Melvin J. Bosma

Rearrangement of antigen receptor genes is defective in mice with severe combined immune deficiency

A process unique to lymphocyte differentiation is the rearrangement of genes encoding antigen-specific receptors on B and T cells. A mouse mutant (C.B-17scid) with severe combined immune deficiency, i.e., that lacks functional B and T cells, shows no evidence of such gene rearrangements. However, rearrangements were detected in Abelson murine leukemia virus-transformed bone marrow cells and in spontaneous thymic lymphomas from C.B-17scid mice. Most of these rearrangements were abnormal: approximately 80% of Igh rearrangements deleted the entire Jh region, and approximately 60% of TCR beta rearrangements deleted the entire J beta 2 region. The deletions appeared to result from faulty D-to-J recombination. No such abnormal rearrangements were detected in transformed tissues from control mice. The scid mutation may adversely affect the recombinase system catalyzing the assembly of antigen receptor genes in developing B and T lymphocytes.

V(D)J recombination : broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes

Lymphoid cells from scid mice initiate V(D)J recombination normally but have a severely reduced ability to join coding segments. Thymocytes from scid mice contain broken DNA molecules at the TCR delta locus that have coding ends, as well as molecules with signal ends, whereas in normal mice we previously detected only signal ends. Remarkably, these coding (but not signal) ends are sealed into hairpin structures. The formation of hairpins at coding ends may be a universal, early step in V(D)J recombination; this would provide a simple explanation for the origin of P nucleotides in coding joints. These findings may shed light on the mechanism of cleavage and suggest a possible role for the scid factor.

The defect in murine severe combined immune deficiency: Joining of signal sequences but not coding segments in V(D)J recombination

Pre-B and pre-T cell lines from mutant mice with severe combined immune deficiency (scid mice) were transfected with plasmids that contained recombination signal sequences of antigen receptor gene elements (V, D, and J). Recovered plasmids were tested for possible recombination of signal sequences and/or the adjacent (coding) sequences. Signal ends were joined, but recombination was abnormal in that half of the recombinants had lost nucleotides from one or both signals. Coding ends were not joined at all in either deletional or inversional V(D)J recombination reactions. However, coding ends were able to participate in alternative reactions. The failure of coding joint formation in scid pre-B and pre-T cells appears sufficient to explain the absence of immunoglobulin or T cell receptor production in scid mice.

V(D)J recombination in mouse thymocytes: Double-strand breaks near T cell receptor δ rearrangement signals

Abstract In the murine T cell receptor δ locus, V(D)J recombination events frequently involve the D2 and J1 elements. Here we report the presence of double-strand breaks at recombination signals flanking D2 in approximately 2% of thymus DNA. An excised linear species containing the sequences between D2 and J1 and a circular product of the joining of D2 and J1 recombination signals were also found. Although broken molecules with signal ends were detected, no species with coding ends could be identified. Observation of these broken molecules in thymus, but not in liver or spleen, provides the first direct evidence for an association between specific cleavage of chromosomal DNA and recombination in mammalian cells, and supports a breakage-reunion model of V(D)J recombination.

DNA-dependent Protein Kinase Activity Is Not Required for Immunoglobulin Class Switching

Class switch recombination (CSR), similar to V(D)J recombination, is thought to involve DNA double strand breaks and repair by the nonhomologous end–joining pathway. A key component of this pathway is DNA-dependent protein kinase (DNA-PK), consisting of a catalytic subunit (DNA-PKcs) and a DNA-binding heterodimer (Ku70/80). To test whether DNA-PKcs activity is essential for CSR, we examined whether IgM+ B cells from scid mice with site-directed H and L chain transgenes were able to undergo CSR. Although B cells from these mice were shown to lack DNA-PKcs activity, they were able to switch from IgM to IgG or IgA with close to the same efficiency as B cells from control transgenic and nontransgenic scid/+ mice, heterozygous for the scid mutation. We conclude that CSR, unlike V(D)J recombination, can readily occur in the absence of DNA-PKcs activity. We suggest nonhomologous end joining may not be the (primary or only) mechanism used to repair DNA breaks during CSR.

V-D-J rearrangements at the T cell receptor σ locus in mouse thymocytes of the αβ lineage

Abstract The T cell receptor (TCR) σ locus lies within the TCR α locus and is excised from the chromosome by Vα-Jα rearrangement. We show here that σ sequences persist in a large fraction of the DNA from mature CD4 + CD8 − αβ + mouse thymocytes. Virtually all σ loci in these cells are rearranged and present in extrachromosomal DNA. In immature αβ lineage thymocytes (CD3 −/lo CD4 + CD8 + ) and in CD4 + CDS − αβ + thymocytes expressing a transgene-encoded αβ receptor, rearranged σ genes are present both in chromosomal and extrachromosomal DNA. Thus, contrary to earlier proposals, commitment to the αβ lineage does not require recombinational silencing of the σ locus or its deletion by a site-specific mechanism prior to Vα-Jα rearrangement.

The catalytic subunit of DNA-protein kinase (DNA-PKcs) is not required for ig class-switch recombination

The joining of DNA ends during Ig class-switch recombination (CSR) is thought to involve the same nonhomologous end-joining pathway as used in V(D)J recombination. However, we reported earlier that CSR can readily occur in Ig transgenic SCID mice lacking DNA-dependent protein kinase (DNA-PK) activity, a critical enzymatic activity for V(D)J recombination. We were thus led to question whether the catalytic subunit of DNA-PK (DNA-PKcs) is essential for CSR. To address this issue, we asked whether class switching to different Ig isotypes could occur in a line of Ig transgenic mice lacking detectable DNA-PKcs protein. The answer was affirmative. We conclude that joining of DNA ends during CSR does not require DNA-PKcs and can occur by an alternative repair pathway to that used for V(D)J recombination.

Development of B cells in scid mice with immunoglobulin transgenes: Implications for the Control of V(D)J recombination

The inability of scid pro-B cells to progress to the pre-B and B cell stages is believed to be caused by a defective recombinase activity that fails to resolve chromosomal breaks resulting from attempted V(D)J recombination. In support of this model, we report that certain immunoglobulin transgenes, specifically those which strongly inhibit endogenous VH-to-DJH and V kappa-to-J kappa rearrangement in wild-type mice, allow scid pro-B cells to progress to the pre-B and B cell stages. This rescue of scid B cell differentiation is associated with a dramatic reduction in expression of the recombination activation genes, RAG1 and RAG2, and with reduced transcription of the kappa locus.

T Cell Leakiness in Scid Mice

FACS analysis showed that the incidence of leaky T cells increases with age, such that virtually all old scid mice (greater than 1 year) contain detectable CD3+ cells. The number of detectable T cells remained very low; individual old scid mice generally contained less than 10(5) CD3+ cells. When CD3+ populations in individual leaky mice were analyzed for expression of the T cell subset markers, CD4 and CD8, the ratios of CD4/CD8 were found to be markedly skewed relative to normal mice. This suggested the presence of very few T cell clones. Indeed, the analysis of TCR gene rearrangements in polyclonally stimulated T cell cultures revealed only 1-5 clones in the pooled spleen and lymph nodes of individual old scid mice. These studies also indicated that TCR gene rearrangements in the majority of the stimulated T cell cultures did not contain abnormal J-associated deletions that are characteristic of antigen receptor genes of scid lymphomas. Four of five alloreactive T cell clones from leaky scid mice also apparently lacked abnormal J-associated deletions in their rearranged TCR alleles. Therefore, most leaky lymphocytes appear to derive from progenitors with normal or near-normal scid recombinase activity. However, one of five leaky T cell clones (S1233) and one Con A stimulated monoclonal culture (8706) contained both normally and abnormally rearranged TCR genes. The configuration of TCR loci in such clones may reflect the ability of the defective scid recombinase to mediate normal rearrangements at a low frequency.

The Scid Mouse

Numerous investigations of the mammalian hematopoietic system in normal and pathologic states have been facilitated by the study of genetically determined immunologic dysfunctions in experimental animals. This article focuses on the scid mutation of the mouse (SCID mouse) that causes severe defects in the development of the immune system. The mutation appears to impair the recombination of antigen receptor genes, causing in the SCID mice a lack of functional T and B lymphocytes. Other hematopoietic cell types appear to develop and function normally. SCID mice readily support normal lymphocyte differentiation and can be reconstituted with normal lymphocytes from syngeneic or allogeneic mice and even partially reconstituted with human lymphocytes. They also support the growth of allogeneic and xenogeneic tumors. Thus, SCID mice might be useful for studies of both normal and abnormal lymphocyte development and function.