The common equine class I molecule Eqca-1*00101 (ELA-A3.1) is characterized by narrow peptide binding and T cell epitope repertoires

Abstract

Here we describe a detailed quantitative peptide-binding motif for the common equine leukocyte antigen (ELA) class I allele Eqca-1*00101, present in roughly 25 % of Thoroughbred horses. We determined a preliminary binding motif by sequencing endogenously bound ligands. Subsequently, a positional scanning combinatorial library (PSCL) was used to further characterize binding specificity and derive a quantitative motif involving aspartic acid in position 2 and hydrophobic residues at the C-terminus. Using this motif, we selected and tested 9- and 10-mer peptides derived from the equine herpesvirus type 1 (EHV-1) proteome for their capacity to bind Eqca-1*00101. PSCL predictions were very efficient, with an receiver operating characteristic (ROC) curve performance of 0.877, and 87 peptides derived from 40 different EHV-1 proteins were identified with affinities of 500 nM or higher. Quantitative analysis revealed that Eqca-1*00101 has a narrow peptide-binding repertoire, in comparison to those of most human, non-human primate, and mouse class I alleles. Peripheral blood mononuclear cells from six EHV-1-infected, or vaccinated but uninfected, Eqca-1*00101-positive horses were used in IFN-γ enzyme-linked immunospot (ELISPOT) assays. When we screened the 87 Eqca-1*00101-binding peptides for T cell reactivity, only one Eqca-1*00101 epitope, derived from the intermediate-early protein ICP4, was identified. Thus, despite its common occurrence in several horse breeds, Eqca-1*00101 is associated with a narrow binding repertoire and a similarly narrow T cell response to an important equine viral pathogen. Intriguingly, these features are shared with other human and macaque major histocompatibility complex (MHC) molecules with a similar specificity for D in position 2 or 3 in their main anchor motif.

Polarisation of equine pregnancy outcome associated with a maternal MHC class I allele: Preliminary evidence

“…The total of 16.7% B2 + (Eqca-1*00101) horses in the current study is comparable with the 13.4% reported by Bergmann et al, (2015).…”

“…Recently, the B2 allele (Eqca-1*00101 using the current nomenclature) has been shown to bind a single 9 residue peptide, namely RDGARFGEL within the immediate early protein (Bergmann et al, 2015). The frequency of the B2 allele is relatively common with estimates ranging from 13.4% to 25% in Thoroughbred horses (Bergmann et al, 2015;Antczak, D.F 1986).…”

“…One EHV-1 CTL target is the immediate early protein (ICP4, encoded by gene 64), which is restricted by the B2 allele expressed on the ELA-A3 haplotype and an unknown allele on ELA-A2 (Kydd et al, 2014). Recently, the B2 allele (Eqca-1*00101 using the current nomenclature) has been shown to bind a single 9 residue peptide, namely RDGARFGEL within the immediate early protein (Bergmann et al, 2015). The frequency of the B2 allele is relatively common with estimates ranging from 13.4% to 25% in Thoroughbred horses (Bergmann et al, 2015;Antczak, D.F 1986).…”

Peptide-binding motifs of two common equine class I MHC molecules in Thoroughbred horses

“…We recently determined a binding motif for the equine MHC class I allele Eqca-1*00101 of the ELA-A3 haplotype using sequencing of endogenous Eqca-1*00101 peptide binders. A more detailed and quantitative motif was further derived by testing the capacity of positional scanning combinatorial libraries (PSCL) to bind Eqca-1*00101 with in vitro assays based on the use of purified MHC molecules (Bergmann et al 2015). …”

“…As detailed previously (Bergmann et al 2015; Sidney et al 2013b), MHC molecules were purified from P815 cell clones transfected with either Eqca-16*00101 or Eqca-1*00201 genes (Azab et al 2014) by immunoaffinity with the equine MHC-I-specific antibody (CZ3) (Rappocciolo et al 2003). Peptides were then eluted from the respective MHC molecules as described previously (Slingluff et al 1993) and analyzed by nanoflow high-performance liquid chromatography (HPLC)/microelectrospray ionization, coupled directly to a Thermo Fisher Scientific Orbitrap Fusion Tribrid mass spectrometer, equipped with a front-end electron transfer dissociation (FETD) source (Earley et al 2013).…”