Foxo1 regulates marginal zone B-cell development

Abstract

A fundamental component of signaling initiated by the BCR and CD19 is the activation of phosphoinositide 3-kinase. Downstream of phosphoinositide 3-kinase, the protein kinase AKT phosphorylates several substrates, including members of the forkhead box subgroup O (Foxo) transcription factor family. Among the Foxo proteins, Foxo1 has unique functions in bone marrow B-cell development and peripheral B-cell function. Here, we report a previously unrecognized role for Foxo1 in controlling the ratio of mature B-cell subsets in the spleen. Conditional deletion of Foxo1 in B cells resulted in an increased percentage of marginal zone B cells and a decrease in follicular (FO) B cells. In addition, Foxo1 deficiency corrected the absence of marginal zone B cells that occurs in CD19-deficient mice. These findings show that Foxo1 regulates the balance of mature B-cell subsets and is required for the marginal zone B-cell deficiency phenotype of mice lacking CD19.

Figure 1

Comparison of splenic B cell subsets by FACS and histology. (A) Flow cytometry was used to distinguish FO, MZ and immature (T1, T2, T3 and MZP) subsets. Spleen cells were stained with antibodies to B220, AA4.1, IgM, CD21 and CD23. The gating strategy is shown on the right for a representative control spleen. Graphs on the left depict mean cell percentages + SD. n = 4 Foxo1^f/+Cd19^Cre and Foxo1^f/fCd19^Cre; n = 2 Foxo1^f/f. *p<0.05; ***p<0.001, unpaired two-tailed t-test. (B) Immunofluorescent staining of spleen sections with antibodies to B cells (anti-B220; red) and macrophages lining the marginal zone (anti-MOMA-1, green) shows increased population of MZ B cells (white arrows) in Foxo1^f/fCd19^Cre mice. Confocal images (10X magnification; scale bar = 100μM) are representative of at least 10 follicles from 2 mice of each genotype.

Figure 2

Comparison of proliferation, apoptosis and gene expression. (A) Purified B cells from Foxo1^f/fCD19^Cre and control Foxo1^f/f mice were labeled with CFSE, then stimulated with anti-IgM or LPS at the indicated concentrations for 66 h at 37 °C. Filled gray histogram shows unstimulated control cells. Similar results were obtained in 4 separate experiments. (B) In 3 of the CFSE labeling experiments, cells were stained at 66 hr with Annexin V to quantitate apoptotic cells among the divided population. Data show mean + SD. Unpaired two-tailed t-tests showed no significant differences between genotypes (p>0.05 for all comparisons; p = 0.055 for comparison labeled “NS”). (C) Quantitative real-time PCR (qRT-PCR) data using RNA extracted from B cells of Foxo1^f/fCD19^Cre and control Foxo1^f/f mice. The numbers indicate the ratio of each gene in Foxo1^f/fCD19^Cre as compared to Foxo1^f/f mice, after normalization to βactin. Data show mean + SD of three separate experiments. *p<0.05; * p<0.01; comparing ratio of each gene to 1.0 using a one-tailed t-test.

Figure 3

Assessment of complementation of the MZ B cell defect in CD19-deficient mice. (A) Flow cytometry was used to compare FO and MZ B cell percentages from representative mice. B220-gated cells were analyzed for CD21 and CD24 to quantitate FO cells (CD21^intCD24^int), or CD21 and CD23 to quantitate MZ cells (CD21^hiCD23^lo). (B) The percentages of FO and MZ cells in the spleens from mice of each genotype are shown. Data represent mean ± SD, n = 3-5 mice. *p<0.05; **p<0.01 vs. Foxo1^f/f, using unpaired, two-tailed t-test. §p = 0.051 vs. Foxo1^f/f. (C) Immunofluorescent staining of spleen sections was done as in Figure 1B.