FIGURE LEGENDS
Figure 1. Flt3 expression by splenic DCs. (A) Representative histograms and quantification of surface Flt3 on splenocytes as determined by flow cytometry. Data are collated from 2 independent experiments, with cells isolated from 2 pooled spleens in each. The gMFI has been normalized to the maximum signal for each cell type. Bars are mean + SEM. (B) Purified cells were analyzed by immunoblotting and probed with anti-Flt3 (A2F10) or anti-actin (A5060) antibodies. Blot is representative of 3 independent experiments. Band intensities of the higher molecular weight (MW) species and lower MW Flt3 were quantified relative to actin. Bars are mean + SEM. (C) Splenic cDCs were isolated and stimulated with (+) or without (–) Flt3L (100 ng/mL) for 1 hour at 37°C. Flt3 surface expression was determined by flow cytometry. Histograms are representative of three independent experiments. Symbols represent individual mice. Bars are mean ± SD, unpaired t-test. **** P <0.0001 (D) Splenic cDCs were isolated and stimulated for 0, 3 or 16 hours with CpG 1668 ODN (0.5 μM) at 37°C. Cell lysates were examined by immunoblotting and probing with anti-Flt3 (A2F10) or anti-tubulin (DM1A) antibodies. Blot is from one experiment.(E) Splenic cDCs were isolated and stimulated overnight with (+) or without (–) CpG 1668 ODN (0.5 μM) at 37°C. (F) Mice were injected intravenously with PBS (-CpG) or CPG 1668 ODN (0.2 μM) and spleens were harvested one day later or (G) at indicated times. (E-G ) Surface expression of Flt3 was determined by flow cytometry. Histograms show representative surface expression. Symbols represent individual mice. (E, F ) Data are from at least 3 independent experiments with the gMFI signal normalized to the maximum gMFI signal. Bars are mean ± SD, unpaired t-test, **** P <0.0001. (G ) Data are from one experiment. Bars are mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons test. *** P <0.001** P <0.01, ns not significant.
Figure 2. Flt3-ITD expands splenic DC populations. (A)Representative gating used to identify Flt3+/+ and Flt3ITD/ITD splenic pDC (BST-2+CD11cint), cDC1 (CD11c+CD8+ CD11b-), cDC2 (CD11c+ CD8-CD11b+), DP cDC1 (CD11c+CD8+ CD11b- CD103+CD86+), NC cDC1 (CD11c+CD8+ CD11b- CD103-CD86-) and SP cDC1 (CD11c+CD8+ CD11b- CD103-CD86+). Quantification of Flt3+/+and Flt3ITD/ITD splenic DC populations. Symbols represent individual mice; data are from one experiment. Bars represent mean ± SEM, unpaired t test, **** P <0.0001*** P <0.001** P <0.01. (B ) Splenic DC were isolated from Flt3+/+, Flt3+/ITD and Flt3ITD/ITD littermates and surface Flt3 expression was determined by flow cytometry. Data are pooled from 2 independent experiments, with symbols representing individual mice. gMFI has been normalized to the maximum signal for each cell type. Bars represent mean ± SEM, one-way ANOVA with Dunnett’s multiple comparisons test, **** P <0.0001*** P <0.001* P <0.05. (C)Spleens from Flt3+/+, Flt3+/ITD and Flt3ITD/ITD littermates were harvested and cDCs isolated. Cells were stimulated with Flt3L (100 ng/mL) for the indicated times at 37ºC. Cells were lysed and Flt3 was subsequently immunoprecipitated using anti-Flt3 antibody (A2F10). Immunoprecipitates were analyzed by immunoblot probed with anti-Flt3 (A2F10) or anti-phospho-tyrosine (4G10) antibodies. Blot is representative of 2 independent repeats.
Figure 3. Flt3-ITD alters splenic DC surface expression.Flt3+/+ (white bars) and Flt3ITD/ITD(grey bars) splenic cDCs were stained for surface markers and analyzed by flow cytometry. gMFI has been normalized to the maximum gMFI signal. Data pooled from at least 2 independent experiments, with symbols representing individual mice. Bars are mean ± SD, multiple unpaired t-tests with Holm-Sidak multiple comparisons test. **** P <0.0001*** P <0.001** P <0.01 * P <0.05, ns not significant. Heat map of surface marker expression of spleen cDC1 or cDC2 isolated from Flt3+/+ and Flt3ITD/ITD mice. The log2 ratio of mean gMFI of Flt3ITD/ITD cells relative to mean gMFI of Flt3+/+ cells is shown. Data pooled from at least 2 independent experiments.
Figure 4. Flt3-ITD impacts DC function. (A) Purified Flt3+/+ and Flt3ITD/ITD cDC1 and cDC2 populations were incubated at 37ºC with 50 μg/mL OVA-Cy5 or 20 μg/mL dextran-A647 10,000 MW for the indicated times. Cells were washed and the intracellular fluorescence was determined by flow cytometry. Data are from one experiment carried out in technical duplicate and representative of two individual experiments. Bars are mean ± SD.(B) Purified Flt3+/+ and Flt3ITD/ITD cDC2 were pulsed with 20 μg/mL DQ-OVA or 20 μg/mL dextran-pHrodo 10,000 MW for 15 mins at 37ºC. Cells were washed before being chased for the indicated times at 37ºC. DQ-OVA and dextran-pHrodo gMFI was determined by flow cytometry. The gMFI signal for DQ-OVA or dextran-pHrodo is normalized to the OVA-Cy5 or dextran-A647 gMFI for the pulse time (15 mins). Data are from one experiment carried out in technical duplicate and representative of two individual experiments. Error bars are ± SD. (C)Flt3+/+ and Flt3ITD/ITD splenic cDC1 and cDC2 populations were isolated and cell sorted to purity. Equal number of cells were incubated with 0.5 µM CpG, 50 ng/mL IFNγ and 20 ng/mL GM-CSF. Supernatants were collected 18 hours later, and cytokine secretion determined using BD™ Cytometric Bead Array (CBA) Mouse Inflammation Kit. Data are pooled from two independent experiments, carried out in duplicate. Bars are mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons test. **** P <0.0001*** P <0.001** P <0.01 * P <0.05, ns not significant.
Figure 5. Flt3-ITD impacts cDC antigen presentation. Equal numbers of purified Flt3+/+ and Flt3ITD/ITD cDC populations were incubated with purified CellTrace Violet (CTV)-labelled (A) OT-I or(B) OT-II cells in the presence of ovalbumin (OVA)-coated splenocytes (OCS) or 100 µg/mL OVA. Three days later, antigen presentation capacity was assessed by flow cytometric analysis. T cell proliferation was normalized to the maximum proliferation. Each symbol represents an individual mouse with data pooled from at least two independent experiments, displayed as mean ± SEM. **** P <0.0001*** P <0.001** P <0.01, one-way ANOVA with Dunnett’s multiple comparisons test.