Analysis of alloantisera against bovine lymphocytes. Joint report of the 1st International Bovine Lymphocyte Antigen (BoLA) Workshop

The results and agreements of the 1st international BoLA workshop, held in Edinburgh, Scotland in August 1978, are reported. Most of these concern the results from a comparison test of 249 alloantisera to bovine lymphocytes, the antisera being contributed by 9 laboratories. These sera were compared directly in Edinburgh on a panel of lymphocytes from 130 cattle of 21 breeds. In the micro-lymphocytotoxicity test used 75% of the sera reacted. Sixty eight of these sera were grouped into clusters according to their reaction patterns against the lymphocyte panel. Eleven of these clusters were clearly defined and were given workshop BoLA designations. In addition 22 sera were assigned to subgroups of the agreed clusters. There was no evidence that the method of production of the sera had any effect on their specificity.
Although genetic data was not available, the phenotypes of the test panel of lymphocytes are consistent with the clusters detecting antigens controlled by multiple alleles at a single autosomal locus. It was agreed to name the genetic region where this putative locus is located BoLA (bovine lymphocyte antigen).     

Joint Report of the Third International Bovine Lymphocyte Antigen (BoLA) Workshop, Helsinki, Finland, 27 July 1986

Summary. Two hundred and eighty-two alloantisera were submitted by 20 participating laboratories from 13 countries and tested against lymphocytes of 1298 cattle. The cell panel consisted of samples from 38 Bos taurus breeds, 11 Bos taurus crossbreeds, 4 Bos indicus breeds, 6 Bos taurus X Bos indicus , and a variety of other crossbred populations. Using a standardized lymphocytotoxicity test, all 17 previously identified BoLA specificities were confirmed. The workshop produced agreement on 16 new lymphocyte alloantigenic specificities. Three of the new specificities behaved as splits of previously identified BoLA specificities. Four of the new specificities behaved as alleles at the agreed BoLA-A locus. Seven new specificities are tentatively assigned to the BoLA-A locus but require further definition. Two new specificities may represent products of a second closely-linked BoLA locus.     

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.     

Sources of bovine lymphocyte antigen (BoLA) typing reagents*

Sera from about 1000 cows were tested for cytotoxicity against a panel of up to 100 lymphocyte samples. Cytotoxic antibodies presumably resulting from trans-placental immunization of the cow by her calf were found in about 45% of these sera. The antibody titers of sera from parous cows rarely exceed 4², some persisted for over one year, but decreased notably at calving. Thirty-five immune sera were also produced by alloimmunization with lymphocytes. They usually reached peak titers of up to 4⁴ at 2 or 3 weeks after the initial immunization. Subsequent immunizations produced sera with very high titers but they were much more polyspecific. High-titered antibodies were also produced by skin graft recipients. Useful cytotoxic antibodies were found in 19 of 111 colostrum whey samples. Studies on 13 dam-calf pairs showed that the newborn calf may acquire cytotoxic antibodies from its mother's colostrum, but the only cytotoxic antibodies detectable in this calf s serum are those not directed against its own lymphocyte antigens. It is concluded that efficient lymphocyte typing requires antibodies from a variety of sources.     

The definition of five B lymphocyte alloantigens closely linked to BoLA class I antigens

B lymphocyte alloantigens in cattle were identified by serological analysis. Alloantisera were raised by skin implant immunization or leucocyte immunization and were absorbed with platelets to reduce class I-specific antibody activity. Leucocyte absorptions were done to reduce the complexity of some antisera. A panning technique was used to prepare B-enriched and B-depleted lymphocytes. Antisera which displayed anti-B cell activity over a number of dilutions were tested against 115 Charolais cattle, and 13 antisera were used to define five B lymphocyte alloantigens. These antigens were present on B lymphocytes but did not appear to be present, at least at the same density, on the majority of T lymphocytes or platelets. Family studies suggested that these antigens are coded by one or two loci which are closely linked to the bovine class I loci. These results suggest the five antigens are class II antigens of the major histocompatibility complex (MHC) of cattle.     

Genetic variation in BoLA microsatellite loci in Portuguese cattle breeds

Major histocompatibility complex (MHC) typing based on microsatellites can be a valuable approach to understanding the selective processes occurring at linked or physically close MHC genes and can provide important information on variability and relationships of populations. Using microsatellites within or in close proximity with bovine lymphocyte antigen (BoLA) genes, we investigated the polymorphisms in the bovine MHC, known as the BoLA, in eight Portuguese cattle breeds. Additional data from non-BoLA microsatellite loci were also used to compare the variability between these regions. Diversity was higher in BoLA than in non-BoLA microsatellites, as could be observed by the number of alleles, allelic richness and observed heterozygosity. Brava de Lide, a breed selected for aggressiveness and nobility, presented the lowest values of observed heterozygosity and allelic richness in both markers. Results from neutrality tests showed few statistically significant differences between the observed Hardy–Weinberg homozygosity ( F ) and the expected homozygosity ( F _E), indicating the apparent neutrality of the BoLA microsatellites within the analysed breeds. Nevertheless, we detected a trend of lower values of observed homozygosity compared with the expected one. We also detected some differences in the levels of allelic variability among the four BoLA microsatellites. Our data showed a higher number of alleles at the BoLA-DRB3 locus than at the BoLA-DRBP1 locus. These differences could be related to their physical position in the chromosome and may reflect functional requirements for diversity.     

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.     

Serological and genetic identification of a bovine B lymphocyte alloantigen system

A two-colour fluorescence micro cytotoxicity test was used to screen antisera for antibodies specific for bovine B lymphocytes. A total of 114 cattle alloantisera were screened against peripheral blood lymphocytes from 100 unrelated individuals. Anti-B lymphocyte activity was detected in 47 antisera. Cytotoxic antibodies to antigens other than B lymphocyte specific antigens were removed by absorbing the antisera with buffy coat cells or platelets isolated from whole blood. Selected antisera were used to type paternal half-sib families. The presence of a polymorphic, MHS-linked antigen system on B lymphocytes was demonstrated. The tissue distribution and MHS linkage of these antigens suggests this system is analogous to the class II or Ia antigens of other species.     

Joint report of the Third International Bovine Lymphocyte Antigen (BoLA) Workshop, Helsinki, Finland, 27 July 1986

Two hundred and eighty-two alloantisera were submitted by 20 participating laboratories from 13 countries and tested against lymphocytes of 1298 cattle. The cell panel consisted of samples from 38 Bos taurus breeds, 11 Bos taurus crossbreeds, 4 Bos indicus breeds, 6 Bos taurus x Bos indicus, and a variety of other crossbred populations. Using a standardized lymphocytotoxicity test, all 17 previously identified BoLA specificities were confirmed. The workshop produced agreement on 16 new lymphocyte alloantigenic specificities. Three of the new specificities behaved as splits of previously identified BoLA specificities. Four of the new specificities behaved as alleles at the agreed BoLA-A locus. Seven new specificities are tentatively assigned to the BoLA-A locus but require further definition. Two new specificities may represent products of a second closely-linked BoLA locus.     

Report of the First Workshop on the Genetic Map of Bovine Chromosome 23

A report of the first workshop on the genetic map of bovine chromosome 23 (BTA23) is given. Five laboratories contributed data from 29 loci, including a total 11586 informative genotypes. The combined pedigrees represented 1930 potentially informative meioses. Eighteen of the 29 loci were common to two or more data sets and were used to construct a framework linkage map of BTA23. Twelve of the 18 could be ordered on the linkage map with a likelihood ratio of greater than 1000:1. Thus, a low resolution consensus map was constructed with a high level of support for order. The sex-averaged, female and male maps span 54.5, 52.7 and 55.8 cM, respectively. Sex-specific differences in recombination frequency were identified for eight pairs of framework loci. Average genetic distance between framework loci on the sex-averaged map is 5.0 cM.     

Joint report1 of the First International Workshop on Lymphocyte Alloantigens of the Horse held 24–29 October 1981

Six equine lymphocyte alloantigen (ELA) specificities were defined by an international antiserum comparison test and workshop held in 1981. Twelve laboratories from four countries submitted 195 antisera for analysis. The antisera were exchanged among the 12 laboratories and tested in a standard lymphocyte microcytoxicity assay against the isolated lymphocytes at 1009 horses of several breeds. The data was pooled and analysed by a single computer analysis. The calculated χ² values of all cells with all antisera provided comparisons between antisera. Fifteen antisera clusters were formed by this analysis, but only six of these clusters met the criteria established by the workshop for the identification of ELA antigens. No horses of the cell panel positively reacted with more than two of these six specificities. The consensus of the participants, although not substantiated in this workshop, was that these six clusters of antisera define alleles of a single genetic region, the ELA region, and it is likely that this genetic region is the major histocompatibility complex of the horse.     

Breed differences in the distribution of BoLA-A locus antigens in American cattle

A total of 627 cattle representing seven breeds from south central Nebraska, USA were tested for 37 BoLA antigens which behave as products of 37 distinct alleles of the class I BoLA-A locus. Four antigens were absent from all breeds tested. The other antigens showed marked and statistically significant differences in breed distribution. There was no evidence for blank (null) alleles. The number of alleles in each breed ranged from 10 to 20. The Hereford and Simmental populations tested were less polymorphic than the Angus, Brown Swiss, Charolais, Gelbvieh and Limousin populations.     

Analysis of alloantisera against bovine lymphocytes. Joint report of the 1st International Bovine Lymphocyte Antigen (BoLA) workshop

The results and agreements of the 1 international BoLA workshop, held in Edinburgh, Scotland in August 1978, are reported. Most of these concern the results from a comparison test of 249 alloantisera to bovine lymphocytes, the antisera being contributed by 9 laboratories. These sera were compared directly in Edinburgh on a panel of lymphocytes from 130 cattle of 21 breeds. In the microlymphocytotoxicity test used 75% of the sera reacted. Sixty eight of these sera were grouped into clusters according to their reaction patterns against the lymphocyte panel. Eleven of these clusters were clearly defined and were given workshop BoLA designations. In addition 22 sera were assigned to subgroups of the agreed clusters. There was no evidence that the method of production of the sera had any effect on their specificity. Although genetic data was not available, the phenotypes of the test panel of lymphocytes are consistent with the clusters detecting antigens controlled by multiple alleles at a single autosomal locus. It was agreed to name the genetic region where this putative locus is located BoLA (bovine lymphocyte antigen).     

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.     

Nomenclature for factors of the BoLA system, 1996: report of the ISAG BoLA Nomenclature Committee

The BoLA (bovine lymphocyte antigen) Nomenclature Committee met during the 1994 and 1996 conferences of the International Society for Animal Genetics to define a sequence-based nomenclature system for genes of the BoLA system. The rules for acceptance of new sequences are described and names are assigned to the sequenced alleles of the class II genes DRA, DRB1, DRB2, DRB3, DQA, DQB, DYA, DIB, DMA and DMB . The assignment of BoLA class I sequences to loci will be considered at a later workshop when further sequencing/mapping data are available.     

Joint Report of the Second International Workshop on Lymphocyte Alloantigens of the Horse, held 3–8 October 1982

The Second International Workshop on Lymphocyte Alloantigens of the Horse was held 3–8 October 1982. At this workshop, the 6 specificities identified at the first workshop were confirmed and an additional 5 new specificities were identified and given workshop nomenclature. Four of the new specificities, products of the ELA locus, were named ELA-W7, W8, W9, and W10. An additional specificity, designated ELY-2.1, is the product of a locus independent of the ELA locus.
Cell isolation methods were compared at this workshop, Technical variation in methods clearly affected reactivity of many reagents. However, when highly selected reagents were used, antigen assignment did not differ regardless of the cell isolation method. Based on the comparison of methods, isolation procedures in which thrombin was used were more effective than those relying on carbonyl iron or slow centrifugation.     

Identification of BoLA-DRB3.2 alleles in Korean native cattle (Hanwoo) and Holstein populations using a next generation sequencer

Bovine leucocyte antigen (encoded by BoLA) has been widely studied to identify the association with many traits related to immunity. Exon2 of BoLA-DRB3 is extremely polymorphic, and more than 100 alleles have been identified. We investigated polymorphisms of BoLA-DRB3.2 in Korean native cattle and Holstein populations using a next generation sequencer of the GS-FLX Titanium system. We found 38 alleles including 11 new alleles (BoLA-DRB3*1303, *4702, *7101, *7501, *7201, *7301, *7601, *1104, *7701, *7401 and *50021) in Hanwoo, and nine alleles including one new allele (BoLA-DRB3*7601) in Holstein. The 454 sequencing method is a promising alternative technology for high throughput genotyping of BoLA-DRB3.2 because of its technical advantages that allow it to overcome the disadvantages of sequence-based typing methods.     

Joint Report of the First International Comparison Test on Swine Lymphocyte Alloantigens (SLA)

The first international comparison test on swine lymphocyte alloantigens (SLA) was held in Helsinki, Finland in July 1986. The results reported were based on a comparison of 157 alloantisera originating from six laboratories. The antisera were tested against a selected panel of 264 lymphocyte samples belonging to four laboratories. The most common breeds in Europe were chosen for this first comparison test (Landrace and Large White). Eighteen of the 31 previously known specificities were confirmed and a new nomenclature was established.     

Characterization of naturally processed and presented peptides associated with bovine major histocompatibility complex (BoLA) class II DR molecules

Differential regulation of genetic resistance to infectious disease may partially be explained by variation in the binding affinity and the repertoire of pathogen-derived antigenic peptides associated with major histocompatibility complex (MHC) molecules. In this study, we investigated characteristics of peptides that bind to the bovine MHC allele BoLA-DRB3*2703, which is associated with occurrence of clinical mastitis in Holstein dairy cattle, and assigned a putative peptide-binding motif to this allele. This was achieved by in vitro expression of allele *2703 as well as a control allele, BoLA-DRB3*1201 which is present at high frequency in Holsteins. Transfected cell lines alone (for allele *1201) or in combination with blood mononuclear cells from an animal homozygous for allele *2703 were used as the source of naturally processed and presented peptides. Subsequent to elution of peptides from BoLA-DR+ cells, their sequences were determined by electrospray ionization mass spectrometry. Eluted peptides were between 13 and 20 amino acids long and the majority were in sets of overlapping sequences. These peptides were derived from intra- and extracellular proteins, as well as foreign proteins present in the culture medium. Some peptides had originated from molecular chaperones present in the endoplasmic reticulum, such as ER-60 and GRP78, pointing to some degree of overlap and cross-sampling between MHC class I and class II antigen presentation pathways. Consistent with reports of human and mouse MHC class II-associated peptides, putative peptide-binding motifs could be assigned to alleles *2703 and *1201, comprising a hydrophobic or an aromatic residue at relative position 1, a hydrophobic residue at position 4 and a small residue at position 6 of the eluted peptides. These findings provide the foundation for future studies of molecular mechanisms of MHC-disease associations of cattle.     

Joint Report of the Fourth International Workshop on Lymphocyte Alloantigens of the Horse, Lexington, Kentucky, 12–22 October 1985

Summary. The workshop consisted of 12 monthly cell exchanges of full-sibling families among the 10 participating laboratories. A total of 33 parents, 52 offspring and five unrelated horses were typed by each laboratory using local antisera. The raw data were submitted for central analysis before any identification of the animals was revealed.
Confidence derived from the consistent agreement between the laboratories on the assignment and segregation of the first 10 ELA-W specificities led to the removal of the W (workshop) notation and acceptance of full status as locus A antigens. The seemingly supertypic W11 specificity, however, remained unchanged.
Ten additional specificities were seen to segregate with the ELA system, suggesting either splits of previously described specificities or products of linked loci. The workshop (W) notation was given to the 10 specificities W12-W21, befitting their status as specificities under study.
The previously described ELY-1.1 specificity, characterized by segregation independent from the ELA system, was confirmed along with a new specificity, ELY-1.2, which behaves as an allele of ELY-1.1. For informative families, the two specificities showed codominant expression and appeared to constitute a closed, autosomal system.
The ELY-2.1 specificity was confirmed to segregate independently from the ELA-A and ELY-1 loci.