Functional analysis of frequently expressed Chinese rhesus macaque MHC class I molecules Mamu-A1*02601 and Mamu-B*08301 reveals HLA-A2 and HLA-A3 supertypic specificities

Abstract

The Simian immunodeficiency virus (SIV)-infected Indian rhesus macaque (Macaca mulatta) is the most established model of HIV infection and AIDS-related research, despite the potential that macaques of Chinese origin is a more relevant model. Ongoing efforts to further characterize the Chinese rhesus macaques' major histocompatibility complex (MHC) for composition and function should facilitate greater utilization of the species. Previous studies have demonstrated that Chinese-origin M. mulatta (Mamu) class I alleles are more polymorphic than their Indian counterparts, perhaps inferring a model more representative of human MHC, human leukocyte antigen (HLA). Furthermore, the Chinese rhesus macaque class I allele Mamu-A1*02201, the most frequent allele thus far identified, has recently been characterized and shown to be an HLA-B7 supertype analog, the most frequent supertype in human populations. In this study, we have characterized two additional alleles expressed with high frequency in Chinese rhesus macaques, Mamu-A1*02601 and Mamu-B*08301. Upon the development of MHC-peptide-binding assays and definition of their associated motifs, we reveal that these Mamu alleles share peptide-binding characteristics with the HLA-A2 and HLA-A3 supertypes, respectively, the next most frequent human supertypes after HLA-B7. These data suggest that Chinese rhesus macaques may indeed be a more representative model of HLA gene diversity and function as compared to the species of Indian origin and therefore a better model for investigating human immune responses.

Fig. 1

The development of the Mamu-A1*02601 and Mamu-B*08301 MHC–peptide-binding assays. The endogenous Mamu-A1*02601 ligand YLPTQQDVL (peptide 3317.02) and HLA-A3 supertype ligand Vaccinia B13R analog KSINKVYGK (peptide 3317.04) were utilized as radiolabeled probes in direct binding dose titration experiments to ascertain binding potential to purified Mamu MHC molecules. a The radiolabeled ligand YLPTQQDVL binds Mamu-A1*02601 (solid circle), but not B*08301 (open triangle). b The radiolabeled ligand KSINKVYGK binds Mamu-B*08301 (solid triangle), but not A1*02601 (open circle). c Inhibition of radiolabeled ligand binding to Mamu-A1*02601 and Mamu-B*08301 by excess unlabeled 3317.02 and 3317.04 ligands in dose titration binding experiments demonstrated specificity of binding

Fig. 2

The relative influence of amino acids at each position on the binding to Mamu-A1*02601. The 9-mer positional scanning combinatorial library was tested for binding to Mamu-A1*02601. Values represent the ratio of the IC₅₀ (nanomolars) of each pool at a given position relative to the geometric mean for the entire library (2,438 nM) and further normalized to the average of libraries tested at each position. Ratios of 0.25 or less are highlighted in orange; >4 are in blue

Fig. 3

Map of the Mamu-A1*02601 motif. Pictorial summary representation of the Mamu-A1*02601 PSCL matrix, indicating residues contributing positive or negative binding potential by position. The total number of residues affecting binding by position is summed, with a double-digit score indicating primary anchor position. Preferred residues at anchor positions, defined as residues showing a 20-fold increase in binding capacity versus the position average, are highlighted in larger font. Positions 2 and the C terminus are identified as main anchor positions

Fig. 4

Mamu-A1*02601 binders tested against HLA-A2 supertype alleles. SIVmac239-derived, Mamu-A1*02601 predicted peptides which had an were tested for binding capacity to HLA-A2 supertype molecules. Affinities highlighted in blue possess an IC₅₀ < 50 nM. Those affinities in the 50–500 nM range are highlighted in green. Dashes indicate binding affinity >20,000 nM

Fig. 5

The relative influence of amino acids at each position on the binding to Mamu-B*08301. The 9-mer positional scanning combinatorial library was tested for binding to Mamu-B*08301. Values represent the ratio of the IC₅₀ (nanomolars) of each pool at a given position relative to the geometric mean for the entire library (518 nM) and further normalized to the average of libraries tested at each position. Ratios of 0.25 or less are highlighted in orange; >4 are in blue

Fig. 6

Map of the Mamu-B*08301 motif. Pictorial summary representation of the Mamu-B*08301 PSCL matrix, indicating residues contributing positive or negative binding potential by position. The total number of residues affecting binding by position is summed, with a double-digit score indicating primary anchor position. Preferred residues at anchor positions, defined as residues showing a 20-fold increase in binding capacity versus the position average, are highlighted in larger font. The C terminus position is identified as the sole main anchor position

Fig. 7

Mamu-B*08301 binders tested against HLA-A3 supertype alleles. SIVmac239-derived, Mamu-B*08301 predicted peptides which had an were tested for binding capacity to HLA-A3 supertype molecules. Affinities highlighted in blue possess an IC₅₀ < 50 nM. Those affinities in the 50–500-nM range are highlighted in green. Dashes indicate binding affinity >20,000 nM

Fig. 8

Phylogenetic tree of HLA and Mamu MHC class I sequences. Phylogenetic analysis of 22 HLA, 14 Indian rhesus, and 14 Chinese rhesus MHC class I sequences is shown. Neighbor-joining tree created based on 1,068 aligned nucleotide sites. The percentage of bootstrap samples supporting the branch is shown (for values >50%). Mamu sequences derived from Indian animals are prepended with In. Sequences identified in Chinese animals (Solomon et al. 2010) are prepended with Ch. The Mamu allele B*08301 demonstrating HLA-A3-like specificity appears in the Mamu-B group and not Mamu-A, indicating this motif specificity between humans and macaques likely did not arise through persistence of a common allele