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*,†,‡ Downloaded from 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 struc- ture. 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 http://www.jimmunol.org/ (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. at University of Melbourne Library on February 18, 2014

he Fc receptors (FcRs) are cell surface molecules primarily play activating and inhibitory roles in normal immune responses expressed on effector leukocytes of the innate immune sys- and immune homeostasis (1, 2), and imbalance in these opposing T tem and by binding Igs provide adaptive immunity with roles is a key contributing factor to the development of patho- a cell-based effector system (1). There are three classes of leukocyte logical inflammation in several autoimmune diseases (3). FcgRIIa IgG FcRs in humans: the high-affinity IgG receptor FcgRI (CD64) and is one of several activating FcRs, but it is the most widespread the low-affinity IgG receptors FcgRII (CD32) and FcgRIII (CD16). and abundant FcgR of humans present on all leukocytes except The two major genes of the human (hu)FcgRII family encode lymphocytes. Despite being a low-affinity receptor, FcgRIIa av- FcgRIIa and FcgRIIb and their splice variants. These receptors idly binds oligovalent Ab-coated targets (immune complexes) to induce cytokine release from inflammatory leukocytes, respiratory burst, Ab-dependent cell-mediated killing, internalization of com- *Centre for Biomedical Research, Burnet Institute, Melbourne, Victoria 3004, Australia; plexes, and platelet aggregation. FcgRIIb in humans is a powerful † Department of Pathology, University of Melbourne, Parkville, Victoria 3056, inhibitor of immune receptor signaling and is critical to the mod- Australia; ‡Department of Immunology, Monash University, Melbourne, Vic- toria 3004, Australia; xDepartment of Surgery, University of Melbourne, Parkville, ulation of humoral immunity and Ab-dependent immune functions { Victoria 3010, Australia; Australian Centre for Blood Diseases, Monash University, (4–6). Its ITIM-dependent modulation of ITAM signaling cascades Melbourne, Victoria 3004, Australia; ‖Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia; and #Department for regulates B cell Ag receptor signaling and consequent Ab respon- Immunology and Microbial Science and International AIDS Vaccine Initiative Neu- ses. FcgRIIb also regulates signaling by the activating FcRs FcgRI, tralizing Antibody Center and Center for HIV/AIDS Vaccine Immunology and Im- FcgRIIa, FcgRIIIa, FcεRI, and FcaRI. In a practical sense, the munogen Discovery, The Scripps Research Institute, La Jolla, CA 92037 IgG–FcgR interaction is a key contributor to the effectiveness of Received for publication June 13, 2013. Accepted for publication November 15, 2013. many vaccines both at the level of immune regulation and induction of effector function. Indeed, it has been suggested that the IgG– This work was supported by National Health and Medical Research Council Fellowship and Project Grant 1002270 and Program Grant 510448, as well as FcgR interaction mediating Ab-dependent cell-mediated cytotox- by the Victorian Operational Infrastructure Support Program. icity may play a role in HIV vaccine–induced protective immunity The cDNA sequences presented in this article have been submitted to GenBank of humans (7). Moreover, the effectiveness of allergen immuno- (http://www.ncbi.nlm.nih.gov/genbank) under accession numbers KF234399, KF234400, therapy (8) and many therapeutic mAbs, particularly anticancer KF234401, KF234402, KF562260, KF562261, KF562262, KF562263, KF234403, and KF234404. mAbs, has been attributed at least in part to successful engagement of Address correspondence and reprint requests to Prof. P. Mark Hogarth, Centre for FcR-dependent effector systems, including FcgRIIa and FcgRIIb (1). Biomedical Research, Burnet Institute, 85 Commercial Road, Melbourne, VIC 3004, In humans, several polymorphisms of the activating IgG re- Australia. E-mail address: [email protected] ceptor, FcgRIIa, are known (9, 10). The most clinically significant Abbreviations used in this article: FcR, Fc receptor; hu, human; MFI, mean fluores- polymorphism encodes amino acid position 131 where either a cence intensity; mnFcgR, Macaca nemestrina FcgR; NHP, nonhuman primate. histidine or an arginine residue may be present, resulting in pro- Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 found effects on binding of huIgG2 (11). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301554 The Journal of Immunology 793

Genetic polymorphisms of huFcRs that affect their capacity to Ethics Committees. Whole venous blood was obtained from animals se- bind IgG or alter the balance of activation over inhibition have been dated with ketamine as previously described (36), and PBMCs were iso- implicated in resistance to HIV and bacterial infection (12–18), lated over Ficoll-Hypaque and stored in liquid nitrogen. susceptibility to autoimmunity (19, 20), and the effectiveness of Cloning of FcgRs therapeutic mAbs, mostly IgG1 but increasingly IgG2 (1). As a result of the success of mAbs, considerable effort has been made Gene transcripts for mnFcgRIIa and mnFcgRIIb were obtained by PCR of cDNA (AffinityScript quantitative PCR cDNA synthesis kit from Agilent to improve their FcR-dependent potency by engineering Fc por- Technologies, Santa Clara, CA) produced from total RNA (RNeasy Mini tions for the purpose of selective engagement of either activating Kit from Qiagen, Melbourne, VIC, Australia) from PBMCs of unrelated FcRs (1), including FcgRIIa (21) or inhibitory FcRIIb (22). Fur- animals. Restriction enzymes and DNA-modifying enzymes were all from thermore, the most clinically significant polymorphism of the New England BioLabs (Beverly, MA), except for PCR applications, which used AccuPrime Pfx DNA polymerase (Life Technologies, Melbourne, activating IgG receptor, FcgRIIa (9, 10), encodes either a histidine VIC, Australia). The primers to generate FcgR PCR fragments were or an arginine residue at amino acid position 131 and influences synthesized by Sigma-Aldrich (Sydney, NSW, Australia). the clinical outcome of Ab therapy (23) and additionally has Because of the absence of any DNA sequence information from profound effects on binding of huIgG2 (11). M. nemestrina, the 59- and 39-amplifying primers for FcgRIIa or FcgRIIb The genetic diversity of nonhuman primate (NHP) FcRs has not were based on sequences of rhesus macaque (M. mulatta,GenBank Downloaded from NM_001257300) or cynomolgus macaque (M. fasicularis,GenBank been extensively characterized, even though NHPs are key animal AF485814.1), respectively. The FcgRIIa 59 primer was complementary models for many diseases, including HIV. Examination of func- to the initiation codon and the following five codons, and the 39 primer tional Fc polymorphisms in NHPs is, as found in humans, pertinent was complementary to the last seven codons and the termination co- to understanding susceptibility to infectious and autoimmune don. The FcgRIIb 59 primer was complementary to the initation codon and the first five codons, and the 39 primer was complementary to the diseases. Interspecies functional substitutions will likely substan- final six codons and the termination codon. The primers also contained tially influence the evaluation of human Abs in NHPs. KpnI and EcoRV sites. The Kpn1/EcoRV digested PCR products were http://www.jimmunol.org/ Indeed, although evolutionarily conserved, the limited infor- cloned into pENTR1A (Life Technologies) followed by Gateway LR mation available to date shows that significant interspecies se- cloning (Life Technologies) into a Gateway-adapted pMXI expression quence differences are apparent between the huFcRs and their vector containing a neomycin resistance cassette (37). The huFcgRIIb2 construct was obtained from PBMC-derived cDNA as orthologs in different NHP species (24–27). Some of this sequence above using 59 and 39 primers based on the sequence of FcgRIIb (38) and diversity occurs at sites that are predicted by homology to be cloned into pMXI as above. essential for the interaction with IgG and may influence the binding The nucleotide sequences of mnFcgR were determined from both of IgG. These differences may result in alterations to the relative PBMC-derived PCR-amplified cDNA and from six FcgR clones for each receptor from each individual animal using BigDye version 3.1 terminator contributions that the different FcR classes make to Ab-dependent

cycle sequencing (Applied Biosystems, Melbourne, VIC, Australia) and at University of Melbourne Library on February 18, 2014 effector function in vivo in macaques compared with humans. separated at the Australian Genome Research Facility (Melbourne, VIC, Correspondingly, differences in species IgG–FcR interactions may Australia). All cDNA sequences have been submitted to GenBank (sub- greatly complicate the interpretation of preclinical studies aimed mission nos. 1637005 and 1655933). at evaluating the functional activity of therapeutic Abs or vaccines Alignments of FcgRII sequences were generated using CLC Sequence Viewer version 6.4 (CLC bio, Aarhus, Denmark). in NHP models such as macaques, including Macaca nemestrina. Isolation and expression of the human FcgRIIa-H131 and FcgRIIa-R131 Additionally, use of outbred populations of macaques may further have been described previously (39–41). complicate interpretation owing to nonsynonymous polymorphisms in the macaque FcR genes. Site-directed mutagenesis Little is yet known of the impact of FcR polymorphism on the Mutated FcgRs were constructed by in vitro site-directed mutagenesis binding of huIgG in NHPs, especially in the three macaque species using a series of mutation primers and the thermostable polymerase Pfx commonly used in medical research: rhesus (Macaca mulatta), (Life Technologies) to obtain the following for mnFcgRIIa-allele 1 (mu- → → cynomolgus (Macaca fascicularis), and pig-tailed (M. nemestrina) tant 1, N135T [N T]; mutant 2, P159L,Y160F [PY LF]), mnFcgRIIa- allele 2 (mutant 3, P131H,M132L,N133D [PMN→HLD]), the double macaques. Furthermore, nothing is known of the activity of FcRs mutants of mnFcgRIIa-allele 2 (mutant 4, PMN→HLD with N→T; mutant of the pig-tailed macaque. Similar to cynomolgus and rhesus mac- 5, PMN→HLD with PY→LF), and for mnFcgRIIb1 (mutant 6, H131R, aques (28, 29), pig-tailed macaques are used in the evaluation of Ab M132S [HM→RS]). immunotherapy (30–32), humoral immune responses to infection, Stable expression of FcgRII and vaccine candidates, including dengue virus (33, 34) and HIV/ simian HIV (35, 36). Furthermore, different species have been used A pMXI retroviral expression system was used to introduce FcgR DNA into in similar models (30, 32). the FcR-deficient IIA1.6 cells. Phoenix packaging cells were maintained in In this study, we define the ligand-binding properties of activating RPMI 1640 containing glutamine and 5% heat-inactivated fetal bovine serum and were transfected with FcgRII plasmids using Lipofectamine FcgRIIa and the inhibitory FcgRIIb of M. nemestrina (mnFcgRIIa 2000 (Life Technologies) to generate retrovirus. The retroviral suspension and mnFcgRIIb). We identify a functionally significant polymor- was overlaid onto IIA1.6 cells as described (39). Transduced IIA1.6 cells phism of FcgRIIa and define the molecular and structural basis were selected for resistance to 0.4 mg/ml Geneticin (Life Technologies). of impaired binding to IgG by mnFcgRIIa and for the enhanced binding and broader specificity of mnFcgRIIb compared with their Abs human orthologs (huFcgRIIa and huFcgRIIb). These data also Purified human myeloma proteins IgG1k, IgG2k, and IgG3k were pur- have implications for the analysis of IgG function in other ma- chased from both Sigma-Aldrich (Castle Hill, NSW, Australia) and The caque species and NHPs. Binding Site (Birmingham, United Kingdom). PE-conjugated F(ab9)2 goat anti-human IgG F(ab9)2-specific polyclonal was from Jackson Immuno- Research Laboratories (West Grove, PA). Human IgG in the form of i.v. Ig Materials and Methods Sandoglobulin was obtained from Novartis (Sydney, NSW, Australia). Animals Abs used to identify populations of peripheral blood leukocytes were anti-CD20, (clone L27; BD Biosciences, Sydney, NSW, Australia), anti- Outbred 3- to 5-y-old pig-tailed macaques (M. nemestrina) were obtained CD14 (clone m5E2; BD Biosciences), anti-CD56 (clone NCAM16.2; from the Australian National Macaque Breeding Facility, and studies were BD Biosciences), anti-CD159a (NKG2) (clone Z199; Beckman Coulter, approved by the University of Melbourne and Commonwealth Scientific Melbourne, VIC, Australia), anti-FcgRIII (clone 3G8; BD Biosciences), and Industrial Research Organization Animal Health Institutional Animal and anti-CD41a (clone HIP8; BD Biosciences). 794 PIG-TAILED MACAQUE FcgR INTERACTION WITH HUMAN IgG

The anti-FcgRIIa mAbs 8.7, 8.2, and IV.3 and the anti-FcgRIIb mono- huIgG1-Fc (Brookhaven Protein Data Bank ID 3RY6) (40) was used as a clonal X63-21/7.2 have been previously described (40, 42, 43). Fab fragments template to generate a homology model of the macaque FcgRIIa/huIgG1-Fc of IV-3 and F(ab9)2 fragments of 8.7, 8.2, and X63 mAbs were generated as complex using the amino acid sequence of mnFcgRIIa-allele 1 (Fig. 1). The described (42) and then biotinylated with EZ-Link Sulfo-NHS-LC-Biotin three-dimensional model of the macaque FcgRIIa/huIgG1-Fc complex was (Thermo Scientific, Rockford, IL). optimized by conjugate-gradient energy minimization against spatial restraints extracted from the 3RY6 template and a probability density IgG ligands function using the Modeler algorithm (46). The protein interface between Two separate methods were used to evaluate the binding of huIgG to cell FcgRIIa and huIgG1-Fc was analyzed using the protein interfaces, surfa- surface–expressed FcRs of M. nemestrina and humans. ces, and assemblies (PISA) server at the European Bioinformatics Institute First, for anti-Fab complexes, complexes of IgG were generated by cross- (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 linking IgG with F(ab9)2 fragments of fluorochrome-conjugated anti-human Fab as previously described (44). Briefly, individual human IgG1, IgG2, or IgG3 were plotted for comparison with the total buried surface area between huFcgRIIa and mnFcgRIIa-allele 1. Because mnFcgRIIa-allele 1 has one subclasses were incubated with F(ab9)2 fragments of PE-conjugated goat anti- additional N-linked glycosylation site compared with huFcgRIIa, in silico human 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 glycosylation of this extra site was performed using the GlyProt Web-based of pooled huIgG were generated using the cross-linker Tris-succinimidyl server (http://www.glycosciences.de/modeling/glyprot/php/main.php) (48). aminotriacetate (Thermo Scientific). Tris-succinimidyl aminotriacetate at 5 mg in 0.5 ml anhydrous DMSO was mixed with 3.85 mg biotin-X- Downloaded from hydrazide (Sigma-Aldrich) in 0.5 ml anhydrous DMSO for 2 h at room Results temperature to generate the intermediate biotinyl bis-succinimidyl ami- Sequence comparison of huFcgRIIa and mnFcgRIIa notriacetate, which was used without purification. Human IgG (4 ml at The mnFcgRIIa sequences were determined from cDNA isolated 25 mg/ml in PBS) was mixed with an 11-fold excess of biotinyl bis- succinimidyl aminotriacetate (0.7 ml) and allowed to react for 1 h at from 10 animals of an outbred colony of M. nemestrina. Replicate room temperature. The resulting IgG dimers/trimers were purified from the sequence analysis of PCR products and of multiple clones from reaction mixture and separated from monomers and multimers by gel fil- independently derived, duplicate PBMC samples were used to http://www.jimmunol.org/ tration on a Sephacryl S-300 column (1.5 3 100 cm). Fractions were establish and then confirm FcR sequences. analyzed by SDS-PAGE, and those corresponding to IgG dimers/trimers were pooled and stored in aliquots at 280˚C. Sequence analysis comparing M. nemestrina sequences to the huFcgRIIa revealed amino acid differences between macaque Flow cytometric detection of immune complex binding and FcgRIIa and humans that were conserved in all animals and also receptor expression identified polymorphic sequence variation between the FcgRIIa The binding of IgG complexes to allelic forms of receptors and mutants of individual macaques (Fig. 1). Thus, 26 aa that differed from thereof was determined as described (40). Briefly, anti-F(ab9)2 PE/IgG huFcgRIIa were conserved in all individuals. Sixteen of these complexes at the indicated concentrations were incubated with 1.2 3 5 aminoaciddifferencesmappedtothe extracellular region, three

10 cells in 50 ml for 1 h on ice and then washed twice and resuspended in at University of Melbourne Library on February 18, 2014 200 ml PBS/0.5% BSA. Similarly, aggregated huIgG or biotinylated IgG to the transmembrane region, and seven to the cytoplasmic tail dimer/trimer binding was detected by indirect fluorescence following in- of mnFcgRIIa (Fig. 1). cubation with cells on ice for 1 h. Cells were then washed and incubated Further sequence analysis among the 10 animals revealed ex- with PE-labeled goat anti-human Ab or allophycocyanin-streptavidin, tensive polymorphism and identified eight allelic products of respectively, for 30 min on ice, washed twice, and then resuspended in 200 ml PBS/BSA. Background binding controls included nonspecific M. nemestrina FcgRIIa (Fig. 1). These allelic forms were distin- binding of IgG to untransfected IIA1.6 cells and nonspecific binding of guished from each other by 16 polymorphic residues, of which conjugate only to FcR-expressing cells. 9 were located in the extracellular domains (at positions 1, 74, 79, Expression levels of FcgR on the transfected cells were determined in 89, 93, 119, 131, 133, and 140), 1 was located in the transmem- each experiment by flow cytometry using the following biotinylated anti- brane region, and 6 were found in the cytoplasmic tail. Interest- FcR mAbs: F(ab9) of 8.7, 8.2, and Fab of IV.3 or the anti-FcgRIIb X63- 2 ingly, the identity of the nine polymorphic amino acids within the 21/7.2 F(ab9)2 (40). Cells were then washed and binding was detected using allophycocyanin-streptavidin as described above. Gating was set to extracellular region suggests that FcgRIIa alleles 1, 3, 4, 6, 7, and record fluorescence of 10,000 viable single cells. The analysis of huIgG 8 are closely related, as they contain the human-equivalent residue binding to each human or macaque FcgR and mutants thereof was per- in many of the polymorphic positions. In contrast, FcgRIIa alleles formed on at least five independent occasions using the anti-Fab complexes and at least three independent occasions using the IgG dimers/trimers. 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 Flow cytometric analysis of Fc receptor expression on blood seven or eight of the nine polymorphic positions, respectively, cells being distinct from huFcgRIIa (Fig. 1). Whole blood was collected from four M. nemestrina as above and from In M. nemestrina, the inhibitory FcgRIIb has 23 nonpolymorphic healthy human volunteers after informed consent as approved by the amino acid differences from huFcgRIIb, and 21 of these occur in the Alfred Health Human Ethics Committee or the Monash University extracellular domains. A single polymorphism resulting in amino Standing Committee on Ethics in Research Involving Humans. PBMCs acid substitution methionine/valine at position 254 was apparent were isolated on a Ficoll density gradient. Total leukocytes were obtained from buffy coats and then erythrocytes were lysed with hypotonic RBC in the cytoplasmic tail (Fig. 2). lysis solution (Miltenyi Biotec, Sydney, NSW, Australia) according to the manufacturer’s instructions. Expression of surface markers was determined Binding of huIgG to mnFcgRIIa using flow cytometry where human and M. nemestrina B lymphocytes were A comparative analysis of the binding of huIgG subclasses to identified with anti-CD20 and monocytes were identified with anti-CD14. Human NK cells were defined with anti-CD56 and macaque NK cells with allelic forms of mnFcgRIIaandtohuFcgRIIa was undertaken in anti-CD159a (NKG2) (45). Neutrophils were identified by forward and side transfected cells where their equal expression was confirmed by scatter profiles. Platelet-rich plasma was isolated by low-speed centrifugation binding of the anti-FcgRIIa mAb F(ab9)2 fragments (Fig. 3A), and platelets were identified by expression of CD41a. The expression of which detects an epitope in the extracellular domains in FcgRIIa FcgRIII was defined using clone 3G8, and FcgRII was identified with mAb 8.7 as described above. and FcgRIIb (40). The binding of huIgG3 complexes to mnFcgRIIa-allele 1 and Homology modeling of the macaque FcgRIIa/huIgG1-Fc huFcgRIIa-His131 was analyzed by flow cytometry and was shown interaction to be essentially identical with relative cell surface staining of Protein homology models were prepared using the Discovery Studio suite, mean fluorescence intensity (MFI) of 70,000 and 71,000, re- version 3.0 (Accelrys, San Diego, CA). The cocrystal structure of huFcgRIIa/ spectively (Fig. 3D). In contrast, mnFcgRIIa-alelle 1 bound IgG1 The Journal of Immunology 795 Downloaded from http://www.jimmunol.org/ at University of Melbourne Library on February 18, 2014

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; mnFcgRIIa- allele 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. or IgG2 complexes with MFIs of 5000 and 360, respectively, and was barely detectable above background (MFI of 30, Fig. 4B). which were 8- to 10-fold lower than their binding to huFcgRIIa- Similarly, IgG2 binding of mnFcgRIIa-allele 2 was also barely His131 (Fig. 3B, 3C). Thus, in comparison with huFcgRIIa, detectable above background levels (Fig. 4C). The binding of IgG3 the mnFcgRIIa-allele 1 has equivalent binding of complexes of complexes (MFI of 11,600) was also reduced 6-fold compared with huIgG3, but greatly diminished binding of complexes of huIgG1 mnFcgRIIa-allele 1 (MFI of 70,300). Thus, not only do interspecies and huIgG2. differences of FcR affect huIgG binding, but polymorphisms (in- In addition to the differences in huIgG binding between human traspecies differences) in mnFcgRIIa also influence the binding of and M. nemestrina FcgRIIa observed above, even greater dif- huIgG. The mnFcgRIIa-allele 2 is greatly impaired in ligand binding ferences were found in huIgG binding to the allelic forms of of human IgG1, IgG2, and IgG3. mnFcgRIIa (Fig. 4). The binding of all huIgG subclasses to Next, we defined the sequence substitutions responsible for the mnFcgRIIa-allele 2 receptor was reduced greatly (Fig. 4B–D). low ligand binding activity of mnFcgRIIa-allele 2. Analysis of the Compared to binding by mnFcgRIIa-allele 1 (MFI of 5000), IgG1 polymorphic amino acid differences in the mnFcgRIIa in the context binding to mnFcgRIIa-allele 2 was reduced 80-fold (MFI of 90) of known functional regions of huFcgRIIa showed that position 131 796 PIG-TAILED MACAQUE FcgR INTERACTION WITH HUMAN IgG Downloaded from http://www.jimmunol.org/

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 poly- morphism 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. at University of Melbourne Library on February 18, 2014 is part of the major contact surface in the huFcgRIIa/IgG inter- spectively (Fig. 4F, 4G; blue filled, dashed line) that was com- action (40, 49, 50). Moreover, in humans, polymorphic FcgRIIa parable to that of mnFcgRIIa-allele 1 (MFI of 5000 for IgG1 and (high and low responder allelic products that differ at position 360 for IgG2) (Fig. 4B, 4C; red filled, dashed line). Similarly, the 131) have very different interactions with mouse IgG1 and huIgG2 binding of IgG3 was also improved from an MFI of 11,600 to an (44). Thus, it seemed possible that the large differences in IgG MFI of 53,400 (Fig. 4D, 4H). However, this increased binding of binding between the two M. nemestrina allelic receptors results IgG was still markedly lower than the binding of IgG1 and IgG2 from the sequence differences in this segment, namely, HMD complexes to the huFcgRIIa-His131; that is, MFIs of 57,200 and (131–133) of the functional receptor encoded by mnFcgRIIa-allele 1 3,600, respectively. Binding of IgG3 by the mnFcgRIIa-allele 2 compared with the PMN(131–133) of the poorly functional receptor with HLD(131–133) remained slightly reduced compared with the encoded by mnFcgRIIa-allele 2 (Fig. 1). human receptor (MFI of 53,400 for mnFcgRIIa-His131 compared Consequently, we attempted to reconstitute binding of huIgG by with 70,100 for huFcgRIIa-His131; compare with Fig. 3). The replacing PMN(131–133) of mnFcgRIIa-allele 2 with HLD(131– failure to fully enable IgG binding to levels observed for the hu- 133) from huFcgRIIa. Note that His131 and Asp133 of mnFcgRIIa- man receptor implied that other amino acid residue differences allele 1 are identical in huFcgRIIa (Fig. 1). Weak binding of IgG1 between mnFcgRIIa and huFcgRIIa contribute to ligand binding. and IgG2 to mnFcgRIIa-allele 2 was demonstrated (Fig. 4B, 4C), Of particular interest in this context were residues Pro159 and and the HLD(131–133) substitution substantially rescued binding Tyr160 in mnFcgRIIa and Leu159 and Phe160 in huFcgRIIa, which are with an MFI of 2900 and an MFI of 300 for IgG1 and IgG2, re- 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). FIGURE 3. Comparative binding of huIgG subclasses to mnFcgRIIa- The role of Pro159 and Tyr160 in macaque receptor binding of allele 1 (red filled histogram with dashed red line) or the huFcgRIIa-His131 (open histogram, solid red line). Cell surface expression of receptor protein IgG1 and IgG2 was further examined in a similar analysis of the was determined using the anti-FcgRII mAb 8.7, which recognizes mnFcgRIIa-allele 1, which contained the HMD(131–133) sequence. 159 160 159 160 huFcgRIIa and mnFcgRIIa. Background binding is shown as a solid black The replacement of Pro and Tyr with Leu and Phe from line. The binding of each IgG subclass to each receptor was tested on at huFcgRIIa also improved IgG1 and IgG2 binding to levels similar least five occasions. to those of huFcgRIIa binding (Fig. 4I–L). Thus, the presence of The Journal of Immunology 797

FIGURE 4. Comparative binding of huIgG sub- classes 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-al- lele 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). Bind- ing 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 Downloaded from (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. http://www.jimmunol.org/

PY(159–160) in mnFcgRIIa results in a significant impairment of Binding of huIgG dimers to FcgRII binding of the human IgG1 and IgG2 subclasses. The binding measurements above were obtained using complexes generated by oligomerization of IgG with an anti-Fab Ab, a Binding of huIgG to mnFcgRIIb technique commonly used for the detection of immune complex The interaction of huIgG subclasses with the inhibitory FcgRIIb of binding to low-affinity receptors (44). To exclude the possibility M. nemestrina was investigated and substantial differences were that the anti-Fab Ab may influence FcR binding of the IgG com- observed in specificity and binding compared with huFcgRIIb plexes, we used a second method where dimers/trimers of huIgG

(Fig. 5). Indeed, unlike the activating FcgRIIa of M. nemestrina, were generated by cross-linking pooled human i.v. Ig with a bio- at University of Melbourne Library on February 18, 2014 the mnFcgRIIb had a 10-fold greater capacity to bind IgG1 tinylated cross-linker (Fig. 6). complexes than did huFcgRIIb (Fig. 5B). The greatest functional Flow cytometry analysis of the binding of the cross-linked IgG divergence was in the binding of huIgG2, which failed to bind to dimers/trimers to FcgRIIa and FcgRIIb and receptor mutants en- huFcgRIIb as expected, but surprisingly was strongly bound by tirely agreed with the binding observed using the complexes mnFcgRIIb (Fig. 5C). FcgRIIb from both species bound IgG3 at generated with the anti-Fab Ab (Figs. 3–5). The mnFcgRIIa-allele similar levels (Fig. 5D). 2 failed to bind the IgG dimers/trimers, but the replacement of This difference in the capacity of the inhibitory mnFcgRIIb to PMN(131–133) with HLD(131–133) enabled binding similar to bind IgG2 was surprising because in humans IgG2 essentially that of allele 1 (Figs 6A, 6B). Further mutation of this construct binds only to the activating FcgRIIa-His131, which is the conse- wherein PY(159–160) was replaced with the LF(159–160) of quence of the Arg/His131 polymorphism dictating specificity for huFcgRIIa resulted in further increase of IgG binding to levels IgG2 in this activating receptor. approaching those of huFcgRIIa-His131 (Fig. 6A). Comparison of the amino acid sequence of the IgG binding One other significant structural difference between humans and regions of human and M. nemestrina FcgRIIb shows that the macaques in this region is a potential N-glycosylation site at po- principal sequence differences occur around position 131 wherein sition 135 in mnFcgRIIa, which is absent from huFcgRIIa but is mnFcgRIIb encodes His131 and Met132, whereas huFcgRIIb has found in both human and macaque FcgRIIb. Because this site sits Arg131 and Ser132 (Fig. 2). Thus, we replaced the HM(131–132) of adjacent to the critical His131, we replaced N135 in mnFcgRIIa mnFcgRIIb with the RS(131–132) of huFcgRIIb and measured the with the human equivalent T135; however, this mutation had little impact on IgG1 and IgG2 binding (Fig. 5B, 5C). The substitution effect on the binding of any huIgG subclass (Fig. 6A). of the human-derived RS(131–132) into mnFcgRIIb resulted in Similarly, the same modifications of mnFcgRIIa-allele 1 (Fig. a marked loss in IgG1 binding, decreasing to levels equivalent to 6B) were made and resulted in similar effects on IgG binding. huFcgRIIb (Fig. 5B). Moreover, IgG2 binding was completely lost Replacement of PY(159–160) with the human LF(159–160) res- (Fig. 5C), which is entirely consistent with the essential role of idues greatly improved IgG binding, but as seen in mnFcgRIIa- His131 in dictating the binding of IgG2. allele 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. 798 PIG-TAILED MACAQUE FcgR INTERACTION WITH HUMAN IgG Downloaded from http://www.jimmunol.org/ at University of Melbourne Library on February 18, 2014

FIGURE 6. The binding of huIgG dimers/trimers to mnFcgRIIa and mnFcgRIIb. Biotinylated huIgG dimers/trimers were titrated on transfectants expressing (A) huFcgRIIa (n)ormnFcgRIIa-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)ormnFcgRIIa-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 (+)ormnFcgRIIb2 (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.

InthecaseofFcgRIIb (Fig. 6C), as was observed with the FIGURE 7. Homology-based model of mnFcgRIIa complex with human anti-Fab Ab complexes, the M. nemestrina receptors bound IgG IgG1-Fc. (A) Comparison of the FG loop and Trp residues of the “Trp dimers/trimers more strongly than did huFcgRIIb, and, as ex- sandwich” of mnFcgRIIa (cyan wire) or huFcgRIIa (magenta wire) in- pected, the substitution of the mnFcgRIIa sequence HM(131–132) teracting with human IgG1-Fc B chain (yellow wire) or A chain (green wire). (B) Solvent-accessible surface view of huFcgRIIa complex with with the human equivalent RS(131–132) profoundly diminished huIgG1-Fc and (C) solvent-accessible surface view of macaque FcgRIIa binding to levels, which were even lower than observed with complex with huIgG1-Fc. Comparison of (B) and (C) indicates the critical huFcgRIIb. Tyr157 of the FG loop has a reduced contact with huIgG1 in mnFcgRIIa. 157 Molecular modeling of mnFcgRIIa The Tyr residues of huFcgRIIa and mnFcgRIIa are shown in magenta and cyan, respectively. The IgG1-Fc chain A is shown in green, chain B in The human FcgRIIa/IgG interaction has been well characterized yellow, and FcgRIIa molecules are in light gray. Models were prepared by extensive mutagenesis (42, 51, 52), x-ray structure studies of based on the cocrystal structure of huFcgRIIa/huIgG1-Fc (Brookhaven huFcgRIIa alone (50) and in complex with human IgG1-Fc (40), Protein Data Bank ID 3RY6) (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 The FG loop of the second domain of FcgRIIa is one major between macaque and human receptors, we generated a molecular contact between receptor and IgG-Fc where Tyr157, conserved in model of mnFcgRIIa-allele 1 interaction with huIgG1 using the all macaque species and humans, makes critical contacts with x-ray structure of the huFcgRIIa/huIgG1-Fc complex (40). This Leu234 and Gly236 in the lower hinge sequence LLGG(234–237) model (Fig. 7) predicts that key contacts defined in the huFcgRIIa/ on the IgG-Fc B chain (Fig. 7A). Compared to the huFcgRIIa/Fc huIgG1-Fc interface are altered in the interaction of mnFcgRIIa complex, the contacts between the Tyr157 of mnFcgRIIa FG loop and huIgG. and the IgG-Fc are reduced in the modeled interface, from a buried The Journal of Immunology 799 surface area of 100 A˚ 2 to 69 A˚ 2, including the loss of a potential hydrogen bond (Fig. 7B, 7C). The interface contact is further diminished at Trp87 of mnFcgRIIa (68 A˚ 2 buried surface) com- pared with the complexed huFcgRIIa (82 A˚ 2 buried surface), which together with Trp110 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 influ- ence 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 con- tact 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 re- Downloaded from placement 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 replace- http://www.jimmunol.org/ ment of Phe160 with alanine substantially increased binding of both huIgG1 and huIgG2 (50, 52). Conversely, the replacement FIGURE 8. Expression of FcgRII on mononuclear leukocytes from of Phe160 with tyrosine reduced binding of IgG (53); note that humans and M. nemestrina. Mononuclear cells were stained with anti- tyrosine occupies this position in mnFcgRIIa (Fig. 1). Thus, it FcgRIIamAb8.7andpopulationswereidentifiedbycostainingwith anti-CD20 (B cells), anti-CD14 (monocytes), anti-CD159a (NKG2) is likely that the improved binding of huIgG by the mutated (M. nemestrina NK cells), or anti-CD56 (human NK cells). Plots are mnFcgRIIa following our replacement of Pro159–Tyr160 with 159 160 representative of three individual experiments involving four different Leu –Phe of huFcgRIIa results from a more favorable orien- animals or human volunteers. tation of the contact Tyr157 and of the Trp87/Trp110 sandwich in the interaction with human IgG-Fc (Fig. 7). at University of Melbourne Library on February 18, 2014 man IgG1 and IgG2. Although the FcgRIIa of both species binds Another major contact of FcgRIIa in complex with Fc is made IgG3 essentially equivalently, IgG1 and IgG2 binding to mnFcgRIIa by the C9E loop, especially residue 131, which determines the is impaired by comparison with its human ortholog. Remarkably, the functional differences of high/low responder polymorphism of converse is the case for FcgRIIb where it is the human receptor that FcgRIIa in humans. Arg131 is accommodated in a shallow de- exhibits impaired binding of IgG1 relative to mnFcgRIIb, and IgG2 pression in the Fc and is somewhat “crowded,” but the His131 is is not detectably bound. Indeed, mnFcgRIIb avidly binds IgG2 and more readily accommodated (40), and in the mnFcgRIIa-allele 1 thereby has a broader specificity for huIgG subclasses than does and mnFcgRIIb, which also contain His131, this also is likely to be huFcgRIIb. These interspecies differences of IgG binding and the case. In the case of mnFcgRIIa-allele 2, the nearly complete specificity to mnFcgR that we describe in this study were deter- loss of binding caused by the presence of proline in position 131 is mined in the physiological context of the cell surface. Similar likely due to a major alteration of the C9E loop structure leading to results were observed in cell-free surface plasmon resonance the disruption of the interface with IgG. analysis of recombinant ectodomains of cynomolgus FcgR, which Expression of FcgRII in M. nemestrina blood cells showed lower affinity of huIgG1 and huIgG2 for cynomolgus The distribution of FcgRII on leukocyte types has been determined FcgRIIa than huFcgRIIa, and conversely showed increased affinity for rhesus and cynomologus macaques (26, 27, 45), and we ana- lyzed expression in M. nemestrina. Flow cytometry analysis of peripheral blood leukocytes from four macaques confirmed the expression of FcgRII on CD20+ Bcells,CD14+ monocytes (Fig. 8), and platelets (not shown) and, as expected was absent from ma- caque 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 ma- caque 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). FIGURE 9. Macaque neutrophils express elevated levels of FcgRII but Discussion lack FcgRIII. Flow cytometry analysis of blood neutrophils from humans In this study, we demonstrate that human and M. nemestrina or M. nemestrina stained for FcgRII with mAb 8.7 or FcgRIII mAb 3G8. FcgRIIa and FcgRIIb have distinct hierarchies of binding of hu- Plots are representive of three individual experiments. 800 PIG-TAILED MACAQUE FcgR INTERACTION WITH HUMAN IgG for IgG1 and a greatly increased affinity of IgG2 for cynomolgus served in other NHPs (Fig. 11), which raises the possibility FcgRIIb compared with its human ortholog (27). that the diminished huIgG binding may be only a feature of To identify key residues that contribute substantially to the macaque FcgRIIa. observed interspecies IgG binding differences, we exchanged In the case of position 131, which forms a major contact with equivalent residues between the human and macaque FcR receptors huIgG, several, but importantly not all, NHPs also have histidine at in site-directed mutagenesis studies (Figs. 4–6). In short, Pro159 this position in FcgRIIa, which could favor huIgG2 binding. His131 and Tyr160 contribute to the lower activity of the mnFcgRIIa, is also found in FcgRIIa of rhesus (M. mulatta) and cynomologus whereas His131 and Met132 are key to the higher activity of (M. fasicularis) macaques, chimpanzee (Pan troglodytes), bonobo mnFcgRIIb compared with huFcgRIIb. Furthermore, we have (Pan paniscus), baboon (Papio anubis), and squirrel monkey also described the profound influence of polymorphism of mnFcgRIIa (Samiri boliviensis),andthusitislikelythattheyalsobind on IgG binding and identified FcgRIIa as highly polymorphic with huIgG2. Indeed, rhesus and cynomolgus macaque FcgRIIa do eight alleles being detected in only 10 individuals. bind huIgG2 as measured by surface plasmon resonance or The present studies have implications for the structural and func- immune complex binding to cells (27). However, orangutan tional analysis of IgG FcR interactions, not only in M. nemestrina but (Pongo abelii)andmarmoset(Callithrix jacchus)haveTyr131 also in the widely used rhesus and cynomolgus macaques and and Arg131, respectively, and therefore may have altered huIgG Downloaded from other NHPs. A comparative analysis of FcgRIIa and FcgRIIb binding compared with macaque FcgRIIa, especially with re- sequences of other NHPs shows that critical residues in the FG spect to huIgG2. and C9E loops identified in this study as affecting the mnFcgRII Our data also describe the profound influence of polymorphism interaction with huIgG are preserved in some but not all NHPs in mnFcgRIIa on IgG binding, which adds additional complexity (Figs. 10–12). to analysis of huIgG interaction with macacque FcR. In M. nemestrina 159 160 The Pro and Tyr residues of the FG loop that adversely affect and interestingly in rhesus macaque, FcgRIIa is the most polymorphic http://www.jimmunol.org/ IgG binding by mnFcgRIIa are conserved in rhesus (M. mulatta) of the FcgRs (24, 54) (Figs. 1, 2) However, only M. nemestrina (Figs. 10, 11) and cynomolgus (M. fasicularis) macaque species showed sequence variation at position 131 wherein proline in this (Fig. 11) (24, 27, 54), suggesting that these residues could be position in allele 2, also present in allele 5, results in ablation of a key interspecies difference that modulates the engagement IgG1 and IgG2 binding. This is the equivalent position to the clin- of huIgG in FcgRIIa of other macaque species such as cyn- ically relevant high and low responder polymorphism in huFcgRIIa omolgus FcgRIIa (27). Notably PY(159–160) are not con- (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 at University of Melbourne Library on February 18, 2014 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, whichinhuFcgRIIa 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 mar- moset (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 posi- tion, which suggests FcgRIIb of these species may behave more similar to huFcgRIIb with reduced immune complex binding and FIGURE 10. Structural location of human and macaque interspecies little IgG2 binding, making the FcgRIIb of macaques unique in sequence diversity and macaque polymorphism of FcgRIIa. The locations this regard. of polymorphic or nonpolymorphic amino acids are shown on the a carbon In humans, subtle affinity differences have indeed been shown to trace of residues 4–170 of huFcgRIIa. Cyan spheres show the location of be critically important in the respective functions of human nonpolymorphic amino acids that are identical to both M. nemestrina (A) and M. mulatta (B) but differ from huFcgRIIa. The red spheres indicate the FcgRIIa and FcgRIIb (53). Because of the altered specificity of location of residues where polymorphism is unique to one macaque spe- mnFcgRIIb and/or the reversed hierarchy of IgG1 and IgG2 cies. The yellow spheres indicate location of residues where polymorphism binding to mnFcgRIIa and mnFcgRIIb, it is conceivable that Ab has been identified in both M. nemestrina and M. mulatta. Sequence therapeutics, especially IgG2, may not behave in M. nemestina or numbering follows that of Fig. 1. other macaque species as they may be expected to in humans. The The Journal of Immunology 801 Downloaded from

FIGURE 11. Alignment of the extracellular domains of NHPs, including rhesus allelic forms and huFcgRIIa. For additional allelic forms of M. nem- http://www.jimmunol.org/ estrina, 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. increased binding to mnFcgRIIb may obscure potent and desirable history of the infection in humans, for example, Ab-dependent en- effector function or alternatively obscure adverse reactions that hancement (57) in dengue infection or skewing of the FcgRIIa/

may otherwise be manifested in humans where affinity for the FcgRIIb expression ratio in HIV infection (12) or resistance to at University of Melbourne Library on February 18, 2014 inhibitory FcgRIIb is lower. Furthermore, with the success of HIV (13, 14). mAb therapy (1), efforts have been made to alter the potency of Thus, the interspecies and polymorphic differences we describe useful therapeutic mAbs that include specific engineered changes in this study may translate to alterations of Ab-induced inflam- to the IgG-Fc region to optimize the interaction with huFcgRs (21, matory outcomes in vivo in NHPs that are distinct from those in 22, 55). However, the FcgRIIa or FcgRIIb selectivity of such humans. engineered Abs may not necessarily exhibit improved binding to When considering the impact of differences in FcgR binding macaque FcgRs (56). function in vivo in NHPs, it is important to consider whether any Thus, our data highlight that the activities of mAbs designed to differences in cell distribution may also confound the interpreta- alter interactions between human Abs and huFcRs may not be tion of in vivo studies of Ab function. The cell distribution of faithfully recapitulated in preclinical studies in nonhuman pri- FcgR is for the most part similar to humans in M. nemestrina mates, or at least in macaques. (Figs. 8, 9) and in rhesus (26, 45) and cynomolgus macaques (27), Similar caveats may apply to viral pathogenesis studies in with the notable exception of neutrophils where FcgRIII is lacking macaques of human infections where FcgRIIa or FcgRIIb are but FcgRII is elevated. Thus, the implication is that differences in involved in significant clinical or biological aspects of the natural 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). 802 PIG-TAILED MACAQUE FcgR INTERACTION WITH HUMAN IgG not affect the evaluation of mAbs except where effector function is 20. Rascu, A., R. Repp, N. A. Westerdaal, J. R. Kalden, and J. G. van de Winkel. 1997. Clinical relevance of Fcg receptor polymorphisms. Ann. N. Y. Acad. Sci. dependent on the FcgRs of neutrophils. 815: 282–295. Although the macaque is a valuable model of human immune 21. Richards, J. O., S. Karki, G. A. Lazar, H. Chen, W. Dang, and J. R. Desjarlais. function, clear differences exist between species. Cautious inter- 2008. Optimization of antibody binding to FcgRIIa enhances macrophage phagocytosis of tumor cells. Mol. Cancer Ther. 7: 2517–2527. pretation of responses involving Ab-induced responses is prudent. 22. Chu, S. Y., H. M. Horton, E. Pong, I. W. Leung, H. Chen, D. H. Nguyen, Clearly analysis of NHP FcgR genotype and binding profiles of C. Bautista, U. S. Muchhal, M. J. Bernett, G. L. Moore, et al. 2012. Reduction of therapeutic IgG to receptors may be useful in the design of ex- total IgE by targeted coengagement of IgE B-cell receptor and FcgRIIb with Fc- engineered antibody. J. Allergy Clin. Immunol. 129: 1102–1115. perimental or preclinical studies. Thus, the use of NHPs as a model 23. Weng, W.-K., D. Czerwinski, J. Timmerman, F. J. Hsu, and R. Levy. 2004. of human immunity or preclinical model for the evaluation of Ab Clinical outcome of lymphoma patients after idiotype vaccination is correlated with humoral immune response and immunoglobulin G Fc receptor genotype. J. FcR interactions will be greatly assisted by identifying and under- Clin. Oncol. 22: 4717–4724. standing the basis for the differences in interaction of macaque and 24. Nguyen, D. C., F. Scinicariello, and R. Attanasio. 2011. Characterization and huFcR with huIgG. allelic polymorphisms of rhesus macaque (Macaca mulatta) IgG Fc receptor genes. Immunogenetics 63: 351–362. 25. Rogers, K. A., F. Scinicariello, and R. Attanasio. 2004. Identification and charac- Disclosures terization of macaque CD89 (immunoglobulin A Fc receptor). Immunology 113: The authors have no financial conflicts of interest. 178–186. Downloaded from 26. Rogers, K. A., F. Scinicariello, and R. Attanasio. 2006. 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