Impaired lymphocyte development and antibody class switching and increased malignancy in a murine model of DNA ligase IV syndrome

Hypomorphic mutations in DNA ligase IV (LIG4) cause a human syndrome of immunodeficiency, radiosensitivity, and growth retardation due to defective DNA repair by the nonhomologous end-joining (NHEJ) pathway. Lig4-null mice are embryonic lethal, and better mouse models are needed to study human LigIV syndrome. We recently identified a viable mouse strain with a Y288C hypomorphic mutation in the Lig4 gene. Lig4^Y288C mice exhibit a greater than 10-fold reduction of LigIV activity in vivo and recapitulate the immunodeficiency and growth retardation seen in human patients. Here, we have demonstrated that the Lig4^Y288C mutation leads to multiple defects in lymphocyte development and function, including impaired V(D)J recombination, peripheral lymphocyte survival and proliferation, and B cell class switch recombination. We also highlight a high incidence of thymic tumors in the Lig4^Y288C mice, suggesting that wild-type LigIV protects against malignant transformation. These findings provide explanations for the complex lymphoid phenotype of human LigIV syndrome.

Nonhomologous end joining (NHEJ) is one of the main pathways for repair of DNA double-strand breaks (DSBs) (1). The pathway comprises 6 core components: Ku70, Ku80, DNA-dependent protein kinase catalytic subunit (DNA-PK_cs), XRCC4, DNA ligase IV (LigIV) (1), and the recently discovered XRCC4-like factor (XLF) (2, 3). The end-joining process starts with the binding of Ku70/Ku80 heterodimers to the DNA ends, followed by DNA-PK_cs and other enzymes involved in end processing. LigIV is subsequently recruited to the DNA ends in complex with XRCC4 and, together with XLF, catalyzes the final end-joining reaction.

At least 10 hypomorphic mutations in the LIG4 gene have been identified in the human population, and in homozygous or compound heterozygous states, they cause a condition known as LigIV syndrome (4–11) (Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI32743DS1). The clinical presentation of the syndrome is complex and heterogeneous and may include radiosensitivity, varying degrees of lymphopenia, growth retardation, and microcephaly (4–12). At the molecular level, all the features of the LigIV syndrome are thought to arise from impaired DNA DSB repair, and the complexity of the syndrome highlights the functions of the NHEJ pathway in many physiological processes.

The embryonic lethality of the Lig4-null mouse strains (13, 14) limits their utility as models of LigIV syndrome. Other NHEJ-deficient strains — Ku70, Ku80, and DNA-PK_cs–null mice — are viable and share some of the phenotypic features of LigIV syndrome (15–19). However, the essential nature of Lig4 but not Ku70, Ku80, or DNA-PKcs attests to differing impact of these genes. Further, at the molecular level, the impact of these alleles is not equivalent to that of Lig4-null or -mutant alleles. For example, the functions of Ku70, Ku80, and DNA-PK_cs are not limited to DNA DSB repair, and all 3 proteins are also required for normal telomere maintenance (20, 21). Additionally, residual DSB rejoining can occur in the absence of DNA-PK_cs and, to a lesser extent Ku, in G₀ phase cells despite being LigIV dependent (19, 22). Furthermore, and possibly underlying the differences in DSB rejoining, the “accessibility” of DNA ends to alternative repair pathways is believed to differ in cells deficient in different NHEJ components (23, 24). The differences between Ku and LigIV deficiencies are further highlighted by the surprising observation that a loss of Ku can partly rescue the lethality of the Lig4-null strain (23). Thus, at least at the molecular level, some features of the previously characterized NHEJ-null mouse strains are distinct from those of the Lig4-null mouse and the human LigIV syndrome, suggesting that a LigIV-mutant mouse strain is a more appropriate model for human LigIV syndrome than the existing NHEJ-deficient lines.

DNA DSBs can form as the result of random DNA damage and also during programmed DNA rearrangement events, namely V(D)J recombination and Ig class switching (25, 26). The phenotypic effects of impaired V(D)J rearrangement are widely known and account for a complete arrest in lymphocyte development in Lig4-null (13) and other NHEJ-null mice (15–19) and most likely also for the lymphopenia in LigIV syndrome (4, 6–8). The phenotypic effects of the impaired repair of nonprogrammed DNA damage are more diverse and difficult to study. They include the increased apoptosis in the developing brain causing the lethality of the Lig4-null mice (13, 14), the progressive decline in hematopoietic stem cells in the Lig4^Y288C strain (27), and the impaired ex vivo lymphocyte proliferation (28, 29) and increased accumulation of chromosome breaks in the lymphocytes in LigIV syndrome (4). Although such defects in cell survival and proliferation are likely to have an impact on the immune system, their contribution to the immunodeficiency of the LigIV syndrome remains unknown.

Another physiological source of DNA DSBs is immunoglobulin class switching (30, 31), which is a somatic rearrangement of the Ig heavy-chain (IgH) locus, occurring in B cells during the immune response and resulting in a switch in the antibody isotype. The DNA rearrangement is initiated by activation-induced deaminase, and the uracil lesions introduced by activation-induced deaminase into the switch-region DNA are processed to DSBs. Overwhelming evidence indicates that NHEJ has a role in the rejoining of the breaks, including evidence from the earlier studies of Ku- and DNA-PK_cs–null mice (28, 29, 32–34) and the recent work on the conditional XRCC4 knockout, Lig4^–/–p53^–/–, and XLF-null mouse strains, and a Lig4-null CH12F3 B cell lymphoma line (35–38). The recent studies indicate that, although NHEJ is involved, an alternative microhomology-mediated repair pathway can to some extent compensate, with residual levels of switching in the NHEJ-null B cells being as high as 25%–50% of the WT level. The alternative pathway was also implicated in class switching in the LigIV syndrome patients, based on the analysis of the structures of switch junctions (39). However, the quantitative impact of hypomorphic LigIV mutations on switching remains unknown.

Aberrant repair of DNA DSBs arising through any of the mechanisms described above could lead to chromosomal translocations, transformations, and cancer. Previous studies in mice indicate that the loss of NHEJ on its own results in only a mild predisposition to cancer in some strains (40–42). However, a combined loss of NHEJ and DNA damage cell-cycle checkpoints leads to a dramatic increase in lymphoid malignancies, as seen in the NHEJ-deficient p53^–/– mouse strains (43–45). In these mice, the tumors arise due to aberrant resolution of DNA DSBs during V(D)J recombination and carry translocations involving IgH and c-myc loci. Lymphomas were also reported in some LigIV syndrome patients (5, 8, 9, 11), although their frequency and the mechanism of carcinogenesis are difficult to ascertain. They may have arisen as a direct result of impaired DNA repair and genomic instability or as a secondary effect of immunodeficiency (8, 9). Furthermore, there may be significant bias in our estimates of lymphoma predisposition in this syndrome as, on the one hand, patients with severe forms of the disease commonly undergo bone marrow transplantation (7, 8, 10), while on the other hand, some patients with milder forms of the condition are diagnosed with LigIV syndrome only following adverse responses to radio- or chemotherapy (5, 11).

We recently identified an ethylnitrosourea-induced (ENU-induced) hypomorphic mutation in the Lig4 gene in a mouse strain that displays the common features of the LigIV syndrome, including lymphopenia and growth retardation (27). The in vivo LigIV activity in the Lig4^Y288C mice is reduced over 10-fold, similar to many of the LigIV syndrome patients (4, 46). One of the effects of the Lig4^Y288 mutation is a progressive attrition of hematopoietic stem cells in aged mice, although this is not limiting in young animals (27). Here, we provide a detailed characterization of the effects of the Lig4^Y288 mutation on the immune system: describing a severe defect in lymphocyte development, a partial arrest in B cell class switching, and a defect in the survival of long-lived lymphocyte populations. Antibody responses to T cell–dependent and –independent antigens are dramatically impaired. We also describe the unexpectedly high incidence of thymic malignancies in the Lig4^Y288 mice and show that these can arise from aberrant resolution of V(D)J breaks leading to translocation of the T cell receptor locus. These findings provide important insights into the underlying basis of immune pathology of LigIV syndrome.

Partial arrest in lymphocyte development in the Lig4^Y288C strain. The Lig4^Y288C mouse line was identified in an ENU mutagenesis program as a recessive C57BL/6 line with growth retardation and peripheral blood lymphopenia (27). The mice carry a Y288C substitution in the catalytic domain of LigIV, which reduces in vivo LigIV activity at least 10-fold and severely impairs the repair of DNA breaks (27). The effects of the mutation at the molecular and cellular levels closely resemble those in human LigIV syndrome patients (4, 27).

We assessed the impact of the mutation on lymphocyte development by comparing primary lymphoid organs of Lig4^Y288C mice with those of age-matched WT and Rag1-null mice, all on a C57BL/6 background. This revealed a severe but incomplete arrest in lymphocyte development in the Lig4^Y288C strain (Figure 1). The total thymic cell count was 100-fold lower than in the WT and comparable to that in the Rag1^–/– mice. The loss of thymocytes increased progressively at each developmental transition, and the proportion of thymocytes at the CD25^hiCD4^–CD8^– stage preceding the onset of TCRβ rearrangement was increased, consistent with impaired V(D)J recombination (Lig4^Y288C = 48% ± 1% and WT = 26% ± 6% of double-negative thymocytes, mean and 95% CI; Figure 1, A and B).

Figure 1

Partial arrest in lymphocyte development in Lig4^Y288C strain. (A) Flow cytometry profiles of the thymus of WT, Rag1^–/–, and Lig4^Y288C mice stained for CD4, CD8, CD44, and CD25 and gated on total thymocytes (upper panels) or CD4^–CD8^– double-negative thymocytes (lower panels). Numbers represent the percentages of cells in the plot that fall within the different regions, corresponding to different stages of thymocyte differentiation. (B) Absolute number of CD4^–CD8^– double-negative (DN), CD4⁺CD8⁺ double-positive (DP), and CD4 and CD8 single-positive cells in the thymus of WT, Rag1^–/–, and Lig4^Y288C mice. Bars represent mean ± 95% CI; n ≥ 6. (C) Flow cytometry of the bone marrow of WT, Rag1^–/–, and Lig4^Y288C mice stained for B220, IgM, IgD (upper panels), or for B220 and CD43 (lower panels) and gated on B220⁺ cells or total lymphocytes, respectively. Numbers represent the percentages of cells in the plot that fall within the different regions, corresponding to different stages of B cell differentiation. (D) Absolute numbers of B220⁺CD43⁺ pro–B, B220⁺CD25⁺ pre–B, and B220⁺IgM⁺IgD^– immature B cells in the bone marrow of WT, Rag1^–/–, and Lig4^Y288C (1 tibia and femur). Bars represent mean ± 95% CI; n ≥ 5.

The absolute numbers of pro–B (B220⁺CD43⁺), pre–B (B220⁺CD25⁺), and immature B cells (B220⁺IgM⁺IgD^–) in the bone marrow of the Lig4^Y288C mice also showed progressive cell loss, and a larger proportion of B220⁺ cells was arrested at the pro–B stage (Lig4^Y288C = 33% ± 11% and WT = 13% ± 4% of B220⁺ bone marrow cells; Figure 1, C and D), consistent with impaired rearrangement of the IgH locus. Mature (B220⁺IgM⁺IgD⁺) B cells in the bone marrow could not be reliably detected but were found in low numbers in the periphery.

Lymphopenia and failure in antibody responses to challenge in the Lig4^Y288C mice. The Lig4^Y288C strain was originally identified based on the decreased frequency of recirculating lymphocytes (27), and the numbers of B and T cells in the blood of the Lig4^Y288C mice were severely reduced (Table 1). Surprisingly, this was associated with elevated serum IgM (Figure 2A), which resembles some features of hyper-IgM immunodeficiencies seen in other DNA repair syndromes, particularly those affecting B cell class switching (12). Elevated IgM levels were also reported in some patients with mutations in LigIV and XLF (2, 8). Serum IgG₃ levels in the Lig4^Y288C mice were significantly reduced, whereas serum IgG₁ and IgA were comparable to those in WT (Figure 2A).

Figure 2

Impaired humoral immunity in Lig4^Y288C mice. (A) Serum antibody isotype levels in naive WT and Lig4^Y288C mice. Data points and bars represent the individual mice and the means of each dataset, respectively. (B) Antigen-specific antibody responses to T cell–independent (NP-Ficoll) and T cell–dependent (CGG) antigens in WT and Lig4^Y288C mice (analyzed by ELISA, optical density plotted against the serum dilution factor). Error bars represent mean ± SEM. **P < 0.01.

Table 1

Reduced lymphocyte numbers in the blood and peripheral lymphoid organs of Lig4^Y288C mice

The numbers of B and T cells in the secondary lymphoid organs, such as the spleen and mesenteric lymph nodes, were severely reduced (Table 1). CD4⁺ and CD8⁺ T cells were 10- to 25-fold down in numbers and stained positive for TCRαβ. Interestingly, most CD4⁺ T cells had an activated phenotype with increased expression of CD44 and reduced CD45RB (Supplemental Figure 1). Such T cell activation is frequently seen in other partial T cell immunodeficiency syndromes in humans and mouse models, for example, in the Omenn syndrome, in which a reduction in T cell numbers caused by hypomorphic mutations in the RAG1/2 genes affects T cell tolerance and activation, probably by reducing competition for limiting amounts of prosurvival cytokines or costimulatory molecules (reviewed in refs. 47, 48).

The absolute numbers of B cells in the secondary lymphoid organs were reduced nearly 50-fold (Table 1), with a large relative increase in the proportion of transitional B cells (B220⁺CD23^–CD21^–). The transitional B cell population constituted 86% ± 7% of B220⁺ splenocytes in the Lig4^Y288C mice compared with only 32% ± 14% in the WT (data not shown). This suggested that further loss of B cells may have been occurring in the peripheral lymphoid organs after the cells progressed from the transitional to the mature stage, as demonstrated later in this study.

In the peritoneal cavity, despite a large reduction in conventional IgM^loB220^hi B2 cells, a considerable population of IgM^hiB220^lo B1 cells was retained (Table 1 and Supplemental Figure 2A). These cells had the typical phenotype of B1 cells — high forward scatter, low expression of IgD, higher expression of CD9, MAC1, and CD43 — and were also CD5⁺ (Supplemental Figure 2B). In scid^DNA–PKcs mice, B1 cells were previously associated with serum autoantibodies (49), and autoantibodies and autoimmune thrombocytopenia were also reported in some LigIV patients (8). Thus, we analyzed Lig4^Y288C for serum autoantibodies. We observed some increase in the incidence of IgM autoantibodies largely targeting cytosolic proteins; however, no IgG autoantibodies were found (Supplemental Figure 2C).

To further characterize the immunodeficiency in the Lig4^Y288C strain, we assessed humoral responses to challenge with T cell–dependent (chicken γ globulin [CGG]) and T cell–independent (4-hydroxy-3-nitrophenylacetyl aminoethylcarboxymethyl–Ficoll [NP-Ficoll]) antigens. We observed an almost complete loss of antibody responses in Lig4^Y288C mice (Figure 2B), consistent with the reported failure of several LigIV patients to mount antibody responses to immunization or infection (8). This highlights the dramatic impairment in humoral immunity in the LigIV syndrome, which likely arises from severe lymphopenia together with additional defects in Ig class switching, cell proliferation, and survival, as reported later in this study.

Increased incidence of thymic tumors in the Lig4^Y288C mice. The Lig4^Y288C mice showed high incidence of lymphoid malignancies. Malignancies were found in 12 out of 45 Lig4^Y288C mice analyzed, compared with none of the WT or Lig4^Y288C/+ littermates. In 8 of 12 of the affected animals, the malignancies were of thymic origin and CD4⁺CD8⁺ cell surface profile, and in 1 mouse, the malignant cells were of B220^loIgM^hi B1 cell phenotype and spread in the peritoneum and spleen. Some of the affected mice were as young as 6 weeks.

To analyze the mechanisms that lead to carcinogenesis, we studied 1 of the thymic tumors for chromosomal translocations involving TCR loci by looking for evidence of separation of red and green FISH probe pairs derived from BACs flanking the TCRα/δ, -β, and -γ loci. In this way, we found chromosomal separation of 54% (216/398) of TCRα/δ probes in the tumor cells, indicative of a translocation (Figure 3A). This observation suggests that thymic tumors in the LigIV syndrome can arise from aberrant resolution of DNA breaks at the TCR locus, probably during V(D)J recombination.

Figure 3

Thymic tumors in Lig4^Y288C mouse. (A) Fluorescence in situ hybridization analysis of a Lig4^Y288C thymic tumor and normal thymus of a WT mouse: loss of pairing of the TCRα/δ flanking probes in cells of the Lig4^Y288C thymic tumor (arrows) and in the inset section is indicative of chromosomal translocation at the locus. Scale bars: 5 μm. (B) Intact G2/M checkpoint in Lig4^Y288C MEFs: WT (Atm^+/+), ataxia telangiectasia–mutated (Atm^–/–), and Lig4^Y288C MEFs were irradiated with 0 to 5 Gy, and allowed to recover for 2 hours before analysis of mitotic index. Percentage of mitotic cells compared with nonirradiated cells is shown. Both Atm^+/+ and Lig4^Y288C cells display an intact G2/M arrest in contrast with Atm^–/– MEFs, which continue to enter mitosis after irradiation. Error bars represent mean ± SD. (C) Lig4^Y288C MEFs activate p53 normally: MEFs were either nonirradiated or irradiated with 10 Gy and allowed to recover for 4 hours before processing. Percentage of cells positive for phosphorylated p53 was scored. Atm^+/+ and Lig4^Y288C MEFs have phosphorylated p53 after irradiation in contrast with Atm^–/– MEFs. Error bars represent means + SD, from 3 independent experiments.

The high incidence and early age of onset of tumors in Lig4^Y288C mice was unexpected. In other NHEJ-deficient mouse strains, a high incidence of cancer typically results from a combined defect in DNA repair and cell-cycle checkpoints, such as in the NHEJ-deficient p53^–/– mouse strains (43–45). Thus, we analyzed the effects of the Lig4^Y288C mutation on G2/M cell cycle checkpoint arrest and on p53 phosphorylation in mouse embryonic fibroblasts (MEFs). The data show that Lig4^Y288C MEFs undergo normal G2/M cell-cycle arrest and phosphorylate p53 normally following irradiation (Figure 3, B and C), which is consistent with previous findings from Lig4^–/– MEFs and the cells of LigIV syndrome patients (4, 13).

The role of LigIV in B cell receptor class switching. To assess the impact of the Lig4^Y288C mutation on B cell class switching, we crossed the following B cell receptor transgenes onto the Lig4^Y288C line to generate Lig4^Y288C-VHL (where VHL indicates variable heavy light) mice: an anti–hen egg lysozyme (anti-HEL) κ light chain transgene and an “IgH knockin” that is capable of switching. To increase litter sizes, the cross was on a mixed C57BL/6×NOD background and age-matched littermates were used as controls in all the comparisons to minimize any differences in genetic background between the groups. In VHL mice, the vast majority of B cells showed specificity for HEL, and the VHL transgene resulted in a partial rescue of B cell development and peripheral B cell numbers in the Lig4^Y288C-VHL mice. Thus, the average number of HEL-binding B cells in the spleen of the Lig4^Y288C-VHL mice was 3.3 × 10⁶, compared with 0.7 × 10⁶ B cells in the nontransgenic Lig4^Y288C and 23 × 10⁶ B cells in the WT-VHL controls. We also measured serum anti-HEL antibody levels in the Lig4^Y288C-VHL mice: IgG₁, IgG_2a, and IgG_2b levels were significantly reduced relative to WT-VHL (P < 0.05), but IgM, IgG₃, and IgA titers were within normal range (Figure 4A).

Figure 4

Partial defect in immunoglobulin class switching in Lig4^Y288C mice. (A) Anti-HEL antibody levels in the serum of WT-VHL (WT) and Lig4^Y288C-VHL (Lig4^Y288C) mice. Bars represent geometric means and 95% CI, n ≥ 3; comparisons by Mann-Whitney test, *P < 0.05. (B–D) Class switching of Lig4^Y288C-VHL and control WT-VHL B cells at day 4 of in vitro stimulation with LPS, LPS plus IL-4, and anti-CD40 plus IL-4. (B) CFSE fluorescence as a measure of the proliferation of Lig4^Y288C-VHL (black lines) and WT-VHL (gray lines) CD19⁺ B cells, representative of 5 mice per group. (C) CD19⁺ versus IgG flow cytometry profiles of WT-VHL and Lig4^Y288C-VHL splenocytes gated on the highly proliferated CFSE-low cells. Numbers indicate the percentages of class-switched B cells within each plot. (D) IgG⁺ cells as a percentage of the highly proliferated CFSE-low CD19⁺ B cells in Lig4^Y288C-VHL, WT-VHL, Lig4^+/Y288C-VHL (Lig4^het), and Rag1^–/–-VHL cultures at day 4 of in vitro stimulation. Error bars represent means ± 95% CI; n = 6 for WT and Lig4^Y288C; n = 3 for Lig4^het and Rag1^–/–. **P < 0.01; ***P < 0.001 using ANOVA.

We chose to assess Ig class switching in an in vitro system, using the previously described protocols that provide a control for reduced B cell number, reduced provision of T cell help, and any changes in B cell proliferation (33, 36, 50, 51). Specifically, cell proliferation in the Lig4^Y288C strain was assessed and controlled for using CFSE labeling. CFSE-loaded Lig4^Y288C-VHL and WT-VHL splenocytes were cultured for 4 days with LPS, LPS plus IL-4, or anti-CD40 plus IL-4. The Lig4^Y288C-VHL B cells showed impaired proliferation in response to LPS and LPS plus IL-4 stimulation, but surprisingly, proliferation in response to anti-CD40 plus IL-4 stimulation was normal (Figure 4B). To control for differences in cell proliferation, highly proliferated CFSE^lo cells were gated, and the proportions of switched IgG⁺ B cells was measured. Lig4^Y288C-VHL B cells showed significantly less switching to IgG₁ and IgG₃ with all 3 classes of stimuli (Figure 4, C and D). Thus, switching to IgG₁ in response to LPS plus IL-4 was reduced from 10% ± 5% in WT-VHL to 1.4% ± 1.5% in Lig4^Y288C-VHL, and switching to IgG₃ in response to LPS was reduced from 3.5% ± 1.9% to 0.7% ± 0.5% (P < 0.001 and P < 0.01, respectively). Importantly, switching in cultures stimulated with anti-CD40 plus IL-4, where there was no difference in cell proliferation, was also significantly reduced, from 25% ± 10% to 10% ± 5% (P < 0.01), confirming that the impaired switching is not a side effect of low proliferation.

To account for the differences in T cell numbers in the cultures and for possible differences in B cell repertoire that could arise through B cell receptor editing, we used Rag1-VHL (VHL-knockin, Rag1^–/–, Lig4^+/+) controls. Using ANOVA, we found no difference in switching between WT-VHL and Rag1-VHL cultures, suggesting that the above factors have no effect, whereas Lig4^Y288C-VHL switching was reduced relative to both control groups (Figure 4D).

Impaired peripheral lymphocyte survival contributes to immunodeficiency. To assess the impact of the impaired repair of background DNA damage on lymphocyte survival, we created a system in which the Lig4^Y288C B cells could develop without undergoing V(D)J rearrangement or Ig class switching. To achieve this, we crossed the MD4 Ig^HEL transgene and the Rag1-null allele onto the Lig4^Y288C strain to generate Lig4^Y288CIg^HELRag1^–/– mice. These mice were compared with age-matched Lig4^+/+Ig^HELRag1^–/– littermates, all on an inbred C57BL/6 background. In these mice, the rearrangement of the endogenous immunoglobulin loci is prevented by the lack of RAG-1, and all B cells express the transgenic MD4 B cell receptor, which does not undergo class switching. Furthermore, both groups of mice lack T cells, controlling for indirect effects of T lymphocytes on B cell turnover.

Analysis of the mice showed no differences in the numbers of immature (B220⁺IgM⁺HSA^hi) B cells in the bone marrow or transitional (B220⁺CD21^–CD23^–) B cells in the spleen between the Lig4^Y288C and Lig4^+/+ groups (Figure 5, A and B), which demonstrates that the impaired B cell development in the Lig4^Y288C mutant is entirely limited by a defect in V(D)J rearrangement. However, the numbers of long-lived B cell populations, such as the follicular (B220⁺CD21⁺ CD23⁺) and marginal zone (B220⁺CD21^hi CD23^lo) B cells in the spleen and mature B cells in the mesenteric lymph nodes (B220⁺IgM⁺IgD⁺), were reduced in the Lig4^Y288CIg^HELRag1^–/– mice compared with the Lig4^+/+Ig^HELRag1^–/– controls (Figure 5B). The number of mature (B220⁺IgM⁺HSA^lo) B cells in the bone marrow was also lower in the mutants, though the difference did not reach statistical significance (Figure 5B). This indicates a biologically important function of NHEJ in the maintenance of genomic stability in peripheral lymphocytes, independent of its roles in V(D)J rearrangement and class switching, and suggests that impaired peripheral lymphocyte survival contributes to the immunodeficiency in the LigIV syndrome.

Figure 5

Impaired peripheral lymphocyte survival contributes to immunodeficiency in Lig4^Y288C mice. (A) Flow cytometry profiles of the spleen of Lig4^+/+Ig^HELRag1^–/– and Lig4^Y288CIg^HELRag1^–/– mice stained for B220, CD21, and CD23 and gated on B220⁺ cells. Numbers indicate the percentages of splenic B cells within the transitional CD21^–CD23^–, follicular CD21⁺CD23⁺, and marginal zone CD21^hiCD23^lo gates. (B) Absolute number of B cells in the bone marrow, mesenteric lymph nodes (MLN), and spleen of Lig4^+/+Ig^HELRag1^–/– (WT) and Lig4^Y288CIg^HELRag1^–/– (Lig4^Y288C) mice. Data points and bars represent individual mice and the mean of each dataset, respectively. Bone marrow immature B cells were gated as B220⁺IgM⁺HSA^hi and mature B cells as B220⁺IgM⁺HSA^lo; splenic transitional B cells were gated as B220⁺CD21^–CD23^–, follicular as B220⁺CD21⁺CD23⁺, and marginal zone as B220⁺CD21^hiCD23^lo; B cells in the MLN were gated as B220⁺IgM⁺IgD⁺. *P < 0.05, 2-tailed Student’s t test.

Increased turnover of lymphocytes in the secondary lymphoid organs of the Lig4^Y288 mice was confirmed by a BrdU-incorporation assay. Nontransgenic WT and Lig4^Y288 mice were fed BrdU at 0.25 mg/ml in drinking water for 1 week, and after this period, 66% ± 23% of B cells and 62% ± 5% of T cells in the spleen of the Lig4^Y288C mice stained BrdU⁺ compared with only 31% ± 6% of B cells and 19% ± 5% of T cells in the WT group; and a similar difference was seen in the mesenteric lymph nodes.

The hypomorphic mouse strain Lig4^Y288C generated in an ENU mutagenesis program is, we believe, the first viable animal model of the LigIV syndrome. Like LigIV syndrome patients, the Lig4^Y288C mice show an over 10-fold reduction in LigIV activity and similar general phenotypic features of growth retardation and lymphopenia. In this study, we provide a detailed analysis of the effects of the hypomorphic LigIV deficiency on the mammalian immune system. This demonstrates how the generation of point mutations by ENU mutagenesis mimics naturally occurring hypomorphic mutations and allows an investigation of gene functions that are masked by embryonic lethality in gene-knockout lines.

We report profound immunodeficiency in Lig4^Y288C mice, with primary and secondary lymphoid organ lymphopenia. Surprisingly, IgG₁ and IgA (but not IgG₃) antibody isotypes in Lig4^Y288C serum are maintained at normal levels, despite the very low numbers of peripheral B cells and impaired Ig class switching, which suggest that compensatory mechanisms must be present. These might operate at the level of B cell selection, T cell activation, competition for other B cell and plasma cell survival factors, or antibody clearance. When immunized, the Lig4^Y288C mice show an almost complete loss of antigen-specific antibody responses to T cell–dependent and T cell–independent antigens. This resembles features of the human LigIV syndrome (6, 8) and highlights the dramatic loss of immune system function caused by the LigIV mutations. We further demonstrate that, in addition to the well-characterized arrest in V(D)J rearrangement, impaired survival of long-lived lymphocyte populations also contributes to immunodeficiency. By creating a system in which Lig4^Y288C B cells develop without undergoing V(D)J rearrangement or class switching and controlling for T cell numbers, we demonstrate the impact of background DNA damage on these long-lived lymphocyte populations and the role of NHEJ in their maintenance. We also report overall activation of peripheral T cells, retention of B1 cells, and some increase in IgM autoantibodies in the Lig4^Y288C mice. These phenotypes have not been previously assessed in any of the LigIV syndrome patients; however, autoimmune thrombocytopenia was seen in at least 1 patient and also in patients with mutations in XLF (2, 8), suggesting that IgM autoantibodies may be relevant in NHEJ deficiency syndromes.

We show impaired class switching in Lig4^Y288C B cells, demonstrating the involvement of LigIV and the NHEJ pathway in the class switching reaction. This confirms the recent conclusions of Yan et al. from studies of Lig4^–/–p53^–/– B cells (35), in this case using LigIV-deficient B cells without additional defects in DNA damage response pathways. Our experimental system directly assesses switching at the cellular level and shows that Lig4^Y288C B cells are defective in class switching to 2 different antibody isotypes with 3 different stimuli, and this is independent of differences in B cell proliferation, B cell receptor specificity, or T cell help. Some switching in Lig4^Y288C is probably due to the residual activity of LigIV, with possible contributions of alternative repair pathways (35, 39). However, despite the contribution of alternative repair pathways to class switching (35, 39), our findings show that hypomorphic mutations in LigIV can significantly impair B cell class switching and contribute to the immunodeficiency of LigIV syndrome.

We further report an unexpectedly high incidence of CD4⁺CD8⁺ thymic tumors in Lig4^Y288C mice, suggesting that LigIV deficiency predisposes to malignancies. The Lig4^Y288C tumors are associated with chromosomal translocations at the TCRα/δ locus, probably caused by aberrant repair of RAG-induced DNA breaks. The strong predisposition to cancer in Lig4^Y288C mice suggests that the mutations that similarly impair LigIV activity in humans will also predispose to cancer and that T cell leukemias reported in some LigIV syndrome patients may arise through similar mechanisms (5, 11). This conclusion is of clinical interest, as the DNA repair deficiency is associated with adverse response to radio- and chemotherapy in this group of cancer patients (5, 11). It also further highlights the possibility that small alterations in LigIV activity in heterozygous carriers of Lig4 mutations may predispose to cancer, as suggested by the fact that Lig4 haploinsufficiency increases the frequency of soft-tissue sarcomas in INK4a/ARF-null mice (52) and by the association of a human LIG4 polymorphism with increased risk of lung carcinomas (53).

The high cancer susceptibility of the Lig4^Y288C line compared with other NHEJ-deficient mouse strains is surprising, as it is widely believed that defects in NHEJ repair lead to carcinogenesis only when combined with loss of cell-cycle checkpoints, as in the NHEJ-deficient p53^–/– strains (42–45). Although Ku70^–/– mice were reported to develop CD4⁺CD8⁺ lymphomas similar to those of Lig4^Y288C mice (54), this was disputed in recent reports arguing that both Ku70^–/– and Ku80^–/– strains are not tumor prone (40, 55). Furthermore, Lig4^–/–p53^R172P/R172P mice with a defect in DNA damage apoptosis induction but normal cell-cycle arrest are tumor free (56). In contrast, we observed high rates of leukemia in Lig4^Y288C mice despite normal G2/M checkpoint arrest and normal p53 activation, and the reason for increased cancer predisposition in Lig4^Y288C compared with other NHEJ-null mouse strains is unknown. We can speculate that because the tumors arise from the CD4⁺CD8⁺ population, progression of some thymocytes to the CD4⁺CD8⁺ stage may be a prerequisite for carcinogenesis. Thus, the increased incidence of malignancies in the Lig4^Y288C mice may be the result of the hypomorphic nature of the phenotype that allows some “leaky” T cell development to take place, as is also seen in the tumor-prone and “leaky” Ku70^–/– line (54). It is also possible to speculate that the accessibility of DNA breaks to alternative error-prone repair pathways may differ between the different NHEJ-deficient mouse lines, leading to increased predisposition to aberrant repair and translocations in some of the lines, such as Lig4^Y288C. Although many questions remain unanswered, our findings with Lig4^Y288C mice provide definitive evidence that hypomorphic LigIV deficiencies can predispose to leukemia.

In summary, access to the ENU-induced hypomorphic Lig4^Y288C mouse strain has made it possible to characterize the effects of LigIV deficiency on the mammalian immune system. Arrest in V(D)J rearrangement is the primary cause of lymphopenia, with impaired cell survival and proliferation due to random DNA damage making additional contributions to the loss of peripheral lymphocytes. We confirm the role of LigIV in normal B cell class switching and suggest that class switching deficiencies contribute to the impaired immune function in LigIV syndrome. We also show that a hypomorphic LigIV mutation can confer strong predisposition to lymphoid malignancies. The study reveals multiple NHEJ-dependent processes in immune system function and helps to explain the complex and multilayered immunodeficiency of the human LigIV syndrome.

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We thank the members of the Cornall, Jeggo, and Goodnow groups for reviews of the manuscript; A. Gennery, A. Enders and C. Jolly for helpful discussions; and the staff of the Australian Phenomics Facility and Oxford Biomedical Services for excellent animal husbandry. This work was funded by the Wellcome Trust and the NIHR Biomedical Research Centre Programme. A. Nijnik was supported by a Wellcome Trust Prize Studentship. P.A. Jeggo is supported by the Medical Research Council, the Association for International Cancer Research, an Integrated Project EU grant, DNA Repair (LSHG-CT-2005-512113), and the United Kingdom Department of Health.

Address correspondence to: Richard Cornall, Henry Wellcome Building of Molecular Physiology, Roosevelt Drive, Oxford OX3 7BN, United Kingdom. Phone: 44-1865-287790; Fax: 44-1865-287787; E-mail: richard.cornall@ndm.ox.ac.uk.

Conflict of interest: The authors have declared that no conflict of interest exists.

Nonstandard abbreviations used: CGG, chicken γ globulin; DNA-PK_cs, DNA-dependent protein kinase catalytic subunit; DSB, DNA double-strand break; ENU, ethylnitrosourea; HEL, hen egg lysozyme; IgH, Ig heavy-chain locus; LigIV, DNA ligase IV; MEF, mouse embryonic fibroblast; NHEJ, nonhomologous end joining; NP, 4-hydroxy-3-nitrophenylacetyl aminoethylcarboxymethyl; TC, Tricolor; VHL, variable heavy light; XLF, XRCC4-like factor.

Reference information: J. Clin. Invest.119:1696–1705 (2009). doi:10.1172/JCI32743

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