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Blood, 15 October 2006, Vol. 108, No. 8, pp. 2669-2677. Prepublished online as a Blood First Edition Paper on June 20, 2006; DOI 10.1182/blood-2006-02-005900.
IMMUNOBIOLOGY c-Myc mediates pre-TCR-induced proliferation but not developmental progressionFrom the Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, MA; the Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; and the Center for Molecular Imaging Research, Massachusetts General Hospital and Harvard Medical School, Charlestown.
Constitutive and cell-autonomous signals emanating from the pre-T-cell receptor (pre-TCR) promote proliferation, survival and differentiation of immature thymocytes. We show here that induction of pre-TCR signaling resulted in rapid elevation of c-Myc protein levels. Cre-mediated thymocyte-specific ablation of c-Myc in CD25+CD44- thymocytes reduced proliferation and cell growth at the pre-TCR checkpoint, resulting in thymic hypocellularity and a severe reduction in CD4+CD8+ thymocytes. In contrast, c-Myc deficiency did not inhibit pre-TCR-mediated differentiation or survival. Myc-/- double-negative (DN) 3 cells progressed to the double-positive (DP) stage and up-regulated TCR   surface expression in the absence of cell proliferation, in vivo as well as in vitro. These observations indicate that distinct signals downstream of the pre-TCR are responsible for proliferation versus differentiation, and demonstrate that c-Myc is only required for pre-TCR-induced proliferation but is dispensable for developmental progression from the DN to the DP stage.
Immature T cells progress through a number of well-defined developmental stages in the thymus. Commitment of bone marrow-derived T-cell progenitors1-6 to the T-cell lineage requires stimulation of Notch signaling,2,3,7,8 and results in the up-regulation of CD25 (double-negative [DN] 2 subset). Survival and proliferation of thymocytes at this stage is supported by cytokines such as interleukin-7 (IL-7) and stem cell factor (SCF). The subsequent CD44-CD25+ (DN3) stage is marked by the rearrangement of the T-cell receptor (TCR) ,  , and  loci. Productive TCR gene rearrangements and synthesis of TCR chains lead to the surface assembly of a functional pre-TCR, comprising the TCR as well as the invariant pT chain and CD3 subunits. Signals emanating from the pre-TCR promote survival and proliferation of immature thymocytes as well as their differentiation to the CD4+CD8+ double-positive (DP) stage, effectively instructing immature thymocytes to the T-cell lineage.9
Despite recent progress in understanding pre-TCR signaling many questions remain unanswered. It is currently known that the pre-TCR is constitutively localized in plasma membrane glycoprotein-enriched microdomains (GEMs) from where it signals in a cell autonomous manner.10,11 Proximal events of pre-TCR signaling include the phosphorylation of Lck and Zap70. Assembly of the pre-TCR and activation of this pathway is accompanied by a biphasic calcium mobilization, which appears to be regulated by cytoplasmic IP3 and plasma membrane store-operated calcium channels (SOCs), resulting in nuclear factor (NF) of activated T cells (NFAT) and NF The basic region/helix-loop-helix/leucine zipper (bHLHZip) transcription factor c-Myc has been described to play a role in lymphocyte development. Members of the Myc family (c-Myc, N-Myc, and L-Myc) play an integral role in proliferation, survival, and differentiation of normal and neoplastic cells. Myc binds E-box DNA motifs as a heterodimer with Max, resulting in cell cycle entry26 and transcriptional activation or suppression of genes.27-30 c-Myc has been implicated in cell proliferation31 as well as the control of cell growth.32-37 Its expression increases rapidly in response to growth factors38,39 and B-cell receptor (BCR)40 or TCR ligation.41 Immature B and T lymphocytes express both c-Myc and N-Myc, while mature cells express only c-Myc. Assessing the requirement for c-Myc in T-cell development was hampered by the embryonic lethality of c-Myc deficient mice prior to the development of lymphocytes.42 To bypass this problem, Douglas and colleagues43 generated chimeric animals from Myc-/- embryonic stem (ES) cells and Rag1-/- blastocysts in which the Rag1-/- cells cannot contribute to the lymphoid lineages. In their study, Myc-/- progenitors populated embryonic thymi but had reduced proliferation and failed to develop beyond the late DN stages, leading to the suggestion that c-Myc is essential for development through the pre-TCR checkpoint. c-Myc-deficient cells did not populate adult thymi at all, indicating additional defects at earlier stages of hematopoietic development. More recently, c-Myc has indeed been reported to control the self-renewal of hematopoietic stem cells (HSCs),44 and its conditional ablation in the bone marrow favored self-renewal over differentiation of HSCs in the stem cell niche.45 The involvement of c-Myc in early hematopoietic development indicates that studying its role at the pre-TCR checkpoint requires conditional animal models that avoid the accumulation of developmental defects resulting from c-Myc deficiency at earlier stages. Here we report that c-Myc is rapidly up-regulated upon induction of pre-TCR signaling. To characterize the role of c-Myc at the pre-TCR developmental checkpoint we used mice that allow conditional Cre-mediated thymocyte-specific ablation of this protein starting at the DN3 stage.46 Our studies indicate that c-Myc is required for the proliferation but not the differentiation or survival signals emanating from the pre-TCR.
Mice
To generate the TetObeta transgenic mice, the cDNA encoding the TCR Flow cytometry and antibodies
Four-color fluorescence-activated cell-sorter (FACS) staining was performed as described.48 Antibodies were from BD PharMingen (San Diego, CA): anti-CD3 DN thymocytes were enriched by depletion of lin+ cells using streptavidin-conjugated magnetic beads (Dynal, Oslo, Norway). Cell sorting was performed on a MoFlo cell sorter (DakoCytomation). Semiquantitative RT-PCR
mRNA was extracted from sorted cells using the High-Pure RNA Isolation Kit (Roche, Indianapolis, IN). cDNA from 50 000 cells was prepared with the Superscript-II RT kit (Invitrogen, Carlsbad, CA). Samples were equilibrated with respect to Quantitative real-time RT-PCR
Quantitative reverse-transcription PCR (qRT-PCR) was performed in real time using an ABI7300 machine (Applied Biosystems, Foster City, CA). p21Cip1 and Gadd45 Western blot Pellets of total thymocytes were lysed in RIPA buffer supplemented with Protease Inhibitor Cocktail (Roche) and 1 mM PMSF. Samples were resolved on Bis-Tris gradient gels (Invitrogen) and transferred onto nitrocellulose membranes. Secondary antibodies were conjugated to horse-radish peroxidase (HRP). The signal was detected using the enhanced chemoluminescence Plus (ECL Plus kit; Amersham Biosciences, Arlington Heights, IL). CFSE labeling and OP9-DL1 coculture Sorted cells (106-1.5 x 106) were resuspended in 100 µL PBS/0.1% BSA and 5 µM CFSE and incubated at 37°C for 10 minutes before washing extensively. Viability after labeling exceeded 60%. CFSE-labeled cells were cultured in 10-cm tissue-culture plates containing a confluent monolayer of OP9-DL1 cells. Cocultures were maintained in the presence of 5 ng/mL Flt3L (PeproTech, Rocky Hill, NJ) and 1 ng/mL IL-7 (R&D Systems, Minneapolis, MN) for 4 to 6 days.
Pre-TCR signaling induces c-Myc expression
We examined the effect of pre-TCR signaling on c-Myc by analyzing a novel mouse strain that allows inducible pre-TCR expression. More specifically, a transgenic strain was generated using a cDNA encoding a TCR
The effect of pre-TCR signaling on c-Myc was examined after induction of TCR
c-Myc expression was further examined after induction of pre-TCR like signaling in Rag-deficient mice by Taken together, these data suggest that c-Myc expression is induced by pre-TCR signaling and emphasize the need for detailed analyses to determine its role at the pre-TCR-dependent stages of thymocyte development. Abnormal thymocyte development upon conditional c-Myc ablation in mice To characterize the role of c-Myc specifically downstream from the pre-TCR, avoiding earlier developmental defects, we used a novel mouse model that allows conditional ablation of c-Myc starting at the DN3 stage of thymocyte development. This was obtained by crossing Mycfl/fl mice46 that carry LoxP sites flanking the coding exons 2 and 3 of the Myc gene with mice expressing Cre under the control of the proximal p56Lck promoter52 (LckCre). Cre-mediated deletion of exons 2 and 3 of the Myc gene in compound mutant LckCre-Mycfl/fl mice was detectable at the DN3 stage (data not shown), and thus ablation of c-Myc was expected to coincide with the onset of pre-TCR signaling. Efficient and stage-specific Cre-mediated ablation of c-Myc in these mice was examined by semiquantitative RT-PCR, using cDNA derived from sorted DN3- and DN4-stage thymocytes. Expression of c-Myc was reduced about 5-fold at the DN3 stage and was completely abrogated at the DN4 stage (Figure 2A). Western blot analyses of extracts from similarly sorted cells showed that LckCre control thymocytes had lower levels of c-Myc protein at the DN3 stage than at the pre-TCR-dependent DN4 stage (Figure 2B). Deletion of the Myc gene in LckCre-Mycfl/fl thymocytes severely diminished the expression of c-Myc protein both in the DN3 and DN4 subsets.
To determine the impact of c-Myc ablation on thymocyte development we compared the thymocyte subset distribution (Figure 2C) and thymic cellularity (Figure 2D) of LckCre-Mycfl/fl and control embryos and adult (5- to 8-week-old) mice. Adult LckCre-Mycfl/fl contained approximately 10 times fewer thymocytes (1.2 x 107 ± 0.11 x 107) than LckCre controls (8.9 x 107 ± 0.66 x 107). This mainly reflected an approximately 30-fold reduction in the number of CD4+CD8+ DPs in LckCre-Mycfl/fl (2.7 x 106 ± 0.17 x 106) compared with the equivalent LckCre control cells (8.4 x 107 ± 0.92 x 107) (Figure 2D), and the reduction of the subsequent SP stages. Cellularity at the DN3 stage of LckCre-Mycfl/fl mice was elevated compared with LckCre controls (P = .02; Figure 2D). Interestingly, the number of LckCre-Mycfl/fl DN4-stage thymocytes was comparable with that of LckCre controls, indicating that development through the pre-TCR checkpoint was not inhibited. Similar results were obtained from embryonic thymi (Figure 2C-D). Control Mycfl/fl embryonic thymi contained on average 4.2 x 106 ± 1.2 x 106 cells. This number was reduced by approximately 50% in LckCre-Mycfl/fl mice (2.3 x 106 ± 0.6 x 106), accounting for an approximately 10-fold decrease in the number of DPs (2.2 x 106 ± 0.4 x 106 versus 1.9 x 105 ± 0.8 x 105). The DN compartment remained unaltered in profile (Figure 2C) and cellularity (Figure 2D). Collectively, these data show that conditional ablation of c-Myc at the pre-TCR checkpoint did not affect the transition to the DN4 stage but resulted in the development of fewer DP cells (19% versus 82% adult, and 8% versus 48% embryonic DP cells; Figure 2C). This could either reflect reduced proliferation or increased apoptosis of cells traversing the pre-TCR checkpoint. c-Myc ablation impairs pre-TCR-dependent proliferation Following assembly of the pre-TCR, developing thymocytes undergo rapid proliferation at the DN4 stage before progressing to the CD4+CD8+ DP stage. To address the role of c-Myc in this wave of proliferation, we compared the fraction of cycling cells in LckCre control and LckCre-Mycfl/fl immature thymocytes. Thymocyte suspensions were stained on the surface to allow identification of the various thymocyte subsets, followed by intracellular staining with the DNA-binding dye 7-amino actinomycin D (7-AAD) and FACS analysis. At the resting DN3 stage, only 4% of LckCre and 2% of LckCre-Mycfl/fl thymocytes were in the G2/S/M phases of the cell cycle. The fraction of LckCre control cells in the G2/S/M phases at the actively proliferating DN4 stage was 29.1% ± 1.96%, while only 12.2% ± 0.78% of the c-Myc-deficient thymocytes were cycling (Figure 3A), suggesting that c-Myc was required for proliferation at the pre-TCR checkpoint. We also observed that LckCre-Mycfl/fl DN4 stage cells were smaller (Figure 3B) than the equivalent LckCre cells, probably reflecting the contribution of c-Myc to cell growth.
To ensure that the ablation of c-Myc did not impact pre-TCR assembly we compared the expression of components of the pre-TCR complex in LckCre control and LckCre-Mycfl/fl thymocytes. Semiquantitative RT-PCR using RNA prepared from sorted DN3- and DN4-stage thymocytes indicated that the expression of pT c-Myc deficiency results in deregulation of cell-cycle inhibitors
Although several genes involved in cell-cycle progression and growth control were shown to be transcriptionally regulated by c-Myc,53,54 the mechanism by which c-Myc mediates cell-cycle progression remains unclear, especially in thymocytes. To trace the impact of c-Myc ablation on genes involved in proliferation, we compared their expression in LckCre-Mycfl/fl and LckCre DN3- and DN4-stage thymocytes by semiquantitative and quantitative RT-PCR as well as Western blots (Figure 4A-C). These analyses revealed strikingly elevated protein levels of the cell-cycle inhibitor p27Kip in LckCre-Mycfl/fl DN3 and DN4 thymocytes (Figure 4C). c-Myc-deficient DN4-stage cells also showed elevated expression of the growth arrest and DNA-damage-inducible factor 45 alpha (Gadd45
These observations indicate that c-Myc impacts proliferation at the pre-TCR checkpoint by affecting the expression of cell-cycle inhibitors, especially p27Kip and Gadd45 , rather than directly affecting the expression of the cell-cycle-promoting cyclins or other genes reported to promote thymocyte expansion downstream of the pre-TCR. c-Myc-deficient thymocytes differentiate without proliferating
LckCre-Mycfl/fl thymocytes developed to the CD4+CD8+ DP stage despite reduced proliferation at the DN4 stage, indicating that c-Myc ablation did not influence their differentiation potential. To precisely address the differentiation potential of c-Myc-deficient immature thymocytes we crossed LckCre-Mycfl/fl mice onto the Rag2-/- background. These mice showed a complete block at the DN3 stage of thymocyte development. We then induced pre-TCR-like signaling in LckCre-Mycfl/fl-Rag2-/- and control LckCre-Rag2-/- mice by injecting
To rule out the possibility that the progressing cells in -CD3 -treated LckCre-Mycfl/fl-Rag2-/- mice may have "escaped" timely Cre-mediated deletion of Myc, we performed semiquantitative PCR analyses using genomic DNA isolated from sorted DN3, DN4, and DP cells. The floxed Myc allele was barely detectable in DN4 and DP stage LckCre-Mycfl/fl-Rag2-/- thymocytes, indicating that these cells had undergone efficient Myc deletion (Figure 5C). Moreover, the LckCre-Mycfl/fl-Rag2-/- DN4 thymocytes were smaller than the LckCre-Rag2-/- (Figure 5D), likewise indicating efficient c-Myc ablation. Thus, LckCre-Mycfl/fl-Rag2-/- thymocytes developed to the DP stage despite the lack of c-Myc.
To determine whether developmental progression required cell division we cocultured immature LckCre-Mycfl/fl and LckCre control thymocytes with OP9-DL18 cells. Independently sorted DN3- and DN4-stage thymocytes from LckCre control and LckCre-Mycfl/fl mice were labeled with CFSE and cocultured with OP9-DL1 cells for 4 days before staining for CD4 and CD8 surface expression and FACS analysis. A substantial fraction of c-Myc-deficient DN3-stage thymocytes up-regulated CD4 (Figure 6A) and CD8 (Figure 6B) surface expression without any cell division, while the developing fraction of LckCre control DN3 cells had undergone 4 to 5 cell divisions. Likewise, c-Myc-deficient DN4 cells did not divide, but up-regulated CD4 and CD8 surface expression. To determine that these DP cells had undergone proper differentiation we examined their surface expression of TCR
c-Myc ablation does not compromise survival of developing thymocytes Immature thymocytes that do not receive pre-TCR signals undergo apoptosis. c-Myc has been reported to control cell survival in other experimental systems. To examine whether c-Myc deficiency had an effect on the survival of developing thymocytes, we compared LckCre-Mycfl/fl and LckCre control thymocytes with respect to their fraction of Annexin V+ cells, as well as the expression levels of the antiapoptotic proteins Bcl-2 and Bcl-xL and the proapoptotic protein p53. To this aim we stained primary thymocytes with Annexin V as well as antibodies directed against surface markers that allow electronic gating of specific thymocyte subsets (Figure 7A). LckCre-Mycfl/fl and LckCre control mice had comparable fractions of Annexin V+ cells in the DN3, DN4, and DP subsets, indicating that the c-Myc deficiency did not affect the survival of developing thymocytes. Intracellular staining with antibodies against Bcl-2 revealed that LckCre-Mycfl/fl and LckCre thymocytes expressed comparable levels of Bcl-2 at the DN3, DN4, and DP stages, further supporting this notion (Figure 7B). We also analyzed the expression levels of Bcl-xL and p53 mRNA using semiquantitative RT-PCR (Figure 7C) and of p53 protein using Western blot (Figure 7D). The expression level of p53 was unchanged, while Bcl-xL expression was modestly elevated in the absence of c-Myc.
In summary, these data indicate that c-Myc ablation at the DN3 stage did not impair the pre-TCR-dependent survival signals, and that the reduced thymic cellularity was entirely the result of reduced proliferation.
Pre-TCR assembly and signaling promotes proliferation, survival, and differentiation of immature thymocytes at the DN3 stage of development, essentially instructing them to the T-cell lineage.9 Using 2 inducible ways to promote this developmental transition we found that an early event following the onset of pre-TCR signaling was the up-regulation of c-Myc. Thus, within hours following induction of pre-TCR signaling, c-Myc protein levels increased, indicating that this molecule was likely involved in the proliferation, survival, or differentiation processes mediated by pre-TCR signaling. We showed that conditional thymocyte-specific ablation of c-Myc impaired cell growth and proliferation of immature thymocytes at the pre-TCR checkpoint. Despite reduced proliferation, the pre-TCR could still signal differentiation and survival to c-Myc deficient thymocytes both in vivo and in vitro. Our findings provide a dissection of pre-TCR signaling, and assign c-Myc specifically downstream of the proliferation but not the differentiation or survival signals.
Three lines of evidence support the suggestion that c-Myc is dispensable for the differentiation signals downstream from the pre-TCR. First, c-Myc-deficient thymocytes progressed efficiently through the DN4 stage, although they yielded a reduced number of DP cells. Second, induction of pre-TCR-like signaling by
It is important to distinguish here between the ability of thymocytes to developmentally progress and the number of cells detected in each developmental stage. Since c-Myc-deficient thymocytes progressed to the DP stage without dividing while the equivalent LckCre cells reached this stage after undergoing 4 to 5 cell divisions, an approximately 32-fold reduction in the number of DP cells would be expected in LckCre-Mycfl/fl mice. This prediction is in line with the reduction in the number of DP cells in LckCre-Mycfl/fl mice. Similarly, fewer LckCre-Mycfl/fl-Rag2-/- thymocytes treated with
The c-Myc-deficient DN4-stage thymocytes were cycling at lower frequencies and were smaller, indicating that the proliferative signals attributed to the pre-TCR were impaired in the absence of c-Myc. This proliferative block was detected both in vivo and in vitro but was more dramatic in OP9-DL1 stromal cell cocultures seeded with sorted thymocytes from either the DN3 or the DN4 stage. c-Myc-deficient thymocytes failed to undergo more than 1 cell division in these cultures. The mechanism by which c-Myc promotes thymocyte proliferation may rely on the regulation of cell-cycle inhibitors such as p27Kip, Gadd45 c-Myc has been shown to promote cell growth in B cells33,37 and thymocytes.34 By contrast, Trumpp and colleagues67 observed that reduced levels of c-Myc do not affect the size of T cells upon activation. Our finding that c-Myc ablation at the DN3 stage of thymocyte development resulted in small cells that failed to develop into blasts at the DN4 stage suggests that while reduced levels of c-Myc may still be sufficient to mediate cell growth, a complete ablation is not. This explanation is in line with recent findings that c-Myc is likely to regulate cellular growth through ribosome biogenesis,27,68,69 and that this regulation requires only low levels of c-Myc in the nucleolus. The reduced thymic cellularity observed in LckCre-Mycfl/fl mice is most likely the result of impaired proliferation, and is not associated with reduced survival. c-Myc has long been thought to sensitize cells to apoptosis, particularly when it is overexpressed. However, conditional ablation of c-Myc was shown not to affect the survival of HSCs45 or primary B lymphocytes.70 In line with these observations, we found that spontaneous apoptosis of immature c-Myc-deficient thymocytes was comparable with that of control thymocytes at the equivalent stages. Moreover, we did not detect deregulated expression of several genes implicated in cell survival/death that have been classified as potential c-Myc target genes, such as p53, Bcl-2, and Bcl-xL.71-73 Here we show that c-Myc is rapidly induced upon activation of the pre-TCR. This could be directly controlled by pre-TCR signals, or it could be an indirect consequence. However, the rapid up-regulation of c-Myc protein levels within 6 hours after induction of the pre-TCR argues in favor of a direct control of c-Myc by the pre-TCR. Irrespective of the mechanism by which pre-TCR induces c-Myc, our study reveals for the first time a bifurcation of signaling pathways at the pre-TCR checkpoint and shows that differentiation of thymocytes occurs efficiently in the absence of c-Myc-dependent proliferation.
The authors thank R. Chang for excellent technical assistance. A. Parmelee and S. Kwok at the Tufts Laser Cytometry facility provided invaluable help with cell sorting. We thank Dr F. Alt for providing the Mycfl/fl mice, Dr P. Sicinski for critical reading of the manuscript, and Dr A. Garbe for helpful discussions.
Submitted February 27, 2006; accepted June 7, 2006.
Prepublished online as Blood First Edition Paper, June 20, 2006; DOI 10.1182/blood-2006-02-005900.
Supported by National Institutes of Health grant R01 AI059676-01 and the Smith Family New Investigator Award from the Medical Foundation (F.G.), and the Claudia Adams Barr Program (K.K.). A.K. is a fellow of the Lymphoma Research Foundation.
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 U.S.C. section 1734.
Reprints: Fotini Gounari, Tufts-New England Medical Center, 750 Washington St, Tufts-NEMC no. 5602, Boston, MA 02111; email: fgounari{at}tufts-nemc.org.
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Abstract
Introduction
B activation.
represents results for LckCre control; 
represents results for LckCre Mycfl/fl mice. Error bars indicate SD. (C) Western blot of sorted cells (2 x 106 per lane) probed with antibodies detecting the indicated proteins. Data are representative of 3 independent experiments.
