Fig. 4.

S1P/Sphk1 upregulates S1PR1 expression in alloantigen-activated CD4⁺ T cells but not CD8⁺ T cells. Purified T cells from FVB mice (H2K^q) were labeled with CFSE and transferred into lethally irradiated WT or Sphk1^−/− (H2K^b) mice at 2 × 10⁶ cells per mouse. Four days after cell transfer, recipient spleens were harvested and analyzed by flow cytometry. A, B Representative flow figures and percentages of CFSE, CFSE-diluted, and IFN-γ⁺ or GM-CSF⁺ and CD25⁺ cells are shown on gated live H2K^q+ CD4⁺ cells. (n = 4 mice/group). C, D The same experiments are shown in Fig. 2E–H. Representative dot plots and the average frequencies of CXCR3, α4β7, and CCR6 expression on gated donor CD4⁺ T cells from the recipient spleen are shown. Purified FVB T cells were cocultured with APCs from WT or Sphk1^−/− mice for three days. E Representative dot plots and F the average levels of CD25 on gated CD4⁺ or CD8⁺ T cells are shown. G Representative histograms and H graphical summary for MFI of S1PR1 on (surface staining) or in (permeable staining) gated CD4⁺ CD25⁺ T cells and CD8⁺ CD25⁺ T cells are shown. PE-conjugated anti-rat IgG was used as the negative control antibody. The experiments were repeated 2 to 3 times. Statistical data are presented as the mean ± 1 SD, and significance was determined by Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001