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. 2015 Jul 1;34(15):2008–2024. doi: 10.15252/embj.201490252

Figure 1.

Figure 1

T cell receptor signalling drives digital expression of Myc

  • A, B CD8+ T cells were purified from OT1 lymph node cells that had been stimulated for 4 h with 10 ng/ml of peptides SIIQFEHL (Q4H7), SIIQFERL (Q4R7), SIIQFEKL (Q4) or SIINFEKL (N4) or were left unstimulated. (A) Western blot data show Myc and ERK1/2 protein expression, representative of 3 biological replicates. (B) qPCR data show Myc mRNA expression relative to unstimulated naïve control (n = 3, mean ± SEM).
  • C–F Flow cytometry data from lymph node cells of OT1 GFP-MycKI mice stimulated through the TCR with peptide or left unstimulated. Data are from at least 3 biological replicates. (C) GFP-Myc expression in CD8+ T cells stimulated for 2 h with 10 ng/ml peptides Q4H7, T4, Q4R7, Q4 or N4 (left panel). The middle panel shows the percentage GFP-Mycpos cells (mean ± SEM, ANOVA was used to determine statistical significance, **P < 0.01). The right panel shows the MFIs of the GFPpos and GFPneg populations (mean ± SEM). (D) GFP-Myc expression in CD8+ T cells stimulated for 2 h with varying concentrations of Q4 (ng/ml) or N4 (10 ng/ml) (left panel). The right panel shows the percentage GFP-Mycpos cells (mean ± SEM, ANOVA was used to determine statistical significance, *P < 0.05, ** P < 0.01). (E) GFP-Myc (top panel) and CD69 (bottom panel) expression on CD8+ T cells stimulated for 20 h with varying concentrations of Q4 and T4 peptides, indicated on the histograms. The right panels show the percentage of GFP-Mycpos or CD69+ populations. (F) GFP-Myc expression in CD69+ or CD69− CD8+ T cells stimulated with 1.1 ng/ml of T4 peptide for 20 h (left panel). The right panel shows the GFP-Myc MFI of the CD69+ populations of cells treated as in (E) (mean ± SEM).
  • G GFP-Mycneg and GFP-Mycpos CD8+ T cells were purified from OT1 GFP-MycKI lymph node cells that had been activated for 2 h with N4 peptide (1 ng/ml) or had been left unstimulated. Graph shows the expression of Myc mRNA measured by qPCR relative to naïve unstimulated CD8+ T cells (*P < 0.05, ANOVA was used to determine statistical significance). Data show mean+SEM of 3 biological replicates.
  • H, I Flow cytometry data from lymph node cells of WT or GFP-MycKI mice stimulated using CD3 and CD28 antibodies or left unstimulated. Data are from at least 3 biological replicates. (H) GFP-Myc expression over time in CD4+ or CD8+ T cells following stimulation with CD3 (1 μg/ml) and CD28 (3 μg/ml) antibodies. (I) The left panel shows the percentage GFP-Mycpos CD8+ T cells stimulated with varying concentrations of CD3 (μg/ml as on graph) and CD28 (3 μg/ml) antibodies for 4 h. The right panel shows the MFIs of the GFP-Mycpos and GFP-Mycneg populations (mean ± SEM, ****P < 0.001, ANOVA was used to determine statistical significance).

Source data are available online for this figure.