FIGURE 2.

Itga4 expression by B cells is crucial for the presence of Bregs in the spleen. Splenocytes were collected from naive CD19^Cre Itga4^fl/fl mice and CD19^Cre mice. (A) B cells were isolated and IL-10 and Ebi3 mRNA relative expressions were determined by quantitative PCR. (B) IL-10 and Ebi3 mRNA relative expression on sorted MZ (B220⁺CD21^highCD23^low), FO (B220⁺CD21⁺CD23⁺), and TN (B220⁺CD21⁻CD23⁻) B cells from CD19^Cre mice. (C) Itga4 expression on MZ (black line), FO (dotted line), and TN B cells (gray histogram) from CD19^Cre mice as identified in (B). (D) Representative CD21 and CD23 expression gated on CD19⁺ B cells from CD19^Cre Itga4^fl/fl mice (bottom) and CD19^Cre mice (top) to define three B cell subsets: MZ (CD21^highCD23⁻), TN (CD21⁻CD23⁺), and FO (CD21⁺CD23⁺). Mean frequency (top) and absolute numbers (bottom) of each B cell subset from CD19^Cre Itga4^fl/fl mice (gray) and CD19^Cre mice (white) determined from three independent experiments with 7–10 mice per group. (E) Absolute numbers of CD1d⁺ MZ B cells from naive CD19^Cre Itga4^fl/fl mice and CD19^Cre mice. (F) Absolute numbers of CD1d⁺ among B cells in the spleen and CNS of CD19^Cre Itga4^fl/fl mice and CD19^Cre mice at the peak of EAE. Statistical significance was designated as follows: *^,**^,#p < 0.05.