Gayle C. Bosma

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.

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.

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.

Limited Clonal Diversity of Serum Immunoglobulin in Leaky Scid Mice

Mice homozygous for the scid mutation are characterized by the absence of functional B and T cells (Bosma et al. 1983; Dorshkind et al. 1984; Custer et al. 1985). Most scid mice do not possess detectable serum immunoglobulin (Bosma et al. 1983). The exact nature of the scid defect is not yet known but considerable evidence suggests that it may be due to a defective recombinase system which precludes the functional rearrangement of antigen receptor genes for B and T cells (Schuler et al. 1986; Kim et al. 1988; Malynn et al. 1988; Okazaki et al. 1988).

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.

Reconstitution of scid mice by injection of varying numbers of normal fetal liver cells into scid neonates.

In our original report (Bosma et al. 1983) we showed that young adult C.B-17scid/scid mice (scid mice) could be successfully engrafted with bone marrow cells of normal BALB/c donors. The reconstituted recipients readily produced serum immunoglobulin heavy (Igh) chain allotype of the donor cells, indicating that they could support the differentiation of normal lymphocytes. However, the extent of reconstitution in these and other similarly reconstituted scid mice was variable and often incomplete (Custer et al. 1985). Furthermore, the extent of reconstitution did not correlate with the number of donor cells injected (1−5 × 106 bone marrow cells) nor with the elapsed time between injection and analysis. In agreement with these findings, Fulop and Phillips (1986) reported that injection of variable numbers of normal bone marrow cells into young adult scid mice seldom resulted in normal numbers of lymphoid cells, surface Ig+ cells or colony-forming B cells. To ensure complete reconstitution, they found it necessary to irradiate scid recipients prior to cell transfer (Fulop and Phillips 1986).

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.

Abnormal V(D)J Recombination in Murine Severe Combined Immune Deficiency: Absence of Coding Joints and Formation of Alternative Products

We have studied the VDJ recombination reaction in early B and T cells from mice homozygous for the severe combined immune deficiency (scid) mutation. Schuler et al. (1986) previously found abnormal deletions by Southern blot analysis at rearranged IgH and TCR β alleles in transformed B and T lymphocytes from seid mice. We have recently completed a study (Lieber et al. 1988a) in which we transfected scid lymphoid cell lines with VDJ recombination substrates that remain extrachromosomal. These substrates allow large numbers of recombinant reaction products to be collected (Hesse et al. 1987; Lieber et al. 1987), thereby permitting detailed analysis of the VDJ recombination reaction in scid lymphoid cells.