Figure 5. Reduced PPARγ Regulates Functionality and Metabolic Commitment of SphK1−/− T Cells.
(A) WT and SphK1−/− T cells were activated for 3 days either in presence or absence of PF543 (1 µM) and used to determine the expression of PPARγ using flow cytometry.
(B and C) Purified T cells from WT and SphK1−/− mice were TCR activated for 3 days in presence or absence of S1P (5 µM). These T cells were used in ELISA-based assay to determine the activity of PPARγ (B) and PPARa (C).
(D) WT and SphK1−/− T cells activated for 3 days in presence or absence of indicated amount of S1P were used to determine PPARγ expression using flow cytometry.
(E) WT and SphK1−/− T cells were stimulated for 3 days in presence or absence of GW9662, TZD, or with S1P. After 3 days of stimulation, supernatant collected after overnight restimulation was used to measure IL-17 secretion by ELISA.
(F and G) WT and SphK1−/− T cells were differentiated under Th17 or Treg polarizing condition for 3 days in either the presence or absence of S1P. The percentage of IL-17-producing cells (F) and FOXP3-expressing T cells were determined (G).
(H) SphK1−/− T cells were transfected with either WT or mutant vector for SphK1 enzyme. Expression of SphK1, PPARγ, and FOXP3 was analyzed by flow cytometry.
(I) Cells in (H) were used for measuring IL-17 secretion by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001. The numerical values within the FACS overlay plots indicate MFI. Adjacent bar diagrams represent cumulative data from at least three repeat experiments. Error bars represent mean ± SD.