Fig 1.
CD11bhi DCs and CD103+ DCs migrated to the lung draining lymph node after CT administration and CD11bhi DCs with the MHCIIloCD86lo phenotype mediated IL-17-favored differentiation of CD4 T cell.
Mice were intranasally administered 2 μg CT, and mediastinal lymph node (medLN) cells were prepared from the mice at day 1, day 2, or day 3 after or before CT administration. (A) Flow cytometry analysis of migratory DC subsets after CT administration. Five mice per group; migratory DC subsets in medLN (CD103+ DC and CD11bhi DC); (B) Cell number of each DC subset after CT administration; (C-F) CD103+ DCs and CD11bhi DCs were isolated from medLN cells of mice 2 day after intranasal CT administration, pulsed with 0.1 μM of OVA323-339 peptide and washed and cultured with OT-II CD4+ T cells for 5 days. Stimulated OT-II CD4+ T cells were analyzed for expression of IL-17A and IFN-γ by flow cytometry after restimulation with PMA and ionomycin. (C) Flow cytometry analysis and (D and E) frequency of IL-17A- and IFN-γ-expressing CD4+ T cells. (F) Ratio of IL-17A+ to IFN-γ+ CD4+ T cells. (G) MHC class II and costimulatory molecules on DC subsets isolated from medLN of mice 2 day after administration of CT or PBS. *p<0.05, **p<0.01 (Student’s t-test). Data are from one experiment representative of two independent experiments with similar results. Dot plots and histograms are representative of four mice in A and G and quadruplicate wells in C and data are average ± SEM.
Fig 2.
DCs with the MHCIIloCD86lo phenotype mediated IL-17-favored differentiation of CD4 T cell.
(A-D) OT-II CD4+ T cell differentiation by untreated BMDCs or BDMCs treated with CT, LPS or CT and LPS. The BMDCs were pulsed with 1 μM of OVA323-339 peptide before co-culture with OT-II CD4+ T cells. Frequency of IFN-γ- or IL-17A-expressing CD4+ T cells (A-C) and ratio of IL-17A-expressing cells to IFN-γ-expressing cells (D). (E) Surface expression of MHC class II and costimulatory molecules between untreated BMDCs and CT-treated BMDCS. BMDCs were treated with CT (2 μg/ml), LPS (100 ng/ml) or CT and LPS or not treated for 24 hr. (F-I) Comparison of OT-II CD4+ T cell differentiation by isolated BMDCs or CT-treated BMDCs based on MHC class II and CD86 expression. Frequency of IFN-γ- or IL-17A-expressing CD4+ T cells (F-H) and ratio of IL-17A-expressing cells to IFN-γ-expressing cells (I). *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test). Dot plots are the representative of at least two independent experiments and data are average ± SEM of triplicate wells in B-D or quadruplicate wells in G-I.
Fig 3.
CT-treated BMDCs downregulate T cell activation and IL-2 production.
OT-II CD4+ T cells were co-cultured with BMDCs or CT-BMDCs pulsed with the indicated concentration of OVA323-339 peptide. At 16 h, OT-II CD4+ T cells were analyzed for expression of CD25 and CD69 (A-C). Histogram of CD25 and CD69 (A); Frequency of CD25-expressing or CD69-expressing CD4+ T cells (B); Intensity of CD25 or CD69 expression (C). CFSE-labeled OT-II CD4+ T cells were cultured alone or with BMDCs or CT-BMDCs pulsed with the indicated concentration of OVA323-339 peptide. At day 4, CFSE dilution was assessed in OT-II CD4+ T cells (D). At day 5, IL-2 was detected in the co-culture of OT-II CD4+ T cells with BMDCs or CT-BMDCs by cytokine bead assay (eBioscience) (E). ***p<0.001 (Student’s t-test). Histograms and data are from one experiment representative of two independent experiments. Data are average ± SEM of triplicate wells.
Fig 4.
Depletion of IL-2 recovers Th17 cell differentiation.
Assessment of OT-II CD4+ T cell differentiation by untreated or CT-treated BMDCs pulsed with the indicated concentration of OVA323-339 peptide in the absence (A and B) or presence of neutralizing IL-2 antibody (10 μg/ml) (A and C). *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test). Dot plots and data are from one experiment representative of at least two independent experiments with similar results and average ± SEM of triplicate wells.
Fig 5.
CT–treated BMDCs mediate Th17 cell differentiation by producing Th17 polarizing cytokines.
(A) RT-PCR analysis of IL-6, IL-1β, TGF-β1 and activin A mRNA in BMDCs. BMDCs were cultured with CT (2 μg/ml) and harvested at 0, 6, or 20 h after CT treatment. mRNA levels of IL-6, IL-1β, TGF-β1 and activin βA were normalized by mRNA expression of GAPDH and β-actin. (B) Determination of cytokines in BMDC-conditioned media. BMDCs were cultured with CT (2 μg/ml) for 2 days, and culture media was removed to measure cytokines. IL-6 and IL-1β were assayed by multiplex bead cytokine assay kit following the manufacturer’s recommended protocol (eBioscience). TGF-β1 in BMDC-conditioned media was assayed for total (left) and active form (right) by ELISA (R&D Systems). (C and D) Intracellular staining of IFN-γ and IL-17A in OT-II CD4+ T cells stimulated with anti-CD3 and anti-CD28 antibody in the presence of BMDC-CM or CT-BMDC-CM for 5 days and restimulated with PMA and ionomycin after 5 days. Neutralizing antibodies and kinase inhibitors were also added in the culture as indicated. (E-G) BMDCs from IL-6-/- mice were used for clarifying a role of IL-6 in Th17 differentiation promoted by CT-treated BMDCs. Frequency of IFN-γ+ or IL-17A+ CD4+ T cells (F) and ratio of IL-17+ cells to IFN-γ+ cells (G). *p<0.5, **p<0.01, ***p<0.001 (Student’s t-test). Data are the representative of at least two independent experiments with similar results and average ± SEM of triplicate wells in A and D or duplicate wells in B, F and G.