Fig 1.
Relative expansion of CD11b+ DCs in DKO mice.
Spleen (SPL) (A) and skin draining lymph nodes (SDLN) (B) of 8 weeks old WT and DKO female mice were assayed for DC populations by flow cytometry. Splenocytes were gated on MHCII+CD11c+ conventional DCs and analyzed for the proportion of CD8+DCs and CD11b+DCs. In SDLNs, MHCIIHiCD11c+ migratory DCs were also examined. Spleen (C) and skin draining lymph nodes (D) of >24 weeks old mice were also assayed for DC populations by flow cytometry. Cells were gated on MHCII+CD11c+B220- conventional DCs and analyzed for the proportion of CD8+DCs and CD11b+DCs. In lymph nodes, MHCIIHiCD11c+ migratory DC frequencies were also examined. Scatter plots show data of individual mice and mean value of at least 3 independent experiments. *: p<0.05; **: p<0.01, ***: p≤0.0001. (E) CD86, PDL2 and PDL1 cell surface expression was analyzed by flow cytometry on conventional CD11b+DCs and CD8+ DCs in the spleen of >24 weeks old WT (red) and DKO (blue) mice. Histograms show relative expression of the indicated marker. Representative data of 2–4 independent experiments with a total of 5–9 mice per group are shown.
Fig 2.
Increased IL-10 production by CD11b+ DKO DCs.
(A) Spleens (SPL) from 14–16 weeks old WT and DKO IL-10 and Blimp1 dual reporter mice were examined by flow cytometry. Splenocytes were gated on MHCII+CD11c+B220- conventional DCs followed by analysis of YFP (Blimp1) and Thy1.1 (IL-10) expression on CD11b+DCs and CD8+DCs. Percentages and numbers of Thy1.1+YFP-, Thy1.1+YFP+ and Thy1.1-YFP+ cells are shown. Scatter plots show data of individual mice and mean value of 4 independent experiments. *: p<0.05; **: p<0.01, ***: p<0.001. (B) IL-10 production by splenic CD11c+ cells stimulated in vitro with 0.1 μg/ml LPS for 24 hours was analyzed by qPCR and ELISA. Representative data of 3 independent experiments are shown **: p<0.01. (C) Skin draining lymph nodes (SDLN) from 14–16 weeks old WT and DKO IL-10 and Blimp1 dual reporter mice were examined by flow cytometry. Cells were gated on MHCII+CD11c+B220- conventional DCs followed by analysis of YFP (Blimp1) and Thy1.1 (IL-10) expression on CD11b+DCs and CD8+DCs. Percentages and numbers of Thy1.1+YFP-, Thy1.1+YFP+, and Thy1.1-YFP+ cells are shown. Scatter plots show data of individual mice and mean value of 4 independent experiments. *: p<0.05; **: p<0.01, ***: p<0.001. (D) PDL2 cell surface expression on DCs from WT and DKO IL-10 and Blimp1 dual reporter mice was analyzed by flow cytometry. Splenic and skin draining lymph node (SDLN) cells were gated on MHCII+CD11c+CD11b+ DCs and PDL2 expression was analyzed on Thy1.1-YFP-, Thy1.1+YFP-, Thy1.1+YFP+ and Thy1.1-YFP+ subsets. Histograms show representative expression of PDL2. Scatter plots show mean florescence intensity (MFI) data of individual mice and mean value of 3 independent experiments: ****: p<0.0001.
Fig 3.
Increased IL-10 and IFNβ expression by TLR-stimulated DKO BMDCs.
(A) WT and DKO BMDCs were generated in vitro in presence of GM-CSF for 7–9 days prior to FACS analysis of MHCII, CD86, CD80, PDL2 and PDL1. Histograms show relative expression of the indicated marker on WT (red) or DKO (blue) CD11c+DCs. One representative experiment out of 1 (CD80) or at least 2 independent experiments (MHCII, CD86, PDL2 and PDL1) is shown. (B-E) BMDCs were generated in vitro for 7 days. CD11c+DCs were purified by magnetic sorting followed by in vitro stimulation with 0.1μg/ml LPS or 3μM CpG for 24h. (B) Cells were harvested and MHCII, CD86 and PDL2 expression on WT and DKO LPS treated CD11c+ DCs analyzed by FACS. One representative experiment out of 2 independent experiments is shown. IL-10 and IFNβ production and gene expression in LPS treated (C) or CpG treated (D) WT and DKO BMDCs were assayed by ELISA and qPCR. One representative experiment out of at least 3 independent experiments is shown. *: p<0.05; **: p<0.01, ***: p<0.001 (E) Thy1.1 expression in BMDCs from IL-10 and Blimp1 dual reporter WT or DKO mice was analyzed by FACS. Histograms show relative expression of Thy1.1 on WT (red) or DKO (blue) CD11c+MHCIIhi or CD11c+MHCIIlo DCs. One representative experiment out of at least 2 independent experiments is shown.
Fig 4.
Selective requirement for IRF4 in TLR stimulated DKO BMDCs.
(A) WT, DKO and CD11c-Cre IRF4fl/fl DKO BMDCs were generated in vitro in presence of GM-CSF for 7–9 days prior to FACS analysis of MHCII, CD86 and PDL2. Histograms show relative expression of the indicated marker on WT (red), DKO (blue) and CD11c-Cre IRF4fl/fl DKO (green) total CD11c+DCs or GFP+CD11c+DCs. One representative experiment out of 3 independent experiments is shown. (B-C) BMDCs were generated in vitro for 7 days. CD11c+DCs were purified by magnetic sorting followed by in vitro stimulation with 0.1μg/ml LPS or 3μM CpG, for 24h. IL-10 and IFNβ gene expression in LPS treated (□) or CpG treated (C) WT, DKO and CD11c-Cre IRF4fl/fl DKO BMDCs were assayed by qPCR. One representative experiment out of 4 independent experiments is shown. *: p<0.05; **: p<0.01, ***: p<0.001.
Fig 5.
Effects of IRF4 deletion in CD11c+ cells on DC, T, and B cell populations in DKO mice.
(A) Spleens of 14–20 weeks old WT, DKO and CD11c-Cre IRF4fl/fl DKO mice were assayed for DC populations by flow cytometry. Splenocytes were gated on MHCII+CD11c+B220- conventional DCs and analyzed for the proportion and numbers of CD11b+DCs and CD8+DCs. (B) CD86 and PDL2 cell surface expression on CD11b+DCs. Histograms show relative expression of the indicated marker on WT (red), DKO (blue) and CD11c-Cre IRF4fl/fl DKO (green) mice. Representative data of at least 2 independent experiments with a total of 3–5 mice per group is shown. (C-F) WT, DKO and CD11c-Cre IRF4fl/fl DKO total splenocytes were analyzed for their CD4+T cell (C-D) and B cell (E-F) populations. Percentages and numbers of activated Tregs (CD4+Foxp3+CD44+), activated T cells (CD4+Foxp3-CD44+), Tfh (CD4+Foxp3-PD1+CXCR5+), germinal center B cells (GC; B220+GL7+Fas+) and plasma cells (PC; B220+CD138+) are shown. Scatter plots show data of individual mice and mean value of 4 independent experiments. *: p<0.05; **: p<0.01, ***: p<0.001, ****: p≤0.0001.