Pamela B. Nakajima

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.

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.

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.

Antigen Receptor Editing in Anti-DNA Transitional B Cells Deficient for Surface IgM

In response to encounter with self-Ag, autoreactive B cells may undergo secondary L chain gene rearrangement (receptor editing) and change the specificity of their Ag receptor. Knowing at what differentiative stage(s) developing B cells undergo receptor editing is important for understanding how self-reactive B cells are regulated. In this study, in mice with Ig transgenes coding for anti-self (DNA) Ab, we report dsDNA breaks indicative of ongoing secondary L chain rearrangement not only in bone marrow cells with a pre-B/B cell phenotype but also in immature/transitional splenic B cells with little or no surface IgM (sIgM−/low). L chain-edited transgenic B cells were detectable in spleen but not bone marrow and were still found to produce Ab specific for DNA (and apoptotic cells), albeit with lower affinity for DNA than the unedited transgenic Ab. We conclude that L chain editing in anti-DNA-transgenic B cells is not only ongoing in bone marrow but also in spleen. Indeed, transfer of sIgM−/low anti-DNA splenic B cells into SCID mice resulted in the appearance of a L chain editor (Vλx) in the serum of engrafted recipients. Finally, we also report evidence for ongoing L chain editing in sIgMlow transitional splenic B cells of wild-type mice.

Two distinct populations of H chain-edited B cells show differential surrogate L chain dependence.

Developing autoreactive B cells may edit (change) their specificity by secondary H or L chain gene rearrangement. Recently, using mice hemizygous for a site-directed VDJH and VJκ transgene (tg) encoding an autoreactive Ab, we reported ongoing L chain editing not only in bone marrow cells with a pre-B/immature B cell phenotype but also in immature/transitional splenic B cells. Using the same transgenic model, we report here that editing at the H chain locus appears to occur exclusively in bone marrow cells with a pro-B phenotype. H chain editing is shown to involve VH replacement at the tg allele or VH rearrangement at the wild-type (wt) allele when the tg is inactivated by nonproductive VH replacement. VH replacement/rearrangement at the tg/wt alleles was found to entail diverse usage of VH genes. Whereas the development of edited B cells expressing the wt allele was dependent on the λ5 component of the surrogate L chain, the development of B cells expressing the tg allele, including those with VH replacement, appeared to be λ5 independent. We suggest that the unique CDR3 region of the tg-encoded μH chain is responsible for the λ5 independence of tg-expressing B cells.

Development of Functional B Cells in a Line of SCID Mice with Transgenes Coding for Anti-Double-Stranded DNA Antibody

Deletion or inactivation of anti-self (DNA) B cells has been reported in non-autoimmune mice bearing Ig transgenes that code for Abs with specificity for dsDNA or ssDNA. However, we report a case in which anti-dsDNA B cells appear to escape both deletion and inactivation. We show that B cells (B220+IgM+) can develop in non-autoimmune SCID mice bearing two site-directed transgenes, 3H9(56R) and Vκ8, that together code for an anti-dsDNA Ab. The B cells appear inactive, because the mice (56RVκ8 SCID mice) generally lack serum Ig. However, 56RVκ8 SCID mice are able to produce IgG Ab with specificity for dsDNA when they become “leaky” for T cells or are reconstituted with exogenous T cells from B cell-deficient JH−/− donors. Thus, anti-dsDNA B cells that escape deletion in 56RVκ8 SCID mice appear fully functional and can differentiate, class switch, and give rise to IgG-producing cells in the presence of T cells and self-Ag.

Variable Diversity Joining Recombination: Nonhairpin Coding Ends in Thymocytes of SCID and Wild-Type Mice

Initiation of V(D)J recombination results in broken DNA molecules with blunt recombination signal ends and covalently sealed (hairpin) coding ends. In SCID mice, coding joint formation is severely impaired and hairpin coding ends accumulate as a result of a deficiency in the catalytic subunit of DNA-dependent protein kinase, an enzyme involved in the repair of DNA double-strand breaks. In this study, we report that not all SCID coding ends are hairpinned. We have detected open Jδ1 and Dδ2 coding ends at the TCRδ locus in SCID thymocytes. Approximately 25% of 5′Dδ2 coding ends were found to be open. Large deletions and abnormally long P nucleotide additions typical of SCID Dδ2-Jδ1 coding joints were not observed. Most Jδ1 and Dδ2 coding ends exhibited 3′ overhangs, but at least 20% had unique 5′ overhangs not previously detected in vivo. We suggest that the SCID DNA-dependent protein kinase deficiency not only reduces the efficiency of hairpin opening, but also may affect the specificity of hairpin nicking, as well as the efficiency of joining open coding ends.

Double-Strand Breaks Associated with V(D)J Recombination at the TCRδ Locus in Murine Thymocytes

Somatic recombination events are responsible for assembling the variable regions of Immunoglobulin and T cell receptor genes from germline-encoded DNA segments (Tonegawa, 1983; Lewis and Gellert, 1989). These rearrangements are mediated by a recombination activity that recognizes signal sequences (consisting of conserved heptamer and nonamer elements separated by nonconserved spacer regions of 12 or 23 nucleotides) located adjacent to the V, D, and J coding segments. Although the mechanism of the reaction remains obscure, recombination is thought to involve either single-stranded or double-stranded cleavage at the border between a signal heptamer and a coding segment, followed by rejoining of the DNA ends in a new configuration (Alt and Baltimore, 1982; Lewis and Gellert, 1989).

Chromosome 14 in B10.A(18R) mice is recombinant and includes Tcra-Va alleles

Analysis of mouse Tcr genes has previously defined at least five different Tcra-V haplotypes among inbred strains of mice. For mice of the Tcra-Vb haplotype, including C57BL/10 (B10), T-cell expression of the Tcra-V11 gene subfamily can be detected with a monoclonal antibody, 1.F2. In the course of further characterizing the specificity of 1.F2, we found that it fails to recognize Tcra-V11-expressing T-cell hybrids derived from the B10 congenic strain, B10.A(18R)/SgIcr. Moreover, staining analysis indicated that the Va11 epitope recognized by 1.F2 is not expressed by peripheral T cells from several different B10.A(18R) colonies with the exception of that at the Research Institute of Scripps Clinic. Nucleotide sequences were determined for cDNA representing rearranged Tcra-V11 genes from two independent, B10.A(18R)/SgIcr derived T-cell hybrids. The two Tcra-V11 gene segments were identical and the predicted amino acid sequence differed by at least five residues from Tcra-V11 sequences previously obtained from B10.A mice. Southern blot analysis of restriction fragment length polymorphisms (RFLP) associated with Tcra-V11, as well as Tcra-V1, subfamily genes revealed that the B10.A(18R) mouse has inherited Tcra-Va alleles rather than the expected Tcra-Vb alleles from the B10 strain. RFLP analysis of the Rib-1 locus, located in close proximity to the Tcra locus on chromosome 14, showed that B10.A(18R) carries the Rib-1b allele from B10. These results indicate that the B10.A(18R) mouse has inherited a recombinant chromosome 14 with a recombination event having occured between the Rib-1 locus and the Tcra-Va gene subfamilies examined. Inheritance of Tcra-Va alleles in B10.A(18R) probably originated from strain 129/J which breeding records show was used in the first cross with B10.A in the production of B10.A(18R) and which we found exhibits Tcra-V11a RFLPs.