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Blood, 1 March 2007, Vol. 109, No. 5, pp. 1887-1896. Prepublished online as a Blood First Edition Paper on October 31, 2006; DOI 10.1182/blood-2006-05-020917.
HEMATOPOIESIS ATM deficiency disrupts Tcra locus integrity and the maturation of CD4+CD8+ thymocytes1 Program in Developmental and Stem Cell Biology, The Hospital for Sick Children Research Institute, University of Toronto, ON, Canada; 2 Department of Medical Biophysics, University of Toronto, ON, Canada; 3 Department of Surgery, University of Toronto, ON, Canada; 4 Department of Immunology, University of Toronto, ON, Canada; 5 Department of Mathematics and Statistics, McMaster University, Hamilton, ON, Canada
Mutations in ATM (ataxia-telangiectasia mutated) cause ataxia-telangiectasia (AT), a disease characterized by neurodegeneration, sterility, immunodeficiency, and T-cell leukemia. Defective ATM-mediated DNA damage responses underlie many aspects of the AT syndrome, but the basis for the immune deficiency has not been defined. ATM associates with DNA double-strand breaks (DSBs), and some evidence suggests that ATM may regulate V(D)J recombination. However, it remains unclear how ATM loss compromises lymphocyte development in vivo. Here, we show that T-cell receptor ß (TCRß)–dependent proliferation and production of TCRßlow CD4+CD8+ (DP) thymocytes occurred normally in Atm–/– mice. In striking contrast, the postmitotic maturation of TCRßlow DP precursors into TCRßint DP cells and TCRßhi mature thymocytes was profoundly impaired. Furthermore, Atm–/– thymocytes expressed abnormally low amounts of TCR  mRNA and protein. These defects were not attributable to the induction of a BCL-2–sensitive apoptotic pathway. Rather, they were associated with frequent biallelic loss of distal Va gene segments in DP thymocytes, revealing that ATM maintains Tcra locus integrity as it undergoes V(D)J recombination. Collectively, our data demonstrate that ATM loss increases the frequency of aberrant Tcra deletion events, which compromise DP thymocyte maturation and likely promote the generation of oncogenic TCR translocations.
Ataxia-telangiectasia (AT) is an autosomal recessive disease with a pleiotropic phenotype that includes cerebellar degeneration, immunodeficiency, sterility, radiosensitivity, and an elevated incidence of lymphoid malignancies.1 AT is caused by mutations in ATM, a gene belonging to the phosphatidylinositol 3-kinase–related family of serine-threonine kinases that function in DNA damage surveillance and repair.2 The radiosensitivity and increased cancer susceptibility of patients with AT and Atm–/– mice are thought to reflect loss of these ATM-dependent DNA damage responses,2 whereas defective DNA repair by homologous recombination likely underlies the sterility of patients with AT.3–5 However, the cause of AT-related immune deficiency has not been defined. Patients with AT are variably lymphopenic and display a range of cellular and humoral immunologic abnormalities,6 leading to recurrent and sometimes fatal sinopulmonary infections. In particular, patients with AT exhibit low output of mature TCRhi CD4 and CD8 single-positive (SP) thymocytes, and their peripheral T-cell pool often exhibits oligoclonal T-cell receptor (TCR) Vß expansions and abnormally low naive/memory T-cell ratios.7 Like patients with AT, Atm-deficient mice also have decreased numbers of mature thymocytes and peripheral T cells, suggesting defective intrathymic T-cell development.8–11
Given ATM's prominent role in regulating DNA damage responses, much interest has focused on the possibility that ATM has critical functions in V(D)J recombination, the somatic rearrangement process by which developing T and B lymphocytes generate a diverse repertoire of antigen receptor variable (V) genes. Tcrb and Tcra recombination occur sequentially during the successive CD4/CD8 double-negative (DN) and CD4/CD8 double-positive (DP) stages of T-cell development, respectively. Signaling through TCRß-containing pre-TCR complexes is required to generate a large pool of DP thymoctyes from DN precursors. Tcra rearrangement begins in postmitotic DP thymocytes.12 However, the Tcra locus can undergo successive rounds of secondary recombination to maximize the opportunity for DPs to produce a TCR V(D)J recombination is initiated by the lymphocyte-specific RAG-1/2 endonuclease, which generates DNA double strand breaks (DSBs) in between recombination signal sequences and adjacent antigen receptor variable (V), diversity (D), or joining (J) gene segments.16 The resulting coding ends (CEs) and signal ends (SEs) are repaired by the nonhomologous end-joining (NHEJ) complex to make coding joints (CJs) and signal joints (SJs), respectively. Defective NHEJ causes abnormal accumulation of CEs17 and induction of p53-dependent apoptosis in thymocytes.18 In NHEJ-p53 double-mutant mice, defective DSB repair during IgH recombination causes frequent deletion of telomeric VH gene segments, facilitating generation of oncogenic translocations with cMyc and initiating IgH/cMyc amplification through repeated cycles of bridge-breakage-fusion.19–21 Thus, defective NHEJ of RAG-induced DSBs can promote lymphoid leukemogenesis. Interestingly, ATM localizes to V(D)J recombination-induced DSBs,22 suggesting that it may monitor recombination intermediates, thus limiting the oncogenic consequences of aberrant V(D)J recombination. Furthermore, 2 ATM substrates, H2AX and NBS1, also localize to V(D)J-induced DSBs, and inactivating mutations in these proteins cause a phenotype reminiscent of ATM deficiency.23–25 Although NHEJ of recombining Tcr loci does not appear to be profoundly defective in ATM-deficient thymocytes, several observations suggest that ATM-deficient T-cell precursors recombine Tcr loci with poor fidelity. T-cell leukemias arising in patients with AT and Atm–/– mice frequently harbor chromosomal translocations involving Tcr and Ig genes.1,26 In addition, peripheral blood lymphocytes from patients with AT display frequent abnormal trans-rearrangements of TCR loci,27 a defect also seen in NHEJ-deficient T-cell precursors.28 Finally, a recent study showed that Atm–/– T- and B-cell precursors display a 50% reduction in normal CJs and a high frequency of abnormal "hybrid" joins between SEs and CEs when rearrangement occurs by inversion, but not deletion.29 During deletional recombination, CJs are made on chromosomes, whereas SJs are joined on extrachromasomal circles that are subsequently lost from the cells. In contrast, inversional recombination requires that both the CJs and SJs be made on the same chromosome. Thus, ATM might be particularly important in stabilizing DSB repair complexes during the more demanding process of inversional V(D)J recombination. However, because most Tcrb and Tcra rearrangements occur by deletional recombination,30 it is not clear whether defects in ATM-induced DSB stabilization can explain the impaired T-cell development observed in ATM-deficient patients and mice.
Here, we address 2 key questions regarding ATM function. Does the T-cell deficiency in Atm–/– mice result from inefficient V(D)J recombination at Tcr loci? Alternatively, does loss of ATM-induced DNA damage checkpoints impair proliferation and/or survival of developing thymocytes? We show that Tcrb recombination was not obviously impaired in Atm–/– thymocytes, enabling the production of a normal-sized pool of TCRßlow DP thymocytes in response to pre-TCR signals. In striking contrast, the postmitotic generation of Atm–/– TCRßint DP thymocytes and their TCRßhi SP progeny was profoundly impaired; neither defect was rescued by BCL-2 overexpression. Although we did not observe Tcra CE accumulation, the hallmark of defective NHEJ, Atm–/– thymocytes expressed dramatically reduced levels of TCR
Mice 129/SvEv-Atmtm1Awb heterozygous mice8 (Jackson Laboratory, Bar Harbor, ME) were bred in our specific pathogen facility (Hospital for Sick Children, Toronto, ON, Canada) to generate Atm+/+, Atm+/–, and Atm–/– progeny. Atm genotypes were determined by polymerase chain reaction (PCR) amplification of tail DNA, using the following primers: Atm-wt antisense, 5'-TCCGAATTTGCAGGAGTTG-3'; Atm-wt sense, 5'-GCTGCCATACTTGATCCATG-3'; neomycin antisense, 5'-AGGTGAGATGACAGGAGATC-3'; neomycin sense, 5'-CTTGGGTGGAGAGGCTATTC-3'. To generate Atm–/–;Bcl2 Tg+ mice, Bcl2 Tg+ mice obtained from Dr Korsmeyer (Sentman et al31) were bred to 129/SvEv-Atmtm1Awb heterozygous mice. Atm+/–;Bcl2 Tg+ mice were backcrossed to 129/SvEv-Atmtm1Awb heterozygous mice to generate the genotypes of interest. The presence of the lckpr-BCL2 transgene was determined by genomic DNA PCR amplification for 30 cycles using the following primers: GH (human growth hormone) sense, 5'-GTAGCCATTGCAGCTAGGTG-3', and BCL2 Tg antisense, 5'-CTTTGTGGAACTGTACGGCCCCAGCATGCG-3', at a final concentration of 1 µM each. BCL2 transgene expression was confirmed by intracellular staining with anti–hBCL-2-PE (6C8; BD Biosciences, San Jose, CA). Three- to 4-week-old Atm–/– mice and Atm+/+ littermates or age-matched controls were used in all experiments, unless otherwise indicated. All animal experiments followed protocols approved by the Hospital for Sick Children Animal Care Committee (Toronto, ON, Canada). Antibodies and flow cytometry
Antibodies used in this study were anti-CD8 Intracellular staining The levels of intracellular TCRß were assessed by flow cytometry using anti–TCRß-APC (H57-597) or control biotinylated hamster IgG isotype followed by SAv-APC. Thymocytes were surface-stained with anti–CD4-PE and anti–CD8-FITC, fixed, permeabilized, and stained intracellularly using the BD Cytofix/Cytoperm Kit (BD Biosciences) as per the manufacturer's instructions. Western blotting/immunoprecipitation
TCR In vitro maturation of DP thymocytes Three million Atm–/– or littermate Atm+/+ thymocytes were cultured in 6-well plates precoated with 7.5 µg/mL purified anti-TCRß (H57-597) or hamster IgG isotype control (Caltag, San Francisco, CA), as previously described.33 BrdU assays Four- to 8-week-old Atm–/– or Atm–/–;Bcl2 Tg+ mice and age-matched controls received 2 intraperitoneal injections of 5-bromo-2'deoxyuridine (BrdU; 1 mg each, 4 hours apart) at day 0. Thymocytes were isolated 1 to 5 days after injection and stained as described in "Antibodies and flow cytometry." BrdU detection was performed using the BrdU Flow Kit per the manufacturer's instructions (BD Biosciences).
Atm–/– DP thymocytes express abnormally low levels of surface TCR
To investigate possible functions of ATM in T-cell development, we examined the steady state phenotype and number of thymocytes in Atm–/– mice compared with Atm+/– and Atm+/+ littermates from 5 to 60 days of age. Consistent with prior reports, we observed a 40% to 60% decrease in thymic cellularity of Atm–/– mice.3,8 In particular, the frequencies and absolute numbers of mature CD4+ and CD8+ thymocyte populations were severely decreased in Atm–/– mice relative to littermate controls (Figure 1A,C). Because production of mature SP thymocytes requires expression of TCR
We next examined the capacity of Atm–/– DP thymocytes to respond to TCR engagement in vitro. Plate-bound anti-TCRß antibody was used to engage the TCR in short-term DP thymocyte cultures. As previously reported,34 within 24 hours most wild-type DP thymocytes responded to TCR signaling by down-regulating CD4 and CD8 coreceptors and up-regulating CD5 and CD69 (Figure 1D). In contrast, less than 30% of the Atm–/– DP thymocytes responded in a similar manner, suggesting that few express sufficient surface TCR ß to initiate positive selection. ATM loss compromises postmitotic generation of TCRßint DP and TCRßhi SP thymocytes The most striking consequence of ATM loss on T-cell development is the reduction in TCRßint DP thymocytes and their TCRßhi progeny (Figure 1). TCRßint DP thymocytes are postmitotic, harbor in-frame TCRß rearrangements, and are actively recombining their Tcra loci.13 These cells are generated from precursors that cycle extensively during the DN3 to DP transition.35 We reasoned that the reduction in Atm–/– TCRßint DP thymocytes could reflect a defect in pre–TCR-induced clonal expansion of DN precursors or reduced survival of postmitotic DP thymocytes. To distinguish between these alternatives, we monitored the initial generation and subsequent maturation of Atm–/– DP thymocytes 1 to 5 days after BrdU pulse labeling in vivo. As expected from previous reports,36 most labeled wild-type cells were TCRßlow DP precursors 1 day after BrdU injection, and their numbers decreased dramatically over the next 5 days, as they either died or matured into SP thymocytes (Figure 2A-B). Similar proportions of the DN and DP subsets were labeled in Atm–/– and Atm+/+ controls on day 1, demonstrating that ATM loss did not affect thymocyte proliferation or the initial generation of TCRßlow DP thymocytes (Figure 2A-B).
Most labeled wild-type DP thymocytes became TCRßint by day 3. In striking contrast, the postmitotic generation of TCRßint DP thymocytes was highly inefficient in Atm–/– mice. The conversion of Atm–/– TCRßint DP into TCRßhi SP cells was also less efficient than in Atm+/+ controls (most evident at day 5 after labeling). Therefore, normal numbers of Atm–/– TCRßlow DP thymocytes were produced, but their postmitotic conversion into TCR -expressing TCRßint DP and mature TCRßhi SP thymocytes was seriously impaired. This loss most likely reflects a survival defect, because we observed a statistically significant 2-fold increase in the numbers of cells expressing active caspase-3 in the cortex of Atm–/– thymi where DP cells reside, but no genotype-dependent variation in the medulla (Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article). Rapid clearance of apoptotic thymocytes by resident macrophages37 likely prevented further accumulation of caspase-3–positive cells in Atm–/– thymic lobes. Collectively, these observations reveal that ATM deficiency does not impair pre–TCR-dependent proliferation during the DN to DP transition. Rather, ATM loss impairs survival during the postmitotic conversion of TCRßlow DP into TCRßint DP thymocytes. ATM regulates postmitotic DP thymocyte survival by a BCL-2–independent pathway
Members of the BCL-2 family regulate thymocyte survival, and ROR
Atm–/– thymocytes express a diverse Tcrb repertoire To determine the basis for the decrease in the TCRßint DP thymocyte population, we examined intracellular TCRß (TCRßic) protein expression. TCRßic protein levels were equivalent in Atm–/– and Atm+/+ DP thymocytes (Figure 4A), suggesting that defects in TCRß rearrangement or expression did not account for the reduction in the TCRßint DP population. However, we reasoned that inefficient V(D)J recombination might limit TCRß chain diversity. Therefore, we carried out complementarity-determining region 3 (CDR3) length analysis to examine the junctional diversity of TCRß transcripts in Atm–/– thymocytes. The CDR3 region is created by V(D)J coding end modifications and greatly contributes to TCRß clonal diversity. Thymocyte cDNA samples from Atm–/– mice and controls were PCR-amplified with primers specific for 1 of 2 widely used Vb gene segments (Vb1 or Vb8), together with an antisense Cb primer. As expected, we obtained a Gaussian distribution of discrete 3-bp (base pair) spaced CDR3 lengths representing in-frame transcripts in wild-type thymocytes (Figure 4B). The CDR3 region length distribution of Atm–/– thymocytes was comparable to Atm+/– and Atm+/+ controls. However, a dominant Vb1 CDR3 length was detected in one 3-week-old Atm–/– animal (Figure 4B, arrow), possibly an early manifestation of the pre–T-cell lymphoblastic leukemia/lymphoma characteristic of murine ATM deficiency.8,39
To exclude the possibility that robust pre–TCR-induced clonal expansion obscured a subtle defect in V(D)J recombination, we examined transcripts from the Vb17 pseudogene which cannot produce a functional TCRß chain in mice carrying the Vb haplotype (Figure 4B-C).40 Even in the absence of selection for in-frame transcripts, Vß17 transcripts displayed CDR3 lengths that were similarly diverse in Atm–/– and control thymocytes (Figure 4B). Finally, we used a panel of Vß-specific monoclonal antibodies to examine the diversity of TCRß proteins expressed on the surface of TCRßhi ATM-deficient thymocytes. We found that Atm–/– and control mature thymocytes exhibited similar frequencies of TCR-Vß3, 5, 6, 7, 8, 9, 10, 11, 12, and 13 (data not shown). Therefore, Atm–/– and wild-type thymocytes express an equally diverse TCRß repertoire, suggesting that ATM loss does not limit Vb gene segment use or junctional diversity during Tcrb recombination.
Reduced TCR
TCR
To determine whether impaired Tcra recombination could account for decreased Tcra expression in Atm–/– thymocytes, we analyzed Tcra recombination intermediates and end products by Southern blotting. We first used a strategy that detects rearrangements and abnormally persisting CEs at Ja50 and Ja49, the most 5' Va-proximal segments of the Ja cluster. These segments are preferentially involved in the initiation of Tcra rearrangement,42 and defective NHEJ causes abnormal CE persistence in this proximal region.43 In contrast to Tcrb-transgenic Prkdcscid/scid thymocytes,43 we observed similar levels of Ja50 rearrangements in Atm–/– and wild-type thymocytes (Figure 5D). Furthermore, we did not detect accumulation of Ja50 CEs in Atm–/– thymocytes. Using a different Southern blot strategy to examine Ja50 and Ja49 SE intermediates and SJ recombination products, we found that SEs were precisely rejoined into SJs in Atm–/– and control thymocytes (Figure S3). In addition, the abundance of Ja50 and Ja49 SEs or SJs was similar in the 2 Atm genotypes (Figure S3). These strategies have previously revealed profound impairment of proximal Ja rearrangement and accumulation of proximal Ja CEs43 in NHEJ-deficient mice. We did not find similar defects in the initiation or processing of proximal Ja recombination intermediates in Atm–/– thymocytes. Therefore, if ATM loss impairs NHEJ during Tcra recombination, it was not detected by these approaches. Analysis of secondary Tcra recombination in Atm–/– thymocytes
We next examined whether secondary Tcra recombination is impaired in Atm–/– thymocytes by a Southern blot strategy predicated on the observation that secondary Tcra rearrangements cause deletion of 5' Ja segments proximal to the Va cluster.13,14 Using probes specific for proximal 5', "middle," and distal 3' regions of the Ja cluster, we found equivalent deletion of the proximal and middle Ja chromosomal regions in Atm-deficient and wild-type thymocytes, consistent with a similar extent of secondary Tcra recombination (Figure 6A). To further assess Ja usage, we sequenced 30 independent Va3-Ca transcripts isolated from Atm–/– and littermate control thymi. These sequences also revealed similar distributions of proximal, middle, and distal Ja segments in Atm–/– and wild-type V
Biallelic deletions of distal Va gene segments in Atm–/– thymocytes Although we observed similar levels of proximal, middle, and distal Ja rearrangements in Atm–/– thymocytes, our strategy could not distinguish between normal rearrangements involving Va gene segments and aberrant rearrangements involving other chromosomal regions. A hallmark of the T-cell leukemias characteristic of both human and murine ATM deficiency is recurrent abnormalities involving the Tcra locus on chromosome 14,26,44 often resulting in loss of Tcra locus material.26 These considerations prompted us to investigate Tcra locus integrity using molecular cytogenetics. Because DP thymocytes undergoing Tcra rearrangement are postmitotic cells, we performed interphase fluorescent in situ hybridization (iFISH) experiments to assess the integrity of the Tcra locus in primary total and purified DP thymocytes isolated from 4-week-old Atm–/– and Atm+/+ mice. The murine TcraC region is separated from the most 5' distal end of TcraV gene cluster on chromosome 14 by about 1.3 megabases.30 In addition, deletional Tcra recombination progresses from proximal to distal Va segments. Therefore, we selected BAC probes containing the TcraC locus and 6 of the most distal TcraV regions, to ensure maximal sensitivity in detecting abnormalities. iFISH signals from TcraC and TcraV probes were analyzed in unfractionated and in sorted DP thymocyte samples isolated from Atm+/+ and Atm–/– mice (Figure 7; Tables S1, S2; Figure S4). The signal distributions in thymocytes from individual mice are shown in Figure 7, and the pooled signal distributions from mutant compared with wild-type thymocytes are shown in Table 1, Table S1, and Table S2. A standard chi-square test of the iFISH signal distributions revealed significant differences in the distribution of TcraC and TcraV signals between Atm genotypes (Tables S1, S2), so we carried out additional comparisons of genotype-specific differences in Tcra locus signals.
One major difference was that compared with wild-type cells, fewer mutant thymocytes had 2 TcraC signals per cell (Figure 7A; P < .001; Table 1) and a greater number had more than 2 TcraC signals per cell (Figure 7B; P < .001; Table 1). We noted a second striking difference in the distribution of TcraV signals between wild-type and mutant thymocytes (Figure 7C-D). Specifically, mutant cells had frequent biallelic loss of TcraV signals, both in total (Figure 7D; P < .001; Table 1) and DP (P < .001) thymocytes. The iFISH images were acquired in a single plane of view potentially obscuring some signals. This technical limitation should equally affect all samples. However, we observed robust genotype-dependent differences in the numbers of TcraV and TcraC signals. Collectively, these data demonstrate that a significant proportion of Atm–/– thymocytes have abnormalities in the TcraV and TcraC regions. Loss of Tcra locus integrity in Atm–/– thymocytes likely underlies the developmental defect in the generation of TCR ß+ cells. Moreover, genomic instability at the Tcra locus is likely progressive during the developmental window of Tcra locus recombination resulting in an accumulation of abnormalities that have been reported in Atm–/– leukemic T cells.
We have demonstrated that the paucity of mature SP thymocytes in Atm–/– mice reflects impaired TCR expression and defective maturation of postmitotic DP thymocytes. Surprisingly, we found no impairment of Tcrb recombination or generation of a diverse primary TCRß repertoire. Furthermore, ATM loss did not compromise proliferation or survival during the DN to DP transition, because BrdU pulse-chase studies showed that normal numbers of TCRßlow DP thymocytes were generated in response to pre-TCR signals. In contrast, the postmitotic generation of TCRßint DP and TCRßhi SP thymocytes was dramatically impaired, and this defect was not rescued by BCL-2 overexpression. However, defective generation of these populations correlated with a high frequency of large biallelic deletions encompassing the distal TcraV region, as well as reduced Tcra mRNA and protein. Thus, our data suggest that, after exiting the cell cycle, many ATM-deficient DP thymocytes undergo aberrant TcraV deletion and die, limiting the pool of TCRßint DP thymocytes available for positive selection. On the basis of these findings, we propose that ATM is vital for maintenance of locus integrity during the extended developmental window of Tcra recombination.
There are several features that could explain why ATM deficiency disrupts Tcra rearrangement without obviously affecting Tcrb recombination. First, the Tcra locus is dispersed over a much larger chromosomal region than the Tcrb locus, and the TcraV region alone covers nearly 1 megabase.30 Such long-range recombination events might be more prone to disruption in the absence of ATM. In support of this notion, ATM deficiency also impairs class-switch recombination,45,46 another event involving rearrangements over long chromosomal distances. Second, the Tcrd locus, which rearranges during the DN stage of development, is contained within the Tcra locus on mouse chromosome 14.30 Moreover, Tcra but not Tcrb gene segments can undergo repeated rounds of secondary recombination on the same chromosome.12,47 Thus, chromosome 14 must remain patent during many rounds of V(D)J recombination at 2 different developmental stages. Finally, signaling through TCRß-containing pre-TCR complexes triggers robust clonal expansion during the DN to DP transition,35 whereas TCR Despite normal generation of TCRß-expressing DP precursors, the pool of postmitotic TCRßint DP thymocytes was dramatically reduced in Atm–/– mice. Surprisingly, although this reduction correlated with a striking reduction in Tcra expression, proximal Ja rearrangement and secondary Ja recombination were not obviously impaired in ATM-deficient thymocytes. However, these approaches did not evaluate whether these rearrangements contained normal Va-Ja coding joints. Because our molecular cytogenetic analyses showed frequent biallelic deletion of the entire distal TcraV gene cluster, it is possible that many of the Ja rearrangements detected by Southern blotting were aberrant. Collectively, these observations reveal that ATM plays a critical role in maintaining Tcra locus integrity during recombination.
Our conclusion accords with the observation that ATM localizes to RAG-induced DSBs,22 as well as with the recent suggestion that ATM stabilizes DSB repair complexes during inversional V(D)J recombination.29 This study used immortalized pre–B-cell lines harboring chromosomally integrated artificial V(D)J substrates to demonstrate that ATM deficiency reduces inversional recombination efficiency by 50%. We observed an even greater defect in TCR Surprisingly, the Bredemeyer et al29 study reported abnormal CE accumulation during rearrangement of the inversion substrate in Atm–/– pre–B-cell lines, whereas we did not observe persistence of Ja50 CEs during deletional recombination in primary ATM-deficient thymocytes. Several possibilities could account for this apparent discrepancy. First, in the cell line model, recombination was temporally synchronized and only one CE could be generated. In contrast, Ja recombination occurs asynchronously in vivo, and many different Ja CEs can be generated across the locus. Indeed, we observed normal frequencies of middle and distal Ja recombination events. Thus, the concentration of particular Ja CEs may be too low in primary ATM-deficient thymocytes to detect by Southern blotting. Moreover, the substrate CEs were short-lived in pre-B cell lines.29 This rapid degradation could make Ja CEs very difficult to detect in asynchronous populations of ATM-deficient thymocytes. We have previously shown that the abnormal accumulation of V(D)J DSBs in NHEJ-deficient thymocytes activates the p53-mediated DNA damage response pathway.18 Analyses of Atm;p53 double-mutant mice have demonstrated that the p53 and ATM-dependent DNA damage response pathways are not entirely overlapping,49–51 and ATM loss can lead to induction of p53-dependent apoptosis.52 Indeed, spontaneous apoptosis was enhanced in ATM-deficient thymocytes (Figure S2; Elson et al10). Therefore, we examined whether an antiapoptotic Bcl2 transgene would rescue the Atm null phenotype. Although BCL-2 overexpression in Atm–/– mice generally enhanced lymphocyte survival and thymic cellularity, it failed to rescue maturation of TCRßint DP thymocytes from TCRßlow precursors. Therefore, impaired maturation of ATM-deficient thymocytes does not reflect activation of a BCL-2–sensitive apoptotic pathway.
In summary, our study provides a molecular and developmental explanation for the T-cell immune deficiency that is characteristic of patients with AT and ATM-deficient mice. In contrast to NHEJ deficiencies which cause profound failure of Tcrb and Tcra recombination,16,43 we showed that ATM loss results in decreased Tcra expression, resulting in a paucity of TCR
Contribution: I.R.M. performed most of the research, analyzed the data, and cowrote the paper; R.A.G. performed some experiments and analyzed the data; L.M.J.N. contributed to the design of the study and supervised the experimental work; A.C. designed and performed the statistical analysis of the cytogenetics data; C.J.G. and J.S.D. designed the study, supervised the experimental work, and cowrote the manuscript. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Jayne S. Danska, The Hospital for Sick Children Research Institute, 101 College St, Toronto Medical Discovery Tower (TMDT) East Tower, Rm 14-313, Toronto, ON M5G 1L7 Canada; e-mail: jayne.danska{at}sickkids.ca.
We thank Dr S. Zhao for expert flow cytometry, the late Dr S. Korsmeyer for providing the lckpr-BCL2 transgenic mice, and Drs H. Petrie and F. Livak for sharing the Ja plasmids and probes. We thank Drs Vikram Jayanth and Martin Lee from The Centre for Applied Genomics (Hospital for Sick Children Research Institute) for performing the iFISH experiments. We also thank Dr Inoul Lee (University of Washington, Seattle) for BAC clones and I. Grandal and C. Webb for animal care. This work was supported by grants from the Leukemia and Lymphoma Society (J.S.D. and C.J.G.), Genome Canada (J.S.D. and C.J.G.), and the Ontario Genomics Institute (J.S.D. and C.J.G.). R.A.G. held Canadian Institutes of Health Research (CIHR) and National Cancer Institute of Canada (NCIC) Fellowship awards. I.R.M. was supported by a RESTRACOMP Studentship from the Hospital for Sick Children Research Institute.
Submitted May 3, 2006; accepted October 19, 2006.
Prepublished online as Blood First Edition Paper, October 31, 2006
DOI: 10.1182/blood-2006-05-020917
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
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Abstract
Introduction
) and littermates (
). The numbers above each bar represent the mean. The graphs display the mean ± SD for 9 Atm+/+ and 12 Atm–/– mice. Percentages and absolute numbers of CD8+ SP cells were determined by gating on TCRßhi CD8+ SP cells to eliminate immature single-positive cells. All differences were statistically significant (P < .001 by Student t test) except for the DN frequency, DN, and TCRßlow DP absolute number comparisons. (D) CD4 versus CD8 expression on thymocytes from 3-week-old Atm+/+ or Atm–/– mice was assessed after overnight culture in the presence of plate-bound anti-TCRß antibody. Fluorescence signals were gated to display CD69 or CD5 staining on CD4+CD8+ DP thymocytes. Expression of the early activation markers CD69 and CD5 by DP thymocytes from Atm–/– mice and littermate controls was evaluated by flow cytometry after overnight culture in the presence (shaded) or absence (open) of stimulation with anti-TCRß.
-dependent expression of BCL-XL supports DP thymocyte survival during secondary Tcra recombination.
17).
