Fig 2: High gene expression levels of ITGAX together with CD8A are prognostic by transcriptomics, irrespective of FOXP3 levels, in human STS. (a) Contrast group analysis of gene expression levels of ITGAX (CD11c) and CD8A (CD8) in the TCGA sarcoma cohort; (b) Contrast group analysis of gene expression levels of ITGAX (CD11c) and CD8A (CD8) in the TCGA sarcoma cohort where FOXP3low and FOXP3high indicate subgroupings according to total FOXP3 levels in the TME. * p < 0.05, ** p < 0.01; p < 0.05 is considered significant.

Fig 3: CD46 co-stimulation provides superior support for CTL activity. a CD46 expression on the surface of resting human CD4+ and CD8+ T cells assessed by FACS analysis (n = 4, gating strategy in Supplementary Fig. 7a). b Comparison of IFN-?, TNF-a and IL-10 secretion by CD3 + CD46-activated T cells. Purified CD4+ and CD8+ T cells from healthy donors were left non-activated (NA) or stimulated with immobilized antibodies to CD3, CD3 + CD28 or CD3 + CD46 and cytokines measured 60 h post activation (n = 5). c, d Degranulation (CD107a staining, (c)) and granzyme B expression (d) by CD8+ T cells upon CD46 co-stimulation. CD8+ T cells were stimulated as in (a) and CD107a and granzyme B expression assessed with left panels showing representative cytometry images and right panels corresponding quantifications (n = 3, gating strategy in Supplementary Fig. 7b). e Killing activity of CD46-activated CD8+ T cells. T cells were stimulated as depicted for 24 h and cytotoxic activity of differently activated CD8+ T cells towards DU145 target cells assessed 24 h post co-culture of T cells and DU145 cells (n = 4, gating strategy in Supplementary Fig. 7c). f Effect of CD46 co-stimulation on CD8+ T-cell proliferation. Purified T cells were activated as indicated for 5 days and cell proliferation measured each day (n = 4) (black circles, non-activated cells; green, blue, and red circles, CD3, CD3 + CD28 or CD3 + CD46-activated cells, respectively). g Effects of CD46 co-stimulation on naive and memory CTLs. Purified human naive or memory CD8+ T cells (sorting gates in Supplementary Fig. 7d) were activated under depicted conditions for 60 h and IFN-? secretion, CD107a and granzyme B surface expression, and cytotoxic activity measured (n = 3). Error bars denote mean ± SEM (standard error of the mean). *p < 0.05; **p < 0.01; ***p < 0.005; ns, statistically not significant. Statistical analyses were performed using One-way ANOVA with Tukey Multiple Comparison test

Fig 4: MAIT cell activation during acute viral infections in vivo.PBMC's from healthy controls (HC, n=5–10), patients suffering from severe dengue (DHF, n=2–10), dengue (DF, n=4–10) or acute, hospitalized patients infected with influenza A virus (n=7–9) were analysed by flow cytometry by gating on live CD3+CD8+CD161++Va7.2+ (MAIT) cells. (a,d,g) Representative flow cytometry plots. (b,e,h) Comparison between acute (day 0) and convalescent phase (day >10) of infection. (c,f,i) Comparison between DHF and DF patients. (j–m) Acute, hospitalized patients infected with influenza virus. (b,c,j,k) MAIT cell frequency as a proportion of the CD8+ T cell population. (e,f) Percentage of CD38 expression by MAIT cells. (h,i,l,m) Percentage of Granzyme B expression by MAIT cells. Statistical significance was determined with a Wilcoxon matched-paired test (b,e,h) or Mann–Whitney test (j,k). Bars represent means±s.e.m. ns>0.05, *P 0.05, **P=0.01, ***P=0.001, ****P=0.0001. conv, convalescent; Gr B, Granzyme B; HC, healthy control; ICU, intensive care unit.

Fig 5: Enhanced Autophagy Levels Are Linked to Effector Function and Mitochondrial Fitness in Human T Cells(A) Example plots of IFN-?, LC3, granzyme B (GzB), and perforin (perf; gated on CD8+ T cells) and histograms of LC3 staining for PBMC ex vivo or after anti-CD3/CD28 stimulation (3 days; see also Figure S3).(B) LC3 staining of CD8+ T cells from unstimulated PBMCs (IFN-?-), IFN-?- and IFN-?+ CD8+ T cells after anti-CD3/CD28 stimulation (3 days; eight biological replicates).(C and D) LC3 staining on GzB and perf-expressing CD8+ T cells ex vivo (C) and after anti-CD3/CD28 stimulation (D) (3 days; eight biological replicates; box whisker, Tukey).(E) Example mitochondrial staining of CD8+ T cells in blood (PBMCs; black) and liver (IHLs; red) and summary data for the ex vivo percentage of total CD8+ T cells with depolarized mitochondria (mitoTracker deep red [MtDR]lo; see also Figure S4; PBMCs, 10; and IHLs, 15 biological replicates).(F) Ex vivo percentage of CD8+ TRM cell subsets in the liver with depolarized mitochondria (14 biological replicates; box whisker, Tukey; outliers shown as dots).(G) The percentage of total CD8+ T cells or CD8+ TRM cell subsets with depolarized mitochondria after overnight culture of IHLs with DMSO (untreated), MRT68921 dihydrochloride (10 µM), bafA1 (0.1 µM), or reagent A (chloroquine diphosphate, 1:1000, FlowCellect LC3 kit; 13–15 biological replicates).Cells were treated with bafA1 (A–D). Bars at mean (B, E, and G). Friedman test (ANOVA) with Dunn’s post hoc test for pairwise multiple comparisons (B, C, D, and F). Kruskal-Wallis test with Dunn’s post hoc test for unpaired multiple comparisons (G). Mann-Whitney unpaired t test for total CD8+ PBMCs versus IHLs (E). Wilcoxon paired t test for untreated versus that treated with MRT68921 (G). Bars at mean (E and G). *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.