Key Points
Rhesus FcγR2A allotypes differ in their interactions with different IgG subclasses.
Rhesus FcγR3A allotypes show little variation in IgG subclass interactions.
Human and macaque IgG1 but not IgG2–4 exhibit similar IgG–FcγR interactions.
Abstract
The rhesus macaque is an important animal model for AIDS and other infectious diseases. However, the investigation of Fc-mediated Ab responses in macaques is complicated by species-specific differences in FcγRs and IgG subclasses relative to humans. To assess the effects of these differences on FcγR–IgG interactions, reporter cell lines expressing common allotypes of human and rhesus macaque FcγR2A and FcγR3A were established. FcγR-mediated responses to B cells were measured in the presence of serial dilutions of anti-CD20 Abs with Fc domains corresponding to each of the four subclasses of human and rhesus IgG and with Fc variants of IgG1 that enhance binding to FcγR2A or FcγR3A. All of the FcγRs were functional and preferentially recognized either IgG1 or IgG2. Whereas allotypes of rhesus FcγR2A were identified with responses similar to variants of human FcγR2A with higher (H131) and lower (R131) affinity for IgG, all of the rhesus FcγR3A allotypes exhibited responses most similar to the higher affinity V158 variant of human FcγR3A. Unlike responses to human IgGs, there was little variation in FcγR-mediated responses to different subclasses of rhesus IgG. Phylogenetic comparisons suggest that this reflects limited sequence variation of macaque IgGs as a result of their relatively recent diversification from a common IGHG gene since humans and macaques last shared a common ancestor. These findings reveal species-specific differences in FcγR–IgG interactions with important implications for investigating Ab effector functions in macaques.
Introduction
The FcγRs are a functionally diverse group of cell surface glycoproteins that bind to the Fc domain of IgG Abs. They include activating and inhibitory receptors with a range of affinities for IgG that are expressed by different hematopoietic cell types. Whereas inhibitory FcγRs have regulatory functions, the activating FcγRs mediate effector functions important for defense against infectious diseases and tumors. Upon recognition of Ab–Ag complexes, these receptors trigger cellular immune responses, including phagocytosis, cell-mediated cytotoxicity, and cytokine release (1, 2).
Humans express up to six different FcγRs that belong to three different classes: FcγR1, FcγR2 (FcγR2A, FcγR2B, and FcγR2C), and FcγR3 (FcγR3A and FcγR3B). Of these, the low-affinity activating receptors FcγR2A and FcγR3A are responsible for cell-mediated Ab effector functions. FcγR2A (CD32a) is expressed by phagocytic cells, such as macrophages and neutrophils, and mediates the uptake of Ab-opsonized Ags or cells by Ab-dependent cellular phagocytosis (1). FcγR3A (CD16) is expressed on the surface of NK cells and some macrophages and serves as the principal receptor for Ab-dependent cellular cytotoxicity (ADCC) (1). FcγR2C (CD32c) may also be expressed on NK cells and can contribute to ADCC but is not present in most individuals because of a premature stop codon in the most common allele for this receptor (3, 4).
FcγR2A and FcγR3A differentially recognize four subclasses of human IgG (huIgG) (huIgG1–4). Both of these receptors preferentially bind to IgG1 and IgG3 but generally exhibit weak or negligible interactions with IgG2 and IgG4 (5, 6). However, polymorphisms in FcγR2A and FcγR3A can affect IgG binding. There are two common variants of FcγR2A with either arginine or histidine at position 131 (7). Whereas the H131 polymorphism enhances binding to IgG2, the R131 variant binds poorly to this IgG subclass (5, 8). Interestingly, homozygosity for FcγR2A R131 has been associated with greater susceptibility to bacterial diseases and more rapid CD4+ T cell decline during HIV-1 infection (9–11). There are also two common variants of FcγR3A with either valine or phenylalanine at position 158. Compared with FcγR3A F158, the V158 variant exhibits higher affinity for IgG1 and IgG3 and is associated with greater efficacy of certain tumor immunotherapies (12, 13).
Although macaques express orthologs of FcγR2A and FcγR3A, sequence comparisons have identified polymorphisms that are not found in human FcγRs (huFcγRs) (14–19). Macaques also express four different subclasses of IgG with species-specific differences relative to huIgGs (20, 21). Similar to huIgGs, the macaque IgGs are numbered according to their relative abundance in serum (IgG1 > IgG2 > IgG3 > IgG4); however, they are more similar to one another than to huIgGs and exhibit more limited sequence and structural diversity (20, 22, 23). As a result of these FcγR and IgG differences, it is not possible to predict FcγR–IgG interactions in macaques based on the interactions of these molecules in humans. Thus, one cannot assume that human Abs will have the same FcγR-mediated functions in macaques as they do in humans or that Ab responses elicited in macaques will accurately reflect Ab effector functions in humans.
As macaques have become increasingly important models for the preclinical evaluation of Ab-based vaccines and therapies for HIV, dengue virus, Zika virus, SARS-CoV-2, and other infectious diseases (24–32, S. Lu, Y. Zhao, W. Yu, Y. Yang, J. Gao, J. Wang, D. Kuang, M. Yang, J. Yang, C. Ma et al., manuscript posted on bioRxiv; DOI: 10.1101/2020.04.08.031807), a better understanding of the molecular interactions of macaque FcγRs with human and macaque Abs is needed for investigating Ab-mediated effector functions in these species. Although biophysical studies with recombinant proteins corresponding to the extracellular domains of rhesus macaque FcγRs (rhFcγRs) have revealed differences in affinity for human and macaque IgGs (33, 34), the functional interactions of Abs with these receptors have not been assessed. This is especially important, as the stimulation of responses through low-affinity FcγRs is dependent on the formation of immune complexes with multiple FcγRs, and cellular studies with huFcγRs and IgG subclass variants have revealed functional interactions that are not detectable with soluble, monomeric FcγRs by biophysical methods such as surface plasmon resonance (35). In the current study, we therefore establish reporter cell lines transduced with selected allotypes of human and macaque FcγR2A and FcγR3A that represent the most common variants of these receptors and quantitatively compare responses to different subclasses and Fc variants of huIgG and rhesus macaque IgG (rhIgG).
Materials and Methods
Ethics statement
Indian-origin rhesus macaques (Macaca mulatta) were used in this study. The animals were housed at the Wisconsin National Primate Research Center according to the Association for the Assessment and Accreditation of Laboratory Animal Care and the University of Wisconsin Research Animal Resources Center standards. Animal experiments were approved by the University of Wisconsin Research Animal Resources Center (protocol number G005141) and conducted in compliance with the principles described in the Guide for the Care and Use of Laboratory Animals (36).
Cell lines
CEM.NKR-CCR5 cells (National Institutes of Health AIDS Reagent Program) (37, 38) and Raji cells (American Type Culture Collection) were cultured in RPMI medium supplemented with 10% FBS, l-glutamine, and penicillin/streptomycin (R10 medium). Jurkat NFAT-luciferase (JNL) cells (Signosis) were cultured in R10 medium plus 0.2 mg/ml Hygromycin B (Dot Scientific). GP2-293 cells (Clontech Laboratories) were cultured in DMEM supplemented with 10% FBS, l37, 39, 40).
JNL cells expressing human and rhesus macaque (M. mulatta) FcγRs were established by retroviral transduction. Full-length cDNA sequences for FCGR2A and FCGR3A
FCGR2A or -FCGR3A clones and pVSV-G using GenJet In Vitro DNA Transfection Reagent (Ver. II) (SignaGen Laboratories) according to the manufacturer’s instructions. The cell culture medium was replaced after overnight incubation, and virus-containing supernatant was collected the following day, centrifuged at 1200 × g to remove cell debris, and concentrated using Amicon Ultra-15 Centrifugal Filter Units with a 50-kDa cutoff (MilliporeSigma). JNL cells (5 × 105) were incubated overnight with 300 μl of concentrated viral supernatant and brought to a volume of 1 ml with 40 μg/ml Polybrene (MilliporeSigma). On the following day, the cells were placed under selection in R10 medium containing 0.8 μg/ml puromycin (Invitrogen). After selection for puromycin resistance, FcγR expression was confirmed by flow cytometry. The GenBank accession numbers for the human FCGR sequences are as follows: FCGR2A H131 (NP_001129691), FCGR2A R131 (AAA35932.1), FCGR3A V158 (HQ447137), and FCGR3A F158 (NM_000569). Accession numbers for rhesus FCGR sequences are as follows: FCGR2A:01:01 (MT304962), FCGR2A:02:01 (MT304964), FCGR2A:08:01 (MT304968), FCGR2A:10:01 (MT304970), FCGR2A:11:01 (MT304972), FCGR2A:12:01 (MT304974), FCGR2A:13:01 (MT304976), FCGR3A:01:01 (MT305008), FCGR3A:01:02 (MT305010), FCGR3A:02:01 (MT305014), and FCGR3A:03:01 (MT305018).mAbs used
For anti-CD20 Abs, cDNA sequences for the variable heavy and variable light domains of rituximab were cloned into separate pCEP4 (Invitrogen) constructs in-frame with expression-optimized sequences for the constant domains of human and rhesus macaque (IgGH1–4), and the corresponding IgG κ L chains (IgGκ) of each species, respectively. Human IgHG1–4 and rhesus macaque IgHG3-4 cDNAs were synthesized (Integrated DNA Technologies). Rhesus macaque IgHG1 and IgHG2 cDNAs were provided by Dr. M. Farzan (Scripps Research, Jupiter, FL). The S239D, I332E, and A330L (DEL) substitutions were introduced into IgGH1 during cDNA synthesis, and the G236A substitution was added to IgGH1 constructs by oligonucleotide-directed PCR mutagenesis. The GenBank accession numbers for Ab sequences are as follows: rituximab variable heavy (AX556949), rituximab variable light (AX556951), huIgG1H (AX556949), huIgG2H (AAG00910.2), huIgG3H (P01860.2), huIgG4H (BC025985.1), huIgGκ (AX556951), rhIgG1H (AAQ57550.1), rhIgG2H (AAQ57565.1), rhIgG3H (ATV90898.1), rhIgG4H (MT310723), and rhIgGκ (ACN96964.1).
mAbs were produced by cotransfecting Expi293 cells (9 × 107
3 for storage and centrifuged prior to use.FCGR genotyping
Rhesus macaques were genotyped for Mamu-FCGR2A and Mamu-FCGR3A transcripts as described previously (1818). After normalization and pooling, SMRTbell libraries were prepared and sequenced on a PacBio Sequel instrument at the University of Wisconsin-Madison Biotechnology Center according to manufacturer’s protocols (Pacific Biosciences). Raw PacBio sequence datasets were processed using SMRT Link v6.0.0 tools (https://www.pacbio.com/support/software-downloads) as described recently by Shortreed et al. (41). The resulting circular consensus sequences from each animal were mapped against a reference database of all known rhesus macaque FCGR2A and FCGR3A sequences using Geneious Prime 2020.0.4 software tools (Biomatters).
Flow cytometry
FcγR staining.
CD20 staining.
Raji cells were washed with PBS and stained with LIVE/DEAD Fixable Aqua ARC. After 20 min, cells were washed in FACS buffer and stained for 30 min with anti-CD20 IgG (5 μg/ml) followed by an Alexa Fluor 647–conjugated goat anti-huIgG polyclonal Ab. As a control for nonspecific staining, cells were also stained with the goat anti-huIgG secondary Ab without the anti-CD20 primary Ab. Samples were washed with FACS buffer and fixed in 2% paraformaldehyde. The MFI of CD20 staining was measured after gating on the live singlet cell population and divided by the secondary-only control to measure the fold-change in MFI. Data were collected using a BD LSR II SORP instrument and analyzed with FlowJo 9.9 software.
JNL reporter cell assay
FcγR interactions with IgGs were measured by the dose-dependent upregulation of luciferase by JNL cells expressing FcγRs in response to Ab-opsonized target cells over a range of IgG concentrations. FcγR-transduced JNL cells (2 × 105) were coincubated with Raji cells (1 × 105) in the presence of serial dilutions of anti-CD20 IgG Abs. JNL cells were incubated overnight or for 4 h with Raji cells in triplicate wells at each Ab concentration (200 μl/well final volume) in opaque white, flat-bottom, 96-well plates (Corning). At the end of the incubation, 50 μl of britelite plus luciferase substrate (PerkinElmer) was added to each well, and luminescence was measured using a VICTOR ×4 multiplate reader (PerkinElmer). Ramos cells (American Type Culture Collection), which do not express FcγR2B, were also tested for Ab-mediated recognition by FcγR-transduced JNL cells. The different IgG subclasses and Fc domain variants of human and rhesus macaque IgG were run in parallel for each experiment.
ADCC assays
37) or macaque PBMCs (2 × 105) were coincubated with CAM-labeled Raji or CD20+ CEM-NKR.CCR5 cells (1 × 104) in the presence of serial dilutions of anti-CD20 IgG. The cells were incubated for 4 h in triplicate wells at each Ab concentration (200 μl/well final volume) in round-bottom, 96-well plates. Wells with only CAM-labeled target cells served as controls for background CAM release, and wells treated with 1% NP-40 (Sigma-Aldrich) indicated maximum CAM release. After 4 h, the plates were centrifuged (500 × g), and 100 μl of supernatant was transferred from each well to corresponding wells of black, clear-bottom, 96-well plates. The release of CAM into the supernatant was measured using a VICTOR ×4 multiplate reader (excitation 485 nm and absorbance 535 nm). Percentage-specific lysis was calculated as (experimental release − spontaneous release)/(maximum release − spontaneous release) × 100.
4) (Phylogenetic analysis of IGHG sequences
Chimpanzee IGHG sequences were located using the National Center for Biotechnology Information Genome Data Viewer, and exons with high similarity to human IGHG sequences were extracted. The loci identifiers for the chimpanzee IGHG sequences are as follows: IgHG1 (LOC749366), IgHG2 (LOC453229), IgHG3 (LOC749390), and IgHG4 (LOC749354). The accession numbers for baboon, pigtail macaque, and mouse IGHG sequences are as follows: baboon IgHG1 (AY125048), baboon IgHG2 (AY125049), baboon IgHG3 (AY125050), baboon IgHG4 (AY125051), pigtail macaque IgHG1 (JQ868732), pigtail macaque IgHG2 (JQ868733), pigtail macaque IgHG3 (JQ868734), pigtail macaque IgHG4 (JQ868735), mouse IgG1 (LT160966), and mouse IgG2a (AB097847). Human and rhesus macaque IGHG accession numbers are provided above. The accession numbers for baboon, pigtail macaque, chimpanzee, and mouse FcγR sequences are as follows: baboon FcγR1 (XP_009178311.1), baboon FcγR2A (XM_021926122.2), baboon FcγR3A (NM_001112647.1), pigtail macaque FcγR1 (XP_011735031.1), pigtail macaque FcγR2A (KF234400), pigtail macaque FcγR3A (XM_011770191.2), chimpanzee FcγR1 (XP_009428355.1), chimpanzee FcγR2A (NM_001009077.1), chimpanzee FcγR3A (XM_024349652.1), and mouse FcγR2B (NM_001077189.1). Human and rhesus macaque FcγR1 accession numbers are P12314 and AFD32558.1, respectively. Human and rhesus macaque FcγR2A and FcγR3A accession numbers are provided above, using the most frequent rhesus alleles. Amino acid sequences were aligned using Geneious and subjected to a substitution model test using Molecular Evolutionary Genetics Analysis X. A phylogenetic tree was constructed with Molecular Evolutionary Genetics Analysis X, using the maximum likelihood method and Jones–Taylor–Thornton substitution model with 1000 bootstrap replicates. Nodes with bootstrap values <0.5 were collapsed using TreeGraph (version 2.15.0-887 β).
Statistical analysis
Graphing and statistical analysis were performed using GraphPad Prism (version 8.2). Ab concentrations for half-maximal responses (EC50) were calculated from sigmoidal curves fitted to mean relative light units data from triplicate wells at each Ab dilution. Mean EC50 values were calculated from at least three independent experiments and were compared using a two-tailed, unpaired Student t test.
Results
rhFcγR2A and rhFcγR3A polymorphisms
Analysis of full-length FCGR cDNA sequences from 17 Indian-origin rhesus macaques identified seven FCGR2A and four FCGR3A allelic variants. Table I provides the relative frequencies of these FCGR sequences in an expanded cohort of 155 rhesus macaques from the Wisconsin National Primate Research Center. The predicted amino acid sequences of these rhesus macaque alleles are aligned with huFcγR2A and huFcγR3A in Fig. 1. In addition to species-specific differences relative to huFcγR2A, polymorphisms in rhFcγR2A were apparent at multiple positions, including residues predicted to contact IgG (Fig. 1A). Whereas five of the seven allotypes of rhFcγR2A have a histidine at position 131, which corresponds to the H131 polymorphism in huFcγR2A that enhances binding to IgG2, two of the rhFcγR2A variants (rhFcγR2A:11 and rhFcγR2A:12) have a proline at this position (P131) (Fig. 1A). Along with these species-specific polymorphisms, all allotypes of rhFcγR2A have a potential N-linked glycosylation site (PNG) at position 135. Two allotypes, rhFcγR2A:01 and rhFcγR2A:13, have an additional PNG at position 128 (Fig. 1A).
Amino acid sequence alignments of human and rhesus macaque FcγR2A (A) and FcγR3A (B) allotypes. Predicted IgG contact sites based on crystal structures of IgG in complex with human FcγR2A and FcγR3A are in shaded gray (55, 56). Amino acid positions are indicated with dots for identity, dashes for deletions, and single-letter amino acid codes for differences.
rhFcγR3A allotypes have fewer amino acid differences relative to huFcγR3A and exhibit considerably less polymorphism than rhFcγR2A. The three most common allotypes of rhFcγR3A differ from huFcγR3A by 20–21 aa, including three positions predicted to contact IgG (Fig. 1B). The only extracellular domain polymorphism among these variants is a conservative isoleucine versus valine difference at position 158, which is at the same position as the V158F polymorphism in huFcγR3A. This residue is an isoleucine (I158) in rhFcγR3A:01 and rhFcγR3A:02 and a valine (V158) in rhFcγR3A:03 (Fig. 1B). Additional polymorphisms previously implicated in the efficiency of B cell depletion with an anti-CD20 Ab were observed in the transmembrane and cytoplasmic domains of rhFcγR3A:01 (M211 and I215) (Fig. 1B) (42).
FcγR2A responses to human and rhesus macaque IgG subclasses and Fc variants
To assess FcγR2A interactions with IgG, reporter cell lines were established by transducing JNL cells with different alleles of human and rhesus macaque FCGR2A. These alleles encoded the H131 and R131 variants of huFcγR2A and five of the most common allotypes of rhFcγR2A (rhFcγR2A:01, rhFcγR2A:02, rhFcγR2A:08, rhFcγR2A:10, and rhFcγR2A:11), which represent 89% of the haplotypes of FCGR2A-genotyped animals (Supplemental Fig. 1A, Table I). rIgGs were also produced with identical variable regions from the anti-CD20 Ab rituximab but different H chain constant domains corresponding to each of the four subclasses of huIgG and rhIgG (huIgG1–4 and rhuIgG1–4) (Supplemental Fig. 2A–D). Additional variants of IgG1 were produced with Fc domain substitutions that selectively enhance binding to FcγR2A (G236A) or FcγR3A (DEL: S239D, I332E, and A330L) (43–45). The purity of these Abs was verified by SDS-PAGE, and similar binding to CD20 on the surface of Raji cells was confirmed by flow cytometry (Supplemental Fig. 2E, 2F). FcγR2A-transduced JNL cells were incubated overnight with Raji cells in the presence of serial dilutions of anti-CD20 Abs, and FcγR ligation was measured by the dose-dependent upregulation of luciferase over a 6-log range of Ab concentrations (Fig. 2A). Ab concentrations for half-maximal responses, or EC50, were compared to determine the relative strength of FcγR–IgG interactions (Table II). Raji cells maintained consistently high levels of CD20 expression during passage in culture, as reflected by similar CD20 staining on three different occasions separated by at least 3-wk intervals (Supplemental Fig. 1C).
FcγR2A-mediated responses. (A) JNL cells expressing individual allotypes of human and rhesus macaque FcγR2A were incubated overnight with Raji cells at a 2:1 E:T ratio in the presence of serial dilutions of anti-CD20 Abs with Fc domains corresponding to each of the four subclasses of human and rhesus macaque IgG and variants of IgG1 that enhance binding to FcγR2A (G236A) or FcγR3A (DEL). Curves were fitted to the data using GraphPad Prism software. Error bars indicate SD of the mean for triplicate measurements at each Ab concentration. (B) Ab concentrations for half-maximal responses (EC50) were calculated from dose response curves. EC50 values that could not be determined within the range of Ab concentrations tested are not shown. Error bars indicate SD of the mean for at least three independent experiments.
As expected, JNL cells expressing huFcγR2A exhibited the strongest responses to huIgG1, and these interactions were significantly enhanced by the G236A substitution (Fig. 2B). The p values for statistical comparisons are shown in Table III. Moreover, consistent with previous observations (5, 8, 46–48), JNL cells expressing huFcγR2A H131 responded significantly better to huIgG2 than the R131 variant of this receptor (Fig. 2B, Table III). Weaker huFcγR2A H131–mediated responses to huIgG4 are also consistent with previous observations (5). However, the responses to huIgG3 were surprisingly weak (Fig. 2A) (5, 34). The reason for this difference is presently unclear because the purity and integrity of huIgG3 was verified by SDS-PAGE (Supplemental Fig. 2F) and this Ab stimulated FcγR3A responses in subsequent experiments (Fig. 3A). These results suggest context-dependent differences in the activity of huIgG3 that may be related to the greater flexibility afforded by the extended hinge region of this Ab.
FcγR3A-mediated responses. (A) JNL cells expressing individual allotypes of human and rhesus macaque FcγR3A were incubated overnight with Raji cells at a 2:1 E:T ratio in the presence of serial dilutions of anti-CD20 Abs with Fc domains corresponding to each of the four subclasses of human and rhesus macaque IgG and variants of IgG1 that enhance binding to FcγR2A (G236A) or FcγR3A (DEL). Curves were fitted to the data using GraphPad Prism software. Error bars indicate SD of the mean for triplicate measurements at each Ab concentration. (B) Ab concentrations for half-maximal responses (EC50) were calculated from dose response curves. EC50 values that could not be determined within the range of Ab concentrations tested are not shown. Error bars indicate SD of the mean for at least three independent experiments.
Consistent with the presence of histidine at position 131 in four of the five allotypes of rhFcγR2A (rhFcγR2A:01, rhFcγR2A:02, rhFcγR2A:08, and rhFcγR2A:10), these receptors exhibited similar responses to huIgG1 and huIgG2 as huFcγR2A H131 (Fig. 2A). With the exception of rhFcγR2A:10, which preferentially recognized huIgG2, these rhFcγR2A variants responded more efficiently to huIgG1 than huIgG2 (Fig. 2B, Table II). All of the rhFcγR2A variants also recognized huIgG4 (Fig. 2B). The G236A substitution had a more modest effect on JNL cells bearing rhFcγR2A than huFcγR2A. Although the G236A substitution reduced the Ab concentration for half-maximal responses for all five allotypes of rhFcγR2A compared with wild-type IgG1 (Fig. 2B, Table II), this difference was only significant for rhFcγR2A:02 (Table III). These observations are in accordance with a recent study showing that the G236A substitution does not increase huIgG1 binding to rhFcγR2A to the same extent as its human counterpart (34). However, contrary to binding studies with rFcγRs, which suggest that the N128 and P131 variants of rhFcγR2A are functionally impaired (33), JNL cells expressing receptors with these polymorphisms respond to all four subclasses of huIgG. As reflected by their EC50 values, rhFcγR2A:01 (N128) and rhFcγR2A:11 (P131) exhibited the strongest and the weakest responses to huIgG1, respectively (Fig. 2B, Table II).
JNL cells expressing rhFcγR2A responded better to rhIgGs than huIgGs and exhibited less variation among subclasses. The pattern of rhIgG recognition by different allotypes of rhFcγR2A also bore a striking resemblance to the pattern of rhIgG recognition by each of the huFcγR2A variants. Similar to huFcγR2A H131, all four allotypes of rhFcγR2A with the H131 polymorphism (rhFcγR2A:01, rhFcγR2A:02, rhFcγR2A:08, and rhFcγR2A:10) responded most efficiently to rhIgG2 (Fig. 2B). These rhFcγR2A variants recognized rhIgG1 and rhIgG4 at similarly low EC50 values (Fig. 2B, Table II). Conversely, the P131 variant of rhFcγR2A (rhFcγR2A:11) responded poorly to rhIgG2, but recognized rhIgG3 as efficiently as rhIgG1 and rhIgG4, which mirrors the recognition of rhIgGs by huFcγR2A R131 (Fig. 2B). Whereas the G236A substitution had a modest effect on rhFcγR2A interactions with huIgG1, the introduction of this substitution into rhIgG1 significantly enhanced interactions with rhFcγRIIa:11 (P131), rhFcγRIIa:02, and huFcγR2A R131 (Table III). This substitution may therefore be used in the context of rhIgG1 to increase interactions with rhFcγR2A. Collectively, these results reveal functional parallels between allotypes of rhFcγR2A and huFcγR2A and indicate that these receptors are less sensitive to differences among rhIgG subclasses than huIgG subclasses.
FcγR3A responses to human and rhesus macaque IgG subclasses and Fc variants
The responses of JNL cells expressing different allotypes of human and rhesus macaque FcγR3A were also tested. These included the V158 and F158 variants of huFcγR3A and the three most common variants of rhFcγR3A (rhFcγR3A:01, rhFcγR3A:02, and rhFcγR3A:03), which represent 98% of the haplotypes of FCGR3A-genotyped animals. Stable JNL cell lines expressing similar levels of these receptors were established (Supplemental Fig. 1B) and incubated with Raji cells in the presence of serial dilutions of anti-CD20 Abs corresponding to each of the human and rhesus macaque IgG subclasses and Fc domain variants (Fig. 3A, Supplemental Fig. 2). As expected, both huFcγR3A variants preferentially responded to huIgG1, and these interactions were significantly enhanced by the DEL substitutions designed to increase binding to huFcγR3A (Fig. 3B). The p values for statistical comparisons are shown in Table IV. The EC50 of the huIgG1 response was also lower for huFcγR3A V158 than for huFcγR3A F158, consistent with the higher IgG1 binding affinity of the V158 variant. Weaker responses to huIgG3 were observed for both receptors, which differs from studies reporting better interactions for IgG3 than IgG1 (5, 6) but is consistent with cytolysis assays done with primary human PBMCs (49–51). As expected, huFcγR3A interactions with huIgG2 and huIgG4 were negligible (Fig. 3B).
JNL cells expressing rhFcγR3A variants exhibited a similar pattern of responses to the huIgGs as huFcγR3A V158. All three allotypes efficiently recognized huIgG1 and mediated measurable responses to huIgG3 and huIgG4 but did not respond to huIgG2 (Fig. 3). However, the DEL substitutions did not significantly enhance the already strong interactions of huIgG1 with these receptors (Fig. 3B, Table IV). Each of the rhFcγR3A variants also responded to huIgG4 better than either of the huFcγR3A variants, suggesting that the stronger cross-species recognition of human Abs of this subclass may trigger FcγR-mediated responses in macaques that are not observed in humans.
JNL cells bearing rhFcγR3A exhibited a similar pattern of responses to the rhIgGs as huFcγR3A V158 but with surprisingly little variation among subclasses. Similar to their responses to huIgGs, all three rhFcγR3A variants responded most efficiently to rhIgG1 (Fig. 3B). Although the DEL substitutions did not improve these interactions to the same extent as they did for huIgG1 (Table II), these substitutions appeared to have a greater effect after a shorter 4-h incubation than after an overnight incubation (Supplemental Fig. 3). Unlike responses to huIgGs, the rhFcγR3A variants also mediated strong responses to rhIgG2, rhIgG3, and rhIgG4 (Fig. 3). The EC50 values of the responses to rhIgG2–4 were in a similar range and ∼2–3-fold higher than the EC50 values for rhIgG1 (Table II). The similarity of responses for rhFcγR3A:02 (I158) and rhFcγR3A:03 (V158), which only differ by a single amino acid at position 158, are consistent with data indicating that this polymorphism does not significantly impact IgG binding (33). The similarity of responses for rhFcγR3A:01 and rhFcγR3A:02, which differ by one residue in the transmembrane domain and one residue in the cytoplasmic domain, also suggests that these polymorphisms do not have a significant effect on signaling. Hence, these results reveal an overall lack of variability in rhFcγR3A-mediated responses to human or rhIgG subclasses, with each allotype exhibiting patterns of IgG recognition most similar to the high-affinity V158 variant of huFcγR3A. Moreover, in accordance with the limited structural diversity among rhIgGs (16, 23), there was little variation in rhFcγR3A-mediated responses to different subclasses of rhIgG.
FcγR3A-mediated responses correlate with ADCC
To further corroborate FcγR3A-mediated responses, we took advantage of a previously established human NK cell line expressing rhFcγR3A (37). KHYG-1 cells transduced with rhFcγR3A:02 were incubated with CAM-labeled Raji cells in the presence of serial dilutions of anti-CD20 Abs, and ADCC was measured as the dose-dependent release of CAM into the culture supernatant (Fig. 4A). The ADCC responses of this NK cell line mirrored luciferase induction from rhFcγR3A:02+ JNL cells. As observed for rhFcγR3A+ JNL cells, ADCC responses were strongest for human and rhesus macaque IgG1 and were further enhanced (significantly so in the case of rhIgG1) by the DEL substitutions (Fig. 4A, Table IV). ADCC responses were also in a similar range for rhIgG2, rhIgG3, and rhIgG4, and weaker responses were detectable for huIgG3 and huIgG4 (Table II). Comparison of the Ab concentrations for half-maximal ADCC and JNL cell activation confirmed a strong correlation between these measures of rhFcγR3A-mediated responses (Fig. 4B).
Correlation of rhFcγR3-mediated ADCC and JNL cell responses. (A) An NK cell line expressing rhFcγR3A:02 was incubated with CAM-labeled Raji cells for 4 h at a 5:1 E:T ratio in the presence of serial dilutions of anti-CD20 Abs. Percentage-specific lysis was calculated from the release of CAM into the supernatant as measured using a VICTOR ×4 multiplate reader (excitation 485 nm and absorbance 535 nm). Error bars indicate SD of the mean for triplicate measurements at each Ab concentration. (B) Mean EC50 values for ADCC responses were calculated from three independent experiments (Table II) and were compared with mean EC50 values for rhFcγR3A:02+ JNL cell responses by two-tailed Pearson correlation.
ADCC assays with primary rhesus macaque PBMC yielded similar results. For these experiments, the activation of rhFcγR3A:02+ JNL cells and the ADCC activity of unstimulated PBMCs from two different macaques were measured against a CD20-transduced CEM.NKR-CCR5 cell line (Fig. 5A, 5B). PBMCs were incubated overnight in plastic flasks to deplete monocytes and other adherent cells, and CD20+ CEM.NKR-CCR5 cells were used as targets because Raji cells are highly susceptible to nonspecific lysis by macaque NK cells in the absence of Abs (52). Area under the curve values were compared because Ab concentrations for 50% ADCC could not be determined in all cases. Although more variable than rhFcγR3A:02+ KHYG-1 cells, ADCC responses measured with PBMCs from two different animals correlated well with rhFcγR3A-mediated JNL cell responses, as indicated by the inverse relationship between ADCC and EC50 values for luciferase induction (Fig. 5C). Among the IgG subclasses, huIgG1 and rhIgG1 consistently resulted in the higher ADCC responses, which were further enhanced by the DEL substitutions (Fig. 5B). Lower ADCC responses were also measurable for rhIgG2, rhIgG3, and rhIgG4 but were negligible for huIgG2 and huIgG4 (Fig. 5B). These results demonstrate a good correspondence between rhFcγR3A-mediated responses of JNL cells and primary macaque NK cells.
Correlation of ADCC by rhesus macaque PBMC and rhFcγR3-mediated JNL cell responses. (A) JNL cells expressing rhFcγR3A:02 were incubated for 6 h with CD20-transduced CEM.NKR-CCR5 at a 1:1 E:T ratio with serial dilutions of anti-CD20 Abs, and luciferase activity was measured using a VICTOR ×4 multiplate reader. (B) Unstimulated PBMCs from two different rhesus macaques (r14094 and r15023) were incubated for 4 h with CAM-labeled CD20+ CEM.NKR-CCR5 cells at a 20:1 E:T ratio in the presence of anti-CD20 Abs. Percentage-specific lysis was calculated from the amount of CAM released in the supernatant, and error bars indicate SD of the mean for triplicate measurements at each Ab concentration. (C) The relationship between the mean rhFcγR3A:02+ JNL cell responses (EC50) from two independent experiments and the mean area under the curve (AUC) values for ADCC responses was determined by two-tailed Pearson correlation.
Responses to an FcγR2B-negative B cell line
Raji cells express FcγR2B, which could interfere with IgG ligation of FcγRs on JNL cells by binding to anti-CD20 Abs in cis (53). To determine whether the presence of FcγR2B on Raji cells interferes with the availability of Abs for the stimulation of FcγR2A and FcγR3A on JNL cells, we compared FcγR-mediated JNL cell responses to Raji cells with responses to Ramos cells, which are another CD20+ B cell line that does not express FcγR2B (53, 54). These experiments were performed with representative alleles of human and rhesus macaque FcγR2A and FcγR3A and each of the IgG subclasses and Fc variants. Although Ramos cells and Raji cells express similar levels of CD20 (Supplemental Fig. 4A), the Ramos cells resulted in a lower magnitude of luciferase induction, possibly reflecting their smaller size and lower surface area for FcγR ligation. Nevertheless, with the exception of slightly higher responses for huIgG3 using Ramos cells, the overall hierarchy and pattern of responses to Raji cells and Ramos cells were very similar (Supplemental Fig. 4B, 4C). This suggests that the presence of FcγR2B on Raji cells did not have a significant impact on the availability of Abs for the stimulation of FcγRs on JNL cells.
Phylogenetic comparisons of the IgG subclasses of hominids and Old World monkeys
The capacity of each of the allotypes of rhFcγR2A and FcγR3A to recognize all four subclasses of rhIgG, albeit with variable efficiency, contrasts with preferential FcγR recognition of huIgG subclasses. To investigate the evolutionary relationships between human and rhesus macaque IgG subclasses, we performed a phylogenetic analysis that included sequences corresponding to the IgG1–4 H chain constant regions of two hominid species (humans and chimpanzees) and three species of Old World monkeys (baboons, pig-tailed macaques, and rhesus macaques) (Fig. 6A). For comparison, we performed a similar analysis of the FcγR1, FcγR2A, and FcγR3A sequences of these species (Fig. 6B).
Phylogenetic analysis of hominid and Old World monkey IgGs and FcγRs. Phylogenetic trees were generated from the amino acid sequences of each of the IgG subclasses (IgG1–4) (A) and three FcγRs (FcγR1, FcγR2A, and FcγR3A) (B) of humans, chimpanzees, baboons, pig-tailed macaques, and rhesus macaques. Trees were constructed with using the maximum likelihood method and Jones–Taylor–Thornton substitution model with 1000 bootstrap replicates. Trees were rooted on murine IgHG1, IgGh2a, and FcγR2B sequences, and nodes with bootstrap values <0.5 were collapsed using TreeGraph software. The scale bars indicate amino acid substitutions per site.
In contrast to the FcγRs, which cluster according to their respective gene products across all five primate species (Fig. 6B), the IgG subclasses of hominids and Old World monkeys segregate into distinct lineages (Fig. 6A). Whereas the human and chimpanzee IgG sequences cluster together according to subclass, reflecting an orthologous relationship among hominid species, the IgG sequences of macaques and baboons form separate branches (Fig. 6A). On one branch, the macaque and baboon IgG1 sequences cluster together with the IgG2 sequences of these species, and on another, the IgG3 and IgG4 sequences of these species intermingle (Fig. 6A). This suggests that, unlike the FcγRs, the IgGs of macaques and baboons are products of gene duplication events that occurred after the divergence of apes and Old World monkeys. Thus, the relatively recent origin of the Old World monkey IgGs explains their limited sequence diversity and lack of orthology with huIgG subclasses.
Discussion
Macaques have become increasingly important animal models for the preclinical evaluation of Ab-based vaccines and therapies for HIV/AIDS and for other infectious diseases. However, the investigation of Fc-mediated Ab responses in macaques is complicated by species-specific differences and polymorphisms in macaque FcγRs and IgG subclasses with respect to their human counterparts. To investigate the effects of these differences on FcγR–IgG interactions, we established JNL reporter cell lines expressing common allotypes of human and rhesus macaque FcγR2A and FcγR3A. We focused on FcγR2A and FcγR3A because these are the receptors responsible for Ab-dependent cellular phagocytosis and ADCC effector functions. Five allotypes of rhFcγR2A and three allotypes of rhFcγR3A were selected, which represent ∼89 and 98% of the alleles for these receptors among FCGR-genotyped animals. FcγR-mediated responses were measured by incubating the JNL reporter cells with Raji cells in the presence of serial dilutions of anti-CD20 Abs with Fc domains corresponding to each of the four subclasses of human and rhesus macaque IgG or with Fc variants of IgG1 that selectively enhance FcγR binding. Our results show that all the allotypes of rhFcγR2A and rhFcγR3A tested are functional and preferentially recognize either IgG1 or IgG2. However, in contrast to the selective recognition of huIgG subclasses, which is a hallmark of FcγR-mediated effector functions in humans, the variability of responses to different rhIgG subclasses was more limited.
Of the rhFcγRs, FcγR2A is the most polymorphic and exhibited the greatest diversity of responses. Consistent with the presence of histidine at position 131, four allotypes of rhFcγR2A (rhFcγR2A:01, rhFcγR2A:02, rhFcγR2A:08, and rhFcγR2A:11) exhibited patterns of IgG recognition similar to huFcγR2A H131. Among the huIgGs, these responses were characterized by efficient recognition of huIgG1 followed by huIgG2, and among the rhIgGs, the responses showed preferential recognition of rhIgG2, efficient recognition of rhIgG1 and rhIgG4, and weaker interactions with rhIgG3. Conversely, rhFcγR2A:11, which has a proline at position 131, exhibited interactions that were more similar to huFcγR2A R131. Although the responses of rhFcγR2A:11 to human and rhesus macaque IgG2 were weaker than the other rhFcγR2A allotypes, responses to rhIgG3 were comparable to rhIgG1. These results suggest that macaques have high- and low-affinity allotypes of FcγR2A that are functionally analogous to huFcγR2A H131 and huFcγR2A R131. Whereas the majority of rhFcγR2A allotypes have the H131 polymorphism and exhibit a pattern of IgG recognition similar to huFcγR2A H131, rhFcγR2A:11 has a P131 polymorphism and exhibits responses more similar to huFcγR2A R131.
These results differ from a previous biophysical characterization of these receptors, which did not detect consistent IgG binding to the N128 and P131 variants of rhFcγR2A (33). In contrast to these experiments, responses to all four subclasses of rhIgG were detectable for rhFcγR2A:01 (N128) and rhFcγR2A:11 (P131). The responses of rhFcγR2A:01 to rhIgG1, rhIgG2, and rhIgG4 were somewhat stronger than the other H131 variants of rhFcγR2A, indicating that the additional PNG of this receptor does not functionally impair interactions with IgG. In the case of rhFcγR2A:11, responses to rhIgG1 and rhIgG2 were weaker than the other allotypes. However, this receptor still mediated measurable responses to all four rhIgGs, including stronger responses to rhIgG3. Hence, our results complement previous binding studies to show that N128 and P131 variants of rhFcγR2A are functional but differ in their recognition of rhIgG subclasses.
Comparatively little variation in IgG recognition was observed among different allotypes of rhFcγR3A. Despite a number of species-specific differences with respect to huFcγR3A, the majority of rhFcγR3A allotypes only differ by a single amino acid in their extracellular domains (I158V) or a pair of residues in their transmembrane and cytoplasmic domains (V211M and I215V). Consistent with biophysical studies (33, 34), I158 and V158 variants of rhFcγR3A (rhFcγR3A:02 and rhFcγR3A:03) exhibited nearly identical responses to each of the subclasses of human and rhesus macaque IgG. Likewise, the same pattern of responses was observed for rhFcγR3A:01, which only differs from rhFcγR3A:02 at positions 211 and 215. The responses mediated by each of the rhFcγR3A allotypes were most similar to the high-affinity V158 variant of huFcγR3, as reflected by preferential recognition of huIgG1, weaker interactions with huIgG3 and huIgG4, and negligible responses to huIgG2. These observations support studies indicating that all allotypes of rhFcγR3A characterized thus far bind IgG1 with high affinity comparable to huFcγR3A V158 and that rhesus macaques lack a low-affinity variant of this receptor analogous to huFcγR3A F158 (16, 34). With the caveat that our assays were performed with human cell lines, the similarity of responses for rhFcγR3A:01 and rhFcγR3A:02 also suggests that polymorphisms in the transmembrane and cytoplasmic domains of these receptors, previously implicated in the efficiency of B cell depletion with rituximab (42), do not have a significant impact on rhFcγR3A-mediated responses.
Compared with FcγR recognition of huIgGs, there was remarkably little variation in FcγR-mediated responses to different subclasses of rhIgG. This was particularly evident for allotypes of rhFcγR3A, which efficiently recognized all four subclasses of rhIgG within a narrow 3-fold range of EC50 values. These observations are consistent with sequence comparisons that indicate different subclasses of rhIgG are more similar to one another than to any of the huIgGs and with recent crystal structures revealing less structural diversity among the Fc domains of rhIgGs (22, 23). To better understand the evolutionary relationships between human and rhesus macaque IgGs underlying these differences in Fc diversity, we performed a phylogenetic comparison of the IgG1–4 H chain constant regions of hominids and Old World monkeys. This analysis suggests that macaque IgGs are products of gene duplication events that occurred after the divergence of apes and Old World monkeys. Thus, macaque and huIgG subclasses are not orthologous, and the limited structural and functional diversity of rhIgGs can be explained by their recent divergence from a common IGHG gene since humans and macaques last shared a common ancestor.
The effects of Fc domain substitutions designed to selectively enhance IgG1 binding to huFcγR2A and FcγR3A are context dependent. The G236A substitution significantly enhanced huFcγR2A-mediated responses to huIgG1, as expected (43, 44). This substitution also enhanced rhFcγR2A-mediated responses to both huIgG1 and rhIgG1. However, in accordance with biophysical measurements (34), G236A consistently resulted in greater increases in rhFcγR2A responses in the context of rhIgG1 than huIgG1. Similar differences were observed in the effects of the DEL substitutions on FcγR3A recognition of human versus rhesus macaque IgG1. Whereas these substitutions significantly enhanced huFcγR3A-mediated responses to huIgG1, the effects of these changes on rhFcγR3A-mediated responses in the context of either huIgG1 or rhIgG1 were more modest. In this case, the smaller effects of the DEL changes on rhFcγR3A recognition may reflect incremental enhancement of already strong IgG interactions with these receptors.
Our findings are generally consistent with studies characterizing IgG binding to macaque FcγRs and have important implications regarding the use of rhesus macaques to model Fc-mediated Ab responses. Although rhesus macaques express orthologs of FcγR2A with high and low affinity for IgG, similar to the H131 and R131 variants of huFcγR2A, and have orthologs of FcγR3A with high affinity for IgG similar to the huFcγR3A V158, the IgG subclasses of macaques are not orthologous to huIgGs and exhibit considerably less diversity in FcγR recognition. Whereas huIgG1 and rhIgG1, which are the most abundant subclasses in their respective species, are efficiently recognized by human and rhesus macaque FcγRs, these interactions differ for other IgG subclasses. Unlike huIgG2–4, rhIgG2–4 are generally well recognized by rhFcγR2A and rhFcγR3A. Although there were some differences in responses to rhIgG2 and rhIgG3 for different allotypes of rhFcγR2A, the responses of the rhFcγR3A allotypes to rhIgGs were remarkably similar. This implies that rhIgG2–4 may contribute to FcγR-mediated effector functions in macaques to a greater extent than huIgG2–4 do in humans.
These differences in FcγR recognition of human versus macaque IgGs are fundamental to the interpretation of studies in macaques involving passive Ab transfer or vaccine-induced Ab responses. Because all allotypes of rhFcγR2A and rhFcγR3A recognize huIgG1 and rhIgG1 with a similar efficiency as their corresponding human receptors, human and rhesus macaque IgG1 are expected to have similar effector functions. The passive transfer of huIgG1 or rhIgG1 to rhesus macaques may therefore be useful for assessing FcγR-mediated functions of certain Abs in humans. Likewise, vaccine-induced IgG1 responses in macaques may have similar effector functions as IgG1 responses in humans.
However, the other subclasses of rhIgG do not follow the same patterns of FcγR recognition as huIgGs. In accordance with the close phylogenetic relationship between rhIgG1 and rhIgG2, rhIgG2 was recognized at least as efficiently as rhIgG1 by four of the five allotypes of rhFcγR2A and nearly as efficiently as rhIgG1 by all three allotypes of rhFcγR3A. rhIgG4 was also recognized as efficiently as rhIgG1 by all allotypes of rhFcγR2A and with similar efficiency by each of the allotypes of rhFcγR3A. In the case of rhIgG3, interactions with rhFcγR2A were variable but detectable for all allotypes, and interactions with rhFcγR3A were similar to the other rhIgG subclasses. In some cases, huIgGs were recognized better by rhFcγRs than huFcγRs. Thus, passive transfer experiments in which human or rhesus macaque IgG2, IgG3, or IgG4 are administered to macaques should be interpreted with caution because rhIgG2–4 may direct rhFcγR2A- and rhFcγR3A-mediated responses with a similar potency as rhIgG1, and huIgG2–4 may trigger stronger responses than would normally occur in humans. Likewise, rhIgG2–4 responses elicited by vaccination are not expected to follow the same paradigms of FcγR recognition as huIgG2–4 and may contribute more broadly to Ab effector functions.
The incubation of FcγR-transduced JNL cells together with anti-CD20 Abs and CD20+ target cells provides a simple, robust, and reproducible system to compare human and rhesus macaque FcγR–IgG interactions under the same conditions. Nevertheless, this approach has some limitations. Because of variation in signaling as result of species-specific differences and polymorphisms in the cytoplasmic domains of the FcγRs and/or epigenetic differences in luciferase expression among independently isolated cell lines, it is difficult to make quantitative comparisons between JNL cells expressing different FcγRs. For this reason, we have avoided quantitative comparisons between cell lines, focusing instead on comparisons of EC50 values for different Abs measured with the same cells and qualitative comparisons of the overall hierarchies and patterns of responses among different cell lines. Additionally, certain features of rituximab Ab binding to CD20 on Raji cells may not translate to other Ab–Ag combinations. For instance, the orientation of Ab binding may affect the accessibility of the Fc domain for FcγR ligation. Smaller targets, such as viral particles, may also stimulate FcγRs differently than Ags expressed on the cell surface. Indeed, the size of immune complexes has been shown to differentially affect functional interactions between huFcγRs and IgG subclass and Fc variants in cellular assays (35).
In conclusion, this study provides a quantitative comparison of the responses of the most common allotypes of rhFcγR2A and rhFcγR3A to each of the four subclasses of human and rhesus macaque IgG. To our knowledge, this is the first functional characterization of individual allotypes of rhFcγRs. Our findings reveal parallels and key differences between the FcγR–IgG interactions of humans and macaques that are important for investigating FcγR-mediated effector functions of Abs in the rhesus macaque.
Disclosures
The authors have no financial conflicts of interest.
Footnotes
This work was supported by National Institutes of Health Grants AI121135, AI095098, AI148379, and AI098485 (to D.T.E.); RR021745 (to D.H.O.); OD011106 (to the Wisconsin National Primate Research Center); and AI155163. D.T.E. is an Elizabeth Glaser Scientist of the Elizabeth Glaser Pediatric AIDS Foundation.
The sequences presented in this article have been submitted to GenBank under accession numbers MT304962, MT304964, MT304968, MT304970, MT304972, MT304974, MT304976, MT305008, MT305010, MT305014, and MT305018.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- ADCC
- Ab-dependent cellular cytotoxicity
- ARC
- amine-reactive compound
- CAM
- calcein acetoxymethyl ester
- DEL
- S239D, I332E, and A330L
- EC50
- half-maximal response
- huFcγR
- human FcγR
- huIgG
- human IgG
- IgGκ
- IgG κ L chain
- JNL
- Jurkat NFAT-luciferase
- MFI
- mean fluorescence intensity
- PNG
- potential N-linked glycosylation site
- rhFcγR
- rhesus macaque FcγR
- rhIgG
- rhesus macaque IgG.
- Received May 4, 2020.
- Accepted October 17, 2020.
- Copyright © 2020 by The American Association of Immunologists, Inc.