Joint Report of the Fourth International Bovine Lymphocyte Antigen (BoLA) Workshop, East Lansing, Michigan, USA, 25 August 1990

Blood samples from 54 animals were exchanged between 15 laboratories in nine countries to improve and expand BoLA class I and class II typing. A total of 27 out of 33 (82%) of previously accepted BoLA-w specificities were represented within the cell panel. Seventeen new serum-defined BoLA specificities were accepted by the workshop participants, thus expanding the number of internationally recognized BoLA specificities to 50. The large number of new specificities detected resulted from the number of serological reagents used (n = 1139) and the genetic diversity of the cell panel. Confidence derived from the high percentage of agreement between the laboratories on antigen detection (97.3%; r = 0.84) permitted the removal of the workshop (w) notation from 23 BoLA-w specificities and their acceptance as full status BoLA-A antigens. Two new non-BoLA antigens were also detected, one completely included within the red blood cell factor S' (BoLy-S'), whereas a second (BoLy-w1) did not show any association with tested red blood cell factors. A comparison between serological, isoelectric focusing (IEF) and DNA typing for BoLA class II polymorphism was conducted with a subset of workshop cells. Correlation between the three methods was significant for three combinations of alleles. Three other serologically defined class II specificities were correlated with DR and/or DQ restriction fragment length polymorphism (RFLP) types, whereas six additional IEF types were correlated with DR and/or DQ RFLP types (r greater than or equal to 0.50). Several new IEF, DRB, DQA and DQB RFLP patterns were identified. In 46 animals that were typed for BoLA-DR and DQ genes by RFLP analysis, 46 different BoLA haplotypes were tentatively defined. These 46 haplotypes were distinguished by 31 serologically-defined BoLA-A alleles (and 2 'blanks'), 15 DRB RFLP types (plus up to 10 new DRB RFLP patterns) and 23 DQA-DQB haplotypes.     

Characterization of class I bovine lymphocyte antigens (BoLA) by one-dimensional isoelectricfocusing

BoLA class I antigens were characterized in a group of British and Dutch Friesian cattle by one-dimensional isoelectric focusing (1D-IEF) and the results compared with serology using alloantisera and microcytotoxicity. For IEF analysis, non-stimulated peripheral blood mononuclear cells (PBM) were metabolically labelled with 35S methionine, detergent lysates were prepared and MHC molecules precipitated with the monoclonal antibodies (mAbs) W6/32 or B1.1G6. Staphylococcus protein A precipitated antigens were separated on a vertical slab gel under denaturing conditions. The banding patterns seen for the W6/32 precipitated molecules obtained by 1D-IEF were compared with the serological specificities. Characteristic banding patterns were observed for most serological specificities as well as workshop undefined haplotypes. These patterns were seen both in families and the outbred population. In families IEF haplotypes segregated with serotypes. Additional MHC class I products were suggested by variable banding patterns for different w10 haplotypes and when using the different mAbs. A pulse chase experiment with a w12 animal also suggested more than one expressed product. The w2 and w5 specificities were not precipitated by either W6/32 or B1.1G6 and w6.2 and w6.4 were precipitated by W6/32 but not by B1.1G6. These results show that 1D-IEF is useful for BoLA typing. For the characterization of class I antigens, however, much depends on the mAbs used.     

The analyses of antigen and DNA polymorphism within the bovine major histocompatibility complex: 1. The class I antigens

Summary. Serology, isoelectric focusing (IEF) of expressed antigens, and restriction fragment length polymorphisms (RFLP) were compared for the identification of BoLA class I haplotypes. Expressed antigens identified as bands by IEF correlated well with serological definition confirming and extending our earlier findings (Joosten et al. 1988). Comparison of serology and isoelectric focusing bands with restriction fragments was more complicated; fragments were found which correlated both with broadly reacting and antigen specific sera. We also found correlation of fragments with two or more sera which showed no cross-reactivity. Fragments unique to particular haplotypes were also observed.
Serology remains the simplest method of typing BoLA class I antigens. Isoelectric focusing generally agrees with serological definition of antigens and detects antigens not yet defined by serology. It may also be useful in defining the products of other expressed BoLA class I loci. In order to identify RFLPs which could be used for typing, comparison with serology or IEF is essential. Haplotype specific RFLPs could be useful in identifying genes linked to the MHC.     

The analyses of antigen and DNA polymorphism within the bovine major histocompatibility complex: 1. The class I antigens

Serology, isoelectric focusing (IEF) of expressed antigens, and restriction fragment length polymorphisms (RFLP) were compared for the identification of BoLA class I haplotypes. Expressed antigens identified as bands by IEF correlated well with serological definition confirming and extending our earlier findings (Joosten et al. 1988). Comparison of serology and isoelectric focusing bands with restriction fragments was more complicated; fragments were found which correlated both with broadly reacting and antigen specific sera. We also found correlation of fragments with two or more sera which showed no cross-reactivity. Fragments unique to particular haplotypes were also observed. Serology remains the simplest method of typing BoLA class I antigens. Isoelectric focusing generally agrees with serological definition of antigens and detects antigens not yet defined by serology. It may also be useful in defining the products of other expressed BoLA class I loci. In order to identify RFLPs which could be used for typing, comparison with serology or IEF is essential. Haplotype specific RFLPs could be useful in identifying genes linked to the MHC.     

Definition of supertypes for HLA molecules using clustering of specificity matrices

Major histocompatibility complex (MHC) proteins are encoded by extremely polymorphic genes and play a crucial role in immunity. However, not all genetically different MHC molecules are functionally different. Sette and Sidney (1999) have defined nine HLA class I supertypes and showed that with only nine main functional binding specificities it is possible to cover the binding properties of almost all known HLA class I molecules. Here we present a comprehensive study of the functional relationship between all HLA molecules with known specificities in a uniform and automated way. We have developed a novel method for clustering sequence motifs. We construct hidden Markov models for HLA class I molecules using a Gibbs sampling procedure and use the similarities among these to define clusters of specificities. These clusters are extensions of the previously suggested ones. We suggest splitting some of the alleles in the A1 supertype into a new A26 supertype, and some of the alleles in the B27 supertype into a new B39 supertype. Furthermore the B8 alleles may define their own supertype. We also use the published specificities for a number of HLA-DR types to define clusters with similar specificities. We report that the previously observed specificities of these class II molecules can be clustered into nine classes, which only partly correspond to the serological classification. We show that classification of HLA molecules may be done in a uniform and automated way. The definition of clusters allows for selection of representative HLA molecules that can cover the HLA specificity space better. This makes it possible to target most of the known HLA alleles with known specificities using only a few peptides, and may be used in construction of vaccines. Supplementary material is available at .     

Production of alloantisera against class II bovine lymphocyte antigens (BoLA) by cross-immunization between class I matched cattle

This paper describes the production of alloantisera directed against bovine major histocompatibility complex (MHC) (BoLA) class II antigens in animals whose MHC phenotypes had been defined by one dimensional isoelectric focusing. Animals of closely matched BoLA class I types were selected by serology and subsequently typed for class I and class II by 1D-IEF of immunoprecipitated antigens. Those with similar class I type by both methods, but differing at the class II locus, were chosen for reciprocal immunization. Cross-immunization was by two skin implantations 6 weeks apart. The resulting antisera showed low titre after the first immunization and elevated titre 3 weeks after the second immunization. The sera reacted strongly with cells expressing specific BoLA class II antigens. The pattern of reactivity correlated well with IEF class II typing on a panel of animals representing all of the class II IEF types present in the Friesian population.     

Close association between DNA polymorphism of bovine major histocompatibility complex class I genes and serological BoLA-A specificities

Class I genes of the bovine major histocompatibility complex (MHC) were investigated by Southern blot hybridization and by serological analysis. A large number of class I restriction fragments and an extensive polymorphism were revealed when genomic DNA samples, digested with the restriction enzyme PvuII, were hybridized with a human cDNA probe. The result indicated the presence of multiple class I genes in cattle. The extensive restriction fragment length polymorphism (RFLP) was interpreted genetically by analysing five paternal half-sib families comprising, besides the bulls, 50 offspring and their dams. No less than 21 RFLP types were distinguished in this limited sample. The class I polymorphism was also analysed using a serological test with sera corresponding to four workshop specificities (w2, w6, w10 and w16) and three locally defined specificities (SRB1, SRB2 and SRB3). There was an excellent agreement between the two typing methods since no RFLP type was associated with more than one specificity and five of the seven specificities were associated with a single RFLP type. Evidence for close genetic linkage between class I and DQ class II genes was obtained since no recombinant was found among 45 informative offspring. Linkage disequilibrium among class I, DQ class II and C4 genes was also observed. The blood group specificity M' was completely associated with the w16 class I specificity and with the haplotype I1DQ1BC4(2) in this material.     

Sequence and evolution of cattle MHC class I cDNAs: concerted evolution has not taken place in cattle

To explore genetic mechanisms responsible for major histocompatibility complex (MHC) class I evolution in the artiodactyls, we cloned and sequenced MHC class I cDNAs from a Bos taurus bull heterozygous for cattle MHC (BoLA) class I serological specificities w2 and w30. Four unique cDNAs were found, indicating the presence of at least two MHC class I loci. Analysis of these four cDNAs and all previously published BoLA cDNA sequences suggested that there may be three cattle MHC class I loci. Additionally, comparison of all of the BoLA class I cDNAs to MHC class I cDNAs of other artiodactyls showed that some of the BoLA class I cDNAs were more similar to certain sheep cDNAs than they were to other cattle cDNAs. These data indicate that each BoLA class I locus has evolved independently after an ancestral gene duplication event and that inter-locus segmental exchange o or concerted evolution has not occurred rapidly enough to cause extensive divergence between the orthologous MHC class I loci of sheep and cattle.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L02832–L02835. Correspondence to: T. L. Garber at the present address.     

Inter-Locus and intea-allelic polymorphisms of HLA class I antigen gene mRNA

We have constructed cDNA clone libraries from two lymphoblastoid cell lines, JY (HLA-A2, B7, C untypeable) and LB (HLA-A28, B40, Cw3), and isolated clones encoding class I HLA antigens. We have characterized short oligonucleotide probes derived from the coding region of the HLA class I antigens which are specific for the HLA-A and -B loci. These probes have been used to subdivide the class I cDNA clones into subclasses. DNA sequencing of several HLA-A and -B related clones has allowed us to extend the primary structural characterization of these cell-surface antigens. This analysis has also detected a sequence polymorphism at the HLA-A locus, indicating that the previously considered homozygous typing cell line LB expresses two alleles of similar, although not identical, serological specificity.     

Analysis of a horse family with a crossing-over between the ELA complex and the A blood group system

A horse family in which a recombination occurred in the chromosome region coding for the serological specificities of the ELA complex and those of the A blood group system of a mare was further analysed by mixed lymphocyte reaction (MLR) and Southern blot hybridization. This family consisted of a stallion, a mare and five full sibs. The stallion and the mare were heterozygous for internationally recognized ELA specificities while only the mare was heterozygous for the A blood group system. MLR between all members of the family confirmed that the stallion possessed two different ELA haplotypes and suggested that recombination in the mare occurred outside the segment delimited by the ELA-A locus and the MLR region. DNA samples from all individuals were investigated by Southern blot analysis using three restriction enzymes (EcoRI, HindIII or TaqI), three human HLA probes (one of class I cDNA and two of class II probes), one cDNA (DR beta) and one genomic (DQ alpha). Class I and class II restriction fragments of the mare segregated in accordance to the ELA specificities and thus clearly confirming that the crossing-over did not occur between the ELA-A gene and the class I, class II region nor between DR beta and DQ alpha subsets. The A blood group genetic determinants would thus be situated outside the ELA region defined by class I and class II genes.     

An analysis of class I antigens of man and other species by one-dimensional IEF and immunoblotting

A procedure for the molecular identification of MHC class I products based on 1-D IEF and subsequent immunoblotting is described. Optimal conditions for 1-D IEF, the electrophoretic transfer of proteins out of denaturing, nonionic detergent-containing gels to nitrocellulose, and the requisite antibodies, both polyclonal and monoclonal, for the visualization of class I heavy chains have been established. Cross-reactivity of antibodies has enabled the biochemical analysis of class I heavy chains in the dog. The procedure reported here requires modest amounts of cells and allows a rapid molecular characterization of class I heavy chain polymorphisms in man and other species without the need for radiochemical methods.Abbreviations used in this paper FCS fetal calf serum - MHC major histocompatibility complex - NP-40 Nonidet P-40 - PBL peripheral blood lymphocytes - PHA phytohemagglutinin - RaHC rabbit anti-heavy chain serum - TX-114 Triton X-114 - 1-D IEF one-dimensional isoelectric focusing     

Transmembrane and cytoplasmic domain sequences demonstrate at least two expressed bovine MHC class I loci

We have used the polymerase chain reaction to amplify cDNA from expressed bovine major histocompatibility complex class I genes. Sequences obtained from transmembrane and cytoplasmic domains were used to identify the number of expressed alleles. Data from three animals suggest that there are four major expressed alleles, representing the products of two (or more) loci. We have also demonstrated the presence of an alternatively spliced mRNA, which has been observed in five animals. The alternative splicing removes exon 7 (the major site of class I phosphorylation), which predicts a truncated molecule with a cytoplasmic portion 16 amino acids shorter than usual. This phenomenon was detected for only a single class I allele within each individual. Correspondence to: S. A. Ellis.     

Serological discrimination of DR4 haplotypes by radioimmunoassay

Three new alloantigenic specificities of human major histocompatibility complex class 11 molecules have been defined by testing the reactivity of alloantisera at the molecular level. Two of these specificities identify different DR4 haplotypes. The Fe75 specificity is associated with the DR4/DW10 haplotype and the CBC/MRG6 specificity with the DR4/DKT2 haplotype. Both are supertypic specificities and are associated with other DR specificities as well. Both specificities are carried by class 11 molecules belonging to the first DR subset. Together with previously described determinants, these specificities contribute to serological discrimination of the different DR4 haplotypes.     

DNA sequence analysis of serologically detected ELA class II haplotypes at the equine DQP locus

The genetic diversity at the ELA DQβ locus was investigated using polymerase chain reaction and DNA sequencing. Based upon serological methods 16 class II homozygous animals were selected and their genomic DNA was used. A DQβ gene from an equine cDNA library was also sequenced. Our methology and the similarity between the genomic and the cDNA sequences suggest that the studied locus is expressed on equine lymphocytes. In the predicted amino acid sequence the most extensive variation is located at residues 56–60. The pattern of these five amino acids is strongly correlated to the serological ELA class II specificities (W13, W22, W23, Be200). The alleles corresponding to the W23 specificity are the most divergent among the equine DQβ alleles and also from other mammalian DQβ sequences.     

A comparative study of major histocompatibility complex antigens in East African and European cattle breeds

An account is given of the serologically defined class I specificities encoded by the bovine MHC (expressed as the BoLA system) in two populations of African cattle and in European breeds. The BoLA typing was performed using alloantisera raised against tissue antigens of both European and African breeds of cattle. All of the specificities agreed in the first two international BoLA workshops were found in the African cattle, although there were significant differences in the frequency of some specificities between the African and European animals. Many of the European antisera, which are operationally monospecific in Bos taurus cattle, were multispecific in the African animals. Subgroups of two specificities (w8 and w10) were demonstrated. Five new BoLA-A locus alleles were detected by means of antisera raised against alloantigens of African cattle. Two of these occurred at an extremely high frequency in the African populations; one being unique to these cattle. Monoclonal antibodies proved to be useful typing reagents, particularly in the elucidation of subgroups.     

MHC class I-related antigen-processing machinery component defects in feline mammary carcinoma

Defects in HLA class I antigen-processing machinery (APM) component expression and/or function are frequent in human tumors. These defects may provide tumor cells with a mechanism to escape from recognition and destruction by HLA class I antigen-restricted, tumor antigen-specific cytotoxic T cells. However, expression and functional properties of MHC class I antigens and APM components in malignant cells in other animal species have been investigated to a limited extent. However, this information can contribute to our understanding of the mechanisms underlying the association of MHC class I antigen and APM component defects with malignant transformation of cells and to identify animal models to validate targeted therapies to correct these defects. To overcome this limitation in the present study, we have investigated the expression of the catalytic subunits of proteasome (Y, X, and Z) and of immunoproteasome (LMP2, LMP7, and LMP10) as well as of MHC class I heavy chain (HC) in 25 primary feline mammary carcinomas (FMCs) and in 23 matched healthy mammary tissues. We found a reduced expression of MHC class I HC and of LMP2 and LMP7 in tumors compared with normal tissues. Concordantly, proteasomal cleavage specificities in extracts from FMCs were different from those in healthy tissues. In addition, correlation analysis showed that LMP2 and LMP7 were concordantly expressed in FMCs, and their expression was significantly correlated with that of MHC class I HC. The abnormalities we have found in the APM in FMCs may cause a defective processing of some tumor antigens.     

Study of the HLA-DQ system by the complement fixation test on lymphocytes stimulated by phytohemagglutinin. Existence of HLA-DQX allele(s)

The complement fixation microtechnique against PHA blasts has been used to study HLA-DQw1, 2, 3 specificities with sera from multiple transfused patients and/or from multiparous women. Several sera (6 or 7) have been used to define each DQ specificity. The sera have been chosen because of their reactivity with cells from HLA-DR 1, 2 or w6 donors (for DQw1), DR3 or 7 donors (for DQw2,) DR4 or 5 donors (for DQw3). Correlation coefficients between DQ and DR specificities were from 0.56 to 0.91. Correlation coefficients between sera were from 0.51 to 0.92 in each cluster of sera. The segregation of DQw1, 2, 3 specificities has been studied in 46 families with 234 children. This study showed haplotypes lacking DQw1, 2, 3 specificities. The segregation of such 11 DQX haplotypes has been observed in 38 children from 8 families; 5 children were DQX/DQX homozygotes. Up to now, no serological reagent defining the specificity (or specificities) corresponding to DQX has been found. No preferential association was observed between DQX and DR specificities. The gene frequencies observed in 170 haplotypes in these 46 families were as follows: DQw1: 0.400; DQw2: 0.252; DQw3: 0.282; DQX: 0.065. Detecting DQ specificities seems easier by CF on PHA blasts than by lymphocytotoxicity microtechnique against B lymphocytes and monocytes from pheripheral blood. This suggests that PHA blasts express larger quantities of DQ molecules than B lymphocytes and monocytes. The results confirm that complement fixation microtechnique against PHA blasts is efficient for HLA-DQw typing.     

Six variants of HLA-1327 identified by isoelectric focusing

Six variant forms of HLA-1327 were identified among 68 unrelated 1327-positive donors by isoelectric focusing (IEF) gel analysis. Each of the six IEF variants was distinguished by charge heterogeneity of desialated B27 heavy chains immunoprecipitated with specific monoclonal antibody (MAb). Charge differences varied from single to several charge units, indicating that these variants may have substantially different amino acid compositions. Informative family study showed that three B27 variant molecules were genetically determined. The uniqueness of these variant molecules was also demonstrable using a panel of alloantisera and MAbs recognizing 1327-associated epitopes. Six distinct serological reactivity patterns were observed. Five of these serological patterns correlated with four of the IEF-defined variants, two of these patterns being associated with one IEF variant form. The sixth serological pattern was shared by the remaining two IEF variants. Combining the results of the electrophoretic and serological analyses, it is apparent that there are more than six structural variants within the B27 alloantigen family. Some B27 variant forms were found only in individuals of particular racial origin, indicating that unique genetic variations might occur in different racial groups. In a preliminary analysis of patients with ankylosing spondylitis, no apparent correlation was observed between any specific B27 variants and disease susceptibility.     

In silico peptide-binding predictions of passerine MHC class I reveal similarities across distantly related species, suggesting convergence on the level of protein function

The major histocompatibility complex (MHC) genes are the most polymorphic genes found in the vertebrate genome, and they encode proteins that play an essential role in the adaptive immune response. Many songbirds (passerines) have been shown to have a large number of transcribed MHC class I genes compared to most mammals. To elucidate the reason for this large number of genes, we compared 14 MHC class I alleles (α1–α3 domains), from great reed warbler, house sparrow and tree sparrow, via phylogenetic analysis, homology modelling and in silico peptide-binding predictions to investigate their functional and genetic relationships. We found more pronounced clustering of the MHC class I allomorphs (allele specific proteins) in regards to their function (peptide-binding specificities) compared to their genetic relationships (amino acid sequences), indicating that the high number of alleles is of functional significance. The MHC class I allomorphs from house sparrow and tree sparrow, species that diverged 10 million years ago (MYA), had overlapping peptide-binding specificities, and these similarities across species were also confirmed in phylogenetic analyses based on amino acid sequences. Notably, there were also overlapping peptide-binding specificities in the allomorphs from house sparrow and great reed warbler, although these species diverged 30 MYA. This overlap was not found in a tree based on amino acid sequences. Our interpretation is that convergent evolution on the level of the protein function, possibly driven by selection from shared pathogens, has resulted in allomorphs with similar peptide-binding repertoires, although trans-species evolution in combination with gene conversion cannot be ruled out.