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Most Abelson murine leukemia virus (A-MuLV)-transformed cell lines derived from scid (severe combined immune deficient) mice actively rearrange their endogenous immunoglobulin (Ig) heavy (H), but not light (L) chain variable region genes. Such cell lines express germline VH segments and other RNA transcripts that are characteristically produced by early precursor (pre)-B lymphocytes, but do not express high levels of transcripts from the germline kappa (k) constant region (C kappa) locus. However, we have derived scid A-MuLV transformants that express germline C kappa transcripts and attempt kappa gene assembly. In one case kappa gene expression and rearrangement occurred in the absence of mu H chain expression, and in another was not induced efficiently by introduction of a mu-expression vector. Although the vast majority of scid H and L chain coding sequence joins are grossly aberrant, scid A-MuLV transformants can form normal coding joints at a very low frequency. In contrast, these cells form generally normal signal sequence joins at an approximately normal efficiency. Thus, these findings mechanistically distinguish coding and signal join formation. Subcloning analyses suggest that scid A-MuLV transformants that do not attempt chromosomal coding sequence joining may have a relative survival advantage, and therefore that these events may often result in unrepaired chromosomal breakage and cell death.

Mice homozygous for an autosomal recessive mutation for the scid gene exhibit a defect that specifically impairs lymphoid differentiation but not myelopoiesis. Such mice can be cured of their lymphoid deficiency by grafts with normal bone marrow, although full reconstitution of lymphoid function is seldom obtained. Long-term bone marrow cultures (LTBMC) are devoid of all mature B and pre-B cells but contain lymphoid stem cells. We therefore reconstituted scid mice with LTBMC cells to study the kinetics of B lymphocyte reconstitution in normal and irradiated (4 Gy) scid recipients and in irradiated (9.5 Gy) co-isogenic C.B-17 mice. Detectable colony-forming B cells rapidly increased in the spleen and bone marrow of irradiated C.B-17 and irradiated scid recipients, reaching normal levels between 4 and 6 wk post-grafting. Unirradiated scid recipients showed limited reconstitution in spleen and very poor reconstitution in bone marrow. Unirradiated scid recipients also had relatively few surface Ig+ cells in spleen or bone marrow, whereas both groups of irradiated recipients had normal numbers between 4 and 6 wk post-reconstitution. Normal levels of cytotoxic T cell activity by 8 wk after reconstitution were observed only in the irradiated C.B-17 and irradiated scid recipients. Analysis of mice reconstituted with cells from LTBMC indicates that these cultures contain lymphoid stem cells with significant proliferative and self-renewal potential, and that full reconstitution of lymphoid function requires prior irradiation of the scid recipient.

scid mice lack detectable B and T lymphocytes; there are no typical pre-B cells as defined by c mu and surface markers in their bone marrow and their thymus contains only 1% of the normal number of cells. In these characters scid mice seem to lack lymphoid stem cells. However, some mice have detectable serum immunoglobulin and others develop thymomas; both observations indicate that the block in lymphoid development is not absolute. To determine whether scid mice have any B-cell precursors, we looked for pre-B cells by their ability to be transformed by Abelson murine leukemia virus (A-MuLV). Surprisingly, scid mice contain as many B-cell precursors transformable with A-MuLV as normal control mice. Cell-surface markers specific for pre-B and B cells were detected on the A-MuLV-transformed bone marrow cells of both scid and normal mice, indicating that the A-MuLV-transformed cells belong to the B lineage. Interestingly, the same surface markers were undetectable on nontransformed scid bone marrow cells. We conclude from these results that scid mice have normal numbers of early B-cell precursors but that their differentiation into functional B cells is severely impaired.

Scid mice are excellent recipients for studying the characteristics of stem cells. Sublethal irradiation not only enhances engraftment of stem cells but enables one to graft limiting numbers of cells without compromising the survival of the recipient. This enables one to estimate the frequency of stem cells by limiting dilution analysis. Compared to fresh bone marrow, LTBMC are slightly enriched for stem cells capable of reconstituting lymphoid function in scid recipients. The stem cells have self-renewal ability since bone marrow from cured primary scid recipients can cure secondary recipients. Our results indicate that lymphoid reconstitution following engraftment with LTBMC occurs from a lymphoid-restricted stem cell; similar restricted stem cells also exist in normal bone marrow.