Fig 2: Analysis of T cell infiltration in 19 tumor types. a T cell infiltration scores (TIS) and the corresponding mutation load in 19 tumor types. TIS is an aggregate score obtained as the average of nine distinct T cell subset scores (CD8+ T, Th1, Th2, Th17, Treg, T effector memory, T central memory, T helper, and T cells). Each circle in the top panel shows the TIS for a tumor sample. In the bottom panel, the vertical bar corresponding to each circle shows the number of somatic missense mutations. Tumor types are ordered from left to right according to increasing median TIS (medians indicated by horizontal gray bars). b Correlation of mutation load with TIS and levels of individual T cell subpopulations. Spearman correlation coefficients are computed between number of somatic missense mutations and ssGSEA-based immune cell infiltration levels. Coefficients are plotted on the y-axis in bar plots and asterisks are added to indicate level of significance, as denoted in the legend. Tumor types are ordered in the same order as in Fig. 3a

Fig 3: T cells from patients with type 1 diabetes exhibited MHP. T cells from peripheral blood of type 1 diabetes patients (T1D; n = 29) exhibited a higher mitochondrial membrane potential (ΔΨm) when compared to T cells from healthy controls (HC; n = 38) in (A) CD4+, (B) CD8+ and (C) CD3+ subsets; (D) In an independent cohort, enriched total T cells from fresh blood of T1D patients (n = 39) showed higher ΔΨm when compared to HC individuals (n = 34) or patients with type 2 diabetes (n = 20). T cell ΔΨm was not correlated with (C) age or (D) HbA1c level. *P < 0.05, **P < 0.005, ****P < 0.001. Mann-Whitney test.

Fig 4: In vitro validation of the immune cell scoring method. a Immune cell populations were sorted from ccRCC patient specimens, and profiled for RNA-Seq gene expression. ssGSEA scores were computed for each sample using Bindea et al. signatures. Each ssGSEA score was the highest for the corresponding tumor-associated immune cell population and also had a significant difference from the other sorted populations (p values are provided above each figure). b Principal component analysis (PCA) of sorted tumor-associated immune cell populations. PCs were computed as a linear combination of 29 immune microenvironment variables (Additional file 2: Table S3). c Immunofluorescence (IF) validation of ssGSEA scores in an MSKCC cohort. The top left panel shows the unsupervised clustering of ssGSEA scores for NK, CD8+ T, and Treg cells in the 10 patients. IF staining for two samples at the opposite ends of the heatmap is shown in the bottom left panel (CD56, CD8, and FOXP3 antibodies respectively). The association of the immune infiltrate levels inferred by these two orthogonal methods (ssGSEA and IF) is shown in the right panel. The IF score (y-axis) represents the ratio of CD56, CD8, and FOXP3 positive cells versus total cells (DAPI-stained) for a given sample and was determined as the average across three representative regions on the slide

Fig 5: Cardiolipin Synthesis Supports CD8+ TM Cell Development, Metabolism, and Function(A) Schematic of CD8+ T cells activated with aCD3/aCD28 + IL-2 for 48 h and then cultured ± AD for 72 h during IL-15 TM cell development.(B) Quantification of total CL content in IL-15 TM cells cultured as in (A).(C) OCR of IL-15 TM cells cultured as in (A) at baseline and after exposure to aCD3/aCD28 coated beads, Oligo, FCCP, and Rot/Ant.(D) IL-15 TM cells were cultured as in (A) and then restimulated with aCD3/aCD28 for 20 h. Percentage of producing IFN-? cells, and IFN-? MFI are shown.(E) Schematic of the activation of CD8+ T cells isolated from Ptpmt1flox/floxErt2-Cre-/- (PTPMT1 iWT) Ptpmt1flox/floxErt2-Cre+/- (PTPMT1 i?T) mice and 4-OHT treatment.(F) CL content in Ptpmt1 iWT and Ptpmt1 i?T CD8+ T cells cultured as in (E).(G) OCR of Ptpmt1 iWT and Ptpmt1 i?T CD8+ T cells cultured as in (E) at baseline and after exposure to PMA/Ionomycin, Oligo, FCCP, and Rot/Ant.(H) Ptpmt1 iWT and Ptpmt1 i?T CD8+ T cells cultured as in (E) and stimulated with aCD3/aCD28 for 20 h. Percentage of producing IFN-? cells and IFN-? MFI are shown.(I) Schematic of WT CD8+ T cells activated with aCD3/aCD28 + IL-2 for 48 h, cultured additional 24 h in IL-2 before performing Ctrl/Ptpmt1 CRISPR. 24 h after CRISPR, equal number of CRISPR Ctrl/Ptpmt1 KO cells were transferred in congenic recipient mice and donor cell frequencies followed for 21 days before LmOVA WT infection.(J) Percentage of CD8+ CD45.2+ Tet+ CRISPR Ctrl/Ptpmt1 KO cells treated as in (I), shown as mean ± SEM.(K) Bacterial burden shown as CFU per µg of liver isolated from mice treated as in (I) 3 days post-LmOVA challenge. Dots represent individual mice.(L) Schematic of WT CD8+ T cells activated and cultured in IL-2 in the presence of BSA-conjugated 50 µM PG(18:1)2 or 50 µM PG(18:2)2 for 48 h.(M) Quantification of CL species in IL-2 TE CD8+ T cell cultured ± PG(18:2)2 for 48 h.(N) Maximal cristae width measured in IL-2 TE CD8+ T cell cultured as in (M).(O) SRC of IL-2 TE CD8+ T cells cultured as in (M) stimulated with aCD3/aCD28 coated beads.(P) Cytokine production (normalized to untreated cells) measured in IL-2 TE CD8+ T cells activated as in (M) and stimulated for 20 h with aCD3/aCD28 + IL-2.(Q) Schematic of adoptive transfer strategy of OT-I IL-2 CD8+ TE cells—pre-treated as in (L)— into congenic recipient mice. Twenty-one days post-transfer, mice were infected i.v. with 1 × 106 CFU of LmOVA ?Acta.(R) Survival of donor cells 21 days post-transfer, expressed as number of donor cells in the spleen. Dots represent individual mice.(S) Percentage of circulating donor cells 21 days post-transfer. Dot represent individual mice.(T) Number of donor cells recovered from spleens of recipient mice 5 days after infection with LmOVA ?Acta. Dots represent individual mice.Data shown as mean ± SEM of =3 independent experiments unless indicated. Statistical comparisons for 2 groups calculated by unpaired two-tailed Student’s t test, *p < 0.05; **p < 0.01; ***p < 0.001.See also Figure S4.