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The Journal of Immunology, 2006, 176: 5871-5879.
Copyright © 2006 by The American Association of Immunologists

Expression of TCR{alpha}beta Partly Rescues Developmental Arrest and Apoptosis of {alpha}beta T cells in Bcl11b–/– Mice1

Jun Inoue2,3,*, Tsutomu Kanefuji2,*, Kiyoshi Okazuka*, Hisami Watanabe{ddagger}, Yukio Mishima*,{dagger} and Ryo Kominami3,*,{dagger}

* Department of Molecular Genetics, Graduate School of Medical and Dental Sciences, {dagger} Center for Transdisciplinary Research, Niigata University, Niigata, Japan; and {ddagger} Division of Cellular and Molecular Immunology, Center of Molecular Biosciences, Ryukyu University, Okinawa, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Bcl11b–/– mice show developmental arrest at the CD44–CD25+ double-negative 3 (DN3) or immature CD8+single-positive stage of {alpha}beta T cell. We have performed detailed analysis of sorted subsets of Bcl11b–/– thymocytes, DN3 and CD44–CD25– double-negative 4 (DN4) cells. Surface expression of TCRbeta proteins was not detected in DN3 thymocytes and markedly reduced in DN4 thymocytes, whereas expression within the cell was detected in both, suggesting some impairment in processing of TCRbeta proteins from the cytoplasm to the cell surface. This lack of expression, resulting in the absence of pre-TCR signaling, could be responsible for the arrest, but the transgenic TCRbeta or TCR{alpha}beta expression on the cell surface failed to promote transition from the DN3 to CD4+CD8+ double-positive stage of development. This suggests that the pre-TCR signal cannot compensate the deficiency of Bcl11b for development. Bcl11b–/– DN3 thymocytes showed normal DNA rearrangements between Dbeta and Jbeta segments but limited DNA rearrangements between Vbeta and DJbeta without effect of distal or proximal positions. Because this impairment may be due to chromatin accessibility, we have examined histone H3 acetylation in Bcl11b–/– DN3 cells using chromatin immunoprecipitation assay. No change was observed in acetylation at the Vbeta and Dbeta gene locus. Analysis of Bcl11b–/– DN4 thymocytes showed apoptosis, accompanied with lower expression of anti-apoptotic proteins, Bcl-xL and Bcl-2, than wild-type DN4 thymocytes. Interestingly, the transgenic TCR{alpha}beta in those cells reduced apoptosis and raised their protein expression without increased cellularity. These results suggest that Bcl11b deficiency affects many different signaling pathways leading to development arrests.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cell development in the thymus is a complex and ordered process. Immature thymocytes that express neither CD4 nor CD8 surface molecules (termed double negative, DN)34 progress to express CD8 (immature CD8+ single positive, ISP). The ISP thymocytes progress to express both CD4 and CD8 concurrently (double positive, DP). The DP thymocytes then differentiate into cells expressing either CD4 or CD8 (single positive, SP). The DN thymocytes can be divided into four subpopulations based on the surface expression of CD44 and CD25, with the developmental progression being CD44+CD25– to CD44+CD25+ to CD44–CD25+ (DN3) and then to CD44–CD25– (DN4) (1). To make the developmental transition from DN3 to DP, proteins produced from productively rearranged TCRbeta genes must be assembled into the pre-TCR complex, which consists of a TCRbeta-chain, the invariant pT{alpha}-chain, and CD3 components (2). Only cells that have a functional pre-TCR survive the transition from DN3 to DP, a process also known as beta selection.

A failure of the pre-TCR signaling, as in scid, RAG-1–/–, or RAG-2–/– mice that cannot rearrange TCR genes, results in an arrest at the DN3 stage of development accompanying with apoptosis (3, 4, 5, 6). As expected, introduction of the functional TCRbeta-chain into those mice can rescue the transition from DN3 to DP stage and prevent apoptosis (6, 7, 8). The mechanism remains unclear by which the pre-TCR signaling controls differentiation, proliferation, and survival of thymocytes. Apoptosis due to the absence of pre-TCR signaling appears to involve the tumor suppressor p53 and Fas-associated death domain (FADD), because the deficiency of p53 (9, 10, 11, 12) or FADD (13) and the expression of a dominantly interfering mutant of FADD in pre-TCR-deficient thymocytes (14) can restore the development and survival. However, it is interesting that introduction of the anti-apoptotic protein Bcl-2 does not promote survival of pre-TCR-deficient DN3 cells in scid mice (15). Thymocytes require other signals of the Wnt signaling pathway for the differentiation in addition to those signals (16). Forced activation of beta-catenin, a central effector of the Wnt signaling cascade, in RAG-2–/– mice also promotes differentiation from the DN3 to DP stage (17).

Bcl11b/Rit1/CTIP2 is a tumor suppressor gene that encodes zinc finger transcription factors (18, 19), homologous to a proto-oncogene, Bcl11a/Evi9 (20). We previously generated Bcl11b/Rit1-deficient mice that die within the first day after birth. Loss of Bcl11b confers interruption of {alpha}beta T cell development at the DN3 or ISP stages and profound apoptosis in the thymus of neonatal mice but does not affect development of cells of the {gamma}{delta} T or B cell lineages (21). Because Bcl11b–/– thymocytes fail to express the pre-TCR on the cell surface, an arrest at the DN3 stage may be ascribed to lack of this expression, although the mechanism for arrest at the ISP stage is not known. Introduction of p53 deficiency into Bcl11b–/– mice restores thymocyte differentiation from the DN3 to ISP, but not DP, stage (22), which is different from the influences in scid or RAG-2–/– mice as described above. This suggests that Bcl11b–/– thymocytes have defects in not only the pre-TCR signaling but also some other signaling required for transition from the ISP to DP stage.

Bcl11b–/– thymocytes exhibit an incomplete rearrangement of the TCRbeta locus (21). Recombination between Dbeta and Jbeta segments normally occurs while recombination between Vbeta and DJbeta segments is reduced in Bcl11b–/– thymocytes. However, the finding was obtained from analysis of unfractionated total thymocytes, so that the stage and mechanism of the impairment was unclear. Also, the stage-dependency of apoptosis in Bcl11b–/– thymocytes remains to be addressed. In this study, we report immunological and biochemical analyses of fractionated subsets of Bcl11b–/– thymocytes. Furthermore, we have generated Bcl11b–/– mice that express functional TCRbeta and TCR{alpha}beta-chains by introducing those transgenes and examined the development and apoptosis of thymocytes. In this study, we show that the expression of either TCRbeta or TCR{alpha}beta fails to promote the transition from the DN3 to DP stage of development, indicating that the pre-TCR signal cannot compensate the deficiency of Bcl11b. Furthermore, Bcl11b–/– DN4 thymocytes show apoptosis and low expression of anti-apoptotic proteins, Bcl-xL, and Bcl-2, and the TCR{alpha}beta transgene expression inhibits the apoptosis possibly due to the premature expression of TCR{alpha}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Bcl11b–/– mice of the BALB/c background were generated as described (21). Rag2/TCRB knockout/microinjected mice of the C57BL/6 background (23) and DO11.10 OVA-TCR transgenic (Tg) mice of the BALB/c background (24) were purchased from Taconic Farms. The former Tg mice express a TCRbeta TCR gene (V°8/D°1/J°1.1 gene) derive from a T cell hybridoma DO11.10, and the latter mice express the same TCRbeta gene and a TCR{alpha} TCR gene (Va13). Those mice were housed in pathogen-free animal facilities at Niigata University.

Flow cytometry and cell sorting

Thymocyte subsets were analyzed and sorted on a FACSAria (BD Biosciences), and data were analyzed using the FlowJo software (Tree Star). We conducted flow cytometric analysis as described previously (21). The following mAbs were purchased from eBioscience: anti-CD4-FITC or anti-CD4-allophycocyanin (RM4-5), anti-CD8-allophycocyanin (53-6.7), anti-CD25-FITC, or anti-CD25-PE (PC61.5), anti-CD44-biotin (IM7), and anti-TCR-{alpha}beta-FITC, or anti-TCR-{alpha}beta-PE (H57-597). Biotinylated Ab was revealed with streptavidin-allophycocyanin-Cy7 (eBioscience). To prevent nonspecific binding of mAbs, we added CD16/32 (93; eBioscience) before staining with labeled mAbs. Dead cells and debris were removed by appropriate gating of forward scatter and side scatter. Anti-TCRbeta-(H57-597) and control hamster Ig-FITC (eBioscience) were used for intracellular staining. Intracellular staining was performed using the Fix and Perm kit (Caltag Laboratories).

Recombination analysis

DNA was isolated using the DNeasy tissue kit (Qiagen) from sorted DN3 thymocytes and total thymocytes. PCR for TCRbeta rearrangements and {alpha}-catenin was performed as described (21). Primers specific for Vbeta2 (5'-GTGGCAGTTTTGCATTCTGTGCCT-3'), Vbeta4 (5'-CCTGATATGCGAACAGTATCTAGGC-3'), Vbeta12 (5'-AGTTACCCAGACACCCAGACATGA-3'), Vbeta6 (5'-GAAGGCTATGATGCGTCTCGAGA-3'), Vbeta3 (5'-GGCTACAAGGCTCCTCTGTTACAC-3'), Vbeta18 (5'-AACAGGGACATCTGTCAAAGTGGC-3'), Vbeta14 (5'-TCATCCTAAGCACGGAGAAGCTGC-3'), Dbeta1 (5'-GTAGACCTATGGGAGGGTCCTTTT-3'), Dbeta2 (5'-GTAGGCACCTGTGGGGAAGAAACT-3'), and a region immediately downstream of the last Jbeta segment in the Jbeta2 gene cluster (5'-TGAGAGCTTGTCTCCTACTATCGATT-3') were used to detect Vbeta-to-DJbeta2 and Dbeta-to-Jbeta2 rearrangements. Template DNA was used at three different concentrations in each experiment to ensure linearity of the PCR signal. As a control for the amount of template DNA, aliquots of a PCR mastermix were amplified with primers specific for {alpha}-catenin (5'-GCATGGCTACAGTTACTAATG-3') and (5'-TGAGCCCGATGGTGAATTTG-3'). The reaction was processed through 32 cycles of 94°C for 30 s, 63°C for 1 min, and 72°C for 5 min (Vbeta-to-DJbeta) or 25 cycles of 94°C for 30 s, 63°C for 1 min, and 72°C for 2 min (Dbeta-to-Jbeta). PCR products were separated on 2% agarose gels and blotted onto a Hybond N+ membrane (Amersham Biosciences) and detected by hybridization with the 32P-labeled Jbeta2 probe. Jbeta2 probe was labeled using the Ramdom Primer DNA labeling kit (Takara Shuzo) and [{alpha}-32P]deoxycytodine triphosphate. Experimental band intensities were normalized to the loading control, which was a PCR product from a nonrecombining locus ({alpha}-catenin), detected by staining with ethidium bromide and quantitated with a Molecular Imager FX (Bio-Rad).

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation assay was performed essentially as described (25, 26). We used ~1 x 106 cells for the fixation with 1% formaldehyde for 30 min at 37°C and the Ab specific to acetylated histone H3 (against residues 1–21 of histone H3, diacetylated at K9 and K14; Upstate Biotechnology). After separation of protein A-Sepharose (Pharmacia) bound and unbound complexes, extensive washing, reverse cross-linking, and RNase A and proteinase K treatment were conducted. DNA was extracted from each of the two fractions, and the size of DNA was 0.5–4 kb in length. We performed multiplex PCR in the presence of 0.1 µl of [{alpha}-32P]dCTP (10mCi/ml) to normalize the relative amount of TCRbeta gene region with respect to the {alpha}-catenin PCR product. The reaction was processed through 35 cycles of 94°C for 30 s, 51°C for 30 s, and 72°C for 30 s. The amount of each PCR product in gel was measured by densitometry (Molecular Imager FX; Bio-Rad) after gel electrophoresis, and the relative fold enrichments (chromatin-bound (B)/chromatin-unbound (U)) were calculated by normalizing with the B:U ratios measured in the amplified products of {alpha}-catenin gene.

Primers were designed to span the RSS elements of specific Vbeta gene segments. Sequences of primers used in this experiment and expected product sizes (indicated as bp in parentheses) are as follows: RVbeta2, 5'-TCACTGATACGGAGCTGAGG-3' and 5'-TAGCACAAGGTGATGGGGAA-3' (210); RVbeta4, 5'-CAGTATCTAGGCCACAATGC-3' and 5'-GCTCAGGTAGACCAGTTACA-3' (301); RVbeta12, 5'-CATCCTTCTCCACTCTGAAG-3' and 5'-CTTCAAGGTCATTTTCCACC-3' (263); RVbeta6, 5'-ACTGTGACATCTGCCCAGAA-3' and 5'-GTACAGTAGTCGGTAGCTAC-3' (200); RVbeta3, 5'-CTCACCTTGCAGCCTAGAAA-3' and 5'-CTGCTGTGGTTGATACAGGT-3' (202); RVbeta18, 5'-GACAGTGAACAATGCAAGGC-3' and 5'-CCCACAGACATATGAACAGG-3' (201); RVbeta14, 5'-ATCCCTAGTGAGGGTTCCTA-3' and 5'-ACTGAACCTCTCAGCTTCCA-3' (191); RDbeta1, 5'-GCAGCTTATCTGGTGGTTTC-3' and 5'-AACACATCTAGGCTTGCGAC-3' (198); and RDbeta2, 5'-AGTCAGACAAACCTCTCTGC-3' and 5'-CATAGACTCCTCCTCACATG-3' (222). All fractionated samples were subjected to PCR with a set of primers that simultaneously amplified the functional p53 gene and the p53 pseudogene to confirm success in the fractionation (27, 28).

Immunoblotting

Immunoblotting was performed as described previously (21). We obtained monoclonal anti-Bcl2 (YTH-10C4) and Bcl-xL (YTH-2H12) from Trevigen. Anti-actin Ab was obtained from Santa Cruz Biotechnology.

Flow cytometric analysis of DNA fragmentation

DNA fragmentation of thymocytes was analyzed using the in situ Cell Death detection kit (Roche Applied Science) according to the manufacturer’s protocol. In brief, sorted cells were treated with 3N HCl, washed in PBS, neutralized with 0.1 M Na2B4O7 (29), and fixed with 2% paraformaldehyde and permeabilized with Triton-X and citric acid after washing twice in PBS. Cells were incubated with TdT enzyme and FITC reactive solution at 37°C for 60 min (22). Those cells were analyzed by a FACSAria (BD Biosciences). We used newborn Bcl11b–/– and control littermates mice that express the transgenes of TCRbeta and TCR{alpha}beta (23, 24).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Decrease of icTCRbeta+ cells in Bcl11b–/– DN thymocytes

Our previous study showed very low expression of the pre-TCR complex on the cell surface of Bcl11b–/– total thymocytes and that this reduction may be responsible for the developmental anomaly (21). To analyze expression of the pre-TCR complex in more details, we examined intracellular TCRbeta (icTCRbeta) and surface TCRbeta expressions in DN3 and DN4 subsets of thymocytes prepared from Bcl11b–/– and wild-type littermates. The proportions of icTCRbeta+ cells in DN3 and DN4 thymocytes were 26.7 and 38.4%, respectively, in Bcl11b–/– mice and 37.1 and 73.9%, respectively, in wild-type littermates (Fig. 1A). In contrast, surface TCRbeta proteins were not detected in DN3 thymocytes from either mice but were in 7.5% of the wild-type DN4 thymocytes and in only a fraction (2.0%) of the Bcl11b–/– DN4 thymocytes. Fig. 1B shows calculation of absolute numbers of icTCRbeta+ and icTCRbeta– cells in DN3 and DN4 thymocytes. The icTCRbeta+ cells were retained in Bcl11b–/– DN3 thymocytes on a level with those of control littermates but significantly reduced in Bcl11b–/– DN4 thymocytes (left). Reduction also was seen in the icTCRbeta– cells (right). Fig. 1C shows calculation of absolute numbers of surface TCRbeta+ and surface TCRbeta– cells in DN3 and DN4 thymocytes. The surface TCRbeta+ cells were seen in control DN4 thymocytes but significantly reduced in Bcl11b–/– DN4 thymocytes (left), and a similar reduction of the surface TCRbeta– cells was seen in Bcl11b–/– DN4 thymocytes (right).


Figure 1
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  		FIGURE 1. Impaired intracellular and surface TCRbeta expression in Bcl11b–/– thymocytes. A, Thymocytes from Bcl11b–/– and wild-type littermates were analyzed by four-parameter flow cytometry for intracellular TCRbeta expression using TCRbeta-FITC (shaded areas) and control Ig-FITC mAb (open areas) in DN3 (CD4–CD8–CD25+CD44–) and DN4 (CD4–CD8–CD25–CD44–) thymocytes and surface TCRbeta expression (TCRbeta-FITC: shaded areas) on DN3 and DN4 thymocytes. The numbers in the squares indicate the percentage ± SD of icTCRbeta+ or surface TCRbeta+ cells for Bcl11b–/– and wild-type littermates where n = 6 for each. B, Absolute cell numbers were calculated for icTCRbeta+ (left) and icTCRbeta– (right) DN3 and DN4 thymocytes in Bcl11b–/– and wild-type littermates. C, Absolute cell numbers were calculated for surface TCRbeta+ (left) and surface TCRbeta– (right) DN3 and DN4 thymocytes in Bcl11b–/– and wild-type littermates. The vertical bars represent mean ± SD values where n = 6 for each. *, p < 0.05, compared with littermate control cells. **, p < 0.01, compared with littermate control cells.

 
Impaired VDJbeta rearrangement in Bcl11b–/– thymocytes

The decrease of icTCRbeta+ DN cell population in Bcl11b–/– thymocytes may be due to impairment of VDJbeta rearrangement. Thus, we assayed Dbeta-to-Jbeta and Vbeta-to-DJbeta rearrangements using a semiquantitative PCR assay in total thymocytes and the DN3 subset that was sorted by flow cytometry. Fig. 2A shows a schematic representation of positions of V, D, and J segments at the TCRbeta locus analyzed in this study. Fig. 2B shows results of the rearrangement of Dbeta1-to-Jbeta2 and Vbeta4.1-to-DJbeta2 in DN3 thymocytes and Fig. 2C summarizes results of these and also of Dbeta2 and other six Vbeta genes. The rearrangement of Dbeta1-to-Jbeta2 or Dbeta2-to-Jbeta2 did not differ between Bcl11b–/– and wild-type DN3 thymocytes, whereas the frequencies of rearrangement of Vbeta-to-DJbeta decreased in Bcl11b–/– DN3 thymocytes (Fig. 2C, top). Similar results were obtained from analysis of total thymocytes (Fig. 2C, bottom). These results indicate that Bcl11b deficiency affects the Vbeta-to-DJbeta, but not Dbeta-to-Jbeta, rearrangements in thymocytes. This impairment may be a cause for the decrease of icTCRbeta+ DN cell population in Bcl11b–/– thymocytes.


Figure 2
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  		FIGURE 2. VDJbeta, but not DJbeta, rearrangements affected by Bcl11b deficiency. A, The TCRbeta locus is schematically represented. The relative location of the Dbeta and Vbeta gene segments analyzed in this study are shown. B, Southern blots of PCR products was performed on DNA of the sorted DN3 cells that were derived from Bcl11b–/– and wild-type littermates. Dbeta1-to-Jbeta2 (top) and Vbeta4-to-DJbeta2 (middle) rearrangements are shown. PCR products using {alpha}-catenin primers for the same DNA are shown as a loading control (bottom). Lanes 1–6 represent 2-fold serial dilutions. C, VDJbeta and DJbeta rearrangements are quantitated in sorted DN3 thymocytes (top, four independent preparations) and total thymocytes (bottom, six independent preparations) that were isolated from Bcl11b–/– and wild-type mice. Results are expressed as percentages of the rearrangements detected.

 
Histone acetylation of TCRbeta V gene segments in Bcl11b–/– DN3 cells

It has been shown that acetylation of histone H3 is tightly correlated with locus-wide accessibility to VDJ recombination at the TCR and IgH loci and is directed by the enhancer and promoter in vivo (30, 31). Thus, we examined accessibility of Vbeta gene segments in sorted DN3 thymocytes using chromatin immunoprecipitation assay. U and B fractions were prepared using the Ab against acetylated histone H3 and DNA in the two fractions was quantitated by measuring respective bands of multiplex PCR assays. DNA amounts used for PCR were adjusted by using {alpha}-catenin primers as a reference so as to give the same amounts of PCR products between the two U and B fractions. Fig. 3A shows gel electrophoresis of PCR products using probes for the functional and pseudo p53 genes. B fraction exhibited the band signal of p53 functional gene on chromosome 11 more intense than the band signal in U fraction in both wild-type and Bcl11b–/– mice, while nonfunctional p53 pseudogene on chromosome 17 displayed a reverse pattern, indicating that B fraction contained chromatin of a more condensed structure than the U fraction did in those preparations.


Figure 3
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  		FIGURE 3. Histone H3 acetylation at the TCRbeta locus in DN3 thymocytes derived from Bcl11b–/– and wild-type littermates. A, Bound and unbound fractions of anti-AcH3 immunoprecipitated DNA were analyzed by PCR using p53 primers sets and {alpha}-catenin primers sets as internal control. U and B represent DNA templates prepared from unbound and bound chromatin fractions, respectively. B, Primers designed span the RSS elements of specific Vbeta gene segments. Multiplex PCR was conducted using RVbeta, RDb, or p53 primers and primer sets for {alpha}-catenin as an internal control. Results are presented as relative fold enrichment of the target sequences were calculated from the B/U ratios. The bars represent mean ± SD values where n = 3 for each.

 
We used PCR primers for locus-wide accessibility to VDJ recombination that spanned the RSS elements of specific Vbeta gene segments. Fig. 3B shows the relative fold enrichment of target sequences that was calculated from the B/U ratios. The relative fold enrichment of Dbeta1 and Dbeta2 was 1.6 and 1.2, respectively, in wild-type mice and 1.6 and 1.1, respectively, in Bcl11b–/– mice. These enrichment levels did not differ between Bcl11b–/– and wild-type mice, and those of Dbeta1 regions were similar to that of the functional p53 locus, indicative of open chromatin conformations. In contrast, Vbeta regions showed similar enrichments between Bcl11b–/– and wild-type thymocytes, and the levels ranged from 0.7- to 1.1-fold, not much skewed depending on positions. These results suggest that Bcl11b deficiency does not affect chromatin accessibility in the whole Vbeta and Dbeta gene region.

Apoptosis in Bcl11b–/– thymocytes

Our previous study showed that total thymocytes are 10-fold less in Bcl11b–/– mice than in wild-type mice (21). In this study, we examined absolute numbers of DN thymocytes and DN3 and DN4 subsets of thymocytes in Bcl11b–/– and wild-type littermates (Fig. 4A). The cellularity of DN3 thymocytes did not differ between them, whereas that of the DN4 subset from Bcl11b–/– mice was one-fifth less than that from wild-type littermates. To test whether or not the reduction of DN4 thymocytes in Bcl11b–/– mice was due to apoptosis, we performed TUNEL analysis for DN3 and DN4 thymocytes. In DN3 thymocytes, TUNEL-positive cells were not detected in either mice. In contrast, TUNEL-positive cells in Bcl11b–/– DN4 cells were detected at a significant level of 6.5%, ~4-fold higher than a background level (1.6%) of wild-type DN4 cells (Fig. 4B).


Figure 4
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  		FIGURE 4. Apoptosis in Bcl11b–/– thymocytes. A, Absolute cell numbers were calculated for DN3 (CD4–CD8–CD25+CD44–), and DN4 (CD4–CD8–CD25–CD44–) cells. The bars represent mean ± SD values for Bcl11b–/– and wild-type littermates where n = 5 for each. B, TUNEL assay was performed on sorted DN3 and DN4 thymocyte derived from Bcl11b–/– and wild-type littermates. The number above each bar indicates the percentage of TUNEL-positive cells. C, Extracts of total thymocyte (left), sorted DN3 (center), and DN4 thymocyte (right) were analyzed by immunoblots for Bcl-xL and Bcl-2. C, wild type; –/–, Bcl11b–/–.

 
Expression of Bcl-xL and Bcl-2 was examined in the DN3 and DN4 thymocytes using Western blot analysis. Their expression levels in total thymocytes from Bcl11b–/– mice were similar to that of scid thymocytes (Fig. 4C, left), consistent with our previous result (21). In Bcl11b–/– DN3 thymocytes, in contrast, the expression of Bcl-xL and Bcl-2 both increased (Fig. 4C, right). Of note is that both expressions decreased in Bcl11b–/– DN4 thymocytes (Fig. 4C, right). These decreases of anti-apoptotic proteins may account for the increased apoptosis in Bcl11b–/– DN4 thymocytes.

Expression of functional TCRbeta or TCR{alpha}beta gene in Bcl11b–/– thymocytes

Reduced expression of the pre-TCR on the cell surface in Bcl11b–/– thymocytes could be the cause for the arrest of T lymphocyte differentiation and apoptosis. To test this possibility, we generated Bcl11b–/– mice with expression of rearranged TCRbeta and TCR{alpha}beta-chains by mating Bcl11b–/– mice to Rag2/TCRB Tg strain expressing the Vbeta8.1 T cel l receptor gene (23) and DO11.10 TCR Tg strain expressing the same Vbeta8.1 and the V{alpha}13 TCR gene (24), respectively. It was reported that introduction of these transgenes in RAG-2–/– mice can promote transition from the DN3 to SP stage of development (8, 23, 32). Fig. 5A shows flow cytometric analysis of DN3 and DN4 thymocytes from TCRbeta-Tg-Bcl11b–/– and control Tg-littermates using the anti-TCRVbeta8 Ab. Intracellular staining showed high expressions, consistent with previous reports (23, 24), and revealed that the percentages of icTCRVbeta8+ thymocytes did not differ between TCRbeta-Tg-Bcl11b–/– mice and TCRbeta-Tg-wild-type littermates in either DN3 or DN4 thymocytes. Also, the proportion of surface TCRVbeta8+ DN4 thymocytes did not differ between them. Fig. 5B shows similar expression patterns in the analysis of TCR{alpha}beta-Tg-Bcl11b–/– thymocytes except for the difference that the percentage of icTCRVbeta8+ DN3 thymocytes in TCRbeta-Tg-Bcl11b–/– mice was twice higher than that of TCRbeta-Tg-wild-type littermates. These results indicated that the pre-TCR complex and probably the TCR{alpha}beta can express on the cell surface of Bcl11b–/– DN4 thymocytes. Analysis using anti-DO11.10 Ab KJ1–26 showed similar results (data not shown). These data suggest no impairment in processing of the Tg Vbeta8 proteins from the cytoplasm onto the cell surface in Bcl11b–/– thymocytes.


Figure 5
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  		FIGURE 5. Intracellular and surface TCRVbeta8 expression in Bcl11b–/–-TCRbeta Tg mice (A) and Bcl11b–/–-TCR{alpha}beta Tg mice (B). Representative histograms are shown for intracellular TCRVbeta8 expression on DN3 and DN4 thymocytes and surface TCRVbeta8 expression on DN4 thymocytes. The number above the bar indicates the percentage ± SD of icTCRbeta+ or surface TCRbeta+ cells for Bcl11b–/–, Bcl11b–/–-Tg, and corresponding littermate controls (n = 4–6).

 
Effect of Tg TCRbeta or TCR{alpha}beta expression on differentiation, cellularity, and apoptosis

We examined T cell differentiation and cellularity of DN3 and DN4 thymocytes in Tg-Bcl11b–/– and Tg-wild-type littermates that were generated using the two different Tg mice. Fig. 6, A and D, show flowcytometric analysis using Abs to CD4 vs CD8 and CD25 vs CD44. Analysis of CD4 vs CD8 showed that introduction of functional TCRbeta did not affect either transition from the DN to DP stage or thymic cellularity in Bcl11b–/– mice. Also, no marked change was observed in Tg-wild-type littermates except for percent increase in the CD4-SP subset, which is consistent with previous reports (32). Similarly, analysis of CD25 vs CD44 exhibited no difference in cell distribution and cellularity between Tg-Bcl11b–/– and Bcl11b–/– littermates (Fig. 6, B and C). These results indicated no promotion by the functional TCRbeta expression of thymocyte differentiation from the DN3 to the DP stage in Bcl11b–/– mice. In contrast, analysis of mice receiving functional TCR{alpha}beta exhibited similar results except for the difference in effect on the DN4/DN3 ratio (Fig. 6, D and E). Although the ratio differed from that in control mice of the experiments using TCRbeta Tg mice possibly reflecting different genetic background (Fig. 6, B and E), introduction of the TCR{alpha}beta transgene increased the ratio that changed from 0.5 ± 0.3 to 1.4 ± 0.3 in Bcl11b–/– mice and from 1.5 ± 0.3 to 2.9 ± 0.8 in wild-type littermates. The absolute number of Tg-Bcl11b–/– DN4 cells was 2-fold higher than that in Bcl11b–/– mice but that of DN3 cells was 2-fold lower (Fig. 6F). The accelerated transition from the DN3 to DN4 stage in the TCR{alpha}beta introduction may be due to the premature TCR{alpha} expression in DN3 thymocytes (33).


Figure 6
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  		FIGURE 6. Phenotypes of thymocytes in Bcl11b–/–-TCRbeta Tg mice (A–C) and Bcl11b–/–-TCR{alpha}beta Tg mice (D–F). A and D, Thymocytes were analyzed for the expression of CD4 and CD8 (top), and the CD4– CD8– DN thymocyte subset was further examined for the expression of CD25 and CD44 (bottom). The percentage of cells is shown in the appropriate quadrant. Absolute cell number of total thymocyte is indicated on the top as mean ± SD values. B and E, The percentages of DN3 (CD4–CD8–CD25+CD44–) and DN4 (CD4–CD8–CD25–CD44–) are shown for Bcl11b–/–, Bcl11b–/–-Tg, and corresponding littermate controls. The bars represent mean ± SD values (n = 4 for TCRbeta Tg mice and n = 6 for TCR{alpha}beta Tg mice). C and F, Absolute cell numbers were calculated for DN3 (CD4–CD8–CD25+CD44–) and DN4 (CD4–CD8–CD25–CD44–) subsets from Bcl11b–/–-TCRbeta or Bcl11b–/–-TCR{alpha}beta Tg mice and indicated control littermates. The bars represent mean ± SD values (n = 4 for TCRbeta Tg mice and n = 5 for TCR{alpha}beta Tg mice).

 
The finding that introduction of TCR{alpha}beta, but not TCRbeta, increased the number of DN4 cells in Bcl11b–/– mice suggests that TCR{alpha}beta expression inhibits apoptosis detected in Bcl11b–/– DN4 cells (see Fig. 4B). To investigate this possibility, we examined DN4 thymocytes from TCR{alpha}beta-Tg-Bcl11b–/– mice with TUNEL assay. TUNEL-positive cells were 1.4 ± 0.6% much lower than 6.5 ± 0.6% in Bcl11b–/– DN4 thymocytes. This decrease in TUNEL-positive cells suggests that the TCR{alpha}beta expression can rescue apoptosis in Bcl11b–/– DN4 thymocytes (Fig. 7A). In contrast, the proportion of TUNEL-positive cells in the ISP subset was as high as ~20%, not affected by the functional TCR{alpha}beta expression. Expression of Bcl-xL and Bcl-2 was both higher in Tg-Bcl11b–/– thymocytes than Bcl11b–/– thymocytes (Fig. 7B). These elevated expressions may prevent apoptosis in DN4 cells of Bcl11b–/– mice.


Figure 7
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  		FIGURE 7. Rescue of apoptosis in Bcl11b–/– DN4 thymocytes by TCR{alpha}beta expression. A, TUNEL assay was performed on sorted DN4 and ISP thymocytes derived from Bcl11b–/– and Bcl11b–/–-TCR{alpha}beta Tg mice. The number above the bar indicates the percentage of TUNEL-positive cells. B, Sorted DN4 thymocytes derived from Bcl11b–/– and Bcl11b–/–-TCR{alpha}beta mice were analyzed by immunoblots for Bcl-xL and Bcl-2.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study analyzes expression of TCRbeta proteins in sorted DN3 and DN4 subsets of thymocytes in Bcl11b–/– mice. The Bcl11b–/– mice show developmental arrest of {alpha}beta T cells and the arrest at DN3 stage could be ascribed to lack of the pre-TCR expression. Bcl11b–/– DN4 thymocytes lack TCRbeta on the surface, though containing it within the cell, and show apoptosis accompanying low expression of anti-apoptotic proteins, Bcl-xL and Bcl-2. Either TCRbeta or TCR{alpha}beta expression by transgene introduction fails to promote transition from the DN3 to DP stage of development, indicating that the pre-TCR signaling cannot compensate the deficiency of Bcl11b. Interestingly, the expression of TCR{alpha}beta inhibits apoptosis of Bcl11b–/– DN4 thymocytes possibly due to the premature TCR{alpha} expression. Furthermore, we investigate DNA rearrangements at the TCRbeta locus in DN3 thymocytes in light of chromatin structure. No change is detected in acethylation of histone H3, although rearrangements between Vbeta and DJbeta segments, but not between Dbeta and Jbeta segments, are reduced in Bcl11b–/– mice.

Expression of TCRbeta proteins and differentiation in Bcl11b–/– mice

Surface expression of TCRbeta proteins to form the pre-TCR complex has been shown to be necessary and sufficient to drive T cell differentiation from the DN to DP stage (34). Signals derived from the pre-TCR complex trigger a maturation program that includes differentiation, proliferation and inhibition of apoptosis. In Bcl11b–/– mice, this surface expression was not observed in either DN4 or DN3 thymocytes whereas expression of TCRbeta proteins within the cell was not much impaired, the level being a half less than that in wild-type mice. This suggests some impairment in processing of TCRbeta proteins from the cytoplasm to the cell surface. However, such impairment was not observed in processing of the TCRVbeta8 proteins in Bcl11b–/– thymocytes that were derived from TCRbeta or TCR{alpha}beta transgenes. The difference and how Bcl11b zinc-finger proteins affect this processing remains to be addressed.

Failure of the TCRbeta expression may be a cause for the developmental arrest at DN3 stage in Bcl11b–/– mice. However, even in the absence of TCRbeta, the development of DN3 thymocytes can be promoted by signals derived from other receptors of TCR{alpha}beta and TCR{gamma}{delta} and only mice that cannot produce any of these receptors will exhibit complete arrest at the DN stage of development, as seen in RAG-1–/– and RAG-2–/– mice (35). However, our rescue experiments using the TCRbeta and TCR{alpha}beta transgenes showed failures to promote the transition from the DN3 to DP stage in Bcl11b–/– mice. This indicates that the developmental arrest in Bcl11b–/– mice is not solely due to lack of TCRbeta proteins on the cell surface. In contrast, the transition from DN3 to DN4 stage was observed in the rescue experiment using TCR{alpha}beta transgenes but not a TCRbeta transgene. This increased DN4/DN3 ratio is probably due to enhanced TCR{alpha}beta signaling in DN3 thymocytes, as seen in TCR{alpha}beta Tg mice (33). Semiquantitative RT-PCR analysis of the pT{alpha} expression that also affects the DN4/DN3 ratio (36) did not show difference in DN3 thymocytes between wild-type and Bcl11b–/– mice (data not shown).

p56lck is a kinase mediating signals downstream of the pre-TCR. p56lck–/– mice that show arrest at the DN stage of thymocyte development (37). Introduction of TCRbeta or TCR{alpha}beta transgenes into p56lck–/– mice does not restore differentiation to DP stage (38), and this failure is ascribed to positioning of p56lck in signaling downstream of the pre-TCR. Accordingly, Bcl11b deficiency may affect signaling downstream of the pre-TCR and possibly of the TCR{alpha}beta. As described in the introduction, developmental arrest and apoptosis due to the absence of pre-TCR signaling can be restored by inhibition of p53 or FADD apoptotic pathways and also by activation of beta-catenin to confer the Wnt signaling cascade. We demonstrated previously that a consequence of Bcl11b deficiency can be rescued by introduction of deficiency of p53 (22). Bcl11b–/–p53–/– mice exhibit further transition from the DN3 to ISP stage probably due to escape of ISP thymocytes from apoptosis. This may be consistent with the above interpretation, i.e., the influence of Bcl11b deficiency to signaling downstream of the pre-TCR. Effect of the beta-catenin activation in Bcl11b–/– mice remains to be examined.

Expression of TCR{alpha}beta proteins and apoptosis in the DN4 subset of thymocytes

Bcl11b–/– DN4 thymocytes exhibited reduced cellularity and elevated apoptosis, although Bcl11b–/– DN3 thymocytes were not affected. BrdU incorporation and cell cycle studies showed that DN4 thymocytes have a high proliferation rate, whereas that of DN3 thymocytes is low (39). Accordingly, the reduced cellularity in Bcl11b–/– DN4 thymocytes is probably attributed to apoptosis of these cells. Decreases in the expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL, were detected in Bcl11b–/– DN4 thymocytes, and therefore these decreases may account for the apoptosis. However, it is not known how Bcl11b deficiency leads to the decrease of those proteins.

DN4 thymocytes with enhanced expression of TCR{alpha}beta in the Tg-Bcl11b–/– mice showed less apoptosis and more expression of Bcl-2 and Bcl-xL proteins than DN4 Bcl11b–/– thymocytes. Therefore, one may predict an increase in the cellularity of Tg-Bcl11b–/– DN4 thymocytes. In fact, the cellularity appeared to increase twice. However, the cellularity of Bcl11b–/– DN3 thymocytes decreased at the same time, and hence the total number of DN thymocytes did not increase. One possibility accounting for this is that DN4 thymocytes further differentiate to the ISP stage, and such ISP thymocytes undergo profound apoptosis. As discussed above, Bcl11b–/–p53–/– mice show transition to the ISP stage and elevated cellularity of ISP thymocytes probably due to escape from apoptosis (22). Therefore, this apoptosis at the ISP stage is controlled by p53 and in the presence of p53 apoptosis is induced in the ISP subset of thymocytes lacking Bcl11b.

VDJ recombination and chromatin structure

DN3 thymocytes lacking expression of Bcl11b undergo limited DNA rearrangements between Vbeta and DJbeta segments, although rearrangements between Dbeta and Jbeta segments was unaffected. This suggests that Bcl11b plays a role in controlling rearrangement of the TCRbeta locus during b selection. Such impairment only affecting Vbeta to DJbeta recombination also is seen in early T cells having tissue-specific inactivation of Notch1 at the CD44+CD25+ DN stage (40). These Notch1-negative thymocytes exhibit not only an impairment in {alpha}beta T cell development but also an inhibition of processing from icTCRbeta– DN4 to icTCRbeta+ DN4 cells. This inhibition is also observed in Bcl11b–/– mice, as discussed above. Therefore, signalings of Bcl11b and Notch1 in immature thymocytes may overlap each other. Although Notch1 stimulation of Pax5–/– pro-B cells is known to activate Vbeta germline transcription of TCRbeta locus that may control chromatin accessibility (41), the molecular mechanism by which Notch1 and Bcl11b regulate VDJ recombination process is largely unknown.

V(D)J recombination is initiated by RAG-1- and RAG-2-mediated cleavage at recognition signal sequence in both the TCR and IgH loci. The cleavage for the Dbeta-to-Jbeta and the Vbeta-to-Dbeta rearrangements is differently regulated in vivo by accessibility to chromatin, the former being enhancer-dependent and the latter enhancer-independent. The failure of Vbeta to Dbeta rearrangements in Bcl11b–/– mice, though leaky, suggested that Bcl11b proteins may function as a chromatin modulator at V segment RSSs. Accordingly, we have examined histone H3 acetylation in Bcl11b–/– DN3 cells using chromatin immunoprecipitation assay. However, no change was observed in histone H3 acetylation at the VDbeta gene locus.

Relationship between VDJ rearrangements and histone H3 acetylation was studied in details at the IgH locus of pro-B cells deficient of IL-R and Pax5. Both of the pro-B cells exhibit normal DJH recombination and impaired VDJH recombination with a progressive 3'- to -5' defect (42, 43, 44), but the two different types of pro-B cells differ in histone H3 acetylation. IL-7R signaling is shown to enhance histone acetylation in the 5' portion of the VH cluster (45), supporting the model that IL-7 plays a key role in controlling the accessibility of this portion of the locus. In Pax5–/– pro-B cells, in contrast, the VH genes are present in accessible chromatin (46). Our study also demonstrated that chromatin at the TCRbeta locus in Bcl11b–/– thymocytes is present in accessible chromatin. These results indicate the presence of other factors controlling VDJ recombination in vivo.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants-in-Aid for scientific research of the administration of Education, Science, Art, and Sports in Japan and Public Trust Haraguchi Memorial Cancer Research Fund. Back

2 J.I. and T. K. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Jun Inoue and Dr. Ryo Kominami, Department of Molecular Genetics, Graduate School of Medical and Dental Sciences, Niigata University, Asahimachi 1-757, Niigata 951-8510, Japan. E-mail addresses: jinoue{at}med.niigata-u.ac.jp or rykomina{at}med.niigata-u.ac.jp Back

4 Abbreviations used in this paper: DN, double negative; ISP, immature CD8+single positive; DP, double positive; SP, single positive; FADD, Fas-associated death domain; ic, intracellular; Tg, transgenic; B, bound; U, unbound. Back

Received for publication April 18, 2005. Accepted for publication February 22, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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