Analysis for the expression of molecules associated with MZ localization on B cells of IRF4-deficient and wild-type mice. (A) Expression of integrins α_L, β₂, α₄, and β₁ on MZ and FO B cells from irf4^+/+ and irf4^−/− mice was assessed by flow cytometry. MZ and FO gates are indicated on the corresponding dot plots. Numbers on plot (top, irf4^−/−; bottom, irf4^+/+) show average mean fluorescence intensity (MFI) value ± SD (n = 3, two independent experiments). Statistical significance was determined by Student’s t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (B) S1P₃ expression in irf4^+/+ and irf4^−/− splenic B cells was assessed by GEP. Data are shown as mean ± SD (n = 4, as in Fig. 3 A). Statistical significance was determined by Student’s t test (***, P < 0.001). (C) The chemotactic response of irf4^+/+ and irf4^−/− MZ and FO B cells to S1P was determined by enumerating splenic cells transmigrated for 3 h across uncoated 5-µm transwells to S1P or media and by analyzing them for CD19, CD21, and CD23 expression by flow cytometry. Chemotactic response is presented as migration index (percentage of input cells that migrated to the lower chamber). Data represent mean ± SD (n = 3 for 0 and 100 nM; n = 2 for 10 nM; three independent experiments). Statistical significance is shown for results obtained with 100 nM S1P and was determined by Student’s t test (**, P < 0.01; ***, P < 0.001).