high levels of CD32 that were capable of triggering cytotoxicity. Differences in expression were correlated with the presence or absence of null alleles. These data show that certain individuals express high levels of functional FcRIIc isoforms on their NK cells.     INTRODUCTION Abstract Introduction Methods Results Discussion References HUMAN NATURAL killer (NK) cells represent a subset of lymphocytes distinct from B cells and T cells, which are capable of mediating spontaneous cytotoxicity against certain target cells without requiring the recognition of antigen in the context of the MHC.1,2 However, major histocompatibility complex (MHC) determinants have been shown to influence cytolytic function.3,4 NK cells also kill immunoglobulin-coated targets through antibody-dependent cellular cytotoxicity (ADCC).5-7 Following activation, NK cells secrete cytokines and growth factors that regulate the physiology and function of other cells of the immune system.8,9 Although the receptors involved in direct recognition and killing of target cells are only now being defined, a receptor that is responsible for triggering NK-cell-mediated ADCC, namely FcRIIIA (CD16), has been extensively studied.7,10 CD16 is a receptor for the Fc portion of IgG and is capable of binding complexed or aggregated IgG and also IgG in monomeric form (mIgG).11-13 Until recently, it was believed that CD16 was the only FcR to be expressed by human NK cells. We, as well as others,14,15 have shown that FcRII (CD32) is also expressed by some human NK cells. FcRII is a glycoprotein of approximately 40 kD that binds IgG with low affinity. It is one of the most widely distributed FcRs, being expressed on cells of both myeloid and lymphoid lineages, as well as on cells of nonhematopoietic origin.16-18 Three genes have been described for FcRII (FcRIIA, B, and C) that encode a total of six transcripts (FcRIIa1, a2, b1, b2, b3, and c) representing alternatively spliced forms of the three genes.19,20 Each protein consists of two IgG-like extracellular domains, encoded in two exons (EC1 and EC2), a transmembrane region encoded by a separate exon (TM), and an intracytoplasmic tail encoded by three separate exons (C1, C2, and C3).19,20 A further polymorphism for FcRIIA has been described, with two codominantly expressed alleles, namely R131 high responder (HR) and H131 low responder (LR), which differ substantially in their ability to bind human IgG2 and murine IgG1.21,22 All of the known FcRII isoforms are highly homologous in the extracytoplasmic domains, but ligand binding specificity differences have been reported.16-18 The intracytoplasmic domains are less homologous, and triggering through different isoforms results in distinct functions.23,24 It has been recently reported that cross-linking of FcRIIa results in upregulation of intracellular Ca2+ concentration, phagocytosis of opsonized particles, as well as internalization of immune complexes.25-27 FcRII is known to associate with the -chain of FcRI, and this results in enhanced activation of these functions.28,29 In contrast, triggering through the FcRIIb isoforms results in downmodulation of a previous state of cell activation triggered via antigen receptors on B cells (BCR), T cells (TCR), or via another FcR.30,31 The FcRIIC gene represents an unequal cross-over event between genes IIA and IIB, as EC1, EC2, TM, and C1 exons are derived from the FcRIIB gene, whereas C2 and C3 exons derive from the FcRIIA gene.32 This generates a hybrid gene product that has been found to be expressed mainly by monocytes/macrophages and polymorphonuclear (PMN) cells.33 Its functional characteristics have not been studied, but it is thought that its binding specificities will be similar to the FcRIIb isoforms but will use similar signaling pathways as FcRIIa.17 Preliminary results reported by our group14 indicated that human NK cells express FcRII at levels that vary among donors. Flow cytometric studies, performed with IV.3 and 41H16 (two anti-CD32 monoclonal antibodies [MoAbs] that bind different epitopes), revealed different patterns of staining ranging from no expression at all to low expression detected using both IV.3 or 41H16 MoAbs. Some other individuals exhibited significant levels of CD32 expression on their NK cells (60% to 90%) when using the 41H16 MoAb, even though the level of IV.3 binding in the same donor was low (6% to 12%). Mantzioris et al15 showed in a different study that approximately 88% of CD3/CD16+/CD56+ cells (which represent the NK population) were positive for 41H16 staining. In addition, we found that the CD32 on NK cells was an activating molecule because Ca2+ flux and reverse ADCC (rADCC) were triggered following anti-CD32 MoAb stimulation of NK cells.14 In this study, we report that human NK cells express four different transcripts of the FcRIIC gene. One of the products, designated FcRIIc1, is the same as the previously described IIc isoform; the other three transcripts, designated IIc2-c4, represent previously undescribed, alternatively spliced products of the IIC gene and possibly include a soluble form. In addition, we describe an allelic polymorphism in the EC1 domain, which in some individuals results in two null FcRIIc alleles that may determine lack of FcRIIc expression. Expression of FcRIIc in individuals with the open reading frame (ORF) allele was shown by flow cytometric and biochemical means on purified NK cells. For the donors expressing FcRIIc on their NK cells, we describe a unique anti-CD32 MoAb binding pattern that, together with the allelic polymorphism, provides an explanation for the previously observed lack of CD32 expression by some NK-cell preparations. We also show that CD32 on NK cells is a functional molecule, capable of triggering cytotoxic events.     MATERIALS AND METHODS Abstract Introduction Methods Results Discussion References MoAbs.   Anti-CD16 and -CD32 MoAbs used were: 3G8 and IV.3 (Medarex, Annandale, NJ); 41H16 (ascites), a gift from Dr M. Longenecker (University of Alberta, Canada) and purified at the UPCI hybridoma facility; KB61, a gift from Dr K. Pulford (University of Oxford, UK); and AT10 (Biosource International, Camarillo, CA). F(ab)2 goat anti-mouse IgG (GAMIgG)-fluorescein isothiocyanate (FITC) was purchased from Tago Inc (Burlingame, CA). MoAbs used for phenotyping the NK cells were purchased from Becton Dickinson (Franklin Lakes, NJ) and consisted of anti-CD56 (Leu 19)-phycoerythrin (PE), anti-CD19 (Leu12)-PE, anti-CD14 (Leu 3M)-FITC, anti-CD3 (Leu 4)-FITC. Human cell lines.   U937 (monocyte cell line), K562 (erythroleukemia), RPMI 8866 (Epstein-Barr virus-transformed B-cell line), Jurkat (T-cell leukemia), and Molt 4 (T-cell lymphoma) were maintained in RPMI 1640 medium (GIBCO-BRL, Gaithesburgh, MD) supplemented with 10% fetal calf serum (FCS; GIBCO-BRL), 2 mmol/L glutamine, 100 U/mL penicillin, and 100 µmol/L streptomycin, subsequently referred to as complete culture medium (CCM). NK cell purification and NK cell analysis from peripheral blood mononuclear cells (PBMC).   Heparinized whole blood or leukocytes from leukapheresed blood from normal donors was centrifuged on a Ficoll-Hypaque (Pharmacia Biotech, Piscataway, NJ) gradient, and PBMC were obtained as previously described.34 PBMC were (1) either directly analyzed by flow cytometry in two color staining, using PE-conjugated anti-CD56 to define the NK population, and indirect staining with anti-CD32 or anti-CD16 MoAbs followed by FITC-GAMIgG or (2) incubated with a cocktail of anti-CD3, anti-CD19, anti-CD14, and anti-CD64 MoAbs, at 4°C for 30 minutes and exposed first to GAMIgG-covered magnetic beads (PerSeptive Bio Systems, Farmingham, MA), and for a second round to GAMIgG-covered Dynabeads M450 (Dynal AS, Oslo, Norway). Using a magnet (Bio-Mag Separator; Advanced Magnetics, Cambridge, MA), highly purified resting NK cells (95% CD3 CD56+ CD16+ cells) were obtained by negative selection. Fourteen-day interleukin-2 (IL-2)- activated human NK cells, referred to as A-NK cells, (generously provided by Dr N. Vujanovic, University of Pittsburgh Cancer Institute) were generally 100% CD3/ CD56+/CD16+ cells.35 The purity of all NK-cell preparations as well as the detection of CD32 expression on these cells was performed by direct and indirect flow cytometric analyses as previously reported.36 RNA isolation and reverse transcription (RT).   Total cellular RNA was isolated from all cell types using the RNAzol B (Biotex Labs Inc, Houston, TX) method. cDNAs were synthesized by RT from 2 µg of total RNA isolated from each cell source using a first-strand cDNA synthesis kit (Pharmacia-Biotech). A human NK cell cDNA library constructed in gt10 phage was a generous gift from Dr S. Ziegler (Darwin Molecular, Inc, Bothell, WA).37 FcRIIa1, FcRIIb1, and FcRIIb2 cDNA clones were a kind gift from Dr J. Ravetch (Sloan-Kettering Institute, New York, NY). PCR, Southern Blotting (SB), and Dot Blotting (DB).   The PCR reactions were performed using sets of primers specific for different FcRII isoforms (Fig 1).EcoRI/Xba I restriction sites were designed into the primers to facilitate cloning. The PCR amplification conditions consisted of a denaturing step at 94°C for 1 minute, annealing at 55°C for 2 minutes, and extension at 72°C for 3 minutes, for a total of 30 cycles. When the human NK cell gt10 library was used as a template, an additional 15-minute denaturation step was introduced before the usual PCR conditions. The PCR products were separated by electrophoresis in an ethidium bromide-containing 2% agarose gel, transferred onto Nytran membranes (Schleicher & Schuell, Keene, NH), and hybridized with digoxygenin-UTP (Boehringer Mannheim, Indianapolis, IN)-labeled oligonucleotides.38 In certain instances, the PCR products were dot blotted onto the Nytran membranes as previously described39 and further hybridized. The probes were designed to specifically hybridize to the known FcRII isoforms or to the novel FcRIIc isoforms (Fig 1). All oligonucleotide primers and probes were synthesized at the University of Pittsburgh, Department of Molecular Genetics and Biochemistry (MGB) core facility, and were purified using NAP-10 columns containing Sephadex G-25 Medium of DNA Grade (Pharmacia Biotech). View larger version (29K): [in this window] [in a new window]   Fig 1. Localization and sequence of FcRII-specific primers and probes. The structure of an FcRII cDNA is shown in (A), with the exons depicted as boxes, and with their names above: S1, first signal exon; S2, second signal exon; EC1, first extracellular exon; EC2, second extracellular exon; TM, transmembrane exon; C2, second intracytoplasmic exon; C3, third intracytoplasmic exon. Primer and probe locations are indicated below the cDNA map. (B) Indicates the primer pairs and probe names, specificities, and sequences. Cloning and DNA sequence analysis.   The full-length FcRIIc cDNAs, obtained from RT-PCR amplifications, were digested with appropriate restriction enzymes, purified, and concentrated using a QIAquick PCR purification column (Qiagen Inc, Chatsworth, CA). They were then cloned into the EcoRI/Xba I (GIBCO-BRL) digested pcDNA3 expression vector (Invitrogen Corp, San Diego, CA). The nucleotide sequence of cloned cDNAs was determined by ABI Prism fluorescent dye dideoxy-terminator cycle sequencing ready reaction kit (Perkin Elmer, Foster City, CA) at the University of Pittsburgh MGB core facility. Stable transfection of Jurkat cells.   Full-length FcRIIc1, IIc3 as well as FcRIIb1 cDNAs were inserted into the pCDNA3 expression vector (Invitrogen Corp). These constructs were transfected by electroporation into Jurkat cells as previously described.40 Briefly, aliquots of 1 × 107 Jurkat cells were transformed with 20 µg plasmid cDNA using an electroporation apparatus (BXT, San Diego, CA) set at 200 V and 1690 µF. Forty-eight hours later, stable transfectants were selected with the neomycin analog G418 (GIBCO-BRL) at 500 µg/mL. After 3 weeks the FcRII expressing cells were purified by panning.41 Briefly, the cells were incubated with an anti-CD32 MoAb (KB61) for 30 minutes on ice, washed three times, and the positive cells were then selected by incubating them in plates precoated with GAMIgG. The transfected cells were stable for CD32 high expression throughout the studies performed. Flow cytometric analyses of CD32 expression in stable transfectants.   Indirect flow cytometric analyses were performed using four different anti-FcRII MoAbs and FITC-labeled F(ab)2 fragment of GAMIgG staining as previously described.36 Data acquisition was performed on a FACStar Plus Cytometer (Becton Dickinson). Cell labeling, immunoprecipitation, and Western blotting.   U937 cells as well as PBLs or highly purified NK cells isolated from donor 2 were biotinylated as described elsewhere.42 Briefly, 2 × 107 cells were incubated in 0.1 mg/mL sulfo-NHS-LC-biotin (Pierce Chemical, Rockford, IL) for 30 minutes at 4°C, washed five times in phosphate-buffered saline (PBS), and lysed in 1% Triton X-100 lysing buffer containing 10 mmol/L EDTA, 2 mmol/L phenylmethanesulfonyl fluoride, 10 µg/mL antipain, 10 µg/mL leupeptin, and 10 µg/mL pepstatin A, for 30 minutes at 4°C. Lysates were centrifuged at 14,000 rpm at 4°C for 15 minutes to remove cell debris and nuclei. F(ab)2 fragments of GAMIgG coupled to CnBr-activated Sepharose 4B (Pharmacia LKB) were incubated with 41H16 MoAb or mIgG2a for 3 hours at 4°C as described.43 Immunoadsorbtion of cell lysates onto antibody-coupled beads was performed overnight at 4°C; the immunoprecipitates were washed with lysis buffer and electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (under nonreducing conditions). The separated proteins were transferred to PVDP membranes (Immunobilon-P, Millipore, Bedford, MA), blocked with a 3% nonfat dry milk and 1% bovine serum albumin (BSA) PBS solution for 1 hour at room temperature and incubated with horseradish peroxidase-conjugated neutravidin (Pierce) for 1 hour at room temperature. The blots were washed in Tris-buffered saline with 0.05% Tween-20, and the protein bands were detected by enhanced chemiluminescence. Cytotoxicity assay.   Cytotoxicity was measured by reverse antibody-dependent cytotoxicity (rADCC) using the 4-hour 51Cr-release microcytotoxicity assay, as previously described in detail.44 The FcR+ target used in the assay was P815 (rat mastocytoma). The spontaneous release was always less than 10%. Results were expressed in percentage of specific cytotoxicity calculated according to the formula     RESULTS Abstract Introduction Methods Results Discussion References NK cells express FcRIIa/IIc transcripts but not FcRIIb.   Our previous findings showed that some human NK cells express CD32, and that this molecule has triggering abilities.14 We, therefore, designed RT-PCR experiments to molecularly characterize the type of FcRII transcript(s) present in NK cells. We isolated RNA from highly purified NK-cell preparations as well as from cell lines known to be positive for FcRII. Sets of primers that would amplify all known CD32 isoforms (RS91-45 and RS91-46), or specifically the FcRIIa/IIc (RS91-45 and IIA) or the FcRIIb isoforms (RS91-45 and IIB), were designed (Fig 1). The specificity of the primers and probes used in these studies was confirmed using cDNA clones of FcRIIa1, FcRIIb1, and FcRIIb2 (data not shown). These primers were used to generate PCR products from the cells described above, and in addition, SB analyses were performed using a probe (RS91-46) that would hybridize with all FcRII cDNAs, to confirm the identity of these amplified products. As shown in Fig 2, the U937 cell line and a B-cell line expressed message for both FcRIIa/c and IIb isoforms, confirming previous reports regarding the coexpression of these isoforms by these cell lines.16,27 In the four NK-cell preparations, only FcRIIa/IIc-specific products could be amplified (Fig 2B), and no bands were visible when the FcRIIb-specific primers were used (Fig 2C). Therefore, these results suggested that NK cells expressed either the FcRIIa and/or the FcRIIc isoforms. View larger version (61K): [in this window] [in a new window]   Fig 2. Southern blot analysis of RT-PCR amplification products using FcRII-specific primers. RNA isolated from the indicated cell sources was used to obtain cDNAs by reverse transcription. PCR amplification was performed using sets of primers specific for all FcRII isoforms (A), Fc RIIa, c isoforms (B), and FcRIIb isoforms (C). A 2% agarose gel was run and Southern blotting was performed as described in Materials and Methods with RS91-46 as the hybridizing probe. Specific bands of expected size for each primer pair were detected in each panel (334 bp for RS9145-RS9146 primer pair, 442 bp for RS9145-FcIIA primer pair, and 441 bp/384 bp for RS9145-FcIIB primer pair). NK cells express message for a hybrid form of FcRII, namely FcRIIc.   To determine which isoform of FcRII was expressed in human NK cells, three oligonucleotide probes were designed that would discriminate among the IIa-HR (HR) and IIa-LR (LR) and IIc (IIB) transcripts (Fig 1). Using the RS91-45 and IIA primer set, RT-PCR was performed on RNA isolated from three IL-2-activated NK-cell preparations (A-NK1-3), K562 and U937 cell lines (positive controls), and from Molt 4 (negative control). SB analyses on these PCR products were performed, and the results presented in Fig 3 show that all of the cells used for this PCR amplification, with the exception of Molt 4, expressed message for FcRIIa and/or IIc (Fig 3A). The HR probe hybridized to both K562 and U937 PCR products, but not to the NK-cell-amplified products (Fig 3B); the LR probe hybridized only to the K562 amplification product, confirming that this cell line is heterozygous for both FcRIIa HR and LR alleles (Fig 3C).33 Furthermore, the IIB-probe hybridized to all PCR products amplified from the NK-cell preparations, as well as from U937 and K562 cells (Fig 3D). These findings indicate that the transcripts expressed by human NK cells represent a hybrid form, similar to the IIb isoform sequence at the 5-end and to the IIa isoform sequence at the 3-end. This type of transcript has been described as the product of the FcRIIC gene,20 shown to be the result of an unequal crossover event between the IIA and IIB genes.32 Our results confirm previous reports that have shown that U937 and K562 cells also express the FcRIIc isoform.33 The lack of amplification of transcripts for the FcRIIA or FcRIIB genes provides additional evidence that the NK preparations were highly pure because transcripts from either of these genes would readily appear from contaminating monocytes or B cells. View larger version (90K): [in this window] [in a new window]   Fig 3. Southern blot analysis to determine the FcRII isoforms expressed by human NK cells. RT-PCR with the FcRIIa/c-specific primer pair on three A-NK cell samples, U937 and K562 cell lines (positive control), or Molt4 (negative control) was performed as described. The Southern blots were probed with oligonucleotides specific for all the FcRII isoforms (RS91-46; [A]), for FcRIIa (HR; [B] and LR; [C]) and for FcRIIc (IIB; [D]), and cell-specific transcripts of the expected sizes were detected with each probe. Human NK cells express four distinct types of FcRIIc transcripts.   To confirm that human NK cells express FcRIIc-specific transcripts, we next designed primers to amplify and clone full-length FcRIIc-specific PCR products. RNA isolated from A-NK cells from 3 donors was used in RT-PCR reactions with STC and 3IIA primers (Fig 4); in addition, a human cDNA library, generated from a highly purified resting NK-cell population, was used as a template with the same set of primers to generate full-length FcRIIc products (data not shown). The RT-PCR reactions were set up using the same amount of RNA from each cell source, and the resulting amplification products are shown in Fig 4. Interestingly, there was substantial donor heterogeneity in the number and abundance of bands amplified from each RT-PCR sample. A-NK2 showed a single band whereas two or three bands were amplified from the other two NK-cell donors. These PCR products were cloned, and 42 positive colonies were selected by colony hybridization using the RS91-46 probe. The purified cDNAs from all the positive clones were analyzed by gel electrophoresis, and four different sizes of inserts were detected (data not shown). Several representatives of each of these clones were sequenced. View larger version (78K): [in this window] [in a new window]   Fig 4. Full-length FcRIIc-specific RT-PCR analysis of NK cell RNA. RNA isolated from the indicated human A-NK cell preparations was reverse transcribed and PCR amplified with an FcRIIc-specific primer pair that would generate full-length products and further analyzed on an EtBr containing 2% agarose gel. The 100-bp DNA ladder was used as a size marker. FcRIIc1 and three novel FcRIIc isoforms are expressed in human NK cells.   From the sequence data, four distinct FcRIIc isoforms were identified that were homologous with the FcRIIC gene, and a schematic map of the coding sequences of the four isoforms is presented in Fig 5. One of the cDNAs, designated FcRIIc1, corresponded to the previously described FcRIIc isoform. The other three cDNAs are likely to represent previously undescribed, alternatively spliced products of the same gene and have several insertions and deletions. A second cDNA (FcRIIc2) had an insertion of 14 bp at the junction between the C2 and C3 intracytoplasmic exons that alters the reading frame of the encoded cytoplasmic tail resulting in a shortened cytoplasmic tail of 22 amino acids (aa) in length. The source of the 14-bp insertion was found at the intron/exon borders of the FcRIIC gene with 3 nucleotides coming from the 5-end of intron 6 and 11 nucleotides from the 3-end of intron 6. The most frequent type of clone sequenced (FcRIIc3) lacked the second intracytoplasmic exon (C2), and this resulted in a truncated 13 aa cytoplasmic tail (Fig 6). Finally, sequence analyses of the fourth cDNA clone, designated FcRIIc4, revealed a deletion of both C1 and C2 exons (similar to the IIc3 isoform) but, in addition, an insertion of 85 bp between the EC2 and TM exons was found that would appear to be an alternatively used exon. This insertion encodes 19 aa followed by a stop codon, which would result in a receptor lacking the TM region and which may represent a soluble form of FcRIIc. Interestingly, the source of this insertion was found within the intron sequence between the EC2 and TM exons, and the flanking nucleotides do consist of appropriate GT-AG junctional splice sequences.45 View larger version (33K): [in this window] [in a new window]   Fig 5. Physical map of the FcRIIc-specific transcripts expressed in human NK cells. Sequence analysis of 16 cDNA clones obtained from four NK cell samples revealed the presence of four different alternatively spliced FcRIIc-specific transcripts (IIc1-c4). IIc1 was identical with the already described FcRIIc isoform; IIc2 transcript had a 14-bp insertion at the junction between the C2 and C3 exons; the IIc3 transcript had spliced out both the C1 and C2 exons; and IIc4 had an 85-bp insertion between the EC2 and TM exons and both the C1 and C2 exons spliced out. View larger version (43K): [in this window] [in a new window]   Fig 6. Predicted aa sequence of the FcRIIc1-4 isoforms as compared with the previously described FcRIIa1, b2, and c isoforms. Four representative FcRIIc isoforms were identified and the translated sequence is shown in comparison to FcRIIa1. The exon borders are demarcated by vertical lines and the numbering follows the aa position as previously described.20 The dashes represent sequence identity with the FcRIIa1 isoform, the pluses represent deleted aa, and the stars indicate a stop codon. At positions 107, 120, and 161 unique changes in the aa sequence of FcRIIc-specific isoforms are indicated. The ITAM within FcRIIa and IIc sequences encompasses aa 254-272. The sequence data are available from GenBank under accesion numbers: U90938-U90941. The aa translation of these clones (Fig 6) revealed several differences in the aa sequence of the FcRIIc isoforms isolated from NK-cell preparations, as compared with the previously described FcRII sequences. For example, at aa 107, because of a point mutation, an arginine (R) found in all FcRII isoforms was changed to a lysine (K) in the IIc2 and IIc4 isoforms. This was found in all five sequenced cDNA clones from two individuals. Furthermore, at aa 120 in the IIc3 sequence, four out of seven cDNA clones had an isoleucine (I) instead of a threonine (T). This change was found in two different individuals and may suggest the presence of another allelic form of FcRIIc. At position 161 all of the IIc isoforms had a tyrosine (Y) instead of a phenylalanine (F). These changes may result in differences in ligand binding specificities of the newly described isoforms. Identification of an FcRIIc allelic polymorphism.   Following translation of the cDNA sequences, an additional allelic polymorphism of FcRIIc was detected in exon 3 (EC1) at aa position 13, with either a CAG = Gln or a TAG = Stop Codon (STP). This results in either a functional open reading frame (ORF) or a null allele (STP; Fig 6). This observation has been previously reported for the FcRIIC gene.20 To detect the potential distribution of the two allelic forms among the four NK cell donors, we designed allele-specific oligonucleotide (ASO) probes based on the polymorphic sequence in EC1. DB analyses of the full-length PCR products generated from the four human NK-cell preparations using ORF and STP probes (Fig 1) determined that A-NK1 and gt10 donors were homozygous for STP (STP/STP), A-NK2 was homozygous for ORF (ORF/ORF) and A-NK3 was heterozygous (ORF/STP) (Table 1).   View this table: [in this window] [in a new window]   Table 1. Results of Southern Blot Analyses on RT-PCR Products From Four NK-Cell Preparations Using ASO and ISO We also designed isoform specific oligonucleotide (ISO) probes (c1-, c2-, c3-, and c4-probes; Fig 1) to detect the distribution of the FcRIIc isoforms within the same RT-PCR amplifications. Results in Table 1 suggested that distribution of the FcRIIc isoforms was donor-dependent. RT-PCR amplification generated from donor A-NK2 resulted in only one major band detected by gel electrophoresis (Fig 4) and consisted exclusively of the FcRIIc1 isoform (confirmed by DB analysis with ISO probes and cloning results). The other three donors expressed all four isoforms. Multiple bands were detected in their RT-PCR amplifications (Fig 4), and these findings were confirmed by DB analysis with ISO probes (Table 1). Transfection of FcRIIc isoforms in Jurkat cells.   We further analyzed the ability of representative FcRIIc cDNAs to be expressed as proteins in stable Jurkat transfectants. Cells transfected with FcRIIb1 cDNA were used as a positive control; cells transfected with the pcDNA3 vector were used as a negative control (mock, data not shown). Following selection, the transfectants were analyzed by flow cytometry for CD32 expression with a panel of four anti-CD32 MoAbs: IV.3 (FcRIIa-specific), 41H16 (FcRIIb and FcRIIa HR-specific), KB61, and AT10 (FcRIIa and FcRIIb-specific). Results were compared with mouse IgG isotype control binding and to the binding pattern of the anti-CD32 MoAbs to the K562 cell line. Two of the FcRIIc isoforms and the FcRIIb1 cDNAs were successfully expressed in Jurkat cells. As shown in Fig 7, the K562 cell line was positive with all four anti-CD32 MoAbs, whereas the FcRIIb1 transfectant was recognized by 41H16, KB61, and AT10 at equal levels (60% to 90%), but not by IV.3 as expected. The cells transfected with FcRIIc1 and FcRIIc3 cDNAs stained positively with 41H16 and KB61 (60% to 90% positive cells). The transfectants were negative for IV.3 binding but interestingly stained poorly with the AT10 MoAb as compared with the FcRIIb1 transfectants. The AT10 antibody was previously described to recognize both FcRIIa and FcRIIb isoforms.46 These results indicate that FcRIIc1 and FcRIIc3 isoforms have a unique staining pattern with the tested anti-CD32 MoAbs, distinct from that described for the previously characterized FcRIIa and FcRIIb isoforms. View larger version (20K): [in this window] [in a new window]   Fig 7. Flow cytometric analysis of FcRIIc1 and IIc3 expression in stable transfections. K562 cell line or Jurkat cells transfected with full-length cDNAs encoding FcRIIb1, FcRIIc1, and FcRIIc3 were analyzed by indirect immunofluorescence staining with four anti-CD32 MoAbs (filled histograms) or with a mouse IgG isotype control (dotted line), followed by staining with FITC-GAMIgG. FcRIIc expression on NK cells varies among donors and correlates with the allelic polymorphism of FcRIIC.   In view of the novel molecular findings and the unique MoAb staining pattern of FcRIIc detected on Jurkat transfectants, we looked for CD32 expression on fresh NK cells from additional donors. In these experiments, PBMCs were analyzed by two color flow cytometry for expression of CD32 and CD16 on CD56+ cells (a marker that is expressed on most NK cells) and compared with the binding of mouse IgG1 isotype control. Results shown in Fig 8A identify different patterns of CD32 expression and staining in two normal individuals. With donor 1, 64% of CD56+ cells were also CD16+, but no CD32 expression was detected by the four anti-FcRII MoAbs used. In contrast, with donor 2, a high level of CD32 expression on CD56+ NK cells was detected. Notably, the percentages of CD56+/CD32+ cells and CD56+/CD16+ cells in this donor were comparable. In addition, the pattern of CD32 staining with the four anti-CD32 MoAbs was comparable to that found in FcRIIc Jurkat transfectants: high levels of staining with the 41H16 and KB61 MoAbs, low levels of staining with AT10 and IV.3. View larger version (34K): [in this window] [in a new window]   Fig 8. Flow cytometric analysis of CD32 expression on human NK cells and its correlation with the allelic polymorphism of FcRIIC. (A) PBLs isolated from donor 1 and donor 2 were analyzed by using PE-conjugated anti-CD56. Gated CD56+ cells (R7 box, panels a) representing NK-cell population, were then assessed for their levels of CD16 (3G8, panels b), or CD32 expression (IV.3, panels c; 41H16, panels d; KB61, panels e; and AT10 panels f). Percentages of positive CD16 and CD32 cells are shown for each histogram and are compared with the binding of mIgG isotype control. (B) SB analyses of FcRIIc-specific RT/PCR amplifications using RS91-46 and ASO probes to detect the genotype of the two donors. The results are representative of three independent experiments. To further test the hypothesis that individuals lacking expression of CD32 on their NK cell are homozygous for STP allele, we investigated the correlation between the level of CD32 expression and the genotype of these two donors. RT-PCR experiments on RNA obtained from NK cells from these two donors were performed using FcRIIc-specific primers (STC-3IIA); the amplified products were further analyzed by DB using ASO probes. As shown in Fig 8B, donor 1 (no detectable CD32 expression) was found to be homozygous for STP (STP/STP), whereas donor 2 (high levels of CD32 expression) was homozygous for ORF (ORF/ORF). We can therefore explain the previous lack of detection of CD32 on NK cells, as a result of donor variability due to an allelic polymorphism within the FcIIC gene, in conjunction with the use of inappropriate anti-CD32 MoAbs for staining. FcRIIc expressed on human NK cells is a protein of approximately 40 kD.   To determine the biochemical features of CD32 expressed on human NK cells, we performed immunoprecipitations from biotinylated cell lysates obtained from U937 cells, as well as from PBL and highly purified NK cells isolated from donor 2 using 41H16 MoAb or a mouse IgG2a as negative control. As shown in Fig 9, a protein of approximately 40 kD was specifically precipitated with the anti-CD32 MoAb from the NK-cell preparation and PBL and was comparable in size with the proteins detected in U937 lysates. The number of cells used to generate lysates was comparable for each cell source. In addition, no material was adsorbed by the beads coupled with the isotype control (mIgG2a). These biochemical findings provide further evidence that the proteins on NK cells detected by flow cytometry with certain anti-CD32 MoAbs are indeed FcRII. View larger version (29K): [in this window] [in a new window]   Fig 9. Immunoprecipitation of FcRIIc from highly purified NK cells. Lysates generated from biotinylated 2 × 106 PBL, NK cells, or U937 cells were incubated with 41H16-, IV.3-, or mIgG2a-coated sepharose-GAM beads o/n at 4°C, eluted by SDS-PAGE, transferred to PVDP membranes, and incubated with HRP-neutravidin. The bands were detected by ECL. Results represent one experiment out of two performed. The molecular markers are shown on margins. FcRIIc expression on NK cells mediates rADCC.   To investigate the functional potential of FcRII expressed on NK cells in certain individuals, we analyzed the role of CD32 ligation in triggering cytotoxic events. The results were compared with the cytolysis triggered through CD16 on the same cells. Fresh, highly-purified NK cells isolated from donors 1 and 2, as well as A-NK cells generated from donor 2, were used in rADCC assays in the presence of intact anti-CD32 MoAb KB61, anti-CD16 MoAb, 3G8 or anti-CD64 MoAb 32.2 against FcR+ target P815. All of these MoAbs were mIgG1 and as a negative control, rADCC in the presence of a nonspecific mIgG1 MoAb was also assessed. Results in Fig 10 show that both fresh or IL-2-activated NK cells isolated from donor 2 (Fig 10B through 10D) could be triggered to kill P815 through either CD16 or CD32, although the killing efficiency of A-NK cells was significantly higher (Fig 10D). In contrast, NK cells isolated from donor 1 (Fig 10A), which lack CD32 expression, were cytotoxic against the P815 target only when triggered via CD16. Addition of anti-CD64 MoAb 32.2 (Fig 10C) did not result in killing of the target cells, further confirming the absence of contaminating monocytes in the NK-cell preparations. These findings provide evidence that CD32 on NK cells is a functional molecule. View larger version (29K): [in this window] [in a new window]   Fig 10. Comparison of rADCC induction through CD16 and CD32 expressed on human NK cells. Fresh, highly-purified NK cells from donor 1 (A) and donor 2 (B and C) as well as A-NK cells generated from donor 2 (D) were tested in the presence of KB61 anti-CD32 (1 µg/mL) or 3G8 anti-CD16 (1 µg/mL) for their ability to kill the FcP+ target P815 in a 4-hour 51Cr-release cytotoxicity test at the indicated E:T ratios. NK cells from donor 2 were also tested in the presence of 32.2 anti-CD64 (1 µg/mL) (C).     DISCUSSION Abstract Introduction Methods Results Discussion References Three classes of receptors for the Fc domain of IgG (FcRI, FcRII, and FcRIII) are known to provide a critical link between the humoral and the cellular compartments of the immune system. Following interaction with IgG, a large array of biologic responses are triggered, such as phagocytosis, endocytosis, ADCC, and cytokine release. A total of eight genes have been described for the FcRs, and further significant transcript heterogeneity results from alternative splicing. In addition, allelic polymorphisms have been described for FcRIa,47 FcRIIa,21 FcRIIc,20 FcRIIIA,48 and FcRIIIB.49 Some of these polymorphisms result in significant functional differences,50-52 whereas others do not appear to influence the functional capabilities of these receptors or to result in obvious clinical manifestations.52,53 Human FcRII (CD32), the low affinity receptor for IgG, has been characterized as a 40-kD glycoprotein, with a putative protein core of 36 kD16-18; it is the most broadly distributed FcR, and numerous reports have previously described coexpression of FcRIIA, B, and C gene products in hematopoietic cells.18,33 The results presented in this study provide conclusive evidence that human NK cells express significant levels of FcRII. In certain individuals, CD32 is expressed on 70% to 80% of their NK cells, at levels comparable to those observed for CD16. The molecular characterization of CD32 isoforms expressed by NK cells revealed that these cells express FcRIIc isoforms. No transcripts from the FcRIIA or FcRIIB genes have been detected, to date, in at least 10 individuals studied, suggesting that NK cells might be relatively unique among hematopoietic cells and only express FcRIIc isoforms. Four FcRIIc transcripts were identified, three encoding transmembrane proteins (IIc1, IIc2 and IIc3), and one encoding a putative soluble form (IIc4). The variability in detection of CD32 expression among donors was found to be related to two main factors: (1) an allelic polymorphism, in the EC1 domain of FcRIIC and (2) a unique pattern of recognition by anti-CD32 MoAbs. These two factors, we believe, are responsible for previous failures to find CD32 on NK cells. The data in this paper show that although certain individuals express only CD16 on their NK cells, others express both CD32 and CD16. The allelic polymorphism results either in a null allele (STP) or a functional one (ORF). An individual who exhibited a high level of CD32 expression was found to be homozygous for ORF/ORF, whereas an individual whose NK cells were CD32 negative was homozygous for STP/STP. We have begun to extend these studies and have been able to identify an additional 5 individuals (out of 10 more tested) that express high levels of CD32 on their NK cells. The precise correlation between the genotypic form (heterozygous v homozygous for STP and/or ORF) and variable (high v low) phenotypic expression of FcRIIc in these donors remains to be established. Immunoadsorbtion with 41H16 MoAb from CD32+ NK-cell lysates revealed a protein of approximately 40 kD, comparable in size with the material precipitated from PBLs and slightly smaller than the proteins adsorbed from U937 cells. This is in good agreement with a previous report providing evidence of discrete differences in gel migration among CD32 isoforms immunoprecipitated from different cell types, including from U937 cell lysates. The proteins ranged from 37 to 42 kD, and that was shown to be dependent on both isoform type and the state of glycosylation.54 In addition, we show that NK-cell populations that express CD32 as well as CD16 may mediate lytic events through either receptor. Interestingly, the levels of killing against P815 when triggered through CD16 were greater as compared with those triggered through CD32, and this is probably related to differences in the levels of expression of these two FcRs on the NK cells used. The mean fluorescence intensity obtained using the anti-CD32 MoAb KB61 is significantly lower than that obtained with anti-CD16 MoAb 3G8 and could reflect either lower levels of expression of CD32 than CD16, or that recognition of CD32 by KB61 is not optimal. In any event, these studies show conclusively that significant functional lytic activity can be triggered through CD32 on NK cells. Transfection experiments using FcRIIc1 and IIc3 revealed that these full-length cDNAs encode functionally expressed proteins and that they display a unique pattern of reactivity with a set of anti-CD32 MoAbs. Most significantly these isoforms were recognized poorly by AT10 (described to be a pan anti-FcRII MoAb) and IV.3, a commonly used anti-CD32 MoAb. The NK-cell-derived FcRIIc isoforms were best recognized by 41H16 and KB61, which are known to predominantly recognize FcRIIb isoforms.46 Most of the studies describing the lack of CD32 expression on NK cells used either IV.3 or AT10 MoAbs. One study in addition to ours, that described CD32+ NK cells, used the 41H16 MoAb. Because it appears that human NK cells only express FcRIIc, the appropriate antibodies to detect this isoform are 41H16 and KB61. Sequence analyses of the Fc