Fig 2: GD2+ NXS2 neuroblastoma growth after allogeneic HSCT and hu14.18-IL2. (A) Lethally irradiated CD45.2+ B6AJF1 mice (H-2b x a) were transplanted with either CD3e cell depleted (T cell depleted) or CD3e depleted BM replenished with 2.5 x 103 T cells (T cell replete) from congenic CD45.1+ B6 mice (H-2b) on Day +0. On Day +10, 2 x 106 NXS2 tumor cells were inoculated into the right flank. On days +14-16, PBS (No IC) or hu14.18-IL-2 (IC) was administered and (B) tumor growth was monitored by using a digital caliper as well as (C) clinical GVHD scores. N=5 mice/group. ** p = 0.01.

Fig 3: Myc induces amino acid transporter expression, a critical step for proteome remodelling.Naïve WT and 24 hr TCR activated MycWT and MyccKO 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 MycWT and MyccKO, 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, MyccKO and Slc7a5cKO 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 Slc7a5cKO uptake level. (G) Forward scatter and CD69 expression of IL-7 maintained or 24 hr TCR activated wild-type and Slc7a5cKO (Cd4Cre+ Slc7a5fl/fl) T cells. (H-K) Quantitative proteomics data of naïve WT and 24 hr TCR activated CD4+ and CD8+ T cells from Ly5.1 (Slc7a5WT) and Slc7a5cKO mice. Baseline naïve WT data is the same as used for the MyccKO dataset. (H) Total protein content (µg/million cells). (I) Heat map of naïve and TCR activated Slc7a5WT and Slc7a5cKO 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 MycWT vs MyccKO and Slc7a5WT vs Slc7a5cKO TCR activated CD4+ T cells. (K) Volcano plots of TCR regulated proteins comparing Slc7a5WT and Slc7a5cKO datatsets. Proteins > 2 fold different between MycWT and MyccKO TCR activated T cells are highlighted in red; proteins reduced in the MyccKO (left panel), proteins higher MyccKO (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 MycWT, naïve WT vs TCR MyccKO, TCR MycWT vs TCR MyccKO, naïve WT vs TCR Slc7a5WT, naïve WT vs TCR Slc7a5cKO and TCR Slc7a5WT vs TCR Slc7a5cKO protein copy number per cell is listed in Supplementary file 1.

Fig 4: Hobit and Blimp‐1 are important for upregulation of CD69 on Trm precursors. (A) The experimental setup of LCMV infection in mixed BM chimeras from HobitWT/CRE and HobitKO/CRE × Blimp‐1flox/flox mice (1:1 ratio) is shown. (B) Representative flow cytometry plots display CD45.1 and CD45.2 expression to identify the contribution of HobitWT/CRE (CD45.1+) and HobitKO/CRE × Blimp‐1flox/flox (CD45.2+) compartments to the GP33+ CD8+ T‐cell population of chimeric mice at day 8 p.i. (C) The log2 ratio of the percentage of GP33+ CD8+ T cells of HobitKO/CRE × Blimp‐1flox/flox mice relative to those of HobitWT/CRE mice was quantified in the indicated tissues. (D) The percentage of tdTomato expression was quantified in the HobitWT/CRE and HobitKO/CRE × Blimp‐1flox/flox compartments of GP33+ CD8+ T cells in the indicated tissues of chimeric mice. (E) Representative flow cytometry plots display tdTomato and CD69 expression within the HobitWT/CRE and HobitKO/CRE × Blimp‐1flox/flox compartment of GP33+ CD8+ T cells in the indicated tissues of chimeric mice. (F, G) The percentage of CD69 expression was quantified in (F) tdTomato+ and (G) tdTomato‐ virus‐specific CD8+ T cells of the HobitWT/CRE and HobitKO/CRE × Blimp‐1flox/flox compartment of chimeric mice. Symbols represent individual mice. Dotted lines connect paired samples. Representative data of one (n = 5) out of two independent experiments. Paired t test. *p < 0.05; **p < 0.01.

Fig 5: Decreased population of iNKT cells in the thymus of LXRα/β-KO mice. (a) Numbers of iNKT cells in thymocytes. (b) Cd1d1 and Zbtb16 mRNA expression and (c) Nr1h3 and Nr1h2 mRNA expression in the thymus. (d) mRNA expression of Nr1h3 and Nr1h2 in iNKT cells, TECs, CD4+ T cells and CD8+ T cells from the thymus. Thymocytes were stained with PE-conjugated CD1d tetramer, FITC-conjugated anti-βTCR, PE-Cy7-conjugated anti-EpCAM and APC-conjugated anti-CD45 to separate iNKT cells (CD45+βTCR+CD1d-tetramer+ cells) and TECs (EpCAM+ cells), or with FITC-conjugated anti-βTCR, PE-conjugated anti-CD4 and PE-Cy7-conjugated anti-CD8a to separate CD4+ T cells (CD45+βTCR+CD4+ cells) and CD8+ T cells (CD45+βTCR+CD8a+ cells). (e) The populations of thymic iNKT cells of stage 0, stage 1, stage 2 and stage 3. Thymocytes were isolated from WT and LXRα/β-KO mice, and stained with FITC-conjugated anti-NK1.1, PE-conjugated CD1d tetramer, PE-Cy5-conjugated anti-βTCR, Brilliant Violet-510-conjugated anti-CD24 and APC-conjugated anti-CD44. iNKT cell stages were determined as stage 0 (CD24+CD44−), stage 1 (CD24−CD44−NK1.1−), stage 2 (CD24−CD44+NK1.1−) and stage 3 (CD24−CD44+NK1.1+) in the population of βTCR+CD1d-tetramer+-gated cells (iNKT cells). Stg, stage. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test). n.d., not detected (around or below detection limits).