Immunoglobulin heavy chain gene diversification in the long‐term bone marrow culture of normal mice and mice with severe combined immunodeficiency

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Abstract
The change of immunoglobulin heavy (H) chain gene configuration during the differentiation of B cells from their early precursors was investigated in long-term cultures of bone marrow cells (LTBC). Hemopoietic stem cells are maintained in LTBC described by Dexter et al. (J. Cell. Physiol. 1977. 91: 335; LTBC-D), which supports the differentiation of myeloid lineage cells but not B lineage cells. By simply shifting the culture condition to that devised by Whitlock and Witte (Proc. Natl. Acad. Sci. USA 1982. 79: 3608; LTBC-B) to support the development of B lineage cells, surface IgM-bearing (sIgM+) B cells became detectable by the 2nd week after the shift and the number quickly increased thereafter, while the number of polymorphonuclear cells and granulocyte-macrophage colony-forming cell (CFU-c) decreased rapidly. H chain gene configuration of the developing cells in the culture was examined by Southern blot analysis of Eco RI-digested DNA with a JH probe. Whereas rearranged JH gene configuration was not detectable in the DNA from LTBC-D cells, it first appeared 2 weeks after the shift, and the level of the rearrangement rapidly increased thereafter as the intensity of JH band of germ-line configuration decreased. Almost all the cells in the culture had undergone H chain gene rearrangement in both chromosomes by the 6th week after the shift. During 2 to 4 weeks after the shift, a cluster of bands spanning around 4.5–5.5 kb appeared dominant among the rearranged configurations of JH gene and then it decreased in intensity as the pattern of JH band became a more homogeneous smear. The distribution of rearranged JH bands observed during 3–4 weeks after the shift was strikingly similar to that observed in normal spleen B cells. By semi-quantitative analysis of the intensity of JH and DSP2 bands remaining in germ-line configuration, it was found that the loss of germ-line JH band was far more rapid than that of germ-line DSP2 bands in the developing B cells in vitro. This result is consistent with the conclusion obtained in B cell tumor lines that D to J assembly occurred first and was followed by V to DJ assembly in this culture system. Taken together, it is likely that the process of H chain gene diversification in this culture system may represent the actual process during B cell differentiation in vivo. Differentiative capacity of bone marrow stem cells from mouse with severe combined immunodeficiency (SCID) was also analyzed in the same culture system. LTBC-D was easily established from the bone marrow of this strain without any defect in cell recovery or the number of CFU-c. By shifting the culture condition, lymphoid cells and B220+ cells were continuously produced, though the generation of sIgM+ cells was not observed in our culture condition. On the other hand, it was shown that H chain gene rearrangement did occur in the cultures of SCID bone marrow cells though the rate was slower than that in the culture of normal bone marrow cells. However, the pattern of the distribution of JH bands on the track of SCID cells was different from that seen on the track of normal cells in that an intense cluster of bands spanning 4.5–5.5 kb in size was undetectable on the track of SCID cells. Moreover, several discrete bands which always coexisted together with the smear on the track of DNA from diversified B cell populations of normal mouse were absent from the track of DNA from the culture of SCID bone marrow. These result suggested that the scid mutation occurred on a gene coding for a molecule which is directly involved in the process of Ig gene assembly.