B cell development in CD154^TGCD22^−/− and CD154^TG mice.

<p>(<b>A</b>) B cell CD154 expression in WT, CD22^−/−, CD154^TG and CD154^TGCD22^−/− mice. Blood and spleen B220⁺ B cells were assessed for CD154 expression by immunofluorescence staining with flow cytometry analysis. Dashed lines delineate borders between CD154⁺ and CD154⁻ cells as determined using WT lymphocytes. (<b>B</b>) Ectopic CD154 expression is B cell-restricted in CD154^TGCD22^−/− mice. CD154 expression by blood and spleen B220⁺ and B220⁻ mononuclear cells was assessed by immunofluorescence staining with flow cytometry analysis. Results represent those obtained in 6 pairs of mice. (<b>C</b>) Increased CD5 expression by B cells from CD22^−/− and CD154^TGCD22^−/− mice. Spleen B cells were assessed for CD5 expression by immunofluorescence staining. Gates indicate percentages of CD5⁺ B cells among total B220⁺ cells. (<b>D</b>) Spleen CD1d^hiB220⁺ B cell localization. Tissue sections from WT, CD22^−/−, CD154^TG, and CD154^TGCD22^−/− mice were stained with B220 (FITC, green) and CD1d (PE, red) mAbs. Merged images highlight CD1d^hiB220⁺ cells (yellow). (A–C) Results are representative of ≥3 mice of each genotype. (<b>E</b>) Spleen CD1d^hiB220⁺ B cell numbers in WT, CD22^−/−, CD154^TG and CD154^TGCD22^−/− mice as determined by immunofluorescence staining. Results represent the mean (±SEM) from ≥3 mice of each genotype. (<b>F</b>) CD154^TGCD22^−/− B cells are hyper-responsive to CD40 signals. Spleen B cells from WT, CD22^−/−, CD154^TG, and CD154^TGCD22^−/− mice were cultured with mitogenic CD40 mAb or anti-IgM Ab for 72 h, with [³H]-thymidine incorporation assessed during the final 18 h of culture. Values represent means (±SEM) of triplicate cultures. (E–F) Means significantly different from WT values are indicated by asterisks (**p<0.01), and between other genotypes by crosses (††p<0.01). (<b>G</b>) CD154^TGCD22^−/− B cell proliferation in response to LPS stimulation. Cell division of CFSE-labeled B cells from WT, CD22^−/−, CD154^TG and CD154^TGCD22^−/− mice following LPS stimulation was quantified after 72 h in culture by flow cytometry. (F–G) Results are representative of 2 independent experiments with similar results. (<b>H</b>) Enhanced survival by CFSE-labeled B cells from CD154^TG and CD154^TGCD22^−/− mice after 10 days in culture without mitogenic stimulation as assessed by flow cytometry. Results represent 3 independent experiments producing similar results.