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

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.

Comparative MHC nomenclature: report from the ISAG/IUIS-VIC committee 2018.

Significant progress has been made over the last decade in defining major histocompatibility complex (MHC) diversity at the nucleotide, allele, haplotype, diplotype, and population levels in many non-human species. Much of this progress has been driven by the increased availability and reduced costs associated with nucleotide sequencing technologies. This report provides an update on the activities of the comparative MHC nomenclature committee which is a standing committee of both the International Society for Animal Genetics (ISAG) and the International Union of Immunological Societies (IUIS) where it operates under the umbrella of the Veterinary Immunology Committee (VIC). A previous report from this committee in 2006 defined the role of the committee in providing guidance in the development of a standardized nomenclature for genes and alleles at MHC loci in non-human species. It described the establishment of the Immuno Polymorphism Database, IPD-MHC, which continues to provide public access to high quality MHC sequence data across a range of species. In this report, guidelines for the continued development of a universal MHC nomenclature framework are described, summarizing the continued development of each species section within the IPD-MHC project.

Cattle NK Cell Heterogeneity and the Influence of MHC Class I.

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.

Definition of the cattle killer cell Ig-like receptor gene family: comparison with aurochs and human counterparts.

Under selection pressure from pathogens, variable NK cell receptors that recognize polymorphic MHC class I evolved convergently in different species of placental mammal. Unexpectedly, diversified killer cell Ig-like receptors (KIRs) are shared by simian primates, including humans, and cattle, but not by other species. Whereas much is known of human KIR genetics and genomics, knowledge of cattle KIR is limited to nine cDNA sequences. To facilitate comparison of the cattle and human KIR gene families, we determined the genomic location, structure, and sequence of two cattle KIR haplotypes and defined KIR sequences of aurochs, the extinct wild ancestor of domestic cattle. Larger than its human counterpart, the cattle KIR locus evolved through successive duplications of a block containing ancestral KIR3DL and KIR3DX genes that existed before placental mammals. Comparison of two cattle KIR haplotypes and aurochs KIR show the KIR are polymorphic and the gene organization and content appear conserved. Of 18 genes, 8 are functional and 10 were inactivated by point mutation. Selective inactivation of KIR3DL and activating receptor genes leaves a functional cohort of one inhibitory KIR3DL, one activating KIR3DX, and six inhibitory KIR3DX. Functional KIR diversity evolved from KIR3DX in cattle and from KIR3DL in simian primates. Although independently evolved, cattle and human KIR gene families share important function-related properties, indicating that cattle KIR are NK cell receptors for cattle MHC class I. Combinations of KIR and MHC class I are the major genetic factors associated with human disease and merit investigation in cattle.

The functional significance of cattle major histocompatibility complex class I genetic diversity.

Current concerns about food security highlight the importance of maintaining productive and disease-resistant livestock populations. Major histocompatibility complex (MHC) class I genes have a central role in immunity. A high level of diversity in these genes allows populations to survive despite exposure to rapidly evolving pathogens. This review aims to describe the key features of MHC class I genetic diversity in cattle and to discuss their role in disease resistance. Discussion centers on data derived from the cattle genome sequence and studies addressing MHC class I gene expression and function. The impact of intensive selection on MHC diversity is also considered. A high level of complexity in MHC class I genes and functionally related gene families is revealed. This highlights the need for increased efforts to determine key genetic components that govern cattle immune responses to disease, which is increasingly important in the face of changing human and environmental demands.

Constraints on haplotype structure and variable gene frequencies suggest a functional hierarchy within cattle MHC class I.

Six major histocompatibility complex (MHC) classical class I genes have been identified in cattle, and up to three of these are expressed in variable combinations on different haplotypes. The origin and functional significance of this genetic complexity is unknown. However, an improved assembly of the cattle genome, an expanded database of full-length cDNA sequences and high-resolution frequency data concerning expressed class I genes in an economically important cattle breed combine to provide a new opportunity to study the significance of cattle MHC class I diversity. Analysis of these new data supports assignment of alleles to six discrete genes and further shows that all these classical genes share a common ancestor with a single non-classical gene, NC1. While haplotype structure is variable, with thirteen gene configurations identified, there are nevertheless clear constraints relating to both the number and combination of genes. Haplotypes expressing two classical genes are most frequently observed, and the classical class I gene 2 is almost invariably present. The frequency data support the dominance of gene 2, showing that close to 100 % of individuals carry at least one copy. This indicates a hierarchy in the functional importance of particular genes and haplotype structures. Haplotype frequency in cattle populations is therefore likely to impact on differential disease susceptibilities. This knowledge will be important for development of informed breeding strategies aimed at increasing the ability of cattle to survive in the face of future unpredictable pathogen exposure.

Cattle MHC nomenclature: is it possible to assign sequences to discrete class I genes?

The cattle major histocompatibility complex (MHC) region contains a variable number of classical class I genes encoding polymorphic molecules involved in antigen presentation. Six classical class I genes have been described, but assigning sequences to these genes has proved problematic. We propose a refinement of the existing nomenclature, which currently names the 97 known classical class I sequences in a single series. Phylogenetic analysis of the 3' portion of the coding region allows segregation of these into six groups; thus, we have prefixed existing names with the appropriate number. Although it is clear that some of these groups correspond to discrete genes, it is currently not possible to state definitively that all do. However, the main groupings are consistent, and in conjunction with other evidence, we feel it is now appropriate to rename the sequences accordingly. Segregation of sequences into groups in this way will facilitate ongoing research and future use of the cattle MHC section of the Immuno Polymorphism Database.

The impact of MHC diversity on cattle T cell responses.

Major histocompatibility complex (MHC) genes play a key role in immunity to infectious pathogens. Their high level of diversity is a functionally important characteristic. In cattle our knowledge of MHC diversity and the functional distinction between genes is limited. Recent studies in commercially important dairy cattle populations reveal that MHC class I diversity is relatively low, although it does not appear to be declining. The presence and frequency of some genes and alleles was markedly different between geographically distinct populations, and trait selection was implicated as an influential force. Functional studies suggest that some alleles may have a disproportionally high impact on T cell responses, thus it may be important to consider their role in both disease resistance and vaccine efficacy. It is clear that increasing our knowledge of the functional capabilities of different cattle MHC class I genes is essential to maintain healthy populations in the future.

MHC class I bound to an immunodominant Theileria parva epitope demonstrates unconventional presentation to T cell receptors.

T cell receptor (TCR) recognition of peptide-MHC class I (pMHC) complexes is a crucial event in the adaptive immune response to pathogens. Peptide epitopes often display a strong dominance hierarchy, resulting in focusing of the response on a limited number of the most dominant epitopes. Such T cell responses may be additionally restricted by particular MHC alleles in preference to others. We have studied this poorly understood phenomenon using Theileria parva, a protozoan parasite that causes an often fatal lymphoproliferative disease in cattle. Despite its antigenic complexity, CD8+ T cell responses induced by infection with the parasite show profound immunodominance, as exemplified by the Tp1(214-224) epitope presented by the common and functionally important MHC class I allele N*01301. We present a high-resolution crystal structure of this pMHC complex, demonstrating that the peptide is presented in a distinctive raised conformation. Functional studies using CD8+ T cell clones show that this impacts significantly on TCR recognition. The unconventional structure is generated by a hydrophobic ridge within the MHC peptide binding groove, found in a set of cattle MHC alleles. Extremely rare in all other species, this feature is seen in a small group of mouse MHC class I molecules. The data generated in this analysis contribute to our understanding of the structural basis for T cell-dependent immune responses, providing insight into what determines a highly immunogenic p-MHC complex, and hence can be of value in prediction of antigenic epitopes and vaccine design.

Induction of a cross-reactive CD8(+) T cell response following foot-and-mouth disease virus vaccination.

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. Current inactivated FMDV vaccines generate short-term, serotype-specific protection, mainly through neutralizing antibody. An improved understanding of the mechanisms of protective immunity would aid design of more effective vaccines. We have previously reported the presence of virus-specific CD8(+) T cells in FMDV-vaccinated and -infected cattle. In the current study, we aimed to identify CD8(+) T cell epitopes in FMDV recognized by cattle vaccinated with inactivated FMDV serotype O. Analysis of gamma interferon (IFN-γ)-producing CD8(+) T cells responding to stimulation with FMDV-derived peptides revealed one putative CD8(+) T cell epitope present within the structural protein P1D, comprising residues 795 to 803 of FMDV serotype O UKG/2001. The restricting major histocompatibility complex (MHC) class I allele was N*02201, expressed by the A31 haplotype. This epitope induced IFN-γ release, proliferation, and target cell killing by αß CD8(+) T cells, but not CD4(+) T cells. A protein alignment of representative samples from each of the 7 FMDV serotypes showed that the putative epitope is highly conserved. CD8(+) T cells from FMDV serotype O-vaccinated A31(+) cattle recognized antigen-presenting cells (APCs) loaded with peptides derived from all 7 FMDV serotypes, suggesting that CD8(+) T cells recognizing the defined epitope are cross-reactive to equivalent peptides derived from all of the other FMDV serotypes.

Cattle MIC is a ligand for the activating NK cell receptor NKG2D.

Major histocompatibility complex (MHC) class I chain-related (MIC) genes encode molecules that are expressed in response to stress, signalling immune system cells primarily via the activating receptor NKG2D. We investigated the expression of receptors for MIC in lymphocyte subsets found in peripheral blood, lymph node and gut in cattle and demonstrated their presence on natural killer (NK) cells, gammadelta T cells and CD8(+) T cells. Recognition of MIC by NKG2D was formally demonstrated using recombinant protein and an ELISA. Staining with a cross-reactive monoclonal antibody recognising both human and cattle MIC showed that MIC is constitutively expressed within cattle intestinal epithelium. A functional response to soluble MIC was observed in receptor-bearing cells in blood, lymph node and gut, the latter requiring relatively high levels of MIC to trigger a response. Results suggest that NKG2D is a functionally important activating receptor in cattle.

Regulation of a bovine nonclassical major histocompatibility complex class I gene promoter.

Studies have shown in humans and other species that the major histocompatibility complex class I (MHC-I) region is involved at a number of levels in the establishment and maintenance of pregnancy. The aim of this study was to characterize how a bovine nonclassical MHC-I gene (NC1) is regulated. Initial serial deletion experiments of a 2-kb fragment of the NC1 promoter identified regions with positive regulatory elements in the proximal promoter and evidence for a silencer module(s) further upstream that cooperatively contributed to constitutive NC1 expression. The cytokines interferon tau (IFNT), interferon gamma (IFNG), and interleukin 4 (IL4) significantly increased luciferase expression in NC1 promoter reporter constructs and endogenous NC1 mRNA levels in a bovine endometrial cell line. In addition, IFNG, IL3, IL4, and progesterone significantly increased Day 7 bovine blastocyst NC1 mRNA expression when supplemented during in vitro embryo culture. Site-directed mutagenesis analysis identified a STAT6 binding site that conferred IL4 responsiveness in the NC1 proximal promoter. Furthermore, methylation treatment of the proximal promoter, which contains a CpG island, completely abrogated constitutive NC1 expression. Overall, the findings presented here suggest that constitutive NC1 expression is regulated positively by elements in the proximal promoter, which are further controlled by upstream silencer modules. The promoter is responsive to IFNT, IFNG, and IL4, suggesting possible roles for these cytokines in bovine preimplantation embryo survival and/or maternal-fetal tolerance. Our studies also suggest that methylation of the proximal promoter, in particular, could play a significant role in regulating NC1 expression.

Cattle Ly49 is polymorphic.

Cattle are the only non-primate species to have an expanded KIR gene family. In addition they have a single Ly49 gene, thought to be monomorphic. We have identified two additional Ly49 cDNA sequences in cattle that encode molecules predicted to differ by 14 and 16 amino acids from the original sequence. All available data indicate the presence of only one Ly49 gene in cattle, thus the divergent sequences reported here may represent ancient allelic lineages, a high level of polymorphism in an ancestral gene or a previously expanded and subsequently contracted Ly49 gene family. Cattle are the only species known to have an expanded polymorphic KIR gene family and a polymorphic Ly49 gene.

Classical and non-classical Major Histocompatibility Complex class I gene expression in in vitro derived bovine embryos.

The role of the Major Histocompatibility Complex class I (MHC-I) genes in human and mouse preimplantation embryo development has received considerable attention. In contrast, information concerning the role of these genes in bovine embryo development is limited. The objective of the current study was to characterize the expression pattern of MHC-I genes during preimplantation embryo development in cattle. To this end, bovine oocytes were harvested from slaughterhouse ovaries, matured, fertilized and cultured in vitro. Samples were collected at immature and mature oocyte, presumptive zygote, 2-4-cell embryo, 8-16-cell embryo, morula, blastocyst and hatched blastocyst stages of development. MHC-I expression was detected using quantitative real-time-PCR, cDNA sequencing, whole mount immunocytochemistry and Western blotting. We report classical and non-classical MHC-I mRNA expression in bovine oocytes and developing embryos. Furthermore, we report that the pattern of MHC-I mRNA expression across development was gene- and stage-specific.

Bovine papillomavirus type 1 oncoprotein E5 inhibits equine MHC class I and interacts with equine MHC I heavy chain.

Bovine papillomavirus type 1 is one of the aetiological agents of equine sarcoids. The viral major oncoprotein E5 is expressed in virtually all sarcoids, sarcoid cell lines and in vitro-transformed equine fibroblasts. To ascertain whether E5 behaves in equine cells as it does in bovine cells, we introduced the E5 open reading frame into fetal equine fibroblasts (EqPalF). As observed in primary bovine fibroblasts (BoPalF), E5 by itself could not immortalize EqPalF and an immortalizing gene, such as human telomerase (hTERT/hT), was required for the cells to survive selection. The EqPalF-hT-1E5 cells were morphologically transformed, elongated with many pseudopodia and capable of forming foci. Equine major histocompatibility complex class I (MHC I) was inhibited in these cells at least at two levels: transcription of MHC I heavy chain was inhibited and the MHC I complex was retained in the Golgi apparatus and prevented from reaching the cell surface. We conclude that, as in bovine cells and tumours, E5 is a player in the transformation of equine cells and the induction of sarcoids, and a potential major cause of MHC I downregulation and hence poor immune clearance of tumour cells.

CD8+ T-cell responses to Theileria parva are preferentially directed to a single dominant antigen: Implications for parasite strain-specific immunity.

Although immunodominance of CD8(+) T-cell responses is a well-recognised feature of viral infections, its role in responses to more antigenically complex pathogens is less clear. In previous studies we have observed that CD8(+) T-cell responses to Theileria parva exhibit different patterns of parasite strain specificity in cattle of different MHC genotypes. In the current study, we demonstrated that animals homozygous for the A10 and A18 MHC haplotypes have detectable responses to only one of 5 T. parva antigens. Over 60% of the responding T cells from the A18(+) and A10(+) animals recognised defined epitopes in the Tp1 and Tp2 antigens, respectively. Comparison of T-cell receptor beta chain expression profiles of CD8(+) T-cell lines and CD8(+) T cells harvested ex vivo confirmed that the composition of the T-cell lines was representative of the in vivo memory CD8(+) T-cell populations. Analysis of the Tp1 and Tp2 antigens revealed sequence polymorphism, which was reflected by differential recognition by T-cell lines. In conclusion, we have demonstrated a profound immunodominance in the CD8(+) T-cell response to T. parva, which we propose is a major determinant of the parasite strain specificity of the response and hence immune protection.

Genomic location and characterisation of MIC genes in cattle.

Major histocompatibility complex (MHC) class I chain-related (MIC) genes have been previously identified and characterised in human. They encode polymorphic class I-like molecules that are stress-inducible, and constitute one of the ligands of the activating natural killer cell receptor NKG2D. We have identified three MIC genes within the cattle genome, located close to three non-classical MHC class I genes. The genomic position relative to other genes is very similar to the arrangement reported in the pig MHC region. Analysis of MIC cDNA sequences derived from a range of cattle cell lines suggest there may be four MIC genes in total. We have investigated the presence of the genes in distinct and well-defined MHC haplotypes, and show that one gene is consistently present, while configuration of the other three genes appears variable.

Genomic location and characterisation of nonclassical MHC class I genes in cattle.

The cattle major histocompatibility complex (MHC) region contains a variable number of classical class I genes encoding polymorphic, ubiquitously expressed molecules with a role in antigen presentation. Class I cDNA sequences have previously been reported that are thought to derive from putative nonclassical class I genes. We have located four nonclassical class I genes within the cattle genome; three are close to the MIC genes, and one is close to the classical class I genes. The genomic position relative to anchor genes is very similar to the arrangement reported in the pig MHC region. We have designed gene-specific oligonucleotide primers with which to investigate the presence of these genes in distinct and well-defined MHC haplotypes and to assess transcription in different cell types. Analysis and comparison of all sequences allows an assessment of allelic variation in each case. Partial characterisation gives an indication of the possible role and likely importance of each of these genes.

An MHC-restricted CD8+ T-cell response is induced in cattle by foot-and-mouth disease virus (FMDV) infection and also following vaccination with inactivated FMDV.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hooved animals that carries enormous economic consequences. CD8(+) cytotoxic T lymphocytes play an important role in protection and disease outcome in viral infections but, to date, the role of the CD8(+) T-cell immune response to FMDV remains unclear. This study aimed to investigate major histocompatibility complex (MHC) class I-restricted CD8(+) T-cell responses to FMDV in vaccinated and in infected cattle. An in vitro assay was used to detect antigen-specific gamma interferon release by CD8(+) T cells in FMDV-infected cattle of known MHC class I genotypes. A significant MHC class I-restricted CD8(+) T-cell response was detected to both FMDV strain O1 BFS and a recombinant fowlpox virus expressing the structural proteins of FMDV. Antigen-specific MHC class I-restricted CD8(+) T-cell responses were also detected in cattle vaccinated with inactivated FMDV. These responses were shown to be directed, at least in part, to epitopes within the structural proteins (P12A region) of the virus. By using mouse cells expressing single cattle MHC class I alleles, it was possible to identify the restriction elements in each case. Identification of these epitopes will facilitate the quantitative and qualitative analysis of FMDV-specific memory CD8(+) T cells in cattle and help to ensure that potential vaccines induce a qualitatively appropriate CD8(+) T-cell response.

Characterization of the fine specificity of bovine CD8 T-cell responses to defined antigens from the protozoan parasite Theileria parva.

Immunity against the bovine intracellular protozoan parasite Theileria parva has been shown to be mediated by CD8 T cells. Six antigens targeted by CD8 T cells from T. parva-immune cattle of different major histocompatibility complex (MHC) genotypes have been identified, raising the prospect of developing a subunit vaccine. To facilitate further dissection of the specificity of protective CD8 T-cell responses and to assist in the assessment of responses to vaccination, we set out to identify the epitopes recognized in these T. parva antigens and their MHC restriction elements. Nine epitopes in six T. parva antigens, together with their respective MHC restriction elements, were successfully identified. Five of the cytotoxic-T-lymphocyte epitopes were found to be restricted by products of previously described alleles, and four were restricted by four novel restriction elements. Analyses of CD8 T-cell responses to five of the epitopes in groups of cattle carrying the defined restriction elements and immunized with live parasites demonstrated that, with one exception, the epitopes were consistently recognized by animals of the respective genotypes. The analysis of responses was extended to animals immunized with multiple antigens delivered in separate vaccine constructs. Specific CD8 T-cell responses were detected in 19 of 24 immunized cattle. All responder cattle mounted responses specific for antigens for which they carried an identified restriction element. By contrast, only 8 of 19 responder cattle displayed a response to antigens for which they did not carry an identified restriction element. These data demonstrate that the identified antigens are inherently dominant in animals with the corresponding MHC genotypes.