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The Journal of Immunology, 2006, 177: 3848-3856.
Copyright © 2006 by The American Association of Immunologists, Inc.

IgG Fc Receptor III Homologues in Nonhuman Primate Species: Genetic Characterization and Ligand Interactions1,2

Kenneth A. Rogers*, Franco Scinicariello{dagger} and Roberta Attanasio3,*

* Department of Biology, Georgia State University, Atlanta, GA 30303; and {dagger} Division of Toxicology Environmental Medicine, Centers for Disease Control and Prevention, Agency for Toxic Substances and Disease Registry, Atlanta, GA 30341


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ig Fc receptors bind to immune complexes through interactions with the Fc regions of specific Ab subclasses to initiate or inhibit the defense mechanisms of the leukocytes on which they are expressed. The mechanism of action of IgG-based therapeutic molecules, which are routinely evaluated in nonhuman primate models, involves binding to the low-affinity FcRIII (CD16). The premise that IgG/CD16 interactions in nonhuman primates mimic those present in humans has not been evaluated. Therefore, we have identified and characterized CD16 and associated TCR {zeta}-chain homologues in rhesus macaques, cynomolgus macaques, baboons, and sooty mangabeys. Similar to humans, CD16 expression was detected on a lymphocyte subpopulation, on monocytes, and on neutrophils of sooty mangabeys. However, CD16 was detected only on a lymphocyte subpopulation and on monocytes in macaques and baboons. A nonhuman primate rCD16 generated in HeLa cells interacted with human IgG1 and IgG2. By contrast, human CD16 binds to IgG1 and IgG3. As shown for humans, the mAb 3G8 was able to block IgG binding to nonhuman primate CD16 and inhibition of nonhuman primate CD16 N-glycosylation enhanced IgG binding. Clearly, differences in interaction with IgG subclasses and in cell-type expression should be considered when using these models for in vivo evaluation of therapeutic Abs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Fc receptors are plasma membrane glycoproteins that bind to the Fc region of one or a few classes of Abs. Cross-linking of Ab Fc receptors by Ab-opsonized Ag complexes initiates cellular immune responses, including phagocytosis, Ab-dependent cell-mediated cytotoxicity (ADCC),4 respiratory burst, release of cytokines and inflammatory mediators, and Ag presentation (1). Fc receptors are therefore crucial for the destruction and clearance of pathogens and tumors. Different Fc receptors with specificity for each of the five classes of Abs (IgM, IgD, IgA, IgE, and IgG) have been identified in mammals. Human IgG Fc receptors include Fc{gamma}RI, Fc{gamma}RII, and Fc{gamma}RIII, which differ for cell-type distributions and affinity for the four subclasses of human IgG (1). Human Fc{gamma}RIII, also known as CD16, is specific for IgG1 and IgG3 (2).

Humans express two 97% identical Fc{gamma}RIII isoforms, CD16a and CD16b, encoded by separate genes consisting of two Ig-like domains and a tail region linking the protein to the plasma membrane (3, 4, 5). CD16a is expressed on monocyte subpopulations, macrophages, NK cells, and select {gamma}{delta} T cells, and can be induced on glomerular mesangial cells (6, 7, 8). Because CD16a is the only Fc receptor expressed on NK cells and is responsible for IgG-initiated ADCC, it has been called the ADCC receptor (9, 10). The CD16a complex consists of three polypeptide chains: one unique ligand-binding chain and two signaling chains. The ligand-binding chain (CD16a or Fc{gamma}RIIIa) spans the plasma membrane and has a short cytoplasmic tail. In humans, the signaling chains of the complex are composed of either a homodimer or a heterodimer of FcR{gamma} and TCR {zeta} that are required for efficient assembly, transport of the receptor from the endoplasmic reticulum to the plasma membrane, and retention on the plasma membrane (4, 11, 12). FcR{gamma} is also a necessary component of the Fc{epsilon}RI and Fc{gamma}RI complexes (1, 13). TCR {zeta}, expressed in T cells and NK cells, is also a component of the TCR complex (14). In contrast to CD16a, CD16b is not associated with signaling chains. The CD16b isoform, exclusively expressed on neutrophils and eosinophils that have been exposed to IFN-{gamma} (1, 15), is linked to the outer plasma membrane by a GPI link, and modulates cellular responses through interactions with the other neutrophil Fc receptors (16, 17). Association of the FcR{gamma}-signaling chain dimer with CD16a may contribute to a higher ligand affinity of CD16a compared with CD16b (18).

Nonhuman primates are widely used in biomedical research. The complex mechanisms of action and pharmacokinetics of therapeutic Abs, usually IgG1 or IgG2 molecules, are routinely evaluated in these species (19, 20, 21, 22). Potential therapeutic cytokines and cytokine receptors are tested in nonhuman primates, and there is interest in extending these studies to Ig-cytokine fusion proteins, as has been done in mice (23, 24, 25). Macaques represent the accepted model for HIV vaccine development and AIDS pathogenesis (26). CD16a is critical for NK cell-targeted destruction of HIV-infected cells through ADCC, although the relative importance of this mechanism in vivo is debated (9). Recently, a small study in SIV-infected macaques indicated that sustained ADCC correlates with delayed onset of AIDS pathogenesis (27). Xenograft rejection, which is also studied in nonhuman primates, is in part mediated through IgG-directed ADCC via CD16 (28, 29, 30, 31). In addition, these species are used in testing mAbs designed to prevent allograft rejection (32, 33, 34, 35, 36, 37). Hence, there is the need to characterize CD16 homologues and the interactions with their ligands in nonhuman primate models.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Samples

Heparinized blood samples were collected from healthy animals of each of the following species: rhesus macaque (Macaca mulatta), cynomolgus macaque (Macaca fascicularis), baboon (Papio hamadryas anubis), and sooty mangabey (Cercocebus torquatus). Rhesus macaque and baboon samples were from animals housed at the Southwest National Primate Research Center (San Antonio, TX). The samples from cynomolgus macaque and sooty mangabey were from animals housed at the Yerkes National Primate Research Center, Emory University (Atlanta, GA). Animal blood was collected under approval of the appropriate institutional review committees.

Determination of CD16 expression on blood leukocytes

Blood from four rhesus macaques, seven cynomolgus macaques, four baboons, and four sooty mangabeys was collected in EDTA Vacutainer tubes (BD Biosciences) by venipuncture under anesthesia. Leukocyte expression of CD16 on macaque cells was analyzed by three-color flow cytometry analysis using CyChrome-conjugated anti-human CD16 (clone 3G8), PE-conjugated anti-human CD89 (clone A59), and FITC-conjugated anti-human CD3 (clone SP34) (BD Biosciences). Simultest Control {gamma}1/{gamma}2 (BD Biosciences) was used to detect nonspecific binding of mouse IgG to cells. Baboon and sooty mangabey leukocytes were analyzed by staining with PE-conjugated anti-human CD16 and FITC-conjugated anti-human CD3. Additionally, sooty mangabey leukocytes were stained with CD89 PE and CD16 FITC. Staining of whole blood was done using a standard procedure (38).

Amplification, cloning, and sequence analysis of nonhuman primate CD16, TCR {zeta}, and FcR{gamma} cDNA

Total RNA was extracted from whole blood using the QIAamp RNA Blood Mini Kit (Qiagen), and reverse transcribed into cDNA using oligo(dT)17 primers, followed by primer extension with the AMV reverse transcriptase (Roche Diagnostic Systems). PCR amplification of the cDNA was performed with Expand High Fidelity polymerase (Roche Diagnostic Systems) with the appropriate primer pair. Primer pairs were FCG3aF (5'-ATGTGGCAGCTGCTCCTCCCA-3') and FCG3aR (5'-TCATTTGTCTTGAGGGTCCTT-3') for CD16, TCRZ3F (5'ATGAAGTGGAAGGCGCTTTTCAC-3') and TCRZ4R (5'-TTAGCGAGGGGGCA-3') for TCR {zeta}, and FcRgamF (5'-ATGATTCCAGCAGTGGTCTTGCT-3') and FcRgam4 (5'-CTACTGTGGTGGTTTCTCATGCTTC-3') for FcR{gamma}. After initial denaturation at 95°C for 10 min, the cDNAs were amplified for 40 cycles, with each cycle consisting of 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min and 30 s. A final step at 72°C for 10 min was used to ensure complete extension. For amplification of the signaling chains, the step at 72°C was reduced to 30 s. All reactions were performed in at least two independent RT-PCRs to verify product sequences. For CD16 of each nonhuman primate species, 10 clones were sequenced from each of two independent PCR. Cloning, sequencing, and sequence analysis were performed using methods previously described (39).

Construction of nonhuman primate CD16 expression vectors

Full-length nonhuman primate CD16 genes were first PCR amplified from cDNAs ligated into the pCR2.1 vector using forward and reverse primers FCG3FHinb (5'-AGATAAGCTTGATATGTGGCAGCTGCTCCTCCCA-3') and FCG3Rbam (5'-TCTAGGATCCTCATTTGTCTTGAGGGTCCTT-3'), which add HindIII and BamHI restriction sites 5' and 3' of the full-length cDNA, respectively. CD16 fragments from clones with the correct sequence were released from vector by sequential digestion with HindIII and BamHI, cleaned up on a 1% agarose gel, and ligated into the HindIII- and BamHI-digested expression vector pcDNA3.1+ (Invitrogen Life Technologies) to create pcCD16 vectors. CD16 expression vectors were then cloned into Top10 Escherichia coli, and colonies were screened by sequencing with primers T7 and T7 reverse.

To improve the stability of the expression vectors in transfected cells, part of the pcDNA3.1 constructs was amplified (containing cDNA of nonhuman primate Fc{gamma}IIIRa, a CMV promoter, a bovine growth hormone polyadenylation signal, and an f1 origin) and inserted into the vector pLSXN (BD Biosciences), which contains long terminal repeats for integration into chromosomes. pLSXN contains a neomycin resistance gene, which allows for transfectant selection. Insertion of a pcDNA-CD16 fragment into pLXSN was conducted by amplifying with primers PcHp (5'-CTGCTGTTAACCGTTAGGGTTAGGCGTTTTGCG-3') and PcSa (5'-ACTTTGTCGACGCTCAGCGGCCGGCCATCGATCCACAGAATTAATTCGCGTT-3'). The resulting fragment was then digested with HpaI and SalI and pLXSN digested with HpaI and XhoI. The two fragments were then ligated together to form pLXSN-CD16.

Generation of HeLa cell clone expressing nonhuman primate rCD16

Large quantities of vector for transfection were prepared using EndoFree Plasmid Maxi kits (Qiagen). A total of 20 µg of expression vector was electroporated into HeLa cells using methods previously described (38). Cells were then grown in DMEM 10% FCS in 5% CO2 at 37°C, and the antibiotic G418 (400 µg/ml) was added to cell 72 h posttransfection to obtain stable transfectants.

Selection and expansion of clones were performed following identification of successful transfections, as indicated by positive stain for CD16 and a positive RT-PCR for CD16. To isolate single clones, cells were diluted serially into 96-well microtiter plates. Cells were grown in 100 µl of DMEM 10% FCS with G418 400 µg/ml, which was 50% fresh medium and 50% conditioned medium collected from flasks of untransfected HeLa cells and filtered with a 0.2-µm filter. Each well was examined by microscopy to identify wells with a single cell. Clones were subsequently expanded in wells of increasing size until cell numbers were sufficient to screen for CD16 expression.

Purification of nonhuman primate IgG

Nonhuman primate IgG was purified from serum from each species. Briefly, 200 µl of serum diluted 1/1 in Pierce ImmunoPure IgG-binding buffer was incubated with 200 µl of ImmunoPure Plus immobilized protein G for 30 min at room temperature (Pierce Biotechnology). The mix was then transferred to a 0.45-µm filter tube and centrifuged for 2 min at 6 x g, and the filtrate was discarded. Following four washes with PBS (pH 7.2) and a 10-min incubation at room temperature with Pierce ImmunoPure IgG elution buffer, the tube was spun for 2 min and the filtrate was collected. Finally, the filtrate was dialyzed against water using Spectral/Por cellulose ester MWCO 50,000 (Spectrum Laboratories), first with two incubations at room temperature for 1 h each and then overnight at 4°C with the buffer being changed each time. Purity of IgG was verified by a reducing SDS-PAGE, and the concentration was checked by spectrophotometry at 280 nm. IgG was frozen at –80°C before use.

IgG subclass ELISA

ELISA was used to verify the purity and identity of the human IgG myeloma proteins used in the CD16-binding assays. Microtiter plates were coated with human myeloma proteins (IgG1, IgG2, IgG3, or IgG4) (The Binding Site) or purified total nonhuman primate IgG from the four species, incubated at 4°C overnight, and blocked with 5% FCS diluted in PBS at 37°C for 30 min. After washing, anti-human IgG1 (clone SG-16), IgG2 (clone HP-6014), IgG3 (clone HP-6050), or IgG4 (clone SK-44) was added to the plate and incubated for 1 h at 37°C (Sigma-Aldrich). Following washing to remove unbound Ab, HRP-labeled goat anti-mouse IgG (H + L) was added to the plate and incubated for 1 h at 37°C (Kirkegaard & Perry Laboratories). The washed plate was developed by the addition of first ABTS/H2O2 and then stop solution, and its absorbance was measured at 405 nm using an automated Benchmark microplate reader (Bio-Rad).

Ig-binding assay

Binding of Ig to CD16 was assessed by flow cytometry using Abs that were heat aggregated at 63°C for 1 h. Human myeloma proteins IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM (The Binding Site), or IgE (Serotec) with Ig{kappa} L chain as well as purified nonhuman primate IgG from different species were added to 0.5 x 106 cells at 20 µg/ml and incubated for 1 h at 4°C. PBS-washed cells were then stained with 5 µl of either FITC-conjugated mouse anti-human {kappa} or FITC-conjugated mouse IgG3 control for 30 min at 4°C (Invitrogen Life Technologies). In addition, in some experiments, cells were stained using FITC goat anti-human IgG (Invitrogen Life Technologies). For dual labeling experiments, mouse anti-human CD16 PE was added as well. Cells were washed and analyzed by flow cytometry, as described above. The ability of mouse mAb 3G8 to block IgG binding was performed by first incubating harvested HeLa cells and mangabey CD16-expressing cells (0.5 x 106 cells/tube) with 20 µl of 3G8 or control mouse myeloma IgG1 at different concentrations (0.5, 0.25, 0.125, and 0 mg/ml) for 30 min at 4°C. Afterward, cells were washed three times with PBS and IgG-binding tests were conducted, as described above, except that human myeloma Abs were added at 40 µg/ml. For N-glycosylation-blocking experiments, tunicamycin was added to half of the cell cultures at 1 µg/ml 30 h before harvesting cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD16 expression on nonhuman primate leukocytes

CD16 is known to be expressed on nonhuman primate NK cells and monocytes (40, 41, 42). Our results show that CD16 is present on monocytes (mean ± SD, 14.67 ± 4.66% rhesus, 28.40 ± 14.00% cynomolgus, 21.38 ± 13.71 baboon, and 25.61 ± 13.43% mangabey; Fig. 1, E, H, K, and N) and CD3– lymphocytes (11.75 ± 4.28% rhesus, 15.31 ± 7.64% cynomolgus, 11.92 ± 6.95% baboon, and 6.70 ± 1.97% mangabey; Fig. 1, D, G, J, and M), corresponding to NK cells (41), as well as on the majority of granulocytes of sooty mangabeys (72.51 ± 14.29% of CD89+CD16+ cells; Fig. 1L), but not granulocytes of macaque and baboons (Fig. 1, C, F, and I).


Figure 1
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  		FIGURE 1. Representative two-color dot plots of whole blood leukocytes from nonhuman primates stained for CD16 expression. A, Forward scatter vs side scatter plot with leukocyte gate indicated; B, staining with mouse isotype controls; C–N, gated leukocyte populations (C, F, I, and L, granulocytes; D, G, J, and M, lymphocytes; E, H, K, and N, monocytes) stained for CD16 and either CD89 or CD3. CD16 expression was examined for rhesus macaques (C–E), cynomolgus macaques (F–H), baboons (I–K), and sooty mangabeys (L–N). At least 10,000 cells were counted for all plots.

 
Cloning and sequencing of nonhuman primate CD16 genes

To verify that both human CD16 genes could be amplified with the primers we designed, human CD16 cDNA was amplified, cloned, and sequenced. All sequences from clones amplified using total RNA from human whole blood matched the CD16b isoform allele FCGR3B*02 (GenBank accession no. AJ581669), probably a result of CD16b transcripts outnumbering CD16a transcripts. Therefore, we stimulated THP-1 monocytic cells with PMA in the absence of estrogen to induce expression of CD16a. Amplification of cDNA from these cells yielded clones all matching a reported CD16a sequence (GenBank accession no. X52645), thus validating our strategy to amplify both human CD16 isoforms. This strategy was then used to amplify CD16 from a single animal of each of four nonhuman primate species.

All nonhuman primate CD16 clones bore greater sequence homology to human CD16a than CD16b. In nonhuman primates, residues at positions that differ between human CD16a and CD16b were conserved with human CD16a, including Gly147, Tyr158, and Phe203 (Fig. 2). Phe203 is substituted by a Ser in CD16b and is critical to the expression of CD16b as a GPI-linked protein on human neutrophils (43). Single sequences were obtained for rhesus macaque, baboon, and sooty mangabey CD16, whereas two sequences differing by a single amino acid (Lys25 or Arg25) were obtained for cynomolgus macaque CD16 (GenBank accession nos. DQ423376-DQ423380). All cysteines involved in forming intrachain disulfide bonds in human CD16 are conserved in nonhuman primates (C47, C89, C128, and C172). Human CD16 (GenBank accession no. CAA34755) has N-glycosylation motifs at Asn56, Asn63, Asn92, Asn180, and Asn187. These motifs are conserved in nonhuman primate CD16 molecules, with the exception of Asn92. An additional motif is present in nonhuman primates at Asn82. Rhesus macaque and cynomolgus macaque CD16 amino acid sequences exhibit 91.7 and 91.3% identity to the human CD16a, respectively. The rhesus macaque CD16 amino acid sequence shows 99.6% identity to the corresponding cynomolgus macaque sequence. Baboon and sooty mangabey CD16 have identical amino acid sequences and share 92.5% identity with human CD16a, 98.4% identity with rhesus macaque, and 98.0% identity with cynomolgus macaque. Therefore, CD16 sequences are highly conserved in nonhuman primates with only three residues distinguishing the African species (baboons and mangabeys) from the Asian species (macaques) (an Asp/Glu substitution at position 122, a Val/Met substitution at position 229, and a Ser/Arg substitution at position 238).


Figure 2
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  		FIGURE 2. Alignment of CD16-derived amino acid sequences obtained by cloning and sequencing rhesus macaque, cynomolgus macaque, baboon, and sooty mangabey cDNA from whole blood and human CD16a and CD16b (GenBank accession nos. CAA34753 and J04162). Amino acid differences between human CD16a and nonhuman primate sequences are underlined. The mature peptide begins at residue 18, as indicated by the arrow. Two vertical arrows indicate the boundaries of exon S2, which is spliced out of one isolated rhesus macaque transcript. Potential N-glycosylation sites and cysteines involved in disulfide bonds are bolded. Shaded amino acids indicate amino acids in human CD16 that interact with human IgG1 (46 ). Domains of the protein are indicated by arrows. The sequence of MafaCD16.2 was found to match an unpublished GenBank sequence (accession no. AF485815). MafaCD16.1 is an experimentally isolated variant of CD16 differing at residue position 25. Hu, human; Soma, sooty mangabey; Paca, baboon; Mamu, rhesus macaque; Mafa, cynomolgus macaque; S1 and S2, signal peptide; Tm, transmembrane. Star indicates the amino acid critical for binding to mAb 3G8.

 
The CD16 membrane-proximal Ig-like domain interacts with the lower hinge and CH2 domain of the IgG Fc region (43, 44, 45). The crystal structure of CD16 reveals that a single Fc{gamma}RIII binds asymmetrically to the two chains of a single IgG1 Fc fragment (46). Numbering from the start of the preprotein, the CD16 residues forming contact with IgG1 are Ile106-Trp108, Trp131-Ala135, His137-Thr140, Asp147-His153, Arg173, and Val176-Lys179 (46). The majority of the human CD16 residues important for binding IgG are conserved in nonhuman primate CD16 molecules (Fig. 2). In all four nonhuman primate species, Ala135, His153, and Val176 are substituted with Leu, Glu, and Ile, respectively. In addition, Asp147 is Gly in all nonhuman primate CD16 molecules as is found in human CD16a.

Expression of sooty mangabey/baboon CD16 and identification of nonhuman primate TCR {zeta}-chains

To characterize the ligand interactions of nonhuman primate CD16, the sooty mangabey gene was selected for expression in HeLa cells. One clone resulted in high CD16 expression as determined by flow cytometry (mean fluorescence intensity (MFI) = 1445.22). Anti-CD16 staining was specific, because staining of untransfected HeLa cells (MFI = 8.12) and staining of the clone with a mouse isotype control (MFI = 11.91) were low (data not shown).

CD16-associated signaling chains FcR{gamma} or TCR {zeta}, which are required for efficient expression of human CD16 (11, 47, 48), have not been reported in HeLa cells. TCR {zeta} is normally only expressed in T cells and NK cells, because its gene is controlled by a tissue-restricted promoter (49). However, we were able to amplify and sequence TCR {zeta} cDNA from HeLa cells. Real time RT-PCR confirmed that TCR {zeta} is transcribed in HeLa cells, albeit at levels lower than those found in Hut-78 cells, a T cell line. By contrast, FcR{gamma} transcripts were amplified successfully using RNA isolated from THP-1 cells (positive control), but not RNA isolated from HeLa cells. These results indicate that high levels of CD16 in HeLa cells may be permissible as a result of endogenous TCR {zeta} expression.

In contrast to human TCR {zeta}, mouse TCR {zeta} acts to down-regulate CD16 as a result of a substitution of Leu46 with Ile46 in the transmembrane domain (50, 51). Therefore, TCR {zeta} cDNA from rhesus macaque, cynomolgus macaque, baboon, and sooty mangabey was cloned and sequenced (Fig. 3) (GenBank accession nos. DQ437667, DQ437669, DQ437663, and DQ437665). Importantly, Leu46 is conserved in monkey TCR {zeta}. In baboons and sooty mangabeys, the transmembrane domain and surrounding residues are completely conserved, whereas two substitutions (Ile41Leu and Val53Ala) are present in macaques, which may influence interactions with CD16. For transient transfection, mangabey and cynomolgus macaque CD16 expression was greater in Hut-78 cells than that achieved in HeLa cells (results not shown). Thus, nonhuman primate CD16 may be positively regulated by TCR {zeta}, similar to human CD16.


Figure 3
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  		FIGURE 3. Alignment of TCR {zeta}-deduced amino acid sequences. ITAM: italics with conserved tyrosine and leucine/isoleucines bolded. Other features are underlined and indicated by symbols that appear below the position of the feature: #, cysteine involved in TCR {zeta} dimerization; $, aspartic acid that pairs with a charged residue of associated ligand-binding chain (48 ); +, lysine and glycine residues in human peptide that bind GTP and GDP (52 ); *, glutamine spliced into a variant that disrupts a G-coupled protein-binding motif, which includes two prolines prior and one after the glutamine (53 ). !, Position 46 of leucine critical for human CD16 association (50 ). GenBank accession numbers for humans and mice are AL031733 and BC052824, respectively.

 
The percentages of identities of the deduced amino acid sequences compared with the human TCR {zeta} sequence for rhesus macaque, cynomolgus macaque, baboon, and mangabey are 92.7, 95.1, 96.3, and 96.3%. The amino acid identity between the two macaque species is 98.2%. Baboon- and mangabey-deduced amino acid sequences are 100% identical, although there are differences at the nucleotide level. The baboon/mangabey amino acid sequence shares 97.0% identity with rhesus macaque and 98.8% identity with cynomolgus macaque. All TCR {zeta}-chains from the nonhuman primates have Asn and Gln inserts between residues 131E and 132R of the corresponding human TCR {zeta}. As a result, nonhuman primate TCR {zeta}-chains are 2 aa longer than their human counterpart (166 aa as compared with 164). In addition to the 164-aa TCR {zeta} polypeptide, humans also produce a 163-aa polypeptide that results from the splicing out of the codon encoding Gln101 (53). Splice variants were isolated for all four nonhuman primate species that were 165 aa long as a result of the same splicing event (GenBank accession nos. DQ437668, DQ437670, DQ437664, and DQ437666).

Mangabey/baboon CD16 binding to Igs

Binding assays were performed to determine the ability of mangabey CD16 to bind the different human and Ab subclasses and polyclonal nonhuman IgG. Heat-aggregated myeloma proteins with an Ig{kappa} L chain or polyclonal Ig were incubated with control HeLa cells or mangabey CD16-expressing cells, followed by staining against bound Ab and analysis by flow cytometry.

All tested subclass-specific Abs did not cross-react with purified nonhuman primate species’ IgG. The staining for human IgG subclasses on mangabey CD16-expressing cells as measured by MFI was: IgG2 (131.78) and IgG1 (75.98) >> IgG3 (17.68) > IgG4 (10.05) (Fig. 4). Staining of control HeLa cell for each subclass was as follows: IgG2 (10.75), IgG1 (10.16), IgG3 (14.08), and IgG4 (9.73). Staining of mangabey CD16-expressing cells using a FITC-conjugated mouse Ab isotype control was negative (12.73). These results indicate that mangabey CD16 binds human IgG2 and IgG1 and only slightly binds IgG3, but not IgG4. These results were independently verified using FITC-conjugated goat anti-human Ig{gamma} (data not shown). No staining for heat-aggregated human IgA1 (11.53), IgM (13.34), or IgE (7.75) was detected, nor for heat-aggregated polyclonal mouse IgG (10.31). Polyclonal IgG from sooty mangabey and baboon bound to CD16, as detected by both FITC-conjugated mouse anti-human Ig{kappa} and FITC-conjugated goat anti-human Ig{gamma} (Fig. 5). Binding was observed also for purified polyclonal IgG of rhesus macaques (anti-human Ig{kappa}, MFI = 78.71; anti-human Ig{gamma}, MFI = 284.56) and cynomolgus macaques (anti-human Ig{kappa}, MFI = 67.65; anti-human Ig{gamma}, MFI = 251.66).


Figure 4
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  		FIGURE 4. Binding of human IgG subclasses to mangabey rCD16 expressed on HeLa cells. Human myeloma Igs of different subclasses all with Ig{kappa} L chain were incubated with HeLa cells expressing mangabey CD16; bound Igs were detected with anti-human Ig{kappa} FITC (IgG1, solid line MFI = 75.98; IgG2, filled MFI = 131.78; IgG3, dashed line MFI = 17.68; IgG4, thin line MFI = 10.05). As negative control, cells were stained with a mouse FITC isotype control (dotted line MFI = 12.73). Ten thousand cells were counted per sample.

 

Figure 5
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  		FIGURE 5. Binding of sooty mangabey (A and B) and baboon (C and D) IgG to HeLa cells expressing mangabey rCD16. A and C, Detection of bound IgG with goat anti-human IgG FITC to mangabey CD16: filled (baboon MFI = 992.63, sooty mangabey MFI = 325.87); control HeLa cells: dotted line (baboon IgG MFI = 29.44, sooty mangabey IgG MFI = 37.17); and goat anti-human IgG bound in the absence of IgG to mangabey CD16: solid line (baboon MFI = 61.20, sooty mangabey MFI = 24.93). B and D, Detection of bound IgG with anti-human Ig{kappa} FITC to mangabey CD16: filled (baboon IgG MFI = 62.04, sooty mangabey MFI = 24.46) and in the absence of IgG (baboon MFI = 7.67, sooty mangabey MFI = 7.69). Ten thousand events were counted per sample.

 
IgG binding correlates with expression of sooty mangabey CD16 and is blocked by mAb 3G8

Taking advantage of the variation in CD16 expression levels among different cells of the clone, IgG binding was correlated to CD16 expression. This was done by incubating HeLa cells expressing rCD16 as well as untransfected HeLa cells with each of the four human IgG subclasses, followed by staining with PE-conjugated anti-human CD16 clone 3G8 and FITC anti-human Ig{kappa}. The 3G8 is an antagonist of IgG binding to human CD16 (44). As observed on dot plots of MFI for anti-human Ig{kappa} vs MFI for anti-human CD16, there was a positive correlation between bound IgG and receptor level for the human subclasses IgG2 and IgG1 (data not shown). As expected, no correlation was observed when using IgG3, IgG4, and IgA1. To ascertain whether or not a similar antagonist effect might exist for mangabey CD16, regression analysis was performed plotting anti-human Ig{kappa} MFI vs anti-human CD16 MFI using different Ig subclasses. A significant negative correlation (r2 = –0.97; p = 0.006) was present, thus indicating that IgG competes with the anti-human CD16 clone 3G8 for binding to mangabey CD16. These results also suggest that IgG did not completely saturate receptor binding sites at the concentration used. To verify that 3G8 blocks mangabey CD16, preventing binding of IgG, IgG-binding experiments were repeated with cells that were first incubated with unlabeled 3G8 at different concentrations. Incubation of cells with 3G8 before addition of human IgG resulted in reduced binding of human IgG1 and IgG2 to mangabey CD16 (Fig. 6). By contrast, incubation with a mouse Ab of irrelevant specificity before addition of human IgG did not alter the ability of mangabey CD16 to bind to human IgG1 and IgG2.


Figure 6
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  		FIGURE 6. Antagonist anti-human CD16 (mouse mAb 3G8) blocks binding of human IgG1 and IgG2 to mangabey rCD16 expressed on HeLa cells. Different concentrations of antagonistic anti-human CD16 (diamonds and squares) or control mouse Ab (triangles) were preincubated with control HeLa cells (squares) or HeLa cells expressing mangabey CD16 (diamonds and triangles). Next, cells were washed of unbound Ab and incubated with either human IgG1 (filled symbols) or human IgG2 (empty symbols). Finally, cells were stained with FITC-labeled anti-human Ig{kappa} to detect bound human IgG and analyzed by flow cytometry (10,000 cells for each condition).

 
Effects of blocking N-glycosylation on the expression and ligand binding of CD16

Human and mangabey CD16 have conserved glycosylation motifs. Therefore, N-glycosylation of mangabey CD16 was blocked to assess the effects on IgG binding. Anti-human CD16 staining with mAb 3G8 of tunicamycin-treated cells decreased 15.43–17.47% compared with untreated cells. Previously, it has been shown that 3G8 binding to human CD16 is unaltered by glycosylation and that blocking N-glycosylation of human CD16 results in a modest decrease in its expression (54). Thus, the decrease in staining for mangabey CD16 most likely represents a similar decrease in receptor expression. By contrast, staining for bound IgG increased for both IgG1 and IgG2 when N-glycosylation was blocked. Adjusting for the decrease in receptor expression, the increase in bound IgG1 and IgG2 ranged from 110 to 129.5% between different experiments. The increase in IgG binding did not favor either isotype over the other. Staining for bound IgG3 and IgG4 was not significant regardless of N-glycosylation (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Although mice have been used extensively to study FcR/Ab interactions, they are unsuitable models to evaluate Ab-based therapeutics for human conditions. Mice have no homologue of CD16b (4). Mouse CD16a does not appear to be a true orthologue of human CD16a because its extracellular region, responsible for binding to IgG, is more conserved with mouse Fc{gamma}RII (55). In addition, mouse Fc{gamma}RIII is expressed on mast cells, whereas human mast cells do not express this receptor (56). A second, not fully characterized mouse receptor (CD16-2), which may be more homologous to human CD16a, has been identified recently (57). In contrast, nonhuman primates represent the currently accepted model to evaluate human therapeutics that may bind CD16. Because the premise that IgG/CD16 interactions in these species mimic those in humans has not been evaluated, we identified CD16 as well as TCR {zeta}-chain genes in four nonhuman primate species. In addition, we generated sooty mangabey rCD16 in HeLa cells, as we demonstrated that these cells transcribe the TCR {zeta}-chain that in humans is necessary for CD16 expression. Our results show that only one CD16 gene, homologous to the human CD16a, is present in nonhuman primates and that the nonhuman primate TCR {zeta}-chain genes are highly conserved with the human counterpart. In all four species, CD16a is expressed on NK cells and monocytes and in sooty mangabeys, is also expressed on granulocytes. We generated sooty mangabey rCD16 in HeLa cells. The sooty mangabey rCD16 was capable of binding to human IgG1 and IgG2, but not human IgG3 and IgG4. Thus, despite the strong conservation of the mangabey CD16 sequence with the human CD16 sequence, mangabey and human CD16 differ in their ability to bind human IgG subclasses. Human CD16 binds to IgG1 and IgG3, but only weakly to IgG2 and IgG4 (2). Mangabey CD16/IgG interactions are specific, as indicated by the positive correlation of cells labeled for both CD16 and bound IgG. Similarly, mAb 3G8, an antagonist of IgG binding to human CD16, blocks binding to mangabey CD16 (Fig. 6). These results indicate that the IgG binding site for mangabey CD16 is most likely conserved with the human CD16 binding site as the sequence homology suggests. The few amino acid substitutions may be sufficient to allow for binding to IgG2.

The IgG1 motif LLGGP located in the lower hinge is particularly important for binding to CD16 (2, 58). In IgG2, this motif is replaced by VAGP. The motif LLGGP is encoded by all four IgG subclass genes in baboons, rhesus macaques, cynomolgus macaques, pigtail macaques, and sooty mangabeys with the exception of baboon IgG2 and pigtail macaque IgG4 (59, 60 and our unpublished results). In this study, we have shown that mangabey rCD16 is capable of binding to rhesus macaque, cynomolgus macaque, baboon, and sooty mangabey IgG. Based on the conservation of the motif LLGGP in most of the nonhuman primate IgG molecules, it is expected that all four subclasses bind CD16. However, IgG/CD16 interactions have been shown to be complex and involve residues outside of the IgG lower hinge (45). Radaev and Sun (61) found that small peptides with the sequences matching those of the lower hinge of human IgG1, IgG2, and IgG4 all have similar affinities for human CD16, and concluded that additional IgG features, such as hinge length, are important. Human IgG3 contains the LLGGP motif, yet we did not detect its binding to mangabey CD16. Therefore, mangabey CD16/IgG interactions are likely to also involve additional IgG subclass differences.

The N-glycan of human CD16 at Asn180 is in close proximity of other residues that interact with the Fc fragment of IgG1 (46). Glycosylation inhibition and mutation of Asn180 to Glu result in increased affinity of CD16 for monomeric IgG, whereas mutations at other CD16 N-glycan sites have no effect (54). CD16a of monocytes, macrophages, and NK cells have different affinities for IgG as a result of differential glycosylation (62). Hence, cells can modulate binding to IgG through modification of the Asn180 N-glycan. CD16 of nonhuman primates has the Asn180 glycosylation motif. Inhibition of N-glycosylation with tunicamycin resulted in a modest increase of human IgG binding to mangabey CD16, just as is reported for human CD16 (54). CD16 glycosylation may be altered during inflammation to modulate affinity of binding to IgG, and hence activation through CD16 (54). Our data support the presence of such a mechanism in nonhuman primates.

In conclusion, our results show that there are extensive similarities shared by human and nonhuman primate CD16 molecules. However, differences in interactions with IgG subclasses as well as in cell-type expression are clearly recognizable. These differences should be carefully considered when using nonhuman primates for the evaluation of therapeutic Abs. Further studies will clarify the extent of these differences and the role that these differences could play in the interpretation of results obtained from in vivo studies conducted in nonhuman primate models.


   Acknowledgments
 
We thank Dr. Frank Novembre and the late Dr. Harold McClure (Yerkes National Primate Research Center) for providing rhesus macaque and sooty mangabey blood samples, and Dr. Jerilyn Pecotte and Dr. Kathy Brasky (Southwest National Primate Research Center) for providing cynomolgus macaque and baboon blood samples.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by National Institutes of Health Grants RR10755 and RR00165, by the Research Program Enhancement from the Georgia State University Office of Research and Sponsored Programs, and by the Georgia Research Alliance. Support for K.A.R. was provided by the Molecular Basis of Disease program at Georgia State University. Back

2 Disclaimer: The findings and conclusions in this report are those of the author and do not necessarily represent the views of the Agency for Toxic Substances and Disease Registry. Back

3 Address correspondence and reprint requests to Dr. Roberta Attanasio, Department of Biology, Georgia State University, P.O. Box 4010, Atlanta, GA 30302. E-mail address: rattanasio{at}gsu.edu Back

4 Abbreviations used in this paper: ADCC, Ab-dependent cell-mediated cytotoxicity; MFI, mean fluorescence intensity. Back

Received for publication March 29, 2006. Accepted for publication June 26, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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