Polymorphisms and tissue expression of the feline leukocyte antigen class I loci FLAI-E, FLAI-H, and FLAI-K

Abstract

Cytotoxic CD8+ T-cell immunosurveillance for intracellular pathogens, such as viruses, is controlled by classical major histocompatibility complex (MHC) class Ia molecules, and ideally, these antiviral T-cell populations are defined by the specific peptide and restricting MHC allele. Surprisingly, despite the utility of the cat in modeling human viral immunity, little is known about the Feline Leukocyte Antigen class I complex (FLAI). Only a few coding sequences with uncertain locus origin and expression patterns have been reported. Of 19 class I genes, 3 loci - FLAI-E, -H and -K – are predicted to encode classical molecules, and our objective was to evaluate their status by analyzing polymorphisms and tissue expression. Using locus-specific, PCR-based genotyping, we amplified 33 FLAI-E, -H, and -K alleles from 12 cats of various breeds, identifying, for the first time, alleles across 3 distinct loci in a feline species. Alleles shared the expected polymorphic and invariant sites in the α1/α2 domains, and full-length cDNA clones possessed all characteristic class Ia exons. Alleles could be assigned to a specific locus with reasonable confidence, although there was evidence of potentially confounding interlocus recombination between FLAI-E and -K. Only FLAI-E, -H and -K-origin alleles were amplified from cDNAs of multiple tissue types. We also defined hypervariable regions across these genes, which permitted the assignment of names to both novel and established alleles. As predicted, FLAI-E, -H, and -K fulfill the major criteria of class Ia genes. These data represent a necessary prerequisite for studying epitope-specific antiviral CD8+ T-cell responses in cats.

Identification of Novel Alleles and Structural Haplotypes of Major Histocompatibility Complex Class I and DRB Genes in Domestic Cat (Felis catus) by a Newly Developed NGS-Based Genotyping Method

“…Sequencing of the FLA genomic region has revealed 19 FLA-I and 13 FLA-II loci within the 2.98 Mb genomic sequence of accession number EU153401 (Yuhki et al, 2008) (Figure 1). In comparison with the human MHC (human leukocyte antigen; HLA) genomic region, the FLA genomic region has the following features: (1) the 19 FLA-I loci are located in genomic regions corresponding to the HLA-B, HLA-C and HLA-E subregions, (2) three FLA-DRA and five FLA-DRB loci are located in the MHC-DR subregion, but the FLA-DRB5 locus, that is not found in DR haplotype 1, is located in the genomic sequence of another MHC-DR haplotype (DR haplotype 2 in Figure 1), (3) a similar gene organization was observed in the DM/DO subregion between both species, (4) no FLA loci are found in orthologous regions corresponding to the HLA-A, HLA-A/G/F, and HLA-DQ subregions, and (5) FLA-DPA1 and FLA-DPB1 loci are located in the orthologous HLA-DP subregion, but both of them are distinct pseudogenes (Yuhki and O'Brien, 1997;Beck et al, 2001;Yuhki et al, 2008;Holmes et al, 2013) (Figure 1).…”

“…Of the 19 classified FLA-I loci, three (FLA-E, FLA-H, and FLA-K) are classical, nine (FLA-A, FLA-C, FLA-F, FLA-J, FLA-L, FLA-M, FLA-O, FLA-Q, and FLA-S) are non-classical and the other seven loci are pseudogene fragments (Yuhki et al, 2008). The FLA-E, FLA-H, and FLA-K loci were characterized as classical class I genes (class Ia) by variability index plot analysis and detailed gene expression analyses (Holmes et al, 2013). However, for the other FLA loci, the gene expression and structural characteristics such as conservation of coding sequence (CDS) is unknown, although a mRNA sequence was identified that is highly similar to FLA-J (97.2% identity with EU915358).…”

“…Some polymorphism analyses were performed in the FLA-I and FLA-DR loci by a PCR -sequence based typing (PCR-SBT) method using locus specific primer pairs in a sample of 12 cats that resulted with 12 allelic sequences for FLA-E, 11 for FLA-H and 10 for FLA-K (Holmes et al, 2013). On the other hand, 82 DRB allele sequences in a total of 176 cats were identified using the methods of PCR-SBT (Yuhki and O'Brien, 1997;Kennedy et al, 2002), PCR -restriction fragment length polymorphism (PCR-RFLP) (Kuwahara et al, 2000;Kuwahara et al, 2001) and PCR -reference strand-mediated conformation analysis (PCR-RSCA) (Kennedy et al, 2003).…”

Major Histocompatibility Complex Class I (FLA-E*01801) Molecular Structure in Domestic Cats Demonstrates Species-Specific Characteristics in Presenting Viral Antigen Peptides

“…FLA-E*01801 (previously known as FLA-B*n06) has been used to study the immune effect of the FIV vaccine (36). To illustrate the structural basis of FIV peptide presentation by FLA I, we solved the crystal structure of FLA-E*01801 in complex with a nonameric peptide derived from an FIV gag protein (RMA9 [RMANVSTGR]).…”

“…c R ϭ ⌺ hkl || F obs | Ϫ k | Fcalc | |⌺ hkl | F obs |, where R free is calculated for a randomly chosen 5% of reflections and R work is calculated for the remaining 95% of reflections used for structure refinement. with the prospective model of polymorphic and conserved sites in the ␣1/␣2 domains (36). Three hypervariable regions (HVRs) were identified, namely, positions 62 to 81 in the ␣-helix of the ␣1 domain, positions 94 to 116 in the ␣-helix, and positions 94 to 116 in the ␤-strand of the ␣2 domain.…”