Fig. 9.

S1P regulates mitochondrial fission of pathogenic T cells via PRKAA1. A Upon soluble anti-CD3 mAb stimulation (1 μg/mL), purified CD25-depleted T cells from WT or PRKAA1^flox/floxCD4-Cre mice were cocultured with APCs from WT or Sphk1^−/− mice for 3 days. CD25 abundance on gated CD4⁺ or CD8⁺ T cells and pS6 abundance on gated CD4⁺CD25⁺ or CD8⁺CD25⁺ T cells were measured. Summary graphs of the average levels are shown. B Purified CD25-depleted T cells from either WT or PRKAA1^flox/floxCD4-Cre mice were cocultured with APCs from BALB/c mice in the presence or absence of PF543 (1 μM). The percentages of CFSE-diluted and IFN-γ⁺ cells on gated CD4⁺ or CD8⁺ T cells were detected after 4 days. Summary graphs of the average frequencies are shown. C Purified CD4⁺ or CD8⁺ T cells from either WT or PRKAA1^flox/floxCD4-Cre mice cocultured with APCs from BALB/c mice in the presence or absence of PF543 (1 μM). Activated T cells were enriched from bulk culture through CD25⁻positive selection after 3 days. Drp1, Drp1-Ser616, Drp1-Ser637, and GAPDH expression in CD4⁺CD25⁺ and CD8⁺CD25⁺ T cells was detected by western blotting. D Purified CD4⁺ or CD8⁺ T cells from FVB mice were cocultured with APCs from either WT or Sphk1^−/− mice for 3 days, and activated CD4⁺ or CD8⁺ T cells were obtained from bulk culture with CD25 using microbeads. AMPKα1 and GAPDH expression in CD4⁺CD25⁺ and CD8⁺CD25⁺ T cells was detected by western blotting. Lethally irradiated BALB/c mice underwent BMT with 5 × 10⁶ TCD-BMCs alone and with or without 0.75 × 10⁶ total T cells isolated from WT or PRKAA1^flox/floxCD4-Cre donors. Recipients were injected i.p. with PF543 at 0.5 mg/kg every other day from Day 0 to Day 14. E Body weight loss, F clinical score, and G survival were monitored over time (n = 10 mice/group). The experiments were repeated 2 to 3 independent times. Log-rank (Mantel‒Cox) test (G) and nonparametric Mann‒Whitney U test (E, F) were performed to compare groups. The gray intensity of each band in the western blot was measured by ImageJ. Statistical data are presented as the mean ± 1 SD, and significance was determined by Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001