2B). The altered response to anti-IgM could arise from a decreased FO/MZ ratio, since BCR engagement causes proliferation of FO cells but apoptosis of MZ cells 14, 15. However, reduced anti-IgM-mediated proliferation was also observed in B cells from Foxo1f/fCd21Cre mice in which no changes in FO/MZ ratios were reported 10, and is consistent with the presence of a prominent IgM− B-cell population (Supporting Information Fig. 1C). Measuring live cell number using a metabolic dye conversion (MTS) assay confirmed the finding of impaired anti-IgM response in Foxo1f/fCd19Cre B cells (Supporting Information Fig. 2A). The LPS response in Foxo1f/fCd19Cre B cells was increased when measured using MTS assays (Supporting

buy BGJ398 Information Fig. 2A), but not using the CFSE assay (Fig. 2A). This might indicate that LPS-stimulated B cells have altered metabolism when Foxo1 is absent, leading to increased MTS conversion despite equivalent cell number. TGF-β is a cytokine with potent anti-proliferative effects in lymphocytes 16. TGF-β signaling activates Smad transcription factors, which in several MEK inhibitor cellular systems cooperate with Foxo proteins to activate target promoters 17, 18. Furthermore,

the TGF-β/Smad signaling axis regulates MZ B-cell development 19. Although we obtained evidence for functional cooperation of Foxo1 and Smad transcription factors in B cells (Supporting Information Fig. 2B and C), Foxo1 was not required for TGF-β-mediated suppression of B-cell proliferation triggered by anti-IgM or LPS (Supporting Information Fig. 2A). CD62L mRNA was consistently reduced about threefold in Foxo1f/fCd19Cre B cells (Fig. 2C), indicating that lower CD62L protein expression on the surface of these cells is at least partly due to reduced steady-state mRNA levels, resulting from altered

transcription and/or RNA processing. Foxo1 also controls expression of the Sell gene encoding CD62L in T cells 20–22. Another Foxo target gene, Klf2, regulates CD62L expression in T cells and might be a link between Foxo1 and CD62L 20, 21, 23–25. Klf2 Wilson disease protein mRNA expression was also significantly reduced in Foxo1-deficient B cells, though less prominently than the reduction in Sell mRNA (Fig. 2C). Previously, we identified Ccng2, Rbl2 and Klf4 as Foxo target genes in B cells 26, 27. By various criteria, including reporter assays, electrophoretic mobility shift assays and chromatin immunoprecipitation, these genes were regulated similarly by Foxo1 and Foxo3a 26, 27. RNA measurements using quantitative real-time PCR showed that none of these genes were differentially expressed in Foxo1-deficient B cells (Fig. 2C), further suggesting that Foxo1 and Foxo3a have redundant functions at these target promoters. The increased population of MZ B cells in Foxo1f/fCd19Cre mice was intriguing, since Foxo factors are turned off by the PI3K/AKT pathway and the opposite phenotype occurs in mice lacking PI3K genes 28–30.