A Single Amino Acid Difference within the α-2 Domain of Two Naturally Occurring Equine MHC Class I Molecules Alters the Recognition of Gag and Rev Epitopes by Equine Infectious Anemia Virus-Specific CTL

Abstract

Although CTL are critical for control of lentiviruses, including equine infectious anemia virus, relatively little is known regarding the MHC class I molecules that present important epitopes to equine infectious anemia virus-specific CTL. The equine class I molecule 7-6 is associated with the equine leukocyte Ag (ELA)-A1 haplotype and presents the Env-RW12 and Gag-GW12 CTL epitopes. Some ELA-A1 target cells present both epitopes, whereas others are not recognized by Gag-GW12-specific CTL, suggesting that the ELA-A1 haplotype comprises functionally distinct alleles. The Rev-QW11 CTL epitope is also ELA-A1-restricted, but the molecule that presents Rev-QW11 is unknown. To determine whether functionally distinct class I molecules present ELA-A1-restricted CTL epitopes, we sequenced and expressed MHC class I genes from three ELA-A1 horses. Two horses had the 7-6 allele, which when expressed, presented Env-RW12, Gag-GW12, and Rev-QW11 to CTL. The other horse had a distinct allele, designated 141, encoding a molecule that differed from 7-6 by a single amino acid within the α-2 domain. This substitution did not affect recognition of Env-RW12, but resulted in more efficient recognition of Rev-QW11. Significantly, CTL recognition of Gag-GW12 was abrogated, despite Gag-GW12 binding to 141. Molecular modeling suggested that conformational changes in the 141/Gag-GW12 complex led to a loss of TCR recognition. These results confirmed that the ELA-A1 haplotype is comprised of functionally distinct alleles, and demonstrated for the first time that naturally occurring MHC class I molecules that vary by only a single amino acid can result in significantly different patterns of epitope recognition by lentivirus-specific CTL.

Cloning and large-scale expansion of epitope-specific equine cytotoxic T lymphocytes using an anti-equine CD3 monoclonal antibody and human recombinant IL-2

“…CTL assays were performed as described (21,25). Briefly, direct CTL activity of cloned CTL was measured using a 51 Cr release assay with a 17-h incubation period using autologous equine kidney (EK) target cells.…”

“…PBMC were isolated from horse A2150 blood (20,25) and initially stimulated in vitro with the Rev-QW11 peptide (20) at a concentration of 2 μM, which was added to PBMC in 10% FBS. Peptide and PBMC were incubated for 2 hours at 37°C with occasional mixing before centrifugation at 250 × g for 10 min.…”

“…Several factors contribute to the protective effects of CTL against EIAV, including epitope specificity and functional avidity (15,20,21,23,24). Importantly, individual EIAV-specific CTL epitopes can be presented by more than one MHC class I molecule, but subtle changes in class I molecules can enhance, diminish, or abolish recognition of these epitopes by CTL (25).…”

“…Because the ELA-A1 haplotype is well-represented in our research herd of Arabian horses that carry the severe combined immunodeficiency (SCID) trait (33)(34)(35)(36), Rev-QW11-specific CTL clones would be excellent candidates for evaluating protective effects by adoptive transfer to SCID foals (19,37). In addition, this clone would be valuable for investigating the differential recognition efficiencies of Rev-QW11 when presented by the 7-6 and 141 class I molecules (25). The cloning procedure utilized an anti-equine CD3 mAb (38,39) and human recombinant IL-2 (rhuIL-2), which stimulates equine lymphocytes (15,19,22,40), such that autologous APC and specific antigen were not required during expansion in culture.…”

“…We chose to clone CTL specific for the conserved EIAV Rev-QW11 epitope, which is recognized by high avidity CTL (20), and is presented by the equine leukocyte antigen (ELA)-A1 haplotype-associated MHC class I molecules 7-6 and 141 (17,25). Because the ELA-A1 haplotype is well-represented in our research herd of Arabian horses that carry the severe combined immunodeficiency (SCID) trait (33)(34)(35)(36), Rev-QW11-specific CTL clones would be excellent candidates for evaluating protective effects by adoptive transfer to SCID foals (19,37).…”