Figure 4. 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-γ 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.