Domenico Bernoco

Monoclonal antibodies that distinguish bovine T and B lymphocytes.

The specificities of three monoclonal antibodies (MAbs) were investigated using microcytotoxicity, fluorescence microscopy and laser flow cytometry (LFC) techniques. By microcytotoxicity, bovine thymocytes (n = 4) were estimated to be 85% B26A+, 4% TH21A+, and 1% H4+. Nylon wool enriched peripheral blood T lymphocytes (n = 3) were 90% B26A+, 10% TH21A+ and 10% H4+. Adherent B cell enriched fractions (n = 3) were 10% B26A+, 90% TH21A+ and 90% H4+. The two fluorochrome method was used to simultaneously identify lymphocytes that were sIg+ and MAb+. In these experiments, 92% of all sIg+ cells were H4+. An identical result was obtained for TH21A. 85% of all sIg- cells were B26A+. Using LFC, the mean percentages of sIg+, H4+ and TH21A+ PBL (n = 5) were not significantly different. B26A recognized a significantly greater population of cells, equivalent to the expected percentage of T lymphocytes. LFC also revealed two relatively discrete sizes of B26A+ PBL. The larger population overlapped the size range in which sIg+, H4+, TH21A+ PBL were found. The more numerous smaller B26A+ PBL were in a size range in which few sIg+, H4+ and TH21A+ PBL were found. In a study of MAb reactions with PBL of 185 cows, it was shown that in 92% of the animals H4 and TH21A were positively correlated (r = +.93), when H4 and TH21A were negatively correlated with B26A (r = -.94 and r = -.92, respectively). These correlation coefficients indicate a converse relationship between B26A and both H4 and TH21A. The remaining 8% of the animals were heterogeneous in their expression of the H4 and TH21A markers but not the B26A marker. These results provide strong evidence that: 1) B26A is a pan-T lymphocyte MAb in cattle, 2) a small but significant degree of heterogeneity exists in the expression of the epitopes recognized by H4 and TH21A. However, both MAbs recognize all B lymphocytes of most individuals, and 3) using a variety of immunological methods these three MAbs can now reliably be used to assay bovine T and B lymphocytes.

HETEROGENEITY AND LINKAGE OF EQUINE C4 AND STEROID 21-HYDROXYLASE GENES

The fourth component of complement (C4) is polymorphic in most species studied, and is encoded by a gene or genes within the MHC. In man and mouse there are two closely linked C4 and steroid 21‐hydroxylase (21‐OH) genes. Therefore we have used Southern blotting to determine whether equine C4 and 21‐OH genes are linked. C4 restriction fragment length polymorphism (RFLP) was found with the enzymes EcoRI and BamHI. Comparison of the sizes of EcoRI‐digested fragments of genomic DNA hybridizing with C4 and 21‐OH probes revealed that equine C4 and 21‐OH genes are separated by no more than 13 kb. Further, there is no evidence of C4 and 21‐OH gene duplication in the horse. Segregation of ELA and different polymorphic forms of equine C4 suggest that C4 and 21‐OH genes are within the MHC. It is likely that equine MHC supratypes will provide improved markers of disease susceptibility.