BoLA class I charge heterogeneity reflects the expression of more than two loci

Internationally recognized allo-antisera in lymphocyte microcytotoxicity assays are thought to detect allelic products of a single highly polymorphic class I locus. A recent report suggested that two bovine lymphocyte antigen (BoLA) class I loci are expressed at the protein level. However, 1D-IEF analysis of BoLA class I molecules reveals multi-band patterns which cannot be reconciled with the reported number of loci. The aim of this study was to investigate the origins of the charge diversity of BoLA class I molecules observed using 1D-IEF. BoLA class I molecules appear to be glycosy-lated at a single N-linked position with a complex type carbohydrate moiety which has up to three terminal sialic acid residues. Class I molecules immunoprecipitated from resting bovine PBL are not phosphorylated. Neither modification is responsible for the observed charge heterogeneity. Peptide mapping reveals that different BoLA charge variants have distinct digestion patterns. Furthermore, a number of different polypeptides are associated with each serological specificity. These polypeptides appear to be encoded by different loci which exist in linkage disequilibrium. The number of charge variants with different peptide maps indicates that the BoLA system has a minimum of three class I loci expressed at the protein level.     

Molecular cloning of bovine class I MHC cDNA

Two cDNA cloned from a Hereford cow B cell line (BL-3) have allowed the determination of the complete coding region for two class I molecules encoded by the bovine MHC (BoLA). The predicted protein sequences have all the features expected of expressed class I molecules that present peptide Ag to cytotoxic T cells. Comparison with class I molecules from other species strongly suggests these cDNA are derived from different genes and provides evidence for the existence of a second expressed class I BoLA locus. The BoLA proteins show greater similarity to HLA than to H-2 molecules, correlating with the cross-reactions of W6/32 and other murine anti-HLA-A,B,C mAb with BoLA molecules. The basis for the W6/32 epitope and the preferential association of H-2 class I H chains with bovine beta 2-m is examined.     

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.     

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.     

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.     

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.     

Mixed lymphocyte culture studies reveal complexity in the bovine MHC not detected by class I serology

The genetic structure of the bovine major histocompatibility complex (MHC) was investigated using the lymphocyte microcytotoxicity test for class I typing and the mixed lymphocyte culture (MLC) assay for class II typing. Using locally produced alloantisera and antisera from the Third International BoLA Workshop, 14 class I BoLA-A locus alleles were identified in the study population, a single herd of approximately 700 Holstein-Friesian cattle. Eleven of these were alleles recognized in the International Workshop and three were new alleles. An MLC titration assay was employed in conjunction with class I typing to define BoLA haplotypes and identify BoLA complex homozygotes. An embryo transfer family consisting of eight full sibling cattle including one BoLA complex homozygote was produced by half sibling mating. Five other BoLA complex homozygotes were subsequently identified in the herd. Six MLC defined class II haplotypes investigated in detail were designated BoLA-D1, D2, D3, D4, D5 and D7. BoLA-D1 was associated with the class I specificity BoLA-Aw6, D2 with Aw6 and the new class I specificity Ac3, D3 with Aw6 and Aw11, D4 with Aw10, D5 with Aw31 and Aw11, and D7 with Aw20. The discovery of four groups of class I identical-class II disparate haplotypes, and three pairs of class I disparate-class II identical haplotypes indicates the presence of considerable complexity in the BoLA complex that is not detected using class I serology.     

Polymorphism of bovine major histocompatibility complex (MHC) class I genes revealed by polymerase chain reaction (PCR) and restriction enzyme analysis

The polymorphic exon 2-exon 3 region of bovine major histocompatibility complex (MHC) class I genes was amplified by polymerase chain reaction (PCR) from genomic DNA samples with characterized class I polymorphism. The primers for amplification were designed in conserved regions at the borders of exons 2 and 3, based on all available cDNA sequences. The primers should, therefore, amplify most expressed class I genes, but may also amplify non-expressed class I genes. The PCR amplified class I gene fragments of 700 bp were characterized on the basis of restriction fragment length polymorphism (RFLP). The PCR-RFLP analysis of class I genes showed that the bands in each digestion could be classified as non-polymorphic, as shared between several bovine lymphocyte antigen (BoLA)-A types, or as specific to a single BoLA-A type. The same primers were then used for amplification of class I gene fragments from eight Sahiwal animals, a breed which originated in the Indian subcontinent. These studies showed that BoLA class I PCR-RFLP could be used to study class I polymorphism in family groups.     

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

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 beta 2m-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 beta 2m 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 beta 2m-specific mAb B1G6 does not recognize the beta 2m-associated molecules (BoT1) precipitated by mAb TH97A and thus the specificity of mAb B1G6 in cattle is for an epitope on bovine beta 2m which is strongly influenced by the nature of the heavy chain with which the beta 2m is associated.     

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.     

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.     

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.     

BoLA class I allele diversity and polymorphism in a herd of cattle

Major histocompatibility complex class I genes are among the most polymorphic genes characterized. The high level of polymorphism is essential for generating host immune responses. In humans, three distinct genomic loci encode human leukocyte antigen (HLA) class I genes, allowing individuals to express up to six different HLA class I molecules. In cattle, the number of distinct genomic loci are currently at least six, and the number of different bovine leukocyte antigens (BoLA) class I molecules that are expressed in individual animals are variable. The extent of allele variation within the cattle population is unknown. In this study, the number and variety of BoLA class I sequences expressed by 36 individuals were determined from full-length BoLA class I cDNA clones. Twenty distinct BoLA class I alleles were identified, with only four being previously reported. The number of expressed BoLA class I alleles in individual animals ranged between one and four, with none of the animals having an identical complement of BoLA class I molecules. Variation existed in the number of BoLA class I alleles expressed as well as the composition of expressed alleles, however, several BoLA class I alleles were found in multiple individual animals. Polymorphic amino acid sites were analyzed for positive and negative selection using the ADAPTSITE program. In the antigen recognition sites (ARS), there were eight positions that were predicted to be under positive selection and three positions that were predicted to be under negative selection from 62 positions. In contrast, for non-antigen recognition sites (non-ARS), there were three positions that were predicted to be under positive selection and 20 that were predicted to be under negative selection from 278, indicating that positive selection of amino acids occurs at a greater frequency within the antigen recognition sites.     

Discrimination between major histocompatibility complex class II DQ and DR locus products in cattle

We have used a panel of anti-major histocompatibility complex (MHC) class II monoclonal antibodies (mAbs) and have assessed their specificity for the products of the individual bovine MHC (BoLA) class II subregions. The mAbs identified two distinct class II molecules by affinity purification and ELISA. Two-dimensional immunoblotting confirmed these data and NH₂-terminal sequencing of the purified class II α chains of one member of each group identified the subregion specificity of the mAbs. The mAbs VPM36, TH22A and TH81A are specific for BoLA DQ, whereas VPM54, TH14B and J11 are specific for BoLA DR. SW73.2 reacts with both MHC subgroups of all cattle tested.     

Comparative analysis of the bovine MHC class IIb sequence identifies inversion breakpoints and three unexpected genes

The bovine major histocompatibility complex (MHC) or BoLA is organized differently from typical mammalian MHCs in that a large portion of the class II region, called class IIb, has been transposed to a position near the centromere on bovine chromosome 23. Gene mapping indicated that the rearrangement resulted from a single inversion, but the boundaries and gene content of the inverted segment have not been fully determined. Here, we report the genomic sequence of BoLA IIb. Comparative sequence analysis with the human MHC revealed that the proximal inversion breakpoint occurred approximately 2.5 kb from the 3' end of the glutamate-cysteine ligase, catalytic subunit (GCLC) locus and that the distal breakpoint occurred about 2 kb from the 5' end from a divergent class IIDRbeta-like sequence designated DSB. Gene content, order and orientation of BoLA IIb are consistent with the single inversion hypothesis when compared with the corresponding region of the human class II MHC (HLA class II). Differences with HLA include the presence of a single histone H2B gene located between the proteasome subunit, beta type, 9 (PSMB9) and DMB loci and a duplicated TAP2 with a variant splice site. BoLA IIb spans approximately 450 kb DNA, with 20 apparently intact genes and no obvious pseudogenes. The region contains 227 simple sequence repeats (SSRs) and approximately 167 kb of retroviral-related repetitive DNA. Nineteen of the 20 genes identified in silico are supported by bovine EST data indicating that the functional gene content of BoLA IIb has not been diminished because it has been transposed from the remainder of BoLA genes.     

Variation in T cell responses to ovalbumin in cattle: evidence for Ir gene control

Genetic control of immune responsiveness in cattle was investigated using an antigen-dependent T cell proliferation assay in vitro. Bovine T cell proliferative responses to ovalbumin were dependent upon major histocompatibility complex (MHC) class II molecules. Responses of an unrelated panel of animals to a limiting concentration of ovalbumin after a single immunization were compared. Two discrete patterns of response were observed. One group of animals had low or non-responses which were not significantly different from the preimmune levels. Another group of animals showed significant responses. After a second immunization the majority of low responders remained low responders. There was no significant correlation between bovine MHC class I BoLA haplotype and magnitude of response within this group of unrelated animals. However, the magnitude of the T cell responses by two half-sib family groups segregated with BoLA haplotypes inherited from the sire. In contrast no significant correlation with antibody responses in vivo could be demonstrated. We suggest that the observed variation in T cell response is linked to bovine MHC class II immune response (Ir) genes.     

Production and characterization of alloantisera specific for bovine class II major histocompatibility complex antigens

Ten alloantisera defining five major histocompatibility complex (MHC) class II specificities of the bovine lymphocyte antigen (BoLA) complex were produced and characterized. Eight antisera defining four of the specificities were generated by immunizing cattle with class I compatible-class II incompatible lymphocytes. The alloantiserum defining the fifth class II specificity was produced by skin implant immunization. A pregnancy serum specific for one of the class II specificities was also identified. The class II antigens recognized by these antisera were designated 'Dx' antigens to indicate that they are BoLA-D region antigens encoded by one or more undetermined class II loci. The molecules identified by the alloantisera are heterodimers composed of a 34-kd alpha and a 26- to 28-kd beta chain, and are expressed on B-lymphocytes but not on resting T-lymphocytes. In family studies the BoLA-Dx antigens segregated in linkage with the BoLA-A locus alleles. Most of the BoLA-A alleles present in the Cornell Holstein herd at a high frequency were found to exist in gametic association with two or more serologically defined class II haplotypes. On the basis of a population study it was determined that three pairs of class I and class II alleles (w10-Dx4, w31-Dx5, and c3-Dx2) were present in the Cornell herd at significantly increased frequencies.     

Diversity of MHC class I haplotypes in cynomolgus macaques

Cynomolgus macaques are widely used as a primate model for human diseases associated with an immunological process. Because there are individual differences in immune responsiveness, which are controlled by the polymorphic nature of the major histocompatibility (MHC) locus, it is important to reveal the diversity of MHC in the model animal. In this study, we analyzed 26 cynomolgus macaques from five families for MHC class I genes. We identified 32 Mafa-A, 46 Mafa-B, 6 Mafa-I, and 3 Mafa-AG alleles in which 14, 20, 3, and 3 alleles were novel. There were 23 MHC class I haplotypes and each haplotype was composed of one to three Mafa-A alleles and one to five Mafa-B alleles. Family studies revealed that there were two haplotypes which contained two Mafa-A1 alleles. These observations demonstrated further the complexity of MHC class I locus in the Old World monkey.     

src-specific immunity in inbred chickens bearing v-src DNA- and RSV-induced tumors

Serological data identify a single major histocompatibility complex (MHC) class I locus in cattle. Molecular data, however, demonstrate the presence of at least two cattle MHC (BoLA) class I loci. To investigate the number of transcribed BoLA class I genes, we amplified cattle cDNA by using a single MHC class I-specific primer that hybridized to a conserved region of exon 4 and a non-specific 3 primer. Six BoLA class I cDNAs have been cloned and sequenced from a Bos taurus bull heterozygous for BoLA class I serological antigens, demonstrating the presence of a minimum of three loci. Sequence comparisons suggested that one of these cDNAs may be an unexpressed allele or the product of a nonclassical locus.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U01186 and U01187.     

Human cytomegalovirus US2 endoplasmic reticulum-lumenal domain dictates association with major histocompatibility complex class I in a locus-specific manner

The human cytomegalovirus-encoded US2 glycoprotein targets endoplasmic reticulum-resident major histocompatibility complex (MHC) class I heavy chains for rapid degradation by the proteasome. We demonstrate that the endoplasmic reticulum-lumenal domain of US2 allows tight interaction with class I molecules encoded by the HLA-A locus. Recombinant soluble US2 binds properly folded, peptide-containing recombinant HLA-A2 molecules in a peptide sequence-independent manner, consistent with US2's ability to broadly downregulate class I molecules. The physicochemical properties of the US2/MHC class I complex suggest a 1:1 stoichiometry. These results demonstrate that US2 does not require additional cellular proteins to specifically interact with soluble class I molecules. Binding of US2 does not significantly alter the conformation of class I molecules, as a soluble T-cell receptor can simultaneously recognize class I molecules associated with US2. The lumenal domain of US2 can differentiate between the products of distinct class I loci, as US2 binds several HLA-A locus products while being unable to bind recombinant HLA-B7, HLA-B27, HLA-Cw4, or HLA-E. We did not observe interaction between soluble US2 and either recombinant HLA-DR1 or recombinant HLA-DM. The substrate specificity of US2 may help explain the presence in human cytomegalovirus of multiple strategies for downregulation of MHC class I molecules.