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Induction of immunoglobulin isotype switching in cultured I.29 B lymphoma cells. Characterization of the accompanying rearrangements of heavy chain genesThe murine B cell lymphoma I.29 contains cells expressing surface IgM or IgA with identical heavy chain variable regions (9, 25, and D. Klein and J. Stavnezer, unpublished data). Purified IgM+ cells from the lymphoma have been adapted to culture and induced to switch to IgA, IgE, or IgG2 by treatment with lipopolysaccharide (LPS) or by treatment with a monoclonal anti-I.29 antiidiotype plus LPS. Clones of IgM+ cells have been obtained and induced to switch. Under optimal conditions, 30% of the cells in the culture expressed IgA 8 d after the inducers were added, and by 15 d 90% of the cells were IgA+. In actively switching cultures, up to 50% of the cells whose cytoplasm stained positively with anti-IgA stained simultaneously with anti-IgM, which indicates that the appearance of IgA+ cells in the cultures was due to isotype switching and not to clonal outgrowth. Examination by Southern blotting experiments of the Ig heavy chain genes in I.29 cells before and after switching revealed that isotype switching was accompanied by DNA recombinations that occurred within or immediately 5' to the tandemly repeated switch sequences. Within 3 d after the addition of inducers of switching, the nonexpressed chromosome underwent a variety of deletions or expansions within the S mu region, and a portion of the S alpha regions had undergone a 0.9-kb deletion. In cultures that contained at least 12% IgA+ cells, rearranged, expressed alpha genes, produced by recombination between the S mu region within the expressed mu gene and the S alpha region, were detected.
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Rearrangements and deletions of immunoglobulin heavy chain genes in the double-producing B cell lymphoma I.29The B cell lymphoma I.29 consists of a mixture of cells expressing membrane-bound immunoglobulin M (IgM) (lambda) and IgA (lambda) of identical idiotypes. Whereas most of the cells express either IgM or IgA alone, 1 to 5% of the cells in this tumor express IgM and IgA simultaneously within the cytoplasm and on the cell membrane (R. Sitia et al., J. Immunol. 127:1388-1394, 1981; R. Sitia, unpublished data). When IgM+ cells are purified from the lymphoma and passaged in mice or cultured, a portion of the cells convert to IgA+. These properties suggest that some cells of the I.29 lymphoma may undergo immunoglobulin heavy chain switching, although it is also possible that the mixed population was derived by a prior switching event in a clone of cells. We performed Southern blotting experiments on genomic DNAs isolated from populations of I.29 cells containing variable proportions of IgM+ and IgA+ cells and on a number of cell lines derived from the lymphoma. The results were consistent with the deletion model for heavy chain switching, as the IgM+ cells contained rearranged mu genes and alpha genes in the germ line configuration on both the expressed and nonexpressed heavy chain chromosomes, whereas the IgA+ cells had deleted both mu genes and contained one rearranged and one germ line alpha gene. In addition, segments of DNA located within the intervening sequence 5' to the mu gene, near the site of switch recombination, were deleted from both the expressed and the nonexpressed chromosomes. Although mu genes were deleted from both chromosomes in the IgA+ cells, the sites of DNA recombination differed on the two chromosomes. On the expressed chromosome, Smu sequences were recombined with S alpha sequences, whereas on the nonexpressed chromosome, Smu sequences were recombined with S gamma 3 sequences.
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Immunoglobulin heavy-chain switching may be directed by prior induction of transcripts from constant-region genesImmunoglobulin heavy-chain switching is effected by a DNA recombination event that replaces the C mu gene with one of the other heavy-chain constant-region (CH) genes located 3' to the C mu gene. How the specificity of this event is controlled is unknown. However, it has been shown that IgM+ cells capable of switching to specific isotypes have the corresponding unrearranged CH genes in an accessible or active chromatin state, as demonstrated by the fact that these specific CH genes are hypomethylated and are transcriptionally active. We now report that the RNAs transcribed from specific unrearranged CH genes are induced prior to switching under conditions that promote switching to these specific CH genes. For example, we find that bacterial lipopolysaccharide, which induces the IgM+ cell line I.29 mu to switch to IgA, induces transcripts from the germ-line C alpha gene(s) in I.29 mu cells prior to switch recombination. Two preparations of T-cell lymphokines (recombinant interleukin 4 and supernatant from the T-cell line 2.19, which contains interleukins 4 and 5) that promote switching to specific isotypes by lipopolysaccharide-treated spleen cells induce transcripts from the corresponding germ-line CH genes prior to expression of the new isotypes. For example, interleukin 4, which appears to be necessary for switching to IgE in vitro and in vivo, induces within 2 days large increases in germ-line C epsilon transcripts in lipopolysaccharide-treated spleen cells and in I.29 mu cells. The most straightforward interpretation of our data is that these lymphokines direct switching to specific isotypes by activating specific CH genes, making them accessible to the putative switch recombinase.
