Figure 1. Myc controls cell growth by selectively remodelling T cell proteomes.

(A) Forward scatter area (FSC-A) of IL-7 maintained or 24 hr anti-CD3 + anti-CD28 (TCR) activated Cd4Cre⁺ (Myc^WT) and Cd4Cre⁺Myc^fl/fl (Myc^cKO) T cells. (B–C, E–O) Quantitative proteomics data of ex vivo naïve WT and 24 hr TCR activated CD4⁺ and CD8⁺ T cells from Myc^WT and Myc^cKO mice. (B) Total protein content (µg/million cells). (C) Mean protein copy number per cell estimated using proteomic ruler (Wiśniewski et al., 2014) of Myc. (D) Myc expression measured by flow cytometry in 24 hr TCR activated Myc^WT and Myc^cKO CD4⁺ and CD8⁺ T cells. Proteins from 24 hr TCR activated Myc^WT (E) CD8⁺ and (F) CD4⁺ T cells were ranked by mass contribution and the mean cumulative protein mass was plotted against protein rank (left panel). Numbers in each quartile indicate total proteins summed with those in the quartiles below. Volcano plots show foldchange in protein copy number between TCR activated Myc^cKO and Myc^WT T cells, with proteins that contribute the top 75% of the T cell mass shown in red (right panel). (G) Heat maps of naïve and TCR activated Myc^WT and Myc^cKO CD8⁺ and CD4⁺ proteomes. Relative protein abundance is graded from low (blue) to high (yellow) per row. Input data for heatmaps is listed in Supplementary file 1. Mean protein copy number per cell for activation markers (H) IL7ra (J) CD69 and (K) CD44 and key transcription factors (I) Klf2, (L) Rel, (M) JunB, (N) Tbet, and (O) Irf4. Symbols on bar charts represent biological replicates: error bars show mean ± S.E.M. Quantitative proteomics was performed on biological triplicates. Fold-change calculations and statistical testing comparing naïve WT vs TCR Myc^WT, naïve WT vs TCR Myc^cKO, and TCR Myc^WT vs TCR Myc^cKO protein copy number per cell is listed in Supplementary file 1.

Figure 1—figure supplement 1. Immune activated Myc-deficient T cells fail to proliferate.

(A) CFSE labelled lymph node cells from Cd4Cre⁺ (Myc^WT) and Cd4Cre⁺ Myc^fl/fl (Myc^cKO) mice were stimulated with anti-CD3 and anti-CD28 (both 0.5 µg/mL) for 48 hr and CFSE dilution was measured. Representative of technical duplicate plots. (B) Lymph node cells from GFP-MycKI mice were stimulated with anti-CD3 (1 µg/ml) and anti-CD28 (3 µg/ml) in the presence of IL-2 receptor-blocking antibody PC61 (2 µg/ml) or isotype control and GFP-Myc expression was measured over time. Symbols show biological replicates.

Figure 1—figure supplement 2. Myc-deficiency has a larger quantitative effect in CD8⁺ T cells.

Naïve WT and 24 hr TCR activated Myc^WT and Myc^cKO proteomic data was generated as described in Figure 1 and Materials and methods. (A) Venn diagram showing that there were a larger number of proteins that were classified as differentially regulated in CD8⁺ T cells compared to CD4⁺ T cells, where differentially regulated is defined as proteins that were more than 2-fold regulated and p<0.05 between Myc^WT vs Myc^cKO TCR activated conditions. (B) Heat maps of proteins that were Myc-regulated in CD4⁺ T cells only, both CD4⁺ and CD8⁺ T cells or CD8⁺ T cells only show similar qualitative effects of Myc-deficiency for most proteins. Relative protein abundance is graded from low (blue) to high (yellow) per row. (C) Mean copy number per cell for examples of proteins classified as Myc-regulated in CD8⁺ T cells only. Protein expression was much higher in Myc^WT CD8 T cells compared Myc^WT CD4⁺ T cells, whereas expression levels in Myc^cKO CD4⁺ and CD8⁺ T cells are reduced to a similar extent thus giving a larger quantitative effect of Myc-deficiency in CD8⁺ T cells. Examples include important transcription factors such as Nfkb1 and Nfkb2, the integrin Icam1 and cytokine receptor components such as Il2rb. Symbols show biological replicates. Mean ± S.E.M. Fold-change calculations and statistical testing comparing naïve WT vs TCR Myc^WT, naïve WT vs TCR Myc^cKO, and TCR Myc^WT vs TCR Myc^cKO protein copy number per cell is listed in Supplementary file 1.