Figure 3.

MSCs and CHIR-99021 induced IL-10⁺ B cells via PKA-CREB signaling of B cells

(A) GSEA enrichment analysis of upregulated and downregulated signaling pathways. (B) The signaling pathways with their gene linkages. (C) Enrichment plots showed the three signaling pathways in CD1c⁺ B cells with MSCs compared with CD1c⁺ B cells individually. (D) Quantitative reverse-transcription-PCR (qRT-PCR) detection of the gene expression of IL10 in B cells alone or with db-cAMP (a PKA agonist, 100 μM) in the presence of CpG and CHIR-99021. (E) qRT-PCR detection of the gene expression of IL10 in B cells alone or with KG-501 (a CREB inhibitor, 10 μM) in the presence of CpG and CHIR-99021. (F) In the presence CpG and CHIR-99021, IL-10 production by B cells alone, B cells cocultured with MSCs, and B cells cocultured with MSCs and KG-501 were detected by flow cytometry. Quantification of IL-10-producing B cells. (G) Phosphorylation of CREB (Ser 133) in B cells alone or stimulated by CpG and CHIR-99021 was analyzed by flow cytometry, and MFI was used for statistical analysis. (H) Phosphorylation of CREB (Ser 133) in B cells alone or with MSCs in the presence of CpG and CHIR-99021 was analyzed by flow cytometry, and MFI was used for statistical analysis. (I) Optimization for a human Breg-inducing system based on PKA-CREB agonist and GSK3β inhibitor. (J) In the presence of CpG and CHIR-99021, IL-10 production by B cells alone, B cells cocultured with MSCs, and B cells treated with db-cAMP were detected by flow cytometry. Quantification of IL-10-producing B cells. Data represent mean ± SEM of three or more independent experiments. not significant (ns) p ≥ 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.