I. Joosten

POLYMORPHISM OF BOVINE MHC CLASS II GENES. JOINT REPORT OF THE FIFTH INTERNATIONAL BOVINE LYMPHOCYTE ANTIGEN (BOLA) WORKSHOP, INTERLAKEN, SWITZERLAND, 1 AUGUST 1992

The objectives of the Fifth International BoLA Workshop were to: standardize nomenclature, compare typing methods, and characterize BoLA haplotypes. The workshop was based on the distribution of blood samples (cells) from 60 selected cattle to 14 laboratories. Results for the class I (BoLA‐A) region are presented in this paper while results for the class II regions are presented in a separate report. Thirty‐six of the 50 previously established serological class I specificities were represented in the cell panel. However, only 30 specificities could be confirmed. Two specificities, A16 and A32, were upgraded from provisional, workshop (w) specificities to BoLA‐A locus specificities and three new specificities, w51(w28), w52 and w53(w28), were defined. The 39 specificities distinguished 30 class I haplotypes in the 60 animals. Class I isoelectric focusing proved to be a useful adjunct to the serology. Isoelectric focusing confirmed several serologically defined splits and detected splits of A15(A8), A18(A6) and A22(w49) that had not been detected by serology. Subsequently, serological support for splits of A15(A8) and A22(w49) was found.

CHARACTERIZATION OF BOVINE MHC CLASS II POLYMORPHISM USING THREE TYPING METHODS: SEROLOGY, RFLP AND IEF

Various methods, with different strengths and weaknesses, are currently used to define polymorphism of the bovine major histocompatibility complex (MHC) class II genes. A more complete characterization of bovine lymphocyte antigen (BoLA) haplotypes can be achieved by combining several of these methods. In this study BoLA class II polymorphism was characterized using three typing methods: serology, restriction fragment length polymorphism (RFLP), and isoelectric focusing (IEF). Twenty six Holstein‐Friesian and 15 Angus cattle that carried an array of serologically defined BoLA haplotypes were selected for the study. The panel included 12 BoLA complex homozygotes. The three class II typing methods recognized polymorphism associated with the same or very tightly linked genes in the DQ‐DR class II subregion. In total 25 BoLA‐A locus (class I) —DQ‐DR subregion (class II) haplotypes were defined. Three of the serological class II specificities, Dx1, Dx3, and Dx4, were associated with more than one RFLP defined DQ‐DR haplotype. The other 4 class II specificities behaved as private specificities. One BoLA haplotype was found in both Holstein and Angus cattle. Two other BoLA haplotypes defined here have previously been described in other breeds. This suggests that these haplotypes exist in strong linkage disequilibrium.

Bovine MHC class II restriction fragment length polymorphism linked to expressed polymorphism.

The present study established the association between bovine MHC class II RFLPs and polymorphism at the product level as defined by IEF of immunoprecipitates. To assess the degree of association between RFLP and expressed polymorphism as revealed by IEF analysis, two distant breeds (Swedish Red and White and Dutch Friesian) were used

Serological and biochemical detection of B-cell antigens in goats.

The production of B‐cell specific alloantisera by lymphocyte transfusion between class I matched, MLR positive goats is described. Furthermore, the usefulness of preceding IEF typing is demonstrated in the selection of immunization pairs. After suitable absorptions and cluster analysis the detected B‐cell specific antigens were designated BeDl‐BeD6. The specificity BeD3 could also be detected by two class II‐specific murine anti‐H2 monoclonal antibodies. IEF class II typing of 34 animals gave concordant results between the two techniques. One additional allelic variant could be detected by IEF typing. The detected products segregated in close linkage to the earlier described caprine class I antigens. One recombinant has been found in 78 offspring. The reactivity of cells in mixed lymphocyte cultures (MLC) was correlated with the compatibility of the test cells for their B‐cell specific antigens. Three of the B‐cell specific sera were characterized by immunoprecipitation. The precipitated antigens corresponded in molecular weight to MHC class II products as described in other species indicating that the here described caprine products are of class II nature.

THE SPECIFICITY OF ANTI‐HLA CLASS II MONOCLONAL ANTIBODIES IN CATTLE

At the Eleventh International HLA Histocompatibility Workshop, numerous anti-HLA class II monoclonal antibodies (mAb) were tested. For several of the polymorphic mAb, one epitope for binding has been mapped within the antigen-binding site of the class II molecules. Screening of the available bovine DRB3 and DQB exon 2 sequences revealed that some of the key amino acid (AA) motifs of these epitopes were present in cattle as well, and the question was raised whether this sharing of key AA motifs might cause interspecies cross-reactivity. Eight polymorphic anti-HLA class II mAb (seven anti-HLA DRB1 and one anti-HLA DQB) were selected for analysis of their reactivity towards bovine lymphocytes. In addition, the monomorphic anti-HLA class II mAb, 7.5.10.1, was selected for analysis, as this mAb was described to detect class II polymorphism in cattle. Flow cytometry and lymphocyte microcytotoxicity testing revealed that five of the polymorphic anti-HLA mAb were reactive with bovine lymphocytes. Furthermore, the anti-bovine reactivity of 7.5.10.1 was confirmed. These findings were supported by biochemical analysis. The anti-bovine reaction of the anti-HLA mAb did not correspond with the expected reaction, which was based on the presence of the AA, postulated to be responsible for recognition. Therefore, we suggest that the patterns of reactivity of the anti-HLA mAb are not always determined by one epitope.