C H gene rearrangements in IgM-bearing B cells and in the normal splenic DNA component of hybridomas making different isotypes of antibody

Abstract

To probe mechanisms operating at the C H gene locus during normal B lymphocyte differentiation, we have used cloned probes for the constant region genes C μ, C γ1, and C α to analyze the immunoglobulin heavy chain genes of three kinds of cell populations in successive stages of B cell development. These are IgM-bearing B lymphocytes from the normal spleen of unprimed mice, hybridomas prepared by fusing spleen cells from antigen-primed mice with the SP2 0 permanent cell line and selected to secrete one of five different isotypes (IgM, IgG3, IgG1, IgG2 and IgA) and a set of plasmacytoma lines. The IgM-bearing B cells carry C μ genes with rearrangements between V H and J H genes on both chromosomes even though only one chromosome is expressed; clearly, allelic exclusion cannot be explained by the lack of C H gene rearrangement on the nonexpressed chromosome. The normal splenic DNA component of antibody-secreting hybridomas displays rearrangements between J H and C μ genes as well as among C H genes other than C μ, with concomitant deletion of C H genes 5′ to those expressed. These C H rearrangements and deletions are likely to accompany the isotype switching process and may occur on both expressed and nonexpressed chromosomes. We used hybridomas (spleen-derived), which secrete primarily IgM, and plasmacytomas (gut-derived), which secrete primarily IgA, to represent plasma cells in early and late stages of differentiation, respectively. A direct comparison of hybridomas and plasmacytomas making the same products (IgG3 or IgG1) indicates that hybridomas display a low frequency ( 2 12 ) of nonexpressed C α gene rearrangements in contrast to the high frequency ( 7 10 ) displayed by plasmacytomas. We propose that C H gene switching rearrangements and deletions may occur successively along the C H gene locus, involving any of the undeleted genes at each step. These can occur on both expressed and nonexpressed chromosomes during the normal clonal outgrowth of a B cell line in vivo, and would result in the accumulation of both productive and nonproductive rearrangements of the presumed last C H gene, C α.