CHARACTERIZATION, SPECIFICITY, AND IGV GENE USAGE OF ANTILYMPHOCYTE MONOCLONAL-ANTIBODIES FROM PERINATAL MICE

Previous studies have shown that CD5+ B cells predominate during development of the immune system and frequently secrete self-reactive antibodies, some of which appear to influence the development of the adult B cell repertoire. In addition, we now show that a high frequency of perinatally derived antibodies react with lymphocytes. Hybridomas derived from perinatal liver and splenic B cells and from spleens of adult BALB/c and C57BL/6 mice were screened by immunofluorescence on thymocytes. Anti-lymphocyte antibodies, all of the IgM isotype, were detected at a high frequency from perinatal fusions, but none were obtained from adult mice. These anti-lymphocyte mAbs were heterogeneous because they stained different subsets of peripheral T and B lymphocytes. Although the antigens recognized by these mAbs were heterogeneous with respect to their sensitivity to a variety of enzymes, 13 of the 19 mAbs recognized epitopes which were modulated by phosphatidylinositol-phospholipase C treatment. Inhibition experiments suggested that six of these 13 mAbs shared the same molecular specificity, and that they recognized the same T cell subset (62% of CD4+ and 98% of CD8+ cells). Furthermore, three of these mAbs immunoprecipitated the same 100 kDa protein from thymocytes (70 kDa in reducing conditions). The related molecular specificity of some antilymphocyte mAbs was also reflected by their restricted V gene usage. Three of the five mAbs specific for the 100 kDa protein used very similar or identical germline SM7 V(H) genes. In addition to using the same germline D and J(H) genes, they also exhibited identical V(H) - D - J(H) joins, despite originating from distinct fusions. Analysis of light chains also showed some restriction by preferential use of germline V(kappa)4 and J(kappa)5 genes. Together, these results suggest that the restricted antibody repertoire characteristic of mouse fetal and neonatal B cells is also reflected in the production of anti-lymphocyte antibodies. These B cells appear consistently in early development, use germline V genes, and express a characteristic V(H) - D - J(H) join.