Figure 1.
LPS induces over-expression of ABCG2 in blood mDCs.
(A) PBDCs were purified from peripheral blood by PBDC isolation kit. Surface ABCG2 expression levels were measured in CD11c−CD123+ pDCs, CD11c+CD123inter mDCs and CD11c−CD123− cells by flow cytometry. (B) Real-time PCR analysis of ABCG2 gene expression, presented relative to that of β-actin, in purified pDCs, mDCs and CD11c−CD123− cells (D/N). Data are representative or the average of analyses of 4 samples from 4 donors for each group. (C) PBDCs were stimulated with LPS for 24 hours. ABCG2 expression in gated pDCs, mDCs and CD11c−CD123− cells were analyzed on a flow cytometry. Data are representative of analyses of 3 samples from 3 donors. (D) Mitoxantrone efflux in purified mDCs and LPS-stimulated mDCs in the presence or absence of Ko143 was analyzed by flow cytometry (left panel) and mean fluorescence intensity (MFI) was shown (right panel). Data are representative or the average of analyses of 3 samples from 3 donors.
Figure 2.
Ko143 suppresses LPS-induced mDC maturation.
(A) Purified CD1c+ mDCs were pre-treated with Ko143 for 1 hour and incubated with or without LPS for 24 hours. CD83 and CD86 expression were analyzed by flow cytometry (left panel). MFI of CD83 and CD86 was shown (right panel). Data are representative or the average of analyses of 3 samples from 3 donors. (B) Intracellular IL-12p40, TNF-α and IL-10 production in purified CD1c+ mDCs were analyzed by flow cytometry. Data are representative or the average of analyses of 3 samples from 3 donors. (C) Concentrations of IL-12p40, IL-12p70, TNF-α and IL-10 in the culture medium of CD1c+ mDCs as measured by ELISA. Data are representative or the average of analyses of 6 samples from 6 donors.
Figure 3.
ABCG2 is required for LPS-induced DC maturation.
MDDCs were transfected with ABCG2 siRNA or control siRNA for 24 hours and then stimulated with LPS for 24 hours. (A) mRNA levels of ABCG2 were measured by real time qPCR. (B) Protein levels of ABCG2 were subjected by western blotting using anti-ABCG2 Abs. (C) The expression levels of CD83 and CD86 were analyzed by flow cytometry. (D) Levels of IL-12p40, IL-12p70, TNF-α and IL-10 in the culture medium as measured by ELISA. All data are representative or the average of analyses of 4 samples from 4 donors for each group.
Figure 4.
LPS plus Ko143-treated mDCs promote expansion of Treg cells.
Ko143-, LPS- or LPS plus Ko143-treated CD1c+ mDCs were co-cultured with CFSE-labeled allogeneic CD4+ T cells (1×105) in a 1∶10 ratio for 4 days. (A) Surface expression of CD25 and CFSE dilution was analyzed in CD4+ T cells by flow cytometry. (B) Flow cytometry of CD4, CD25 and FOXP3 expression was shown. Data are representative of analyses of 3 samples from 3 donors. (C) Flow cytometry of intracellular IL-10 and IFN-γ production in gated CD4+ T cells was shown. (D) Intracellular IL-10 expression in CD4+FOXP3+ T cells (left panel) and mean percentage of IL-10+FOXP3+ or IL-10+FOXP3− cells (right panel) were shown. Data are representative or the average of analyses of 3 samples from 3 donors. (E) CFSE-labeled PBMCs were incubated with soluble anti-CD3 and CD28 Abs for 4 days in the presence or absence of CD25+ Treg cells. CFSE dilution was analyzed in CD4+CD25− and CD8+ T cells (left panel) and mean percentage of proliferating cells (right panel) was shown. Data are representative or the average of analyses of 4 samples from 4 donors. (F) CD1c+ mDCs were cultured with LPS and CFSE-labeled syngenic CD4 T cells, in the presence or absence of Staphylococcal Enterotoxin B (SEB) and Ko143 for 4 days. CFSE dilution was analyzed in CD4+TCRVβ3+ T cells by flow cytometry. Data are representative of analyses of 3 samples from 3 donors.
Figure 5.
Generation of Treg cells induced by LPS plus Ko143-treated mDCs is dependent on IL-10.
CD1c+ mDCs and CD4 T cells were co-cultured as described in Figure 4 in the presence of anti-IL-10 or control IgG Abs. (A) Expression of CD25 and CFSE dilution were analyzed in CD4+ T cells. (B) CD4, CD25 and FOXP3 expression was analyzed by flow cytometry. (C) IFN-γ (left panel) and IL-10 (right panel) concentrations in the culture medium were measured by ELISA. Data are representative or the average of analyses of 3 samples from 3 donors.
Figure 6.
ERK pathway is essential for optimal induction of tolerogenic mDCs by LPS and Ko143.
(A) MDDCs (1×106) were pre-treated with or without Ko143 for 1 hour and then incubated with LPS for 10 minutes. The samples were then subjected to Western blotting using anti-phosphorylated p38 (p-p38), ERK (pERK), AKT (p-AKT) and IKK (p-IKK) Abs. (B) MDDCs were treated with Ko143, LPS or Ko143 plus LPS for indicated amount of time. Phosphorylation of MEK and ERK were subjected to Western blotting. (C) MDDCs were pre-incubated with PD98059 for 10 minutes and further cultured with Ko143, LPS or Ko143 plus LPS for 30 minutes. Phosphorylation of ERK was measured by Western blotting. (D) CD1c+ mDCs were stimulated with LPS together with Ko143 as described in Figure 2 in the presence or absence of PD98059. Flow cytometry of CD83 expression in CD1c+ mDCs is shown. (E) Concentrations of IL-12p40, IL-12p70, TNF-α and IL-10 in the culture medium. (F) CD1c+ mDCs were co-cultured with allergenic CD4 T cells as described in Figure 4. Expression of CD25 and CFSE dilution were analyzed in CD4+ T cells (left panel). Mean percentage of proliferating CD4+CD25− cells (middle panel) or CD4+CD25+ cells (right panel) was shown. (G) CD4, CD25 and FOXP3 expression was analyzed by flow cytometry. Data in A–C are the representative of at least three independent experiments, and data in D–G are representative or the average of analyses of 4 samples from 4 donors for each group.