Mauritian cynomolgus macaques share two exceptionally common major histocompatibility complex class I alleles that restrict simian immunodeficiency virus-specific CD8+ T cells

Abstract

Vaccines that elicit CD8(+) T-cell responses are routinely tested for immunogenicity in nonhuman primates before advancement to clinical trials. Unfortunately, the magnitude and specificity of vaccine-elicited T-cell responses are variable in currently utilized nonhuman primate populations, owing to heterogeneity in major histocompatibility (MHC) class I genetics. We recently showed that Mauritian cynomolgus macaques (MCM) have unusually simple MHC genetics, with three common haplotypes encoding a shared pair of MHC class IA alleles, Mafa-A*25 and Mafa-A*29. Based on haplotype frequency, we hypothesized that CD8(+) T-cell responses restricted by these MHC class I alleles would be detected in nearly all MCM. We examine here the frequency and functionality of these two alleles, showing that 88% of MCM express Mafa-A*25 and Mafa-A*29 and that animals carrying these alleles mount three newly defined simian immunodeficiency virus-specific CD8(+) T-cell responses. The epitopes recognized by each of these responses accumulated substitutions consistent with immunologic escape, suggesting these responses exert antiviral selective pressure. The demonstration that Mafa-A*25 and Mafa-A*29 restrict CD8(+) T-cell responses that are shared among nearly all MCM indicates that these animals are an advantageous nonhuman primate model for comparing the immunogenicity of vaccines that elicit CD8(+) T-cell responses.

FIG. 1.

Determination of MHC class I restriction. SIV-specific CD8⁺ T-cell lines were cocultured with peptide-pulsed antigen-presenting cells. (A) Gating strategy for measuring activation of CD8⁺ T-cell lines. Activation was measured by intracellular staining of IFN-γ-FITC and TNF-α-PE. (B) Three SIV-specific CD8⁺ T-cell lines were tested against autologous B-lymphoblastoid cells or 721.221 transferents expressing Mafa-A*25, -A*29, or -B*44. MHC class I restriction was confirmed in at least three separate experiments.

FIG. 2.

Determination of optimal CD8⁺ T-cell epitopes. 721.221 transferents expressing the appropriate restricting MHC class I allele were pulsed with serial dilutions of prospective optimal peptides. SIV-specific CD8⁺ T-cell lines were added, and activation was measured by intracellular staining of IFNγ-FITC and TNF-α-PE. CD8⁺ T-cell responses are displayed in terms of the percent maximum response. The actual percentage of CD3⁺ CD8⁺ cells responding to 10³ nM of peptide is listed beneath each peptide. Each condition was performed in triplicate, and optimal epitopes were confirmed in at least two separate experiments.

FIG. 3.

An IFN-γ ELISPOT assays reveals responses in six SIVmac239-infected MCM expressing Mafa-A*25 and -A*29. A total of 10⁵ PBMC were pulsed with 10 μM peptide for 18 to 24 h in a Mabtech monkey IFN-γ ELISPOT plate. Wells were imaged with an AID ELISPOT reader, and spots were counted by an automated system with set parameters for size, intensity, and gradient. Responses were considered positive if the mean number of SFC of triplicate sample wells exceeded the background plus two standard deviations. Responses of <50 SFC per one million PBMC were not considered positive (limit of detection).

FIG. 4.

Viral sequencing reveals variation in six SIVmac239-infected MCM expressing Mafa-A*25 and -A*29. Cell-free plasma was obtained from each animal at 2, 6, 8, 14, and 20 wpi. Viral RNA was isolated, reverse transcribed, amplified, and sequenced. Dashes indicate identity with the wild-type sequence. Complete nucleotide substitutions that changed the resulting amino acid are denoted with uppercase one-letter amino acid abbreviations. Lowercase one-letter amino acid abbreviations indicate positions where mixtures of wild type and a single variant sequence were detected. Numbers indicate positions where mixtures of bases in the viral sequence encode three or more amino acids. WT, wild-type.