Characterization of Mauritian Cynomolgus Macaque FcγR Alleles Using Long-Read Sequencing

Abstract

The FcγRs are immune cell surface proteins that bind IgG and facilitate cytokine production, phagocytosis, and Ab-dependent, cell-mediated cytotoxicity. FcγRs play a critical role in immunity; variation in these genes is implicated in autoimmunity and other diseases. Cynomolgus macaques are an excellent animal model for many human diseases, and Mauritian cynomolgus macaques (MCMs) are particularly useful because of their restricted genetic diversity. Previous studies of MCM immune gene diversity have focused on the MHC and killer cell Ig-like receptor. In this study, we characterize FcγR diversity in 48 MCMs using PacBio long-read sequencing to identify novel alleles of each of the four expressed MCM FcγR genes. We also developed a high-throughput FcγR genotyping assay, which we used to determine allele frequencies and identify FcγR haplotypes in more than 500 additional MCMs. We found three alleles for FcγR1A, seven each for FcγR2A and FcγR2B, and four for FcγR3A; these segregate into eight haplotypes. We also assessed whether different FcγR alleles confer different Ab-binding affinities by surface plasmon resonance and found minimal difference in binding affinities across alleles for a panel of wild type and Fc-engineered human IgG. This work suggests that although MCMs may not fully represent the diversity of FcγR responses in humans, they may offer highly reproducible results for mAb therapy and toxicity studies.

Figure 1

Macaque FCGR family. Due to the proximity of FCGR2A, 3A, and 2B, linkage disequilibrium occurs; FCGR1A is located further away and is not frequently inherited with the others. Locations are based on the Mafa5 reference genome.

Figure 2

FCGR polymorphisms by allele. Every possible nucleotide variant for each region is listed, and the presence or absence of that variant for each allele is noted. If the variant is present and causes a change in an amino acid, it is indicated; otherwise, if it is present but does not change the amino acid at that position, “syn” is used to indicate a synonymous variant. Nucleotides are numbered from the start of the leader peptide sequence using the longest isoform. (A) FCGR1A polymorphisms; longest isoform is X02. (B) FCGR2A polymorphisms; longest isoform is X02. (C) FCGR2B polymorphisms; longest isoform is X01. (D) FCGR3A polymorphisms; longest isoform is X01.

Figure 3

Summary of the alleles, and the proteins they form, comprising each FCGR haplotype. “Rec” indicates recombinant haplotypes.

Figure 4

FCGR protein binding to fourteen different IgG isotypes, with two different monoclonal antibodies per isotype, was tested by surface plasmon resonance assay. SPR screening data showing percentage of theoretical Rmax is shown for (A) FCGR2A, (B) FCGR2B, and (C) FCGR3A. The sequences used to generate the 2A:01 protein were the same as the 2A:06 protein; likewise for 2A:04 and 2A:05. (D) KD_apparent values are shown for representative IgG isotypes binding to select FCGR2A proteins, obtained from 2-fold serially diluted IgG titrations on captured FCGR surfaces, fit to a 1:1 steady-state model.