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. 2006 Oct 15;108(8):2669-77.
doi: 10.1182/blood-2006-02-005900. Epub 2006 Jun 20.

c-Myc mediates pre-TCR-induced proliferation but not developmental progression

Marei Dose  1 , Irum Khan, Zhuyan Guo, Damian Kovalovsky, Andreas Krueger, Harald von Boehmer, Khashayarsha Khazaie, Fotini Gounari
Affiliations

c-Myc mediates pre-TCR-induced proliferation but not developmental progression

Marei Dose et al. Blood. .

Abstract

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 TCRalphabeta 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.

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Figures

Figure 1.
Figure 1.
c-Myc expression in response to pre-TCR stimulation. (A) Developmental progression to the DP stage after pre-TCR induction in TetOβ-LTH-Rag1-/- thymocytes. FACS analysis of thymocytes from neonatal thymic organ cultures derived from mice that had been treated with tetracycline during gestation. Thymic lobes were kept in organ cultures in the absence of tetracycline for the indicated times. Top dot plots show CD4 versus CD8 and bottom dot plots show CD44 versus CD25 of gated lin- cells. Histograms show intracellular (ic) TCRβ expression in the DP and the DN4 subsets. (B) c-Myc Western blot of thymocytes from LTH-Rag1-/- and TetOβ-LTH-Rag1-/- mice that had been treated with tetracycline prior to coculturing with OP9-DL1 cells in tetracycline-free growth medium for 24 hours. Thy1+ cells were sorted from the cocultures and total cell lysates were used for Western blotting. Lane 1 shows LTH-Rag1-/-; lane 2, TetOβ-LTH-Rag1-/-. (C) FACS analysis of α-CD3 mAb-induced thymocyte development in Rag-/- mice. Dot plots show thymocyte expression profiles for CD4 versus CD8 (top) and CD44 versus CD25 (bottom, data gated on lin- cells) from Rag-/- mice 0, 2, and 4 days after intraperitoneal injection of α-CD3 antibody. (D) c-Myc Western blot from Rag-/- mice that were injected with 50 μg of α-CD3 mAb at the indicated time points prior to killing. Total thymic lysates were obtained for Western blotting. Results are representative of 4 independent experiments.
Figure 2.
Figure 2.
c-Myc ablation at the DN3 stage impacts thymic cellularity and subset distribution. (A-B) Efficiency of c-Myc ablation at the DN3 and DN4 stages of thymocyte development. Semiquantitative c-Myc RT-PCR with 5-fold serial dilutions (A) and c-Myc Western blots (B) were performed on FACS-sorted cells from the indicated mice and subsets. Data shown are representative for 3 independent experiments. (C) FACS analyses for CD4/CD8 (top) and CD44/CD25 (bottom, gated on lin- events) surface expression in LckCre-Mycfl/fl and LckCre (Adult = 5-8 weeks old) or Mycfl/fl (E 16 = Embryonic day 16) mice. Numbers given indicate the percentage of events in the respective quadrant. Data shown represent observations from more than 10 independent experiments (Adult) and 2 independent experiments (E 16). (D) Cellularity was determined by multiplying the number of total thymocytes with the percentages from panel A (for DN3, DN4 also considering the percentage of lin- cells). Error bars indicate SD; Thy, total number of thymocytes; and γδ, TCRγδ+ thymocytes. Numbers of adult animals analyzed to obtain these statistics were as follows (NLckCre-Mycfl/fl, NLckCre): total thymocytes (n = 27, n = 11), DP/CD4+/CD8+/DN (n = 13, n = 6), DN3/DN4 (n = 9, n = 7), and TCRγδ+ thymocytes (n = 10, n = 5). Embryo data are based on 3 Mycfl/fl control embryos and 11 LckCre-Mycfl/fl.
Figure 3.
Figure 3.
c-Myc ablation inhibits proliferation of DN4-stage thymocytes. (A) 7AAD staining of permeabilized thymocytes. Thymocytes from the indicated mice were surface stained with anti-lin antibodies as well as anti-CD44 and anti-CD25, followed by staining with 7AAD and FACS analysis. Histograms are electronically gated lin-/CD44-/CD25+ (DN3) or lin-/CD44-/CD25- (DN4) cells. Percentages in histograms represent cells in S/G2/M phases of the cell cycle. Histogram bars represent cumulative measurements of 8 control and 7 LckCre-Mycfl/fl cycling DN4 cells. Error bars indicate SD. (B) Cell size. Forward scatter (FSC) profiles of the indicated mice and subsets are shown. (C) Semiquantitative RT-PCR for pTα and TCRβ mRNA expression in DN3 and DN4 thymocytes. RT-PCR for β-actin is used as quantity control. Similar results were observed in 3 independent experiments. (D) Intracellular TCRβ expression in DN3 and DN4 thymocytes. Cells were surface stained as in panel A followed by permeabilization and staining with anti-TCRβ antibodies and FACS analysis. Similar results were obtained in more than 5 independent experiments.
Figure 4.
Figure 4.
Elevated levels of cell-cycle inhibitors in LckCre-Mycfl/fl thymocytes. (A) Semiquantitative RT-PCR with 5-fold serial dilutions for cell-cycle-related genes performed on c-DNA obtained from FACS-sorted DN3 and DN4 thymocytes. Data sets are representative of observations obtained in 3 independent experiments. (B) Quantitative RT-PCR analyses using RNA prepared from similarly sorted cells were performed in triplicate for the indicated genes. □ represents results for LckCre control; ▪ represents results for LckCre Mycfl/fl mice. Error bars indicate SD. (C) Western blot of sorted cells (2 × 106 per lane) probed with antibodies detecting the indicated proteins. Data are representative of 3 independent experiments.
Figure 5.
Figure 5.
Pre-TCR-like signals induce differentiation of Lck-Cre-Mycfl/fl-Rag2-/- thymocytes. (A) FACS profiles for CD4/CD8 (top panels) and lin-/CD44/CD25 expression 4 days after injection (intraperitoneally) of 50 μg α-CD3 mAb. Numbers given indicate the percentages of cells in the respective quadrants. FACS plots are representative of observations obtained in at least 3 independent experiments. (B) Cellularity was calculated from total thymocyte numbers and the fraction of the indicated subsets. Error bars indicate SD. Eleven LckCre-Mycfl/fl-Rag2-/- and 6 LckCre-Rag2-/- were analyzed to obtain statistics. (C) Semiquantitative PCR with 5-fold serial dilutions was performed to detect the floxed Myc allele. Genomic DNA was obtained from FACS-sorted DN3, DN4, and DP cells. (D) Cell size of DN4 stage thymocytes. FSC as observed by FACS.
Figure 6.
Figure 6.
c-Myc-deficient thymocytes differentiate without proliferation. FACS-sorted DN3 and DN4 cells from the indicated mice were labeled with CFSE and cocultured with OP9-DL1 cells for 4 days. Cell suspensions from the OP9-DL1 cocultures were stained with antibodies against CD4, CD8, or TCRαβ and analyzed by flow cytometry. Empty histograms represent results for LckCre Mycfl/fl; filled gray histograms represent results for LckCre control mice. (A) Top panels show 2-parameter dot plots of CD4 versus CFSE staining of the indicated subsets and mice. Bottom panels show histogram overlays comparing CFSE in LckCre versus LckCre-Mycfl/fl cells after the coculture. (B) Top panels show 2-parameter dot plots of CD4 versus CD8 surface staining of the indicated cells and mice. Numbers represent the percentage of total events in the shown gates. (C) TCRαβ surface expression in the gates shown in the 2-parameter dot plots. Arrows depict the starting populations used in the cocultures. In vitro differentiation was observed in 3 independent experiments.
Figure 7.
Figure 7.
c-Myc ablation does not affect survival of developing thymocytes. (A) Annexin V staining of primary thymocytes. Thymocytes of the indicated mice were stained with antibodies against CD4 and CD8 or against lin, CD44, and CD25 followed by Annexin V and FACS analysis. Histograms of Annexin V staining are electronically gated on the indicated subsets. (B) The same subsets were also analyzed with respect to the expression of intracellular Bcl-2 levels. (C) Expression levels of Bcl-xL and p53 mRNA in FACS-sorted DN3 and DN4 cells as determined by semiquantitative RT-PCR with 5-fold serial dilutions. Semiquantitative RT-PCR for β-actin was used as quantity control (Actin). Similar results were obtained in 3 independent experiments. (D) Protein levels of p53 in FACS-sorted cells (2 × 106 per lane) as determined by Western blot.
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