Agonist-induced S1PR1 internalization is impaired in moesin-deficient T cells.

(A) S1PR1 localization in CD4⁺ T cells. Lymph node CD4⁺ T cells from Msn^+/Y and Msn^−/Y mice were treated with or without 100 nM S1P or 100 nM FTY720P for the indicated times. Cells were fixed, permeabilized, and stained with anti-S1PR1 antibody. Representative confocal images are shown. Scale bars, 5 µm. (B) Quantification of S1PR1 internalization. Lymph node CD4⁺ T cells from Msn^+/Y and Msn^−/Y mice were incubated with or without 100 nM S1P, 100 nM FTY720P, or 1 µM SEW2871 for 1 h. Cells were stained as in (A). Cell percentages with the indicated S1PR1 localization pattern are shown. (C) The effect of W146 on S1P-induced S1PR1 relocalization. Lymph node CD4⁺ T cells from WT mice were incubated with or without 1 µM W146 for 30 min and then stimulated with 100 nM S1P for 10 or 60 min. Cells were stained as in (A). Representative confocal images are shown. Scale bar, 5 µm. (D) Quantification of the effect of W146. Percentages of cells with the indicated distribution patterns of S1PR1 were determined. (B and D) n>50 cells for each group. ***, P<0.001; **, P<0.01; n.s., not significant (Fisher's exact test).