Scid Cells Research Articles

Strand breaks without DNA rearrangement in V (D)J recombination.

Somatic gene rearrangement of immunoglobulin and T-cell receptor genes [V(D)J recombination] is mediated by pairs of specific DNA sequence motifs termed signal sequences. In experiments described here, retroviral vectors containing V(D)J rearrangement cassettes in which the signal sequences had been altered were introduced into wild-type and scid (severe combined immune deficiency) pre-B cells and used to define intermediates in the V(D)J recombination pathway. The scid mutation has previously been shown to deleteriously affect the V(D)J recombination process. Cassettes containing a point mutation in one of the two signal sequences inhibited rearrangement in wild-type cells. In contrast, scid cells continued to rearrange these cassettes with the characteristic scid deletional phenotype. Using these mutated templates, we identified junctional modifications at the wild-type signal sequences that had arisen from strand breaks which were not associated with overall V(D)J rearrangements. Neither cell type was able to rearrange constructs which contained only a single, nonmutated, signal sequence. In addition, scid and wild-type cell lines harboring cassettes with mutations in both signal sequences did not undergo rearrangement, suggesting that at least one functional signal sequence was required for all types of V(D)J recombination events. Analysis of these signal sequence mutations has provided insights into intermediates in the V(D)J rearrangement pathway in wild-type and scid pre-B cells.

Aberrant immunoglobulin gene rearrangement in scid mouse bone marrow cells.

We have analyzed Ig gene rearrangement in the immunodeficient mutant mouse, CB-17 scid. Bone marrow stem cells from scid mice were cultured in the in vitro culture system of Whitlock and Witte. Ig gene rearrangement in the scid cells was studied by DNA cloning. Seven DNA clones of Ig H chain JH and DH regions were analyzed by DNA sequencing, and all the clones contained a failure in D-J joining. In the rearranged structure, both DH and JH coding sequences are either partly or completely deleted. Molecular mechanisms causing the aberrant DNA rearrangement are discussed.

PURINE ENZYME ACTIVITIES AS MARKERS OF LYMPHOCYTIC DIFFERENTIATION: STUDIES OF LYMPHOCYTES FROM HORSES HORSES WITH SEVERE COMBINED IMMUNODEFICIENCY (SCID): 116

Foals with SCID lack both identifiable B lymphocytes and functional T lymphocytes. Morphological characterization of SCID lymphocytes showed them to be large granular lymphocytes (LGL), a morphology associated with natural killer (NK) cells in human and rat systems. Like NK and T lymphocytes, SCID LGL could be maintained in continuous culture in the presence of interleukin 2 (I1-2). The distribution of cell surface antigens and purine enzymatic activities were evaluated. Surface markers on fresh SCID LGL resembled normal peripheral blood lymphocytes (PBL) except for the pan-T marker (26%, SCID; 62%, Normal). Incubation of SCID and normal PBL with IL-2 for 2 weeks increased the percentage of cells carrying the lymphoid precursor (from 10% to 55%) and the monocytic (from 3% to 52%) markers. Normal PBL, however, lost the pan-T marker (from 62% to 2%) while no change was detected for SCID cells. Purine enzymatic activities (6 enzymes) for fresh SCID cells were 2 to 10 times higher than for normal PBL. When normal PBL were maintained in culture with IL-2 for several weeks, morphology and enzymatic activities had changed to those of fresh SCID LGL. These results suggest that LGL from SCID horses may be related to NK or T lymphocytes. (Supported by USPHS grant HD 08886.)

Functional status of cells from lymphoid and myeloid tissues in mice with severe combined immunodeficiency disease.

Cells from mice with severe combined immunodeficiency disease (SCID) were tested in assays that measure myeloid and lymphoid function. Results showed that C.B-17 scid and their normal counterparts (C.B-17) have similar levels of spleen colony-forming units. The frequency of in vitro myeloid colony-forming units in C.B-17 scid spleen is elevated, but the absolute number of colony-forming units in C.B-17 scid and C.B-17 spleen is similar. The absolute number of bone marrow colony-forming units in C.B-17 scid and C.B-17 mice is comparable. Cells from C.B-17 scid spleen are consistently negative in all tests of B and T cell function. C.B-17 scid splenocytes fail to proliferate in response to T and B cell mitogens or to allogeneic lymphocytes in a one-way MLR; C.B-17 scid cells do serve as stimulators in MLR. B lymphocyte colony-forming units are absent, as are cytotoxic lymphocyte precursors and cells that can generate T cell colonies with cytotoxic progenitors. The microenvironment of the C.B-17 scid mouse is conducive to lymphocyte differentiation, because functional B and T cells are easily detectable in mice reconstituted with normal bone marrow cells. The results of this study indicate that scid specifically impairs the differentiation of stem cells into mature lymphocytes; myeloid cell differentiation is not affected.