Cattle NK Cell Heterogeneity and the Influence of MHC Class I

Abstract

Primate and rodent NK cells form highly heterogeneous lymphocyte populations owing to the differential expression of germline-encoded receptors. Many of these receptors are polymorphic and recognize equally polymorphic determinants of MHC class I. This diversity can lead to individuals carrying NK cells with different specificities. Cattle have an unusually diverse repertoire of NK cell receptor genes predicted to encode receptors that recognize MHC class I. To begin to examine whether this genetic diversity leads to a diverse NK cell population, we isolated peripheral NK cells from cattle with different MHC homozygous genotypes. Cytokine stimulation differentially influenced the transcription of five receptors at the cell population level. Using dilution cultures, we found that a further seven receptors were differentially transcribed, including five predicted to recognize MHC class I. Moreover, there was a statistically significant reduction in killer cell lectin-like receptor mRNA expression between cultures with different CD2 phenotypes and from animals with different MHC class I haplotypes. This finding confirms that cattle NK cells are a heterogeneous population and reveals that the receptors creating this diversity are influenced by the MHC. The importance of this heterogeneity will become clear as we learn more about the role of NK cells in cattle disease resistance and vaccination.

FIGURE 1.

Ex vivo NK cells are predominantly CD2^high, and CD8 expression increases upon stimulation with IL-2. PBMCs from 14 MHC class I–defined cattle were assessed for the expression of NCR1, CD2, and CD8 immediately ex vivo (unstimulated, filled bars) or after 7-d in vitro IL-2 stimulation (open bars). (A) The percentage of CD2^high and CD8⁺ cells within the NCR1⁺ population. Statistical significance was determined using a linear mixed model, with expression as the response variable and CD2/CD8 status and stimulated/unstimulated as fixed effects, plus an interaction between them and the animal as a random effect. (B) Overall change in the percentage of CD2^high and CD8⁺ within the NCR1⁺ population after cytokine stimulation. Statistical significance was determined using paired t tests. *p < 0.001.

FIGURE 2.

The variable transcription of NK cell receptor genes in ex vivo NK cells. The transcription of 25 NK cell receptor and associated genes was determined by PCR in NK cells from 14 MHC class I–defined animals (4 A18, 4 A14, and 6 A31). Genes that showed consistent variation are not shown. The dots represent transcription in immediately ex vivo NK cells (black), NK cells stimulated for 7 d in recombinant bovine IL-2 (orange), recombinant bovine IL-12, and recombinant human IL-18 (gray) or recombinant human IL-15 (blue).

FIGURE 3.

Individual dilution cultures have variable CD2 and NCR1 expression. Representative plots from six dilution cultures showing NCR1 and CD2 cell surface expression that was assessed by flow cytometry for each of the 66 limiting dilution cultures. Quadrants were set based on isotype matched control mAbs as described in Materials and Methods, with percentages shown in each quadrant.

FIGURE 4.

A summary of NK receptor transcription patterns from ex vivo, in vitro stimulated, and limiting dilution culture NK cells. Immediately ex vivo (NCR1⁺), NK cells that were stimulated in vitro in IL-2, IL-12, and IL-18 or IL-15 for 7 d (cytokine stimulated), and limiting dilution cultures (dilution cultures) from each animal are shown. Each square displays the percentage of transcription of that gene for that sample.

FIGURE 5.

The frequency of dilution cultures transcribing NK cell receptors correlates with MHC class I genotype (A) and CD2 expression (B). Gene transcription is displayed as a normalized percentage of the total number of cultures within each population. Samples in which expression was mixed and genes were transcribed by all the dilution cultures were omitted. Statistical significance was determined using Fisher’s exact tests. *p < 0.05.

FIGURE 6.

MHC class I haplotype influences the number of KLR receptors transcribed on individual dilution cultures. Dilution cultures were separated by MHC class I haplotype (A14, filled bars, and A18, open bars) and assessed for the total number of transcribed genes (top left) and also transcription of each NK receptor gene group: KIR, KLR, and CKM. The frequency has been calculated based on a normalized percentage of the dilution cultures with statistical significance between MHC class I haplotypes determined by a Fisher exact test. *p < 0.05.