Polymorphisms and interspecies differences of the activating and inhibitory FcγRII of Macaca nemestrina influence the binding of human IgG subclasses.

This information is current as of December 3, 2014.

Polymorphisms and Interspecies Differences of the Activating and Inhibitory Fc γRII of Macaca nemestrina Influence the Binding of Human IgG Subclasses

J Immunol 2014; 192:792-803; Prepublished online 16 December 2013; doi: 10.4049/jimmunol.1301554 http://www.jimmunol.org/content/192/2/792

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Halina M. Trist, Peck Szee Tan, Bruce D. Wines, Paul A. Ramsland, Eva Orlowski, Janine Stubbs, Elizabeth E. Gardiner, Geoffrey A. Pietersz, Stephen J. Kent, Ivan Stratov, Dennis R. Burton and P. Mark Hogarth

The Journal of Immunology

Polymorphisms and Interspecies Differences of the Activating and Inhibitory FcgRII of Macaca nemestrina Influence the Binding of Human IgG Subclasses Halina M. Trist,* Peck Szee Tan,* Bruce D. Wines,*,†,‡ Paul A. Ramsland,*,‡,x Eva Orlowski,* Janine Stubbs,* Elizabeth E. Gardiner,{ Geoffrey A. Pietersz,*,†,‡ Stephen J. Kent,‖ Ivan Stratov,‖ Dennis R. Burton,# and P. Mark Hogarth*,†,‡

T

he Fc receptors (FcRs) are cell surface molecules primarily expressed on effector leukocytes of the innate immune system and by binding Igs provide adaptive immunity with a cell-based effector system (1). There are three classes of leukocyte IgG FcRs in humans: the high-affinity IgG receptor FcgRI (CD64) and the low-affinity IgG receptors FcgRII (CD32) and FcgRIII (CD16). The two major genes of the human (hu)FcgRII family encode FcgRIIa and FcgRIIb and their splice variants. These receptors

*Centre for Biomedical Research, Burnet Institute, Melbourne, Victoria 3004, Australia; † Department of Pathology, University of Melbourne, Parkville, Victoria 3056, Australia; ‡Department of Immunology, Monash University, Melbourne, Victoria 3004, Australia; xDepartment of Surgery, University of Melbourne, Parkville, Victoria 3010, Australia; {Australian Centre for Blood Diseases, Monash University, Melbourne, Victoria 3004, Australia; ‖Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia; and #Department for Immunology and Microbial Science and International AIDS Vaccine Initiative Neutralizing Antibody Center and Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037 Received for publication June 13, 2013. Accepted for publication November 15, 2013. This work was supported by National Health and Medical Research Council Fellowship and Project Grant 1002270 and Program Grant 510448, as well as by the Victorian Operational Infrastructure Support Program. The cDNA sequences presented in this article have been submitted to GenBank (http://www.ncbi.nlm.nih.gov/genbank) under accession numbers KF234399, KF234400, KF234401, KF234402, KF562260, KF562261, KF562262, KF562263, KF234403, and KF234404. Address correspondence and reprint requests to Prof. P. Mark Hogarth, Centre for Biomedical Research, Burnet Institute, 85 Commercial Road, Melbourne, VIC 3004, Australia. E-mail address: [email protected] Abbreviations used in this article: FcR, Fc receptor; hu, human; MFI, mean fluorescence intensity; mnFcgR, Macaca nemestrina FcgR; NHP, nonhuman primate. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301554

play activating and inhibitory roles in normal immune responses and immune homeostasis (1, 2), and imbalance in these opposing roles is a key contributing factor to the development of pathological inflammation in several autoimmune diseases (3). FcgRIIa is one of several activating FcRs, but it is the most widespread and abundant FcgR of humans present on all leukocytes except lymphocytes. Despite being a low-affinity receptor, FcgRIIa avidly binds oligovalent Ab-coated targets (immune complexes) to induce cytokine release from inflammatory leukocytes, respiratory burst, Ab-dependent cell-mediated killing, internalization of complexes, and platelet aggregation. FcgRIIb in humans is a powerful inhibitor of immune receptor signaling and is critical to the modulation of humoral immunity and Ab-dependent immune functions (4–6). Its ITIM-dependent modulation of ITAM signaling cascades regulates B cell Ag receptor signaling and consequent Ab responses. FcgRIIb also regulates signaling by the activating FcRs FcgRI, FcgRIIa, FcgRIIIa, FcεRI, and FcaRI. In a practical sense, the IgG–FcgR interaction is a key contributor to the effectiveness of many vaccines both at the level of immune regulation and induction of effector function. Indeed, it has been suggested that the IgG– FcgR interaction mediating Ab-dependent cell-mediated cytotoxicity may play a role in HIV vaccine–induced protective immunity of humans (7). Moreover, the effectiveness of allergen immunotherapy (8) and many therapeutic mAbs, particularly anticancer mAbs, has been attributed at least in part to successful engagement of FcR-dependent effector systems, including FcgRIIa and FcgRIIb (1). In humans, several polymorphisms of the activating IgG receptor, FcgRIIa, are known (9, 10). The most clinically significant polymorphism encodes amino acid position 131 where either a histidine or an arginine residue may be present, resulting in profound effects on binding of huIgG2 (11).


Little is known of the impact of Fc receptor (FcR) polymorphism in macaques on the binding of human (hu)IgG, and nothing is known of this interaction in the pig-tailed macaque (Macaca nemestrina), which is used in preclinical evaluation of vaccines and therapeutic Abs. We defined the sequence and huIgG binding characteristics of the M. nemestrina activating FcgRIIa (mnFcgRIIa) and inhibitory FcgRIIb (mnFcgRIIb) and predicted their structures using the huIgGFc/huFcgRIIa crystal structure. Large differences were observed in the binding of huIgG by mnFcgRIIa and mnFcgRIIb compared with their human FcR counterparts. MnFcgRIIa has markedly impaired binding of huIgG1 and huIgG2 immune complexes compared with huFcgRIIa (His131). In contrast, mnFcgRIIb has enhanced binding of huIgG1 and broader specificity, as, unlike huFcgRIIb, it avidly binds IgG2. Mutagenesis and molecular modeling of mnFcgRIIa showed that Pro159 and Tyr160 impair the critical FG loop interaction with huIgG. The enhanced binding of huIgG1 and huIgG2 by mnFcgRIIb was shown to be dependent on His131 and Met132. Significantly, both His131 and Met132 are conserved across FcgRIIb of rhesus and cynomolgus macaques. We identified functionally significant polymorphism of mnFcgRIIa wherein proline at position 131, also an important polymorphic site in huFcgRIIa, almost abolished binding of huIgG2 and huIgG1 and reduced binding of huIgG3 compared with mnFcgRIIa His131. These marked interspecies differences in IgG binding between human and macaque FcRs and polymorphisms within species have implications for preclinical evaluation of Abs and vaccines in macaques. The Journal of Immunology, 2014, 192: 792–803.


Materials and Methods Animals Outbred 3- to 5-y-old pig-tailed macaques (M. nemestrina) were obtained from the Australian National Macaque Breeding Facility, and studies were approved by the University of Melbourne and Commonwealth Scientific and Industrial Research Organization Animal Health Institutional Animal

Ethics Committees. Whole venous blood was obtained from animals sedated with ketamine as previously described (36), and PBMCs were isolated over Ficoll-Hypaque and stored in liquid nitrogen.

Cloning of FcgRs Gene transcripts for mnFcgRIIa and mnFcgRIIb were obtained by PCR of cDNA (AffinityScript quantitative PCR cDNA synthesis kit from Agilent Technologies, Santa Clara, CA) produced from total RNA (RNeasy Mini Kit from Qiagen, Melbourne, VIC, Australia) from PBMCs of unrelated animals. Restriction enzymes and DNA-modifying enzymes were all from New England BioLabs (Beverly, MA), except for PCR applications, which used AccuPrime Pfx DNA polymerase (Life Technologies, Melbourne, VIC, Australia). The primers to generate FcgR PCR fragments were synthesized by Sigma-Aldrich (Sydney, NSW, Australia). Because of the absence of any DNA sequence information from M. nemestrina, the 59- and 39-amplifying primers for FcgRIIa or FcgRIIb were based on sequences of rhesus macaque (M. mulatta, GenBank NM_001257300) or cynomolgus macaque (M. fasicularis, GenBank AF485814.1), respectively. The FcgRIIa 59 primer was complementary to the initiation codon and the following five codons, and the 39 primer was complementary to the last seven codons and the termination codon. The FcgRIIb 59 primer was complementary to the initation codon and the first five codons, and the 39 primer was complementary to the final six codons and the termination codon. The primers also contained KpnI and EcoRV sites. The Kpn1/EcoRV digested PCR products were cloned into pENTR1A (Life Technologies) followed by Gateway LR cloning (Life Technologies) into a Gateway-adapted pMXI expression vector containing a neomycin resistance cassette (37). The huFcgRIIb2 construct was obtained from PBMC-derived cDNA as above using 59 and 39 primers based on the sequence of FcgRIIb (38) and cloned into pMXI as above. The nucleotide sequences of mnFcgR were determined from both PBMC-derived PCR-amplified cDNA and from six FcgR clones for each receptor from each individual animal using BigDye version 3.1 terminator cycle sequencing (Applied Biosystems, Melbourne, VIC, Australia) and separated at the Australian Genome Research Facility (Melbourne, VIC, Australia). All cDNA sequences have been submitted to GenBank (submission nos. 1637005 and 1655933). Alignments of FcgRII sequences were generated using CLC Sequence Viewer version 6.4 (CLC bio, Aarhus, Denmark). Isolation and expression of the human FcgRIIa-H131 and FcgRIIa-R131 have been described previously (39–41).

Site-directed mutagenesis Mutated FcgRs were constructed by in vitro site-directed mutagenesis using a series of mutation primers and the thermostable polymerase Pfx (Life Technologies) to obtain the following for mnFcgRIIa-allele 1 (mutant 1, N135T [N→T]; mutant 2, P159L,Y160F [PY→LF]), mnFcgRIIaallele 2 (mutant 3, P131H,M132L,N133D [PMN→HLD]), the double mutants of mnFcgRIIa-allele 2 (mutant 4, PMN→HLD with N→T; mutant 5, PMN→HLD with PY→LF), and for mnFcgRIIb1 (mutant 6, H131R, M132S [HM→RS]).

Stable expression of FcgRII A pMXI retroviral expression system was used to introduce FcgR DNA into the FcR-deficient IIA1.6 cells. Phoenix packaging cells were maintained in RPMI 1640 containing glutamine and 5% heat-inactivated fetal bovine serum and were transfected with FcgRII plasmids using Lipofectamine 2000 (Life Technologies) to generate retrovirus. The retroviral suspension was overlaid onto IIA1.6 cells as described (39). Transduced IIA1.6 cells were selected for resistance to 0.4 mg/ml Geneticin (Life Technologies).

Abs Purified human myeloma proteins IgG1k, IgG2k, and IgG3k were purchased from both Sigma-Aldrich (Castle Hill, NSW, Australia) and The Binding Site (Birmingham, United Kingdom). PE-conjugated F(ab9)2 goat anti-human IgG F(ab9)2-specific polyclonal was from Jackson ImmunoResearch Laboratories (West Grove, PA). Human IgG in the form of i.v. Ig Sandoglobulin was obtained from Novartis (Sydney, NSW, Australia). Abs used to identify populations of peripheral blood leukocytes were anti-CD20, (clone L27; BD Biosciences, Sydney, NSW, Australia), antiCD14 (clone m5E2; BD Biosciences), anti-CD56 (clone NCAM16.2; BD Biosciences), anti-CD159a (NKG2) (clone Z199; Beckman Coulter, Melbourne, VIC, Australia), anti-FcgRIII (clone 3G8; BD Biosciences), and anti-CD41a (clone HIP8; BD Biosciences).


Genetic polymorphisms of huFcRs that affect their capacity to bind IgG or alter the balance of activation over inhibition have been implicated in resistance to HIV and bacterial infection (12–18), susceptibility to autoimmunity (19, 20), and the effectiveness of therapeutic mAbs, mostly IgG1 but increasingly IgG2 (1). As a result of the success of mAbs, considerable effort has been made to improve their FcR-dependent potency by engineering Fc portions for the purpose of selective engagement of either activating FcRs (1), including FcgRIIa (21) or inhibitory FcRIIb (22). Furthermore, the most clinically significant polymorphism of the activating IgG receptor, FcgRIIa (9, 10), encodes either a histidine or an arginine residue at amino acid position 131 and influences the clinical outcome of Ab therapy (23) and additionally has profound effects on binding of huIgG2 (11). The genetic diversity of nonhuman primate (NHP) FcRs has not been extensively characterized, even though NHPs are key animal models for many diseases, including HIV. Examination of functional Fc polymorphisms in NHPs is, as found in humans, pertinent to understanding susceptibility to infectious and autoimmune diseases. Interspecies functional substitutions will likely substantially influence the evaluation of human Abs in NHPs. Indeed, although evolutionarily conserved, the limited information available to date shows that significant interspecies sequence differences are apparent between the huFcRs and their orthologs in different NHP species (24–27). Some of this sequence diversity occurs at sites that are predicted by homology to be essential for the interaction with IgG and may influence the binding of IgG. These differences may result in alterations to the relative contributions that the different FcR classes make to Ab-dependent effector function in vivo in macaques compared with humans. Correspondingly, differences in species IgG–FcR interactions may greatly complicate the interpretation of preclinical studies aimed at evaluating the functional activity of therapeutic Abs or vaccines in NHP models such as macaques, including Macaca nemestrina. Additionally, use of outbred populations of macaques may further complicate interpretation owing to nonsynonymous polymorphisms in the macaque FcR genes. Little is yet known of the impact of FcR polymorphism on the binding of huIgG in NHPs, especially in the three macaque species commonly used in medical research: rhesus (Macaca mulatta), cynomolgus (Macaca fascicularis), and pig-tailed (M. nemestrina) macaques. Furthermore, nothing is known of the activity of FcRs of the pig-tailed macaque. Similar to cynomolgus and rhesus macaques (28, 29), pig-tailed macaques are used in the evaluation of Ab immunotherapy (30–32), humoral immune responses to infection, and vaccine candidates, including dengue virus (33, 34) and HIV/ simian HIV (35, 36). Furthermore, different species have been used in similar models (30, 32). In this study, we define the ligand-binding properties of activating FcgRIIa and the inhibitory FcgRIIb of M. nemestrina (mnFcgRIIa and mnFcgRIIb). We identify a functionally significant polymorphism of FcgRIIa and define the molecular and structural basis of impaired binding to IgG by mnFcgRIIa and for the enhanced binding and broader specificity of mnFcgRIIb compared with their human orthologs (huFcgRIIa and huFcgRIIb). These data also have implications for the analysis of IgG function in other macaque species and NHPs.

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PIG-TAILED MACAQUE FcgR INTERACTION WITH HUMAN IgG

The anti-FcgRIIa mAbs 8.7, 8.2, and IV.3 and the anti-FcgRIIb monoclonal X63-21/7.2 have been previously described (40, 42, 43). Fab fragments of IV-3 and F(ab9)2 fragments of 8.7, 8.2, and X63 mAbs were generated as described (42) and then biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific, Rockford, IL).

IgG ligands

Flow cytometric detection of immune complex binding and receptor expression The binding of IgG complexes to allelic forms of receptors and mutants thereof was determined as described (40). Briefly, anti-F(ab9)2 PE/IgG complexes at the indicated concentrations were incubated with 1.2 3 105 cells in 50 ml for 1 h on ice and then washed twice and resuspended in 200 ml PBS/0.5% BSA. Similarly, aggregated huIgG or biotinylated IgG dimer/trimer binding was detected by indirect fluorescence following incubation with cells on ice for 1 h. Cells were then washed and incubated with PE-labeled goat anti-human Ab or allophycocyanin-streptavidin, respectively, for 30 min on ice, washed twice, and then resuspended in 200 ml PBS/BSA. Background binding controls included nonspecific binding of IgG to untransfected IIA1.6 cells and nonspecific binding of conjugate only to FcR-expressing cells. Expression levels of FcgR on the transfected cells were determined in each experiment by flow cytometry using the following biotinylated antiFcR mAbs: F(ab9)2 of 8.7, 8.2, and Fab of IV.3 or the anti-FcgRIIb X6321/7.2 F(ab9)2 (40). Cells were then washed and binding was detected using allophycocyanin-streptavidin as described above. Gating was set to record fluorescence of 10,000 viable single cells. The analysis of huIgG binding to each human or macaque FcgR and mutants thereof was performed on at least five independent occasions using the anti-Fab complexes and at least three independent occasions using the IgG dimers/trimers.

Flow cytometric analysis of Fc receptor expression on blood cells Whole blood was collected from four M. nemestrina as above and from healthy human volunteers after informed consent as approved by the Alfred Health Human Ethics Committee or the Monash University Standing Committee on Ethics in Research Involving Humans. PBMCs were isolated on a Ficoll density gradient. Total leukocytes were obtained from buffy coats and then erythrocytes were lysed with hypotonic RBC lysis solution (Miltenyi Biotec, Sydney, NSW, Australia) according to the manufacturer’s instructions. Expression of surface markers was determined using flow cytometry where human and M. nemestrina B lymphocytes were identified with anti-CD20 and monocytes were identified with anti-CD14. Human NK cells were defined with anti-CD56 and macaque NK cells with anti-CD159a (NKG2) (45). Neutrophils were identified by forward and side scatter profiles. Platelet-rich plasma was isolated by low-speed centrifugation and platelets were identified by expression of CD41a. The expression of FcgRIII was defined using clone 3G8, and FcgRII was identified with mAb 8.7 as described above.

Homology modeling of the macaque FcgRIIa/huIgG1-Fc interaction Protein homology models were prepared using the Discovery Studio suite, version 3.0 (Accelrys, San Diego, CA). The cocrystal structure of huFcgRIIa/

Results Sequence comparison of huFcgRIIa and mnFcgRIIa The mnFcgRIIa sequences were determined from cDNA isolated from 10 animals of an outbred colony of M. nemestrina. Replicate sequence analysis of PCR products and of multiple clones from independently derived, duplicate PBMC samples were used to establish and then confirm FcR sequences. Sequence analysis comparing M. nemestrina sequences to the huFcgRIIa revealed amino acid differences between macaque FcgRIIa and humans that were conserved in all animals and also identified polymorphic sequence variation between the FcgRIIa of individual macaques (Fig. 1). Thus, 26 aa that differed from huFcgRIIa were conserved in all individuals. Sixteen of these amino acid differences mapped to the extracellular region, three to the transmembrane region, and seven to the cytoplasmic tail of mnFcgRIIa (Fig. 1). Further sequence analysis among the 10 animals revealed extensive polymorphism and identified eight allelic products of M. nemestrina FcgRIIa (Fig. 1). These allelic forms were distinguished from each other by 16 polymorphic residues, of which 9 were located in the extracellular domains (at positions 1, 74, 79, 89, 93, 119, 131, 133, and 140), 1 was located in the transmembrane region, and 6 were found in the cytoplasmic tail. Interestingly, the identity of the nine polymorphic amino acids within the extracellular region suggests that FcgRIIa alleles 1, 3, 4, 6, 7, and 8 are closely related, as they contain the human-equivalent residue in many of the polymorphic positions. In contrast, FcgRIIa alleles 2 and 5, found in 2 of the 10 animals investigated, are closely related to each other but are the most different to huFcgRIIa, with seven or eight of the nine polymorphic positions, respectively, being distinct from huFcgRIIa (Fig. 1). In M. nemestrina, the inhibitory FcgRIIb has 23 nonpolymorphic amino acid differences from huFcgRIIb, and 21 of these occur in the extracellular domains. A single polymorphism resulting in amino acid substitution methionine/valine at position 254 was apparent in the cytoplasmic tail (Fig. 2). Binding of huIgG to mnFcgRIIa A comparative analysis of the binding of huIgG subclasses to allelic forms of mnFcgRIIa and to huFcgRIIa was undertaken in transfected cells where their equal expression was confirmed by binding of the anti-FcgRIIa mAb F(ab9)2 fragments (Fig. 3A), which detects an epitope in the extracellular domains in FcgRIIa and FcgRIIb (40). The binding of huIgG3 complexes to mnFcgRIIa-allele 1 and huFcgRIIa-His131 was analyzed by flow cytometry and was shown to be essentially identical with relative cell surface staining of mean fluorescence intensity (MFI) of 70,000 and 71,000, respectively (Fig. 3D). In contrast, mnFcgRIIa-alelle 1 bound IgG1


Two separate methods were used to evaluate the binding of huIgG to cell surface–expressed FcRs of M. nemestrina and humans. First, for anti-Fab complexes, complexes of IgG were generated by crosslinking IgG with F(ab9)2 fragments of fluorochrome-conjugated anti-human Fab as previously described (44). Briefly, individual human IgG1, IgG2, or IgG3 subclasses were incubated with F(ab9)2 fragments of PE-conjugated goat antihuman IgG F(ab9)2 at a 2:1 ratio for 30 min at 37˚C and then on ice for 5min. Second, for IgG dimers/trimers, biotinylated IgG dimer/trimer complexes of pooled huIgG were generated using the cross-linker Tris-succinimidyl aminotriacetate (Thermo Scientific). Tris-succinimidyl aminotriacetate at 5 mg in 0.5 ml anhydrous DMSO was mixed with 3.85 mg biotin-Xhydrazide (Sigma-Aldrich) in 0.5 ml anhydrous DMSO for 2 h at room temperature to generate the intermediate biotinyl bis-succinimidyl aminotriacetate, which was used without purification. Human IgG (4 ml at 25 mg/ml in PBS) was mixed with an 11-fold excess of biotinyl bissuccinimidyl aminotriacetate (0.7 ml) and allowed to react for 1 h at room temperature. The resulting IgG dimers/trimers were purified from the reaction mixture and separated from monomers and multimers by gel filtration on a Sephacryl S-300 column (1.5 3 100 cm). Fractions were analyzed by SDS-PAGE, and those corresponding to IgG dimers/trimers were pooled and stored in aliquots at 280˚C.

huIgG1-Fc (Brookhaven Protein Data Bank ID 3RY6) (40) was used as a template to generate a homology model of the macaque FcgRIIa/huIgG1-Fc complex using the amino acid sequence of mnFcgRIIa-allele 1 (Fig. 1). The three-dimensional model of the macaque FcgRIIa/huIgG1-Fc complex was optimized by conjugate-gradient energy minimization against spatial restraints extracted from the 3RY6 template and a probability density function using the Modeler algorithm (46). The protein interface between FcgRIIa and huIgG1-Fc was analyzed using the protein interfaces, surfaces, and assemblies (PISA) server at the European Bioinformatics Institute (http://www.ebi.ac.uk/pdbe/prot_int/pistart.html) (47). The buried surface area of residues at the interface of FcgRIIa and the bound huIgG1-Fc were plotted for comparison with the total buried surface area between huFcgRIIa and mnFcgRIIa-allele 1. Because mnFcgRIIa-allele 1 has one additional N-linked glycosylation site compared with huFcgRIIa, in silico glycosylation of this extra site was performed using the GlyProt Web-based server (http://www.glycosciences.de/modeling/glyprot/php/main.php) (48).


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or IgG2 complexes with MFIs of 5000 and 360, respectively, which were 8- to 10-fold lower than their binding to huFcgRIIaHis131 (Fig. 3B, 3C). Thus, in comparison with huFcgRIIa, the mnFcgRIIa-allele 1 has equivalent binding of complexes of huIgG3, but greatly diminished binding of complexes of huIgG1 and huIgG2. In addition to the differences in huIgG binding between human and M. nemestrina FcgRIIa observed above, even greater differences were found in huIgG binding to the allelic forms of mnFcgRIIa (Fig. 4). The binding of all huIgG subclasses to mnFcgRIIa-allele 2 receptor was reduced greatly (Fig. 4B–D). Compared to binding by mnFcgRIIa-allele 1 (MFI of 5000), IgG1 binding to mnFcgRIIa-allele 2 was reduced 80-fold (MFI of 90)

and was barely detectable above background (MFI of 30, Fig. 4B). Similarly, IgG2 binding of mnFcgRIIa-allele 2 was also barely detectable above background levels (Fig. 4C). The binding of IgG3 complexes (MFI of 11,600) was also reduced 6-fold compared with mnFcgRIIa-allele 1 (MFI of 70,300). Thus, not only do interspecies differences of FcR affect huIgG binding, but polymorphisms (intraspecies differences) in mnFcgRIIa also influence the binding of huIgG. The mnFcgRIIa-allele 2 is greatly impaired in ligand binding of human IgG1, IgG2, and IgG3. Next, we defined the sequence substitutions responsible for the low ligand binding activity of mnFcgRIIa-allele 2. Analysis of the polymorphic amino acid differences in the mnFcgRIIa in the context of known functional regions of huFcgRIIa showed that position 131


FIGURE 1. Alignment of the eight allelic forms of FcgRIIa of M. nemestrina with their human counterpart (huFcgRIIa-His131, UniProt P12318.4) (41). Amino acids identical to M. nemestrina allele 1–encoded receptor are shown as dots, and positions of polymorphism are indicated by inverted triangles; the transmembrane region is shown as a dashed underline. Critical residues analyzed in this study are boxed. Trp87 and Trp110 are indicated with stars. These sequences were derived from 10 animals. The M. nemestrina sequences are available in GenBank (http://www.ncbi.nlm.nih.gov/genbank) as follows: mnFcgRIIa-allele 1, accession no. KF234399; mnFcgRIIa-allele 2, accession no. KF234400; mnFcgRIIa-allele 3, accession no. KF234401; mnFcgRIIaallele 4, accession no. KF234402; mnFcgRIIa-allele 5, accession no. KF562260; mnFcgRIIa-allele 6, accession no. KF562261; mnFcgRIIa-allele 7, accession no. KF562262; and mnFcgRIIa-allele 8, accession no. KF562263.

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is part of the major contact surface in the huFcgRIIa/IgG interaction (40, 49, 50). Moreover, in humans, polymorphic FcgRIIa (high and low responder allelic products that differ at position 131) have very different interactions with mouse IgG1 and huIgG2 (44). Thus, it seemed possible that the large differences in IgG binding between the two M. nemestrina allelic receptors results from the sequence differences in this segment, namely, HMD (131–133) of the functional receptor encoded by mnFcgRIIa-allele 1 compared with the PMN(131–133) of the poorly functional receptor encoded by mnFcgRIIa-allele 2 (Fig. 1). Consequently, we attempted to reconstitute binding of huIgG by replacing PMN(131–133) of mnFcgRIIa-allele 2 with HLD(131– 133) from huFcgRIIa. Note that His131 and Asp133 of mnFcgRIIaallele 1 are identical in huFcgRIIa (Fig. 1). Weak binding of IgG1 and IgG2 to mnFcgRIIa-allele 2 was demonstrated (Fig. 4B, 4C), and the HLD(131–133) substitution substantially rescued binding with an MFI of 2900 and an MFI of 300 for IgG1 and IgG2, re-

FIGURE 3. Comparative binding of huIgG subclasses to mnFcgRIIaallele 1 (red filled histogram with dashed red line) or the huFcgRIIa-His131 (open histogram, solid red line). Cell surface expression of receptor protein was determined using the anti-FcgRII mAb 8.7, which recognizes huFcgRIIa and mnFcgRIIa. Background binding is shown as a solid black line. The binding of each IgG subclass to each receptor was tested on at least five occasions.

spectively (Fig. 4F, 4G; blue filled, dashed line) that was comparable to that of mnFcgRIIa-allele 1 (MFI of 5000 for IgG1 and 360 for IgG2) (Fig. 4B, 4C; red filled, dashed line). Similarly, the binding of IgG3 was also improved from an MFI of 11,600 to an MFI of 53,400 (Fig. 4D, 4H). However, this increased binding of IgG was still markedly lower than the binding of IgG1 and IgG2 complexes to the huFcgRIIa-His131; that is, MFIs of 57,200 and 3,600, respectively. Binding of IgG3 by the mnFcgRIIa-allele 2 with HLD(131–133) remained slightly reduced compared with the human receptor (MFI of 53,400 for mnFcgRIIa-His131 compared with 70,100 for huFcgRIIa-His131; compare with Fig. 3). The failure to fully enable IgG binding to levels observed for the human receptor implied that other amino acid residue differences between mnFcgRIIa and huFcgRIIa contribute to ligand binding. Of particular interest in this context were residues Pro159 and Tyr160 in mnFcgRIIa and Leu159 and Phe160 in huFcgRIIa, which are located in the G strand of the second domain adjacent to the critical FG loop that contacts IgG. Thus, allele 2–encoded mnFcgRIIa, which we had mutated to contain the HLD(131–133) of huFcgRIIa-His131 (Fig. 4E–H, dashed line), was further modified by replacing Pro159 and Tyr160 with the equivalent Leu159 and Phe160 from huFcgRIIa (Fig. 4E–H). This substitution increased IgG1 binding 5-fold to levels similar to huFcgRIIa-His131 and increased the binding of IgG2 almost 10-fold. Interestingly, there was no further improvement in the IgG3 binding beyond that already achieved by the substitution of PMN(131–133) for HLD(131–133). The role of Pro159 and Tyr160 in macaque receptor binding of IgG1 and IgG2 was further examined in a similar analysis of the mnFcgRIIa-allele 1, which contained the HMD(131–133) sequence. The replacement of Pro159 and Tyr160 with Leu159 and Phe160 from huFcgRIIa also improved IgG1 and IgG2 binding to levels similar to those of huFcgRIIa binding (Fig. 4I–L). Thus, the presence of


FIGURE 2. Alignment of the allotypic form of FcgRIIb of M. nemestrina with the splice variants of its human counterpart (huFcgRIIb1, GenBank NM_001002275.2; huFcgRIIb2, GenBank NM_001002273.2) (38). Amino acids identical to M. nemestrina are shown as dots, and positions of polymorphism are indicated by inverted triangles; the transmembrane region is shown as underlined dashed line, and the deletions within the cytoplasmic tail of FcgRIIb2 resulting from mRNA splicing or within the membrane stalk are indicated by solid lines. Critical residues analyzed in this study are boxed. Trp87 and Trp110 are indicated with stars. M. nemestrina sequences are available in GenBank (http://www.ncbi.nlm.nih.gov/genbank) as follows: mnFcgRIIb1, accession no. KF234403; and mnFcgRIIb2, accession no. KF234404.


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PY(159–160) in mnFcgRIIa results in a significant impairment of binding of the human IgG1 and IgG2 subclasses. Binding of huIgG to mnFcgRIIb The interaction of huIgG subclasses with the inhibitory FcgRIIb of M. nemestrina was investigated and substantial differences were observed in specificity and binding compared with huFcgRIIb (Fig. 5). Indeed, unlike the activating FcgRIIa of M. nemestrina, the mnFcgRIIb had a 10-fold greater capacity to bind IgG1 complexes than did huFcgRIIb (Fig. 5B). The greatest functional divergence was in the binding of huIgG2, which failed to bind to huFcgRIIb as expected, but surprisingly was strongly bound by mnFcgRIIb (Fig. 5C). FcgRIIb from both species bound IgG3 at similar levels (Fig. 5D). This difference in the capacity of the inhibitory mnFcgRIIb to bind IgG2 was surprising because in humans IgG2 essentially binds only to the activating FcgRIIa-His131, which is the consequence of the Arg/His131 polymorphism dictating specificity for IgG2 in this activating receptor. Comparison of the amino acid sequence of the IgG binding regions of human and M. nemestrina FcgRIIb shows that the principal sequence differences occur around position 131 wherein mnFcgRIIb encodes His131 and Met132, whereas huFcgRIIb has Arg131 and Ser132 (Fig. 2). Thus, we replaced the HM(131–132) of mnFcgRIIb with the RS(131–132) of huFcgRIIb and measured the impact on IgG1 and IgG2 binding (Fig. 5B, 5C). The substitution of the human-derived RS(131–132) into mnFcgRIIb resulted in a marked loss in IgG1 binding, decreasing to levels equivalent to huFcgRIIb (Fig. 5B). Moreover, IgG2 binding was completely lost (Fig. 5C), which is entirely consistent with the essential role of His131 in dictating the binding of IgG2.

Binding of huIgG dimers to FcgRII The binding measurements above were obtained using complexes generated by oligomerization of IgG with an anti-Fab Ab, a technique commonly used for the detection of immune complex binding to low-affinity receptors (44). To exclude the possibility that the anti-Fab Ab may influence FcR binding of the IgG complexes, we used a second method where dimers/trimers of huIgG were generated by cross-linking pooled human i.v. Ig with a biotinylated cross-linker (Fig. 6). Flow cytometry analysis of the binding of the cross-linked IgG dimers/trimers to FcgRIIa and FcgRIIb and receptor mutants entirely agreed with the binding observed using the complexes generated with the anti-Fab Ab (Figs. 3–5). The mnFcgRIIa-allele 2 failed to bind the IgG dimers/trimers, but the replacement of PMN(131–133) with HLD(131–133) enabled binding similar to that of allele 1 (Figs 6A, 6B). Further mutation of this construct wherein PY(159–160) was replaced with the LF(159–160) of huFcgRIIa resulted in further increase of IgG binding to levels approaching those of huFcgRIIa-His131 (Fig. 6A). One other significant structural difference between humans and macaques in this region is a potential N-glycosylation site at position 135 in mnFcgRIIa, which is absent from huFcgRIIa but is found in both human and macaque FcgRIIb. Because this site sits adjacent to the critical His131, we replaced N135 in mnFcgRIIa with the human equivalent T135; however, this mutation had little effect on the binding of any huIgG subclass (Fig. 6A). Similarly, the same modifications of mnFcgRIIa-allele 1 (Fig. 6B) were made and resulted in similar effects on IgG binding. Replacement of PY(159–160) with the human LF(159–160) residues greatly improved IgG binding, but as seen in mnFcgRIIaallele 2, the removal of the 135 glycosylation site had little effect.

FIGURE 5. Comparative binding of huIgG subclasses (B–D) to mnFcgRIIb (solid blue line) or huFcgRIIb (solid red line) compared with a mutant of mnFcgRIIb (blue filled histogram) wherein sequence HMD(131–133) was replaced with the human derived sequence RSD(131–133). Expression of receptor protein was determined using the huFcgRIIa mAb 8.7 (A). The binding of IgG to each receptor or mutated receptor was tested on at least five occasions.


FIGURE 4. Comparative binding of huIgG subclasses to allelic forms and mutants of mnFcgRIIa . Binding of anti-receptor Ab mAb8.2 (A) or IgG (B–D) to mnFcgRIIa- allele 2 (blue filled histogram, solid line) or the mnFcgRIIa-allele 1 (red filled histogram, dashed line) and background binding (black line). Binding of anti-FcgRII mAb 8.7 (E) or IgG (F–H) to mnFcgRIIa-allele 2 HLD where sequence PMN(131– 133) was replaced by human sequence HLD(131–133) (blue filled histogram, dashed line) or mnFcgRIIa-allele 2 HLD plus LF where both PMN(131–133) and PY (159–160) were replaced by human HLD(131–133) and LF(159–160) (open histogram, solid blue line). Binding of anti-receptor mAb 8.7 (I) or IgG (J–L) to mnFcgRIIa- allele 1 (red filled histogram, dashed line as in (B)–(D)) or mnFcgRIIa-allele 1 LF where PY (159–160) was replaced with human LF(159–160) (open histogram, solid red line). The binding of each IgG subclass to each allelic receptor was tested on at least five occasions, and the binding to mutated receptors was tested on at least three occasions.

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In the case of FcgRIIb (Fig. 6C), as was observed with the anti-Fab Ab complexes, the M. nemestrina receptors bound IgG dimers/trimers more strongly than did huFcgRIIb, and, as expected, the substitution of the mnFcgRIIa sequence HM(131–132) with the human equivalent RS(131–132) profoundly diminished binding to levels, which were even lower than observed with huFcgRIIb. Molecular modeling of mnFcgRIIa The human FcgRIIa/IgG interaction has been well characterized by extensive mutagenesis (42, 51, 52), x-ray structure studies of huFcgRIIa alone (50) and in complex with human IgG1-Fc (40), which have collectively identified four structurally contiguous regions that form the IgG binding surface in the second domain of FcgRIIa. To understand the key interspecies sequence differences between macaque and human receptors, we generated a molecular model of mnFcgRIIa-allele 1 interaction with huIgG1 using the x-ray structure of the huFcgRIIa/huIgG1-Fc complex (40). This model (Fig. 7) predicts that key contacts defined in the huFcgRIIa/ huIgG1-Fc interface are altered in the interaction of mnFcgRIIa and huIgG.

FIGURE 7. Homology-based model of mnFcgRIIa complex with human IgG1-Fc. (A) Comparison of the FG loop and Trp residues of the “Trp sandwich” of mnFcgRIIa (cyan wire) or huFcgRIIa (magenta wire) interacting with human IgG1-Fc B chain (yellow wire) or A chain (green wire). (B) Solvent-accessible surface view of huFcgRIIa complex with huIgG1-Fc and (C) solvent-accessible surface view of macaque FcgRIIa complex with huIgG1-Fc. Comparison of (B) and (C) indicates the critical Tyr157 of the FG loop has a reduced contact with huIgG1 in mnFcgRIIa. The Tyr157 residues of huFcgRIIa and mnFcgRIIa are shown in magenta and cyan, respectively. The IgG1-Fc chain A is shown in green, chain B in yellow, and FcgRIIa molecules are in light gray. Models were prepared based on the cocrystal structure of huFcgRIIa/huIgG1-Fc (Brookhaven Protein Data Bank ID 3RY6) (40).

The FG loop of the second domain of FcgRIIa is one major contact between receptor and IgG-Fc where Tyr157, conserved in all macaque species and humans, makes critical contacts with Leu234 and Gly236 in the lower hinge sequence LLGG(234–237) on the IgG-Fc B chain (Fig. 7A). Compared to the huFcgRIIa/Fc complex, the contacts between the Tyr157 of mnFcgRIIa FG loop and the IgG-Fc are reduced in the modeled interface, from a buried


FIGURE 6. The binding of huIgG dimers/trimers to mnFcgRIIa and mnFcgRIIb. Biotinylated huIgG dimers/trimers were titrated on transfectants expressing (A) huFcgRIIa (n) or mnFcgRIIa-allele 2 (♦) and mutants thereof wherein PMN(131–133) was replaced with HLD(131–133) of huFcgRIIa ( ) and then further modified by additional replacement of PY(159–160) with LF(150–160) of huFcgRIIa (▴) or N135 replaced with T135 of huFcgRIIa (d); (B) huFcgRIIa (n) or mnFcgRIIa-allele1 ( ) and mutants thereof wherein PY(150–160) was replaced with LF(159–160) of huFcgRIIa (O) or where N135 was replaced with T135 of huFcgRIIa (s) or binding to untransfected cells only (3); (C) huFcgRIIb1 (+) or mnFcgRIIb2 (s) or mnFcgRIIb1 (N) and mutants thereof wherein HM(131–132) was replaced with RS(131–132) of huFcgRIIb ()). The binding of IgG to each receptor or mutated receptor was tested on at least three occasions.


Expression of FcgRII in M. nemestrina blood cells The distribution of FcgRII on leukocyte types has been determined for rhesus and cynomologus macaques (26, 27, 45), and we analyzed expression in M. nemestrina. Flow cytometry analysis of peripheral blood leukocytes from four macaques confirmed the expression of FcgRII on CD20+ B cells, CD14+ monocytes (Fig. 8), and platelets (not shown) and, as expected was absent from macaque CD159a+ NK cells and human CD56bright NK cells, which is in agreement with cell distribution in rhesus and cynomolgous macaques (26, 27, 45) and humans (reviewed in Refs. 1, 2). Considerable differences in FcgR expression were observed on neutrophils wherein FcgRII and FcgRIII were both expressed on human neutrophils (Fig. 9) but only FcgRII was present on M. nemestrina neutrophils and also at a level 5-fold greater than that detected on human cells. The absence of FcgRIII from macaque neutrophils has also been reported for rhesus and cynomolgus macaques (26, 45), and increased expression of FcgRII has also been shown in cynomologus macaques (27).

Discussion In this study, we demonstrate that human and M. nemestrina FcgRIIa and FcgRIIb have distinct hierarchies of binding of hu-

FIGURE 8. Expression of FcgRII on mononuclear leukocytes from humans and M. nemestrina. Mononuclear cells were stained with antiFcgRIIa mAb 8.7 and populations were identified by costaining with anti-CD20 (B cells), anti-CD14 (monocytes), anti-CD159a (NKG2) (M. nemestrina NK cells), or anti-CD56 (human NK cells). Plots are representative of three individual experiments involving four different animals or human volunteers.

man IgG1 and IgG2. Although the FcgRIIa of both species binds IgG3 essentially equivalently, IgG1 and IgG2 binding to mnFcgRIIa is impaired by comparison with its human ortholog. Remarkably, the converse is the case for FcgRIIb where it is the human receptor that exhibits impaired binding of IgG1 relative to mnFcgRIIb, and IgG2 is not detectably bound. Indeed, mnFcgRIIb avidly binds IgG2 and thereby has a broader specificity for huIgG subclasses than does huFcgRIIb. These interspecies differences of IgG binding and specificity to mnFcgR that we describe in this study were determined in the physiological context of the cell surface. Similar results were observed in cell-free surface plasmon resonance analysis of recombinant ectodomains of cynomolgus FcgR, which showed lower affinity of huIgG1 and huIgG2 for cynomolgus FcgRIIa than huFcgRIIa, and conversely showed increased affinity

FIGURE 9. Macaque neutrophils express elevated levels of FcgRII but lack FcgRIII. Flow cytometry analysis of blood neutrophils from humans or M. nemestrina stained for FcgRII with mAb 8.7 or FcgRIII mAb 3G8. Plots are representive of three individual experiments.


˚ 2 to 69 A ˚ 2, including the loss of a potential surface area of 100 A hydrogen bond (Fig. 7B, 7C). The interface contact is further ˚ 2 buried surface) comdiminished at Trp87 of mnFcgRIIa (68 A ˚ 2 buried surface), pared with the complexed huFcgRIIa (82 A 110 which together with Trp form the so-called “Trp-sandwich” that binds Pro329 of the Fc A chain (Fig. 7A). These alterations to contact residues (Tyr157 and Trp87) can be attributed to the influence of the interspecies substituted residues Pro159 and Tyr160 in the G strand of mnFcgRIIa. The model predicts that the mnFcgRIIa Pro159 kinks the G strand and repositions Tyr157, reducing its contact with the Fc (Fig. 7A). Furthermore, the Tyr160 side-chain packs against Trp87, reducing contact in the Trp sandwich of Trp87 with Pro329 of the IgG-Fc A chain (Fig. 7A). The structural effect of these residues PY(159–160) on the critical contact residues agrees with the mutagenesis data (Figs. 4, 6), which showed that the replacement of the PY(159–160) of mnFcgRIIa with LF(159–160) of huFcgRIIa optimized the interaction with huIgG1 and huIgG2. Importantly, previous studies of huFcgRIIa support our binding and modeling data and are consistent with a role for Tyr160 in the macaque receptor in modulating interactions with IgG. Varying the sequence at position 160 of the human receptor by replacement of Phe160 with alanine substantially increased binding of both huIgG1 and huIgG2 (50, 52). Conversely, the replacement of Phe160 with tyrosine reduced binding of IgG (53); note that tyrosine occupies this position in mnFcgRIIa (Fig. 1). Thus, it is likely that the improved binding of huIgG by the mutated mnFcgRIIa following our replacement of Pro159–Tyr160 with Leu159–Phe160 of huFcgRIIa results from a more favorable orientation of the contact Tyr157 and of the Trp87/Trp110 sandwich in the interaction with human IgG-Fc (Fig. 7). Another major contact of FcgRIIa in complex with Fc is made by the C9E loop, especially residue 131, which determines the functional differences of high/low responder polymorphism of FcgRIIa in humans. Arg131 is accommodated in a shallow depression in the Fc and is somewhat “crowded,” but the His131 is more readily accommodated (40), and in the mnFcgRIIa-allele 1 and mnFcgRIIb, which also contain His131, this also is likely to be the case. In the case of mnFcgRIIa-allele 2, the nearly complete loss of binding caused by the presence of proline in position 131 is likely due to a major alteration of the C9E loop structure leading to the disruption of the interface with IgG.

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FIGURE 10. Structural location of human and macaque interspecies sequence diversity and macaque polymorphism of FcgRIIa. The locations of polymorphic or nonpolymorphic amino acids are shown on the a carbon trace of residues 4–170 of huFcgRIIa. Cyan spheres show the location of nonpolymorphic amino acids that are identical to both M. nemestrina (A) and M. mulatta (B) but differ from huFcgRIIa. The red spheres indicate the location of residues where polymorphism is unique to one macaque species. The yellow spheres indicate location of residues where polymorphism has been identified in both M. nemestrina and M. mulatta. Sequence numbering follows that of Fig. 1.

served in other NHPs (Fig. 11), which raises the possibility that the diminished huIgG binding may be only a feature of macaque FcgRIIa. In the case of position 131, which forms a major contact with huIgG, several, but importantly not all, NHPs also have histidine at this position in FcgRIIa, which could favor huIgG2 binding. His131 is also found in FcgRIIa of rhesus (M. mulatta) and cynomologus (M. fasicularis) macaques, chimpanzee (Pan troglodytes), bonobo (Pan paniscus), baboon (Papio anubis), and squirrel monkey (Samiri boliviensis), and thus it is likely that they also bind huIgG2. Indeed, rhesus and cynomolgus macaque FcgRIIa do bind huIgG2 as measured by surface plasmon resonance or immune complex binding to cells (27). However, orangutan (Pongo abelii) and marmoset (Callithrix jacchus) have Tyr131 and Arg131, respectively, and therefore may have altered huIgG binding compared with macaque FcgRIIa, especially with respect to huIgG2. Our data also describe the profound influence of polymorphism in mnFcgRIIa on IgG binding, which adds additional complexity to analysis of huIgG interaction with macacque FcR. In M. nemestrina and interestingly in rhesus macaque, FcgRIIa is the most polymorphic of the FcgRs (24, 54) (Figs. 1, 2) However, only M. nemestrina showed sequence variation at position 131 wherein proline in this position in allele 2, also present in allele 5, results in ablation of IgG1 and IgG2 binding. This is the equivalent position to the clinically relevant high and low responder polymorphism in huFcgRIIa (11, 23) wherein allotypic receptor with Arg131 is hypofunctional with respect to IgG2 binding. While the functional importance of FcR polymorphism on IgG binding in other macaque species has not been investigated, it is noteworthy that in rhesus macaques, polymorphism in the C9E loop at position 128 results in the presence/absence of a possible N-glycosylation site that replaces the Lys128 adjacent to Phe129, which in huFcgRIIa is a critical IgG contact (Fig. 10) (40). Interestingly, the same site is found in baboon and a unique site of possible N-glycosylation is present at position 133 in marmoset (Fig. 11), but whether these positions are polymorphic or functionally important in these species is unknown. Thus, polymorphism, at least in FcgRIIa, is extensive (eight alleles in 10 animals), and it is likely that more alleles exist in M. nemestrina. Importantly, the presence of null or hypofunctional allotypic receptors is sufficiently frequent in M. nemestrina (two of eight allotypic receptors among 10 individuals) and potentially other species to warrant caution when interpreting results of studies involving models of Ab-based effects. The inhibitory FcgRIIb also contains His131 in all major macaque species widely used in research, including pig-tailed (M. nemestrina), rhesus (M. mulatta), and cynomologus (M. fasicularis) macaques as well as the baboon (P. anubis) (Fig. 12). Based on the M. nemestrina data in this study, the FcgRIIb of these species is likely to bind human IgG1, and importantly IgG2, more avidly than huFcgRIIb. Indeed, avid binding of IgG2 has been observed in cynomologus FcgRIIb (27). In all other NHP species, arginine is preserved at this position, which suggests FcgRIIb of these species may behave more similar to huFcgRIIb with reduced immune complex binding and little IgG2 binding, making the FcgRIIb of macaques unique in this regard. In humans, subtle affinity differences have indeed been shown to be critically important in the respective functions of human FcgRIIa and FcgRIIb (53). Because of the altered specificity of mnFcgRIIb and/or the reversed hierarchy of IgG1 and IgG2 binding to mnFcgRIIa and mnFcgRIIb, it is conceivable that Ab therapeutics, especially IgG2, may not behave in M. nemestina or other macaque species as they may be expected to in humans. The


for IgG1 and a greatly increased affinity of IgG2 for cynomolgus FcgRIIb compared with its human ortholog (27). To identify key residues that contribute substantially to the observed interspecies IgG binding differences, we exchanged equivalent residues between the human and macaque FcR receptors in site-directed mutagenesis studies (Figs. 4–6). In short, Pro159 and Tyr160 contribute to the lower activity of the mnFcgRIIa, whereas His131 and Met132 are key to the higher activity of mnFcgRIIb compared with huFcgRIIb. Furthermore, we have also described the profound influence of polymorphism of mnFcgRIIa on IgG binding and identified FcgRIIa as highly polymorphic with eight alleles being detected in only 10 individuals. The present studies have implications for the structural and functional analysis of IgG FcR interactions, not only in M. nemestrina but also in the widely used rhesus and cynomolgus macaques and other NHPs. A comparative analysis of FcgRIIa and FcgRIIb sequences of other NHPs shows that critical residues in the FG and C9E loops identified in this study as affecting the mnFcgRII interaction with huIgG are preserved in some but not all NHPs (Figs. 10–12). The Pro159 and Tyr160 residues of the FG loop that adversely affect IgG binding by mnFcgRIIa are conserved in rhesus (M. mulatta) (Figs. 10, 11) and cynomolgus (M. fasicularis) macaque species (Fig. 11) (24, 27, 54), suggesting that these residues could be a key interspecies difference that modulates the engagement of huIgG in FcgRIIa of other macaque species such as cynomolgus FcgRIIa (27). Notably PY(159–160) are not con-


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increased binding to mnFcgRIIb may obscure potent and desirable effector function or alternatively obscure adverse reactions that may otherwise be manifested in humans where affinity for the inhibitory FcgRIIb is lower. Furthermore, with the success of mAb therapy (1), efforts have been made to alter the potency of useful therapeutic mAbs that include specific engineered changes to the IgG-Fc region to optimize the interaction with huFcgRs (21, 22, 55). However, the FcgRIIa or FcgRIIb selectivity of such engineered Abs may not necessarily exhibit improved binding to macaque FcgRs (56). Thus, our data highlight that the activities of mAbs designed to alter interactions between human Abs and huFcRs may not be faithfully recapitulated in preclinical studies in nonhuman primates, or at least in macaques. Similar caveats may apply to viral pathogenesis studies in macaques of human infections where FcgRIIa or FcgRIIb are involved in significant clinical or biological aspects of the natural

history of the infection in humans, for example, Ab-dependent enhancement (57) in dengue infection or skewing of the FcgRIIa/ FcgRIIb expression ratio in HIV infection (12) or resistance to HIV (13, 14). Thus, the interspecies and polymorphic differences we describe in this study may translate to alterations of Ab-induced inflammatory outcomes in vivo in NHPs that are distinct from those in humans. When considering the impact of differences in FcgR binding function in vivo in NHPs, it is important to consider whether any differences in cell distribution may also confound the interpretation of in vivo studies of Ab function. The cell distribution of FcgR is for the most part similar to humans in M. nemestrina (Figs. 8, 9) and in rhesus (26, 45) and cynomolgus macaques (27), with the notable exception of neutrophils where FcgRIII is lacking but FcgRII is elevated. Thus, the implication is that differences in cell distribution in M. nemestrina or other macaques species may

FIGURE 12. Alignment of the extracellular domains of NHPs and huFcgRIIb. Amino acids identities are shown as dots. Critical residues analyzed in this paper are boxed. Shown are pig-tailed macaque (M. nemestrina), rhesus macaque (M. mulatta) (24), cynomologus macaque (M. fasicularis) (27), baboon (P. anubis, GenBank XM_003892974.1), chimpanzee (P. troglodytes, GenBank XM_ 001153863.2), bonobo (P. paniscus, GenBank XM_003824761.1), orangutan (P. abelii, GenBank XM_003892974.1), squirrel monkey (S. boliviensis, GenBank XM_003943370.1), and gorilla (Gorilla gorilla, GenBank XM_004027772).


FIGURE 11. Alignment of the extracellular domains of NHPs, including rhesus allelic forms and huFcgRIIa. For additional allelic forms of M. nemestrina, see Fig. 1. Amino acids identities are shown as dots. Critical residues analyzed in this study are boxed. Species shown are pig-tailed macaque (M. nemestrina), rhesus macaque (M. mulatta) (24), cynomologus macaque (Macaca fasicularis, GenBank AF485813) (27), baboon (P. anubis, GenBank XM_003892972.1), chimpanzee (P. troglodytes, UniProt Q8SPV8.1), bonobo (P. paniscus, GenBank XM_003804397.1), orangutan (P. abelii, GenBank XM_002809886.2), squirrel monkey (S. boliviensis, GenBank XM_003943372.1), and marmoset (C. jacchus, GenBank XM_003735165.1). The known allelic forms with polymorphisms in the extracellular region are shown for M. nemestrina and M. mulatta.

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not affect the evaluation of mAbs except where effector function is dependent on the FcgRs of neutrophils. Although the macaque is a valuable model of human immune function, clear differences exist between species. Cautious interpretation of responses involving Ab-induced responses is prudent. Clearly analysis of NHP FcgR genotype and binding profiles of therapeutic IgG to receptors may be useful in the design of experimental or preclinical studies. Thus, the use of NHPs as a model of human immunity or preclinical model for the evaluation of Ab FcR interactions will be greatly assisted by identifying and understanding the basis for the differences in interaction of macaque and huFcR with huIgG.

Disclosures The authors have no financial conflicts of interest.

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Sex steroid binding protein in the plasma of Macaca nemestrina.

Corticosteroid binding protein in the plasma of Macaca nemestrina.

Allotype-associated differences in concentrations of human IgG subclasses.

The binding of human and guinea-pig IgG subclasses to homologous macrophage and monocyte Fc receptors.

Differences between total IgG and sum of the IgG subclasses in clinical samples.

The profile of IgG and IgG subclasses of onchocerciasis patients.

Functional polymorphism of IgG FcRII (CD32) on human neutrophils.

The subclasses of human IgG antibodies against tetanus toxoid.

Antigammaglobulin (rheumatoid factor) activity of human IgG subclasses.

Fc receptor binding affinity.

The complete mitochondrial genome of Southern pig-tailed macaque, Macaca nemestrina, and comparative mitochondrial genomics of Macaca species.

Human triclonal anti-IgG gammopathy. II. Determination of the antigenic specificity patterns of the IgG, IgA and IgM autoantibodies for the subclasses of IgG.

Investigation of the binding site of mouse IgG subclasses to homologous peritoneal macrophages.

The antigenic interrelations of some mammalian IgG subclasses detected with cross-reacting fowl antisera to human and mouse IgG-Fc.

The sex difference in dimensional communalities in Macaca nemestrina.

The N-Glycosylation of Mouse Immunoglobulin G (IgG)-Fragment Crystallizable Differs Between IgG Subclasses and Strains.

Ultracentifuge studies of the binding of IgG of different subclasses to the Clq subunit of the first component of complement.

Mhc-DRB genes of the pigtail macaque (Macaca nemestrina): implications for the evolution of human DRB genes.

The cranial base in fetal Macaca nemestrina: a quantitative analysis of size and shape.

Comparative functional characterization of canine IgG subclasses.

IgG subclasses in human periodontal disease. I. Distribution and incidence of IgG subclass bearing lymphocytes and plasma cells.

Interspecies differences in the pharmacokinetics of kanamycin and apramycin.

Interspecies differences and scaling for the pharmacokinetics of xanthine derivatives.

IgG Subclasses and Isotypes of VH4-34 Encoded Antibodies.