Keith T. Ballingall

ISAG/IUIS-VIC Comparative MHC Nomenclature Committee report, 2005

Nomenclature for Major Histocompatibility Complex (MHC) genes and alleles in species other than humans and mice has historically been overseen either informally by groups generating sequences, or by formal nomenclature committees set up by the International Society for Animal Genetics (ISAG). The suggestion for a Comparative MHC Nomenclature Committee was made at the ISAG meeting held in Göttingen, Germany (2002), and the committee met for the first time at the Institute for Animal Health, Compton, UK in January 2003. To publicize its activity and extend its scope, the committee organized a workshop at the International Veterinary Immunology Symposium (IVIS) in Quebec (2004) where it was decided to affiliate with the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS). The goals of the committee are to establish a common framework and guidelines for MHC nomenclature in any species; to demonstrate this in the form of a database that will ensure that in the future, researchers can easily access a source of validated MHC sequences for any species; to facilitate discussion on this area between existing groups and nomenclature committees. A further meeting of the committee was held in September 2005 in Glasgow, UK. This was attended by most of the existing committee members with some additional invited participants (Table 1). The aims of this meeting were to facilitate the inclusion of new species onto the database, to discuss extension, improvement and funding of the database, and to address a number of nomenclature issues raised at the previous workshop.

Cattle MHC: evolution in action?

Summary: Because major histocompatibility complex (MHC) genes play a major role in the development of acquired immune responses, it is essential to obtain comparative information on their organisation, expression and possible functional dichotomies in different species. In human, three classical, polymorphic class I genes (HLA‐A, B‐ and ‐C) and four expressed A/B class II gene pairs (HLA‐DM, ‐DP, ‐DQ and ‐DR) are each present on all haplotypes. With the exception of the HLA‐DRB loci, it has been assumed that a similar rigid organisational situation exists in other mammalian species. However, extensive analysis of the bovine MHC (BoLA) at both the genomic and transcriptional levels has revealed a degree of genetic fluidity not described in other species. None of the four (or more) classical class I genes identified is consistently expressed, and haplotypes differ from one another in both the number and composition of expressed class I genes. Similarly, in the class II region, the number of DQ genes varies between haplotypes in both number and composition. These variations in both class I and II (which appear to reflect differences at the genomic level) are likely to play an important role in cattle immune responses. The observed phenotypic differences in cattle demonstrate very clearly the dynamic nature of the MHC region. This review addresses the functional impact of such variation in different breeds and populations, and its significance in terms of MHC evolution.

The DY sub-region of the sheep MHC contains an A/B gene pair.

The major histocompatibility complex (MHC) class II region of ruminants appears to have a structure broadly similar to that of the human class II or HLA-D region. Restriction fragment length polymorphism (RFLP) studies of class II genes in cattle (Andersson et al. 1988; Anderson and Rask 1988; Sigurdardottir et al. 1988, 1991 b), and in sheep (Scott et al. 1987), have provided an estimate of the number and type of class II genes in these species. The subsequent cloning and sequencing of sheep and cattle class II genes (Muggli-Cockett and Stone 1989; Groenen et al. 1990; van der Poel et al. 1990; Andersson et al. 1991; Scott et al. 1991 a, b; Ballingall et al. 1992; Sigurdardottir et al. 1991 a, 1992), have demonstrated that they are highly homologous to their human counterparts. Of more interest, therefore, are loci within the ruminant MHC which differ from the HLA class II region.Three distinguishing features of the ruminant class II region described to date are, firstly, the apparent absence of a DP-like isotype, secondly, the variability in the number of DQ genes between haplotypes (Andersson and Rask 1988), and thirdly, the presence of class II genes presumed to be unique to the ruminant (Andersson et al. 1988). The presence of two such genes, designated DYA and DYB, was deduced from RFLP studies of cattle DNA. These genes were shown to segregate together with the DOB gene in one region separated by a recombination distance of 17 cM from the region which contains the DQA, DQB, DRB, DRA, and C4 loci (Andersson et al. 1988). Subsequently, Bota-DYA was cloned from a phage library and sequenced (van der Poel et al. 1990; Acc. Nos. m30119 and m30118). The sequence of part of a similar gene in the goat, obtained by PCR by using primers derived from the cattle sequence, has recently been reported (Mann et al. 1993; Acc. No. m94325). However, there has been no report of the cloning of a B gene partner for the DYA gene. A novel cattle class II B gene designated Bota-DIB was cloned from a phage library and sequenced by Stone and Muggli-Cockett (1990). This was shown to be a single copy gene of limited polymorphism, which on the basis of RFLP analysis was probably not Bota-DYB but did appear to be distinct from other known cattle class II genes. The species distribution of this B gene was shown to be restricted to Cervidae, Giraffidae, and Bovidae (Stone and Muggli-Cockett 1993). However, it is not known whether any of these novel genes are functional.Expressed human class II genes usually occur as A/B gene pairs situated close to each other on the chromosome. This is also the case with Bota-DQ genes (Groenen et al. 1990) and Ovar-DQ genes (Deverson et al. 1991; Wright and Ballingall 1994). We used the techniques of cosmid cloning and DNA-mediated gene transfection to determine whether there is a sheep equivalent of the Bota-DYA gene, whether there is a DYB gene partner, and whether there is a protein product.A cosmid library was constructed from DNA prepared from a Finnish Landrace ram. The library was screened with Ovar-DQA, Ovar-DQB, HLA-DQA, and HLA-DQB gene probes at low stringency. A cosmid clone, 365, was obtained which hybridized weakly to both the Ovar gene probes. Restriction maps of the clone were produced for the enzymes Eco R1, Bam HI, Hin dIII, Sac I and Sma I. When the maps were compared to those published for the phage clones containing the Bota-DYA (van der Poel et al. 1990) and the Bota-DIB gene (Stone and Muggli-Cockett 1990), there was an imperfect match (Figure 1 shows the Eco RI maps). However, the sequence data for the A and B genes in cosmid 365 are more convincing. The sequences of exons 2 and 3 of the A gene in cosmid 365 and the Bota-DYA gene, together with the partial sequence from the third exon of the Cahi-DYA gene are shown in Figure 2 A. The predicted amino acid translations of these genes together with those of other published sheep MHC class II A genes are shown in Figure 2 B. The A gene in cosmid 365 had all the salient features of an MHC class II A gene. It showed a high sequence similarity to the cattle and caprine DYA genes and much less so to the Ovar-DRA gene (Ballingall et al. 1992; Acc. No z11600) and the Ovar-DQA1 and DQA2 (Scott et al. 1991 a; Acc. Nos. m33304 and m33305), as detailed in Table 1. The cosmid A gene showed low sequence similarity to the sheep DNA (formerly DZA) gene (unpublished observations). The A gene described here is clearly the sheep homologue of the Bota-DYA gene.The sequences of the second, third, and fourth exons of the B gene in cosmid 365 are shown in Figure 3 A together with those of the Bota-DIB gene (Stone and Muggli-Cockett 1990). Unfortunately, the presence of a Bam HI site in exon 2 of the sheep gene caused a truncation at this point, during the cloning procedure and so a part of exon 2, the whole of exon 1, and all the upstream regulatory elements were missing. The predicted amino acid translations of exons 2, 3, and 4 are shown together with those of an Ovar-DQB (Scott et al. 1991 a; Acc. No. m33323) and an expressed Ovar-DRB gene (Ballingall et al. 1992; Acc. No. z11522) in Figure 3 B.

Analysis of genetic diversity at the DQA loci in African cattle: evidence for a BoLA-DQA3 locus

Abstract We describe the development of a polymerase chain reaction (PCR)-based approach for analysis of genetic diversity at the DQA loci in African Bos indicus and Bos taurus cattle. This approach, equally effective in European and Asian cattle breeds, detects the presence or absence of DQA1 and most duplicated DQA2 genes. Nucleotide and predicted amino acid sequence analysis of the highly polymorphic second exons, in addition to analysis of the locus-specific and relatively non-polymorphic transmembrane, cytoplasmic, and 3-prime untranslated regions, has provided evidence for considerable diversity between each of the duplicated DQA2 genes. Therefore, we propose the designation BoLA-DQA3 for the previously unpublished alleles at the second DQA2 locus. Fourteen distinct PCR restriction fragment length polymorphism (RFLP) patterns, each identifying families of alleles at three DQA loci, can be distinguished. Nucleotide sequence analysis of new PCR-RFLP patterns from 193 Kenyan Boran, Ethiopian Arsi (B. indicus), and Guinean N’Dama (B. taurus) cattle identified 13 DQA1 alleles within eight major allelic families, five DQA2 alleles within a single allelic family, and seven DQA3 alleles within three major allelic families.

Recombinant bovine interferon gamma inhibits the growth of Cowdria ruminantium but fails to induce major histocompatibility complex class II following infection of endothelial cells

Recombinant bovine IFN gamma is a potent inhibitor of Cowdria ruminantium growth in vitro irrespective of the rickettsial stock, or the origin of the endothelial cells. These results suggest an important role for IFN gamma in protective immune responses against C. ruminantium infections. Here we also show that IFN gamma can induce the expression of MHC class II molecules on the surface of endothelial cells. However, treatment of endothelial cells with IFN gamma following infection with Cowdria fails to induce MHC class II expression. The implications of this pathogen-specific effect on class II expression by endothelial cells with regard to its recognition by the host immune system are discussed.

Haplotype characterization of transcribed ovine major histocompatibility complex (MHC) class I genes

The ovine major histocompatibility complex (MHC) remains poorly characterized compared with those of other livestock species. Molecular genetic analysis of the bovine MHC has revealed considerable haplotype and allelic diversity that earlier serological analysis had not detected. To develop cellular and molecular tools to support development of vaccines against intracellular pathogens of sheep, we have undertaken a molecular genetic analysis of four distinct ovine MHC haplotypes carried by two heterozygous Blackface rams. We have identified 12 novel class I transcripts and used a class I sequence-specific genotyping system to assign each of these transcripts to individual haplotypes. Using a combination of phylogenetic analysis, haplotype and transcript expression data, we identified at least four distinct polymorphic class I MHC loci, three of which appear together in a number of combinations in individual haplotypes. The haplotypes were further characterized at the highly polymorphic Ovar-DRB1 locus, allowing selection of the progeny of the two founder rams for the establishment of an MHC-defined resource population.

In vitro infection with Theileria parva is associated with IL10 expression in all bovine lymphocyte lineages.

The protozoan parasite Theileria parva infects and transforms bovine lymphocytes, giving rise to a fatal lymphoproliferative condition known as East Coast fever. Although immune cattle mount strong cytolytic T lymphocyte responses to the parasite, naive animals appear unable to respond and develop severe immunopathological lesions. We have investigated the patterns of cytokine mRNA expressed by 19 bulk and cloned parasite‐infected lymphoblast cell lines using a multiplex PCR system. Considerable variation was observed in the cytokine profiles of these lines and only IL10 was universally expressed. Investigation of cloned lines representing the major bovine lymphocyte populations failed to reveal a lineage‐specific pattern of cytokine mRNA expression that could be associated with infection. Nonetheless, analysis of a CD4+ T cell clone before and after transformation with the parasite indicated that infection does alter the pattern of cytokine expression, with apparent upregulation of IL10. These observations raise the possibility that IL10 derived from infected cells may influence the immune responses of naive cattle to challenge.

Trans-species polymorphism and selection in the MHC class II DRA genes of domestic sheep.

Highly polymorphic genes with central roles in lymphocyte mediated immune surveillance are grouped together in the major histocompatibility complex (MHC) in higher vertebrates. Generally, across vertebrate species the class II MHC DRA gene is highly conserved with only limited allelic variation. Here however, we provide evidence of trans-species polymorphism at the DRA locus in domestic sheep (Ovis aries). We describe variation at the Ovar-DRA locus that is far in excess of anything described in other vertebrate species. The divergent DRA allele (Ovar-DRA*0201) differs from the sheep reference sequences by 20 nucleotides, 12 of which appear non-synonymous. Furthermore, DRA*0201 is paired with an equally divergent DRB1 allele (Ovar-DRB1*0901), which is consistent with an independent evolutionary history for the DR sub-region within this MHC haplotype. No recombination was observed between the divergent DRA and B genes in a range of breeds and typical levels of MHC class II DR protein expression were detected at the surface of leukocyte populations obtained from animals homozygous for the DRA*0201, DRB1*0901 haplotype. Bayesian phylogenetic analysis groups Ovar-DRA*0201 with DRA sequences derived from species within the Oryx and Alcelaphus genera rather than clustering with other ovine and caprine DRA alleles. Tests for Darwinian selection identified 10 positively selected sites on the branch leading to Ovar-DRA*0201, three of which are predicted to be associated with the binding of peptide antigen. As the Ovis, Oryx and Alcelaphus genera have not shared a common ancestor for over 30 million years, the DRA*0201 and DRB1*0901 allelic pair is likely to be of ancient origin and present in the founding population from which all contemporary domestic sheep breeds are derived. The conservation of the integrity of this unusual DR allelic pair suggests some selective advantage which is likely to be associated with the presentation of pathogen antigen to T-cells and the induction of protective immunity.

IPD-MHC 2.0 : an improved inter-species database for the study of the major histocompatibility complex

The IPD-MHC Database project (http://www.ebi.ac.uk/ipd/mhc/) collects and expertly curates sequences of the major histocompatibility complex from non-human species and provides the infrastructure and tools to enable accurate analysis. Since the first release of the database in 2003, IPD-MHC has grown and currently hosts a number of specific sections, with more than 7000 alleles from 70 species, including non-human primates, canines, felines, equids, ovids, suids, bovins, salmonids and murids. These sequences are expertly curated and made publicly available through an open access website. The IPD-MHC Database is a key resource in its field, and this has led to an average of 1500 unique visitors and more than 5000 viewed pages per month. As the database has grown in size and complexity, it has created a number of challenges in maintaining and organizing information, particularly the need to standardize nomenclature and taxonomic classification, while incorporating new allele submissions. Here, we describe the latest database release, the IPD-MHC 2.0 and discuss planned developments. This release incorporates sequence updates and new tools that enhance database queries and improve the submission procedure by utilizing common tools that are able to handle the varied requirements of each MHC-group.

The CD45 locus in cattle: allelic polymorphism and evidence for exceptional positive natural selection

Abstract. Cattle in Africa are a genetically diverse population that has resulted from successive introduction of Asian Bos indicus and European B. taurus cattle. However, analysis of mitochondrial genetic diversity in African cattle identified three lineages, one associated with Asian B. indicus, one with European B. taurus, and a third ascribed to an indigenous African sub-species of cattle. Due to their extended co-evolution, indigenous African herbivores are generally tolerant to endemic African pathogens. We are interested in identifying alleles derived from the indigenous African cattle that may be associated with tolerance to African pathogens. An analysis of the locus which encodes the abundant plasma membrane-associated tyrosine phosphatase, CD45, identified three highly divergent allelic families in Kenya Boran cattle. Analysis of allelic distribution in a diverse range of cattle populations suggests a European B. taurus, an Asian B. indicus, and an African origin. This demonstrates not only significant allelic polymorphism at the CD45 locus in cattle but also convincing autosomal evidence for a distinct African sub-species of cattle. Furthermore, maximum-likelihood analysis of selection pressures revealed that the CD45 locus is subject to exceptionally strong natural selection which we suggest may be pathogen driven.