Alloreactive T-cell recognition of bovine major histocompatibility complex class II products defined by one-dimensional isoelectric focusing

T-cell recognition of bovine MHC (BoLA) class II antigens was investigated in relation to BoLA class II polymorphisms defined by one-dimensional isoelectric focusing (1D-IEF). One-way mixed lymphocyte reactions (MLRs), and allospecific cell lines and clones were used. In general, T-cell responses correlated with the 1D-IEF defined haplotypes (EDF types). However, with MLRs some responses appeared to be associated with BoLA class I differences. All combinations of responder-stimulator pairs produced alloreactive T-cell responses both in MLR and in generation of allolines/clones. Thus allospecific lines and clones were generated to all EDF types tested. Splits in the IEF typing were observed with EDF6 and EDF3, indicating that distinct BoLA class II haplotypes are not necessarily distinguished by 1D-IEF alone. Furthermore, the patterns of reactivity with EDF3 expressing cells were complex with the T-cell specificities splitting EDF3 into several distinct types. Also, in some cases it was clear that more than one T-cell specificity per EDF type was detectable. Thus, allospecific lines and clones provide complementary and additional information to the 1D-IEF typing for polymorphism of the BoLA class II complex. This extra information is particularly important in terms of the functional significance of the BoLA complex for antigen presentation and immune response gene effects.     

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.     

Biochemical characterization of activation-associated bovine class I major histocompatibility complex antigens

Summary. Utilizing a 'sandwich' ELISA assay we have been able to demonstrate that mAb W6/32, B1G6 and IL-A19 are reactive with three different monomorphic determinants on bovine class I major histocompatibility complex (MHC) molecules. Sequential immunoprecipitations performed with the mAb revealed that class I molecules on PBM comprise a single population with respect to reactivity with the mAb in that the β2m-associated proteins bear all three epitopes. By contrast, TCGF-driven lymphoblasts and cells transformed by Theileria parva (Tp) additionally express molecules of Mr 45000 bound to β2m which are recognized by mAb B1G6 and IL-A19 but not by W6/32. These two subclasses of molecules were further distinguished on the basis that, when tunicamycin was added to cultures in the preparation of cells for analysis, mAb W6/32 precipitated class I heavy chains of Mr 39000 while the extra molecules detected only by mAb B1G6 and IL-A19 were of Mr 37000 and 39000. On thymocytes, the mAb W6/32-non-reactive class I molecules are present in low amounts and are expressed by cells in the medulla area, unlike BoT1 (analogous to human CD1) molecules which are expressed by the cortical cells. Our studies also revealed that the supposed β2m-specific mAb B1G6 does not recognize the β2m-associated molecules (BoT1) precipitated by mAb TH97A and thus the specificity of mAb B1G6 in cattle is for an epitope on bovine β2m which is strongly influenced by the nature of the heavy chain with which the β2m is associated.     

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.     

One-dimensional isoelectric focusing and immunoblotting of bovine major histocompatibility complex (BoLA) class I molecules and correlation with class I serology

One-dimensional isoelectric focusing followed by immunoblotting and development of the immunoblots with the monoclonal antibody HC-10, raised against denatured HLA class I heavy chains, was used to demonstrate biochemical variation in cattle MHC (BoLA) class I molecules. The bands obtained correlated well with BoLA-A specificities. Two or three bands were identified for the specificities w7, w8, w16, w18, w21, cph43 and cph49, whereas no bands were observed for the specificity w2. Two serologically indistinguishable subtypes of specificity w18 were identified.     

Biochemical evidence of the expression of two major histocompatibility complex class I genes on bovine peripheral blood mononuclear cells

For a long time, the bovine major histocompatibility complex (MHC) (BoLA) class I region was characterized, rather uniquely among mammalian species, as having one expressed locus. Recent reports have suggested otherwise. Selective immunoprecipitation and molecular characterization of products enable a decisive answer to the question of whether there is indeed more than one locus expressed. Therefore, we characterized serologically defined w10 encoding haplotypes in European and African cattle by immunoprecipitation of [35S]-methionine-labelled peripheral blood mononuclear cells (PBMC), followed by one- and two-dimensional isoelectric focusing (1D/2D-IEF) of cell lysates. Monoclonal antibodies (mAb) used were directed against either human class I monomorphic determinants (W6/32 and B1.1G6) or bovine polymorphic determinants expressed on products encoded by serologically defined w10 encoding haplotypes of Boran and Friesian cattle. Sequential immunoprecipitations with W6/32 and B1.1G6 using lysates of PBMC of British Friesian cattle, revealed that from this haplotype W6/32 precipitated one product, whereas B1.1G6 precipitated two products. The product precipitated in addition appeared to be the one that was selectively precipitated by the mAb directed against polymorphic determinants on a product of w10 encoding haplotypes. Additionally, peptide maps of protease V8-digested precipitates showed that this particular 'w10' associated product was distinctly different from the product recognized by W6/32. Thus, we suggest that the two products are distinct gene products and that the product with higher pI is associated with the serologically defined A-locus product, whereas the product with lower pI is the putative second locus product. In the African Boran breed, variants of the serologically defined w10 specificity were found on the basis of IEF typing. These variants appeared to be associated with different second locus products. Therefore, we conclude that serologically defined w10 encoding haplotypes encode at least two independent class I locus products, expressed on normal bovine PBMC. In IEF analysis the additional use of mAb recognizing polymorphic determinants on serologically defined A-locus products highly facilitated the detection and typing of second locus products.     

Molecular characterization of major histocompatibility complex class II alleles in the common frog, Rana temporaria

Major histocompatibility complex (MHC) class II genes, which play a major role in the immune system response, are some of the most polymorphic genes in vertebrates. We developed polymerase chain reaction primers for part of the second exon of an expressed MHC class II gene in the common frog, Rana temporaria. We genotyped this locus in five frog populations in southeast England and detected eight alleles in 215 individuals. Five or six alleles were detected in each population with a maximum of two alleles per individual, indicating that only a single locus was amplified. We also inferred the possible existence of a null allele. There were 23 variable nucleotide sites (out of 136) and 13 variable amino acid sites (out of 44), many of which corresponded to amino acids involved in antigen recognition. We detected a significant excess of nonsynonymous substitutions at antigen binding sites, indicating that this gene is under positive selection. The level of variation we found was similar to that in other amphibian MHC class II loci, such as those in Bombina bombina, Xenopus laevis and Ambystoma tigrinum.     

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.     

Expression and characterization of ovine major histocompatibility complex class II (OLA-DR) genes

Previous work made use of nucleic acid probes corresponding to different subtypes of the class II regions of the human and murine major histocompatibility complex (MHC) to isolate seven different alpha and 24 different beta genes of the ovine MHC from two cosmid libraries. In an attempt to identify pairs of alpha and beta genes capable of cell surface expression, all permutations of alpha and beta genes were in turn transfected into mouse L-cells. Two pairs of alpha and beta genes co-expressed and stable ovine MHC class II L-cell lines were developed. The expressed alpha genes had previously been defined as DR-alpha homologues (DRA) by differential Southern hybridization to human subtype specific class II probes. The expressed ovine beta genes were also assigned as ovine DR-beta homologues (DRB) on the basis of their sequence having a higher degree of similarity with human DRB than any other subtype. A total of eight out of 23 anti-sheep class II specific monoclonal antibodies were typed OLA-DR specific by FACScan analysis using the L-cell lines.     

Cloning of the bovine major histocompatibility complex class II genes

Summary. Class II genes of the bovine major histocompatibility complex (MHC) have been cloned from a genomic library. The library was constructed in the bacteriophage Λ vector EMBL3 and comprises approximately 10 times the equivalent of the haploid genome. Half the library was screened with the human DQA, DQB, DRA and DRB cDNA probes. Of the 100 positively hybridizing phage clones, 37 were eventually fully characterized and mapped by means of Southern blot analysis. The exons encoding the first, second and transmembrane domain of all different A and B genes were subcloned and mapped in more detail. These analyses showed that these 37 clones were derived from five different A and 10 different B genes. The hybridization studies indicate that we have cloned and mapped two DQA genes, one DRA gene, two other A genes, four DQB genes, three DRB genes and three other B genes. Since the library was made from a heterozygous animal, this would suggest that there are at least one DQA, one DRA one other undefined A, two DQB, two DRB and one or two other undefined B genes in the haploid genome of Holstein Friesian cattle.     

Studies on the bovine major histocompatibility class I and class II antigens using homozygous typing cells and antigen-specific BoT4+ blast cells

Animals were identified from two sire lines as being homozygous for the class I bovine lymphocyte antigen (BoLA-A) w23. These animals were also shown to be homozygous for class II antigens (BoLA-D) which, however, differed between the two sire lines. Lymphocytes from these animals were then used either as stimulator cells in one-way mixed lymphocyte reactions (MLR) with all animals in the herd carrying the w23 antigen or as antigen presenting cells to bovine T4+ cell blasts. It was shown that, within each sire line, the genes encoding the MHC class I and class II antigens were closely linked. There were no detected recombinations between the MHC class I and class II regions nor within the BoLA-D region responsible for mixed lymphocyte reactivity. MLR typing of MHC class II antigens correlated with the results from T-lymphocyte proliferation studies. Cells from these cattle, which are homozygous at the class I and II MHC loci but differ in the class II antigen expressed, could be used to type the BoLA-D of other cattle.     

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.     

Restriction fragment length polymorphism analysis of major histocompatibility complex class II genes from inbred chicken lines

Summary. High molecular weight DNA was extracted from sperm from chickens of 14 inbred lines. The DNA was digested with each of four restriction enzymes ( Pvu II, Hind III, Bg /II, and Bam HI), electrophoresed for 18 or 45h, blotted onto nitrocellulose, and hybridized to a chicken major histocompatibility complex (MHC, B complex) class II β-chain probe (β2-exon specific). Restriction fragment length polymorphisms (RFLPs) were found with each of the restriction enzymes used. Birds with the same B haplotype always showed the same RFLP pattern; however, some birds of different B halotypes also shared the same RFLP pattern. To test for the Mendelian inheritance of the RFLP patterns, the F2 progeny of an informative cross were analysed. The RFLP patterns corresponded with the serologically determined B haplotypes of the F2 birds, thereby showing the Mendelian inheritance of the polymorphic bands.     

Partial nucleotide sequence of a novel bovine major histocompatibility complex class II β-chain gene, BoLA-DIB

A bovine genomic clone that hybridized to HLA-DQ beta cDNA was isolated and fragments containing the beta 1, beta 2 and transmembrane (TM) exons subcloned. The nucleotide sequences of the exons and flanking intron regions were determined. Comparisons of these exon nucleotide sequences and derived amino acid sequences to human class II beta-chain sequences showed that this gene is only 77% identical to HLA-DQ beta and about 75% identical to bovine DQ beta-like genes. The exon sequences were more divergent from other class II beta-chain genes. However, structural features such as conserved cysteines and regions of amino acids strongly suggest this to be a class II beta-chain gene. When exon-containing fragments were used as hybridization probes on Southern blots of bovine genomic DNA digested with Eco RI or Pvu II, each exon hybridized to a single band. Based on these results we have referred to this gene as a novel bovine class II beta-chain gene, BoLA-DIB.     

Improved discrimination of bovine class II DRβ-chains polymorphisms using immunoblotting

An immunoblotting technique is reported that reveals electrophoretic variants in the β-chains of class II antigens of the bovine major histocompatibility complex. One monoclonal antibody, mAb VPM57, reacted on immunoblots with an epitope present in approximately half of the haplotypes investigated. This reagent is especially useful in discriminating electrophoretic variants that have similar isoelectric points.     

Assembly of major histocompatibility complex class II subunits with invariant chain

The highly polymorphic major histocompatibility complex class II (MHCII) polypeptides assemble in the ER with the assistance of invariant chain (Ii) chaperone. Ii binds to the peptide-binding pocket of MHCII heterodimers. We explored the mechanism how MHCII subunits attach to Ii. Expression with single alpha or beta subunits from three human HLA and two mouse H2 class II isotypes revealed that Ii co-isolates predominantly with the alpha polypeptide. Co-isolation with alpha chain requires the groove binding Ii-segment and depends on M91 of Ii. Immunoprecipitation of Ii from pulse chase labeled cells showed sequential assembly of alpha and beta chains.     

Isolation and characterization of cDNA clones for chicken major histocompatibility complex class II molecules

The chicken major histocompatibility complex (MHC), the B complex, is being intensively analysed at the DNA level. To further probe the molecular structure of chicken MHC class II genes, cDNA clones coding for chicken MHC class II (B-L) p chain molecules were isolated from an inbred G-B2 Leghorn chicken spleen and liver. Twenty-nine cDNA clones were isolated from the spleen and eight cDNA clones were isolated from the liver. Based on restriction maps, most clones could be clustered into one family of genes. Four cDNA clones were sequenced (S7, S10 and S19 from the spleen and L1, which was identical to S19, from the liver). Complete amino acid sequences of B-Lβ chain molecules were predicted from the nucleotide sequences of the cDNA clones. Although both the nature and the location of the conserved residues were similar in chicken and mammalian sequences, some species-specific differences were found, suggesting that the structures of the B-L molecules of this haplotype are similar, but not identical, to their mammalian counterparts.     

Biochemical identification of the bovine blood group M' antigen as a major histocompatibility complex class I-like molecule

Absorption and elution experiments showed that it was impossible to separate antibodies against blood group factor M' from antibodies against bovine lymphocyte antigen (BoLA) A16 in an antiserum showing haemolytic activity against M' as well as lymphocytotoxic activity against BoLA-A16. To elucidate the structural relationship between BoLA-A16 and blood group antigen M', immunoprecipitation experiments on red and white cell lysates isolated from M'-A16 positive and negative cattle were carried out. These results showed that M_r 44 000 and M_r 12000 polypeptides can be precipitated from both red and white cells isolated from M'-A16 positive animals, whereas no bands were seen in M'-A16 negative animals in precipitations with the same antibody. Precipitation with a crossreacting human β₂-microglobulin (β₂-m) specific antibody confirmed a class-I-like structure associated with β₂-m on M' positive red cells and the absence of such a structure on M' negative red cells. Sequential precipitations gave analogous results. Proteolytic degradation by papain and V8 protease did not reveal any substantial difference between red and white M'-A16 positive cells, but a slight difference in the pI of the immunoprecipitable components of red and white cells was observed. All together, this indicates that either the blood group antigen M' is the BoLA-A16 class I antigen or M' and BoLA-A16 are two different class I polypeptides with the same relative mass, sharing identical epitopes and both associated with β₂-m. Comparable results were obtained with M₁ and BoLA-A24.     

Restriction fragment length polymorphisms in bovine major histocompatibility complex class II ß-chain genes using bovine exon-containing hybridization probes

Restriction fragment length polymorphisms (RFLPs) have been identified in the bovine MHC class II region using five hybridization probes constructed from two bovine genomic clones. Four probes were constructed from a bovine DR beta-like gene, BoLA-DRB2. These included a probe containing the complete beta 1 exon (R2-beta 1), a probe containing the last 129 base pairs of the beta 2 exon (R2-beta 2), a probe containing intron immediately 5' of the beta 2 exon (R2-5' beta 2), and a probe containing the complete transmembrane exon (R2-TM). A fifth probe was constructed from a novel bovine beta-chain gene, BoLA-DIB, and contained the entire TM exon (I1-TM). R2-beta 1 defined very little polymorphism. R2-beta 2 hybridized to several fragments but one or two fragments hybridized much stronger on all Southern blots and it was presumed these corresponded to BoLA-DRB2 fragments. By using R2-5' beta 2 as a probe, these BoLA-DRB2 fragments were confirmed: 6.4 and 2.7-kb Eco RI alleles, 1.7- and 1.5-kb Pvu II alleles, 5.9-, 5.4-, 3.7- and 1.9-kb TaqI alleles, and a non-polymorphic 22.5-kb BamHI fragment. I1-TM identified three alleles with TaqI. To investigate the linkage between the RFLP alleles, 166 offspring of five sires were tested. Complete linkage was found for all RFLPs identified with the BoLA-DRB2 probes. However, the RFLP patterns of 13 calves out of 58 indicated recombination between BoLA-DRB2 and BoLA-DIB.