Evidence for Ig light chain isotype exclusion in shark b lymphocytes suggests ordered mechanisms

Unlike most vertebrates, the shark IgL gene organization precludes secondary rearrangements that delete self-reactive VJ rearranged genes. Nurse sharks express four L chain isotypes, k, l, s, and s-2, encoded by 35 functional minigenes or clusters. The sequence of gene activation/expression and receptor editing of these isotypes have not been studied. We therefore investigated the extent of isotypic exclusion in separated B cell subpopulations. Surface Ig (sIg)k-expressing cells, isolated with mAb LK14 that recognizes Ck, carry predominantly nonproductive rearrangements of other L chain isotypes. Conversely, after depletion with LK14, sIgM+ cells contained largely nonproductive k and enrichment for in-frame VJ of the others. Because some isotypic inclusion was observed at the mRNA level, expression in the BCR was examined. Functional l mRNA was obtained, as expected, from the LK14-depleted population, but was also in sIgk+ splenocytes. Whereas l somatic mutants from the depleted sample displayed evidence of positive selection, the l genes in sIgk+ cells accumulated bystander mutations indicating a failure to express their products at the cell surface in association with the BCR H chain. In conclusion, a shark B cell expresses one L chain isotype at the surface and other isotypes as nonproductive VJ, sterile transcripts, or in-frame VJ whose products may not associate with the H chain. Based on the mRNA content found in the B cell subpopulations, an order of L chain gene activation is suggested as: s-2 followed by k, then s and l.