Figure 6.

Evidence of marginal zone skewing within T1 and T2 subsets in FL-/- mice. (A) Flow cytometric analysis of splenic CD19⁺ B cells (pre-gated) from a representative wild-type (WT) and FL-/- mouse further stained by CD21/35 and CD1d to examine CD1d^hi and CD1d^lo peripheral B cell subsets. (B) Splenic cells were stained using CD19 and AA4.1 to initially resolve CD19⁺ AA4.1⁺ TS B cells. To examine T1 and T2 B cells, CD19⁺ AA4.1⁺ cells were further characterized using CD21/35 and CD23. T1 are defined as CD21/35^-/lo CD23⁻ and T2 are CD21/35^lo/int CD23⁺ within CD19⁺ AA4.1⁺ TS B cells. T1 and T2 were subsequently analyzed with CD1d to determine the percentage of CD1d^hi cells within these TS subsets. CD1d^hi gates were set based on expression of CD1d in the MZ as in (A). (A and B) Data are representative of 9–11 mice/genotype and four independent experiments. (C) Scatter plots depict the frequency of CD1d^hi cells within T1 and T2 B cells in each WT and FL-/- mouse in the analysis (n = 11 WT and 9 FL-/- mice). (D) Bar graphs illustrating percentages of TS, MZ, and FO B cells generated from WT CD45.1⁺ T1 B cells adoptively transferred into CD45.2 WT (WT T1 into WT) or CD45.2 FL-/- (WT T1 into FL-/-) mice. The bars labeled CD45.1 WT represent TS, MZ, and FO B cells from uninjected CD45.1 WT mice analyzed to ensure correct CD45.1⁺ and peripheral B cell subset gating. Bars labeled FL-/- PBS represent endogenous CD45.2⁺ TS, MZ, and FO B cells from PBS-injected CD45.2 FL-/- mice. CD45.1 background staining (controlled by PBS-injected CD45.2 FL-/- mice) was very minimal, averaging 0.0003 ± 0.0003% or 12 ± 11 CD45.1⁺ cells (data not shown). Data are representative of 5–9 mice/condition and five independent experiments. (C and D) * and ** represent statistically significant differences measured using the Student's t-test at P ≤ 0.05 and P ≤ 0.005, respectively.