Figure 3. Myc induces amino acid transporter expression, a critical step for proteome remodelling.

Naïve WT and 24 hr TCR activated Myc^WT and Myc^cKO CD4⁺ and CD8⁺ T cell proteomic data was generated as described in Figure 1 and Materials and methods. (A) Mean copy number per cell of abundant amino acid transporters in T cells. (B) Fold-change in amino acid transporter protein copy number from naïve WT to 24 hr TCR activated Myc^WT and Myc^cKO, mean (min, max). Transport by system L amino acid transporters was measured by uptake of fluorescent (emission 450 nm when excited at 405 nm) Tryptophan metabolite, Kynurenine (Kyn) (Sinclair et al., 2018) in (C) 2 hr IL-7 maintained and (D-E) 4 hr IL-7 maintained or TCR activated splenic CD4⁺ and CD8⁺ WT, Myc^cKO and Slc7a5^cKO T cells or (F) 20 hr TCR activated Myc-GFP reporter CD4⁺ T cells. In (C,E) system-L uptake is represented as the ratio of BCH (a system L inhibitor) untreated: treated T cells. In (E) dotted line indicates Slc7a5^cKO uptake level. (G) Forward scatter and CD69 expression of IL-7 maintained or 24 hr TCR activated wild-type and Slc7a5^cKO (Cd4Cre⁺ Slc7a5^fl/fl) T cells. (H-K) Quantitative proteomics data of naïve WT and 24 hr TCR activated CD4⁺ and CD8⁺ T cells from Ly5.1 (Slc7a5^WT) and Slc7a5^cKO mice. Baseline naïve WT data is the same as used for the Myc^cKO dataset. (H) Total protein content (µg/million cells). (I) Heat map of naïve and TCR activated Slc7a5^WT and Slc7a5^cKO CD4⁺ T cell proteomes. Relative protein abundance is graded from low (blue) to high (yellow) per row. Input data for heatmaps is listed in Supplementary file 1. (J) Venn diagram showing the overlap in TCR regulated proteins that are more than 2-fold regulated and p<0.05 in Myc^WT vs Myc^cKO and Slc7a5^WT vs Slc7a5^cKO TCR activated CD4⁺ T cells. (K) Volcano plots of TCR regulated proteins comparing Slc7a5^WT and Slc7a5^cKO datatsets. Proteins > 2 fold different between Myc^WT and Myc^cKO TCR activated T cells are highlighted in red; proteins reduced in the Myc^cKO (left panel), proteins higher Myc^cKO (right panel). Symbols in bar charts represent biological replicates: error bars show mean ± S.E.M. Dot plot in (F) is representative of biological triplicate data. 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, TCR Myc^WT vs TCR Myc^cKO, naïve WT vs TCR Slc7a5^WT, naïve WT vs TCR Slc7a5^cKO and TCR Slc7a5^WT vs TCR Slc7a5^cKO protein copy number per cell is listed in Supplementary file 1.

Figure 3—figure supplement 1. Myc-deficient T cells fail to induce protein translation machinery.

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. Diagram and mean copy number per cell of (A) translation initiation complex and (B) 43S preintiaition complex components. (C) Protein content of 40S and 60S ribosome (µg/million cells). 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.

Figure 3—figure supplement 2. Amino acid transport capacity corresponds with transporter number.

Transport by system L amino acid transporters was measured by uptake of fluorescent (emission 450 nm when excited at 405 nm) Tryptophan metabolite, Kynurenine (Kyn) in 6 or 24 hr IL-7 maintained or anti-CD3/anti-CD28 (TCR) activated CD4⁺ and CD8⁺ WT and Myc^cKO T cells. Histograms are representative of at least three biological replicates.

Figure 3—figure supplement 3. Myc-deficient T cells fail to induce Branched-chain amino acid and Methionine metabolism.

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. Diagram and mean copy number per cell of proteins involved in (A) Methionine metabolism and (B) Branched-chain amino acid metabolism. 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.

Figure 3—figure supplement 4. Ribosome expression is reduced in both Myc and Slc7a5 deficient T cells, but other proteins are differentially regulated between Myc and Slc7a5 deficient T cells.

(A) Protein content of ribosomes (excluding mitochondrial) from naïve WT and 24 hr TCR activated Myc^WT and Myc^cKO proteomics data (generated as described in Figure 1 and Materials and methods). (B) Protein content of ribosomes (excluding mitochondrial) and (C) Slc2a3 mean copy number per cell from naïve WT and 24 hr TCR activated Slc7a5^WT and Slc7a5^cKO proteomics data (generated as described in Figure 3 and Materials and methods). 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, TCR Myc^WT vs TCR Myc^cKO, naïve WT vs TCR Slc7a5^WT, naïve WT vs TCR Slc7a5^cKO and TCR Slc7a5^WT vs TCR Slc7a5^cKO protein copy number per cell is listed in Supplementary file 1. Splenocytes from Myc^WT, Myc^cKO and Slc7a5^cKO were stimulated with anti-CD3 and anti-CD28 for 24 hr and; (D) Golgi plug with or without PdBU and ionomycin was added to the cultures for the last 4 hr prior to harvest and IFNgamma expression was measured in CD8⁺ T cells by flow cytometry; or (E) Granzyme B was measured in CD8⁺ T cells by flow cytometry. Histograms are representative of at least three biological replicates.