Fig 2: Fatty acid metabolism-associated gene expression and lipid uptake in T cells. a The cellular mechanism underlying aCD3/F/AN-induced enhancement of lipid metabolism is illustrated. The left half of the schematic cell depicts the untreated condition, and the right half shows treatment with aCD3/F/ANs. aCD3/F/AN treatment is proposed to activate PPARa in T cells, leading to over-expression of fatty acid metabolism-associated proteins, including CD36 (fatty acid translocase), CPT1B, LCAD and MCAD. The increased fatty acid metabolism in mitochondria generates higher energy via the TCA cycle. b The corresponding levels of PPARa protein in T cells (PPARa+/CD3+), measured by flow cytometry. c The levels of CD36 protein in T cells (CD36+/CD3+), measured by flow cytometry. d Protein expression levels of CPT1B, LCAD and MCAD in T cells were measured by western blot. e, f T cells treated with different nanoparticle preparations were incubated with fluorescent lipid BODIPY C16, and the association of fluorescent lipid with T cells was determined by flow cytometry (e) and expressed as mean fluorescence intensity (f) (***P < 0.001). g mRNA expression levels of the fatty acid metabolism-associated genes, CPT1B, LCAD and MCAD, determined by RT-PCR (***P < 0.001)

Fig 3: Regulatory T Cells Utilize Lipids for Their Mitochondrial Metabolism under Hypoxia and Prefer Lipids for Metabolism within Glioma(A and B) Flow-cytometry-sorted and expanded HIF-1α WT or HIF-1α KO Tregs were placed under 1% O2 overnight before being adhered to microplates, and extracellular flux analysis was performed over time (A) and analyzed as bar graphs (B). Eto treatment (20 μm) was added immediately before assay to determine reliance on lipids for mitochondrial metabolism.(C–F) Wild-type C57/Bl6 mice were implanted with 4 × 105 GL-261 astrocytoma cells, and after 2 weeks of tumor growth, T cell expression of surface fatty acid transporters was analyzed via flow cytometry.(C) Percent palmitic acid uptake and MFI of conventional CD4+, CD8+, and Treg subsets.(D) Percent 2-NDBG and MFI of 2-NBDG conventional CD4+, CD8+, and Treg subsets.(E) Tumor interstitial fluid was obtained from either the tumor hemisphere or the non-tumor hemisphere of mice, and FFA content was measured via colorimetric readout.(F) Data show the expression of fatty acid transporters CD36, SLC27A1, and SLC27A4 across different T cell subsets in the brains, DLN, and spleens of tumor-bearing mice (left); representative flow cytometry plots are shown on the right.Statistics were calculated as percent positive population ± SEM. Data in (A) and (B) were analyzed using Wave software from Agilent. Statistics were calculated as percent positive population ± SEM, n = 5 per group; results are representative of three experiments in (A) and (B) and two experiments in (C)–(F). One-way ANOVA followed by Tukey’s post hoc analysis was used to calculate significance. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.See also Figure S4.

Fig 4: PPAR?-Deficient T Cells Show Improved Antitumor Potential(A–C) Three-day activated WT and PPAR?-deficient T cells were used to determine (A) cell surface expression of stem cell antigen (Sca1), CD122, and CD127; (B) ECAR time course in response to glucose, oligomycin, and 2DG; and (C) WT and PPAR?-/- T cells were activated either in presence or absence orlistat (100 µM). OCR under basal condition and in response to indicated mitochondrial inhibitors was determined.(D) WT and PPAR?-/- T cells were activated either in presence or absence of orlistat (100 µM) and used to determine cell viability with 7AAD staining.(E) Bodipy conjugated FL C12 uptake; and expression of CD36 by flow cytometry in day 3 activated WT and PPAR?-/- T cells.(F) Day 3 activated WT and PPAR?-deficient T cells were cultured overnight ± oleate (30 µM), followed by 24 h of starvation in low glucose (2 mM) media, and then stained with Bodipy. The numerical values within the FACS overlay plots indicate MFI. Adjacent bar diagrams represent cumulative data from at least three repeat experiments.(G) C57BL/6 HLA-A2+ mice (n = 12 mice/group) were inoculated (s.c.) with 0.3 × 106 B16F10-A2+ melanoma cells for 9 days, after which mice either kept untreated or adoptively transferred with melanoma reactive 1 × 106 TIL1383I tyrosinase epitope-reactive TCR transduced T cells generated using splenocytes from WT or PPAR?creCD4flox mice. Tumor growth was measured using digital calipers twice weekly. Left: estimated growth curves for each experimental condition. Right: survival curve (p value WT versus PPAR?-/-: 2e–04). (A–G) Error bars represent mean ± SD. *p < 0.05, **p < 0.01.See also Figure S6.

Fig 5: SphK1-/- T Cells Preferentially Depend on Oxidative Phosphorylation and Lipolysis for Energy Need(A) Three days TCR activated WT and SphK-/- T cells were used to determine ECAR time course in response to glucose, oligomycin, and 2DG.(B) OCR in presence or absence of etomoxir (200 µM) followed by the addition of various mitochondrial inhibitors as indicated in the figure. Graph on left represents time course, and bar diagram on right represents basal OCR values. 0.5 × 106 cells/well were used to perform the study.(C) mRNA from 3 days TCR-activated WT and SphK1-/- T cells were used to determine the expression of CPT1a using qPCR.(D and E) Three days activated WT and SphK1-/- T cells were used for (D) determining ATP levels and (E) Bodipy-conjugated FL C12 uptake (left panel), and cell surface expression of CD36 (right panel) by flow cytometry.(F) Three days TCR activated WT and SphK1-/- T cells were cultured overnight ± oleate (30 µM), followed by 24 h of starvation in low glucose (2 mM) media and then stained with Bodipy.(G) Activated WT and SphK1-/- T cells were used to determine the Tip47 expression by flow cytometry.(H) Activated WT and SphK1-/- T cells were used to determine the abundance of lipid signature molecules by mass spectroscopy.(I) WT and SphK1-/- T cells were activated either in presence or absence orlistat (100 µM), and cell viability was determined using 7AAD staining.(J) Cells in (I) were also used for determining IFN-? secretion by intracellular cytokine staining using flow cytometry.(K) OCR was measured using real-time flux analysis using Seahorse and with mitochondrial inhibitors. 0.4 × 106 cells/well were used to perform the study. The numerical values within the FACS overlay plots indicate MFI, and the bar diagrams represent cumulative data from at least three repeat experiments in (A)–(K).(L) C57/BL6 mice (n = 15 mice/group) were inoculated (s.c.) with 0.3 × 106 B16-F10 melanoma cells for 9 days, after which mice either kept untreated or adoptively transferred with 1 × 106 pMel and pMel-SphK-/- T cells cultured in presence or absence of orlistat (100 µM). Tumor growth was measured using digital calipers twice weekly. Upper panel: growth curves for each experimental condition. Lower panel: survival curve (p value; pMel versus pMel SphK1-/-: 2e–06, pMel SphK1-/- + Orlistat versus pMel SphK1-/-: 5e–07). *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent mean ± SD.See also Figure S4.