Two Lineages of KLRA with Contrasting Transcription Patterns Have Been Conserved at a Single Locus during Ruminant Speciation

Abstract

Cattle possess the most diverse repertoire of NK cell receptor genes among all mammals studied to date. Killer cell receptor genes encoded within the NK complex and killer cell Ig-like receptor genes encoded within the leukocyte receptor complex have both been expanded and diversified. Our previous studies identified two divergent and polymorphic KLRA alleles within the NK complex in the Holstein-Friesian breed of dairy cattle. By examining a much larger cohort and other ruminant species, we demonstrate the emergence and fixation of two KLRA allele lineages (KLRA*01 and -*02) at a single locus during ruminant speciation. Subsequent recombination events between these allele lineages have increased the frequency of KLRA*02 extracellular domains. KLRA*01 and KLRA*02 transcription levels contrasted in response to cytokine stimulation, whereas homozygous animals consistently transcribed higher levels of KLRA, regardless of the allele lineage. KLRA*02 mRNA levels were also generally higher than KLRA*01 Collectively, these data point toward alternative functional roles governed by KLRA genotype and allele lineage. On a background of high genetic diversity of NK cell receptor genes, this KLRA allele fixation points to fundamental and potentially differential function roles.

FIGURE 1.

SNP diversity over the extended KLRA locus. Enriched genomic data from B. taurus (feral species names and SNPs in gray) and three B. indicus (names underlined and SNPs in white) were mapped to the KLRA locus in the UMD3.1 cattle genome. SNPs are indicated with a dot, and KLRA genotype is given according to the previously established or predicted cDNA sequencing.

FIGURE 2.

Phased KLRA alleles from each animal indicate a common recombination point. (A) Enriched genomic data were phased and broken down by intron and exon for phylogenetic analysis. Assignment to KLRA*01 or KLRA*02 is indicated by a 1 or 2 (shaded), respectively. B. indicus animal names are underlined. Predicted recombination points are boxed with a broken line. (B) Neighbor joining phylogenetic trees drawn using Molecular Evolutionary Genetics Analysis software (39) with 1000 bootstraps. Branch support is shown, and the clear grouping between the KLRA*01 and KLRA*02 can been seen. The 5′ and 3′ trees were constructed separately, excluding exon 2 as this was the predicted recombination point. The KLRA*02 group is shaded, and the recombinant alleles are boxed.

FIGURE 3.

KLRA allele lineage frequency and diversification. (A) The KLRA genotype of a group of British and Canadian bulls was determined by PCR. KLRA*01- and KLRA*02-specific primers were used to establish the lineage frequency within a cohort of 73 animals. The different genotypes in each population of bulls are represented as a percentage of the total number of animals assessed within that population. Black and white bars represent KLRA*01 and KLRA*02 homozygous animals, respectively. Gray bars represent heterozygous animals. (B) KLRA allele lineage diversification was investigated in a group of 10 ruminant species. Intron 4 was amplified by PCR from genomic DNA samples in seven of the species, as indicated, and PCR products were sequenced. The KLRA sequence was acquired from six of the species examined in which full genome sequences were available, as indicated. Intron 4 sequences from all 10 ruminant species were aligned to produce neighbor-joining phylogenetic trees using Molecular Evolutionary Genetics Analysis software with 1000 bootstraps. Branch support is indicated at each node.

FIGURE 4.

KLRA transcription is predominant in NCR1⁺ cells over other lymphocytes, and transcription within NCR1⁺ cells is influenced by different cytokines. KLRA*01 (top) and KLRA*02 (bottom) transcription was assessed in ex vivo (A and B) PBMCs, NCR1^– and NCR1⁺ cells from 14 animals. In addition, ex vivo NCR1⁺ cells were compared with NCR1⁺ cells that had been stimulated for 7 d in vitro with IL-2, IL-12, and IL-18 or IL-15 (C and D). Statistical significance is indicated between samples. Open circles represent homozygous animals, and filled circles represent heterozygous animals. *p < 0.05.

FIGURE 5.

KLRA allele transcription exhibits an allelic dosage effect, which is dependent upon KLRA genotype. Sixty-seven single-cell dilution cultures were generated from six animals: two animals were homozygous for KLRA*01 (252 and 405; white bars), two homozygous for KLRA*02 (375 and 882; black bars), and two were heterozygous for KLRA alleles (598 and 818; gray bars). Dilution cultures were assessed for transcription of KLRA*01 (A) and KLRA*02 (B). Each bar represents a single dilution culture, and samples from individual animals are separated by a dashed line, with KLRA haplotypes separated by a solid line.