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Figure S1. Depletion of pDCs from PBMCs abrogates IFN-α production induced by CpG-A (A) PBMCs were stained with PE-labeled anti-BDCA2 mAb (Miltenyi Biotec) and propidium iodide, and PE- and propidium iodide-negative viable cells were obtained by FACSAria cell sorter. All the propidium iodide-negative cells were obtained as total PBMCs. The percentages of BDCA-2+ cells are indicated on the plot. (B) Total PBMCs or PBMCs depleted of pDCs were stimulated with ODN2216 for 24 h. The Concentration of IFN-α in the supernatants was measured in duplicate by ELISA. The data shown are representative of 2 experiments.

Figure S2. Dasatinib suppresses IFN-α production by pDCs stimulated with CpG-A when calculated at a single cell level PBMCs were stimulated with ODN2216 in the absence or presence of the indicated concentrations of TKIs for 24 h. The concentration of IFN-α in the supernatants was measured in duplicate by ELISA, and the absolute amounts of IFN-α secreted from a single pDC were calculated. The cytokine concentrations were normalized to the maximum value obtained without the TKIs. The data are shown as means +/- SE of 3 independent experiments. *P < 0.05; ***P < 0.001. P values refer to the comparison between the data obtained without TKIs and those obtained with each concentration of TKIs. The means and ranges of absolute concentrations of IFN-α obtained without TKIs are 1.21 pg (0.92-1.47 pg).

Figure S3. Dasatinib reduces the frequency of pDCs bearing intracellular IFN-α and TNF-α pDCs were enriched by depleting lin+ cells from PBMCs using anti-CD3, anti-CD14, and anti-CD16 mAbs, and were stimulated with ODN2216 in the absence or presence of the indicated concentrations of dasatinib for 6 h. Brefeldin A was added during the last 2 h. After stimulation, the cells were stained with FITC-labeled anti-CD3, CD14, CD16, CD20, PE-Cy7 labeled anti-CD4, and APC-labeled anti-CD11c mAbs. Then the cells were fixed, permeabilized, and stained with PE-labeled anti-IFN-α and anti-TNF-α mAbs (BD Biosciences). Lin-CD4+CD11c- cells were gated and intracellular IFN-α and TNF-α were detected by FACSCalibur. The percentages in each quadrant are indicated on the plot. The data shown are representative of 2 experiments.

Figure S4. Dasatinib suppresses upregulation of CD86 induced by CpG-A but not by CpG-B Purified pDCs were stimulated with ODN2216 or ODN2006 in the absence or presence of the indicated concentrations of TKIs for 24 h, stained with FITC-conjugated anti-CD86 mAb (BD Biosciences), and were analyzed by FACSCalibur. Open histograms represent cells stained with isotype-matched control mAbs. The numbers shown with each histogram represent ratios of mean fluorescence intensity of CD86 to that of isotype-matched control. Data are representative of 3 experiments.

Figure S5. Dasatinib suppresses IL-6 production by pDCs stimulated with virus Purified pDCs were stimulated with HSV-1 or influenza virus in the absence or presence of the indicated concentrations of dasatinib for 24 h, The concentration of IL-6 in the supernatants was measured in duplicate by ELISA, and were normalized to the maximum value obtained without dasatinib. The data are shown as means +/- SE of 3 experiments. *P < 0.05; **P < 0.01. The significance of differences was determined by paired two-tailed t-test. The means and ranges of absolute concentrations of IL-6 obtained without dasatinib are as follows: HSV-1 219 pg/mL (182-260 pg/mL); influenza virus 253 pg/mL (157-421 pg/mL).

Figure S6. Dasatinib and imatinib do not reduce the viability of pDCs Purified pDCs were stimulated with ODN2216 (A) or ODN2006 (B) in the presence of the indicated concentrations of dasatinib or imatinib for 24 h. Percentages of Annexin V- and propidium iodide-double negative viable cells were measured by flow cytometry, and were normalized to the value obtained without TKIs. The data are shown as means +/- SE of 3 independent experiments. The significance of differences was determined by paired two-tailed t-test, and was not detected between the viability obtained without and with TKIs.

Figure S7. Dasatinib does not inhibit endocytosis of CpG ODN by pDCs Purified pDCs were cultured with FITC-conjugated ODN2216 in the absence or presence of dasatinib for 3 h. The cells were stained with PE-conjugated anti-HLA-DR mAb. pDCs cultured with FITC-conjugated ODN2216 in the absence of dasatinib on ice were used as a negative control for endocytosis. The cells were observed by confocal microscopy. The data are representative of 3 experiments. Scale bars, 10 μm.

Figure S8. Dasatinib does not inhibit the trafficking of TLR9 from the ER to early endosomes Purified pDCs were stimulated with ODN2216 in the absence or presence of dasatinib for 2 h. ER-Tracker Red was added for the last 30 min. Alternatively, the cells were stained with anti-EEA1 or anti-Rab5 mAb followed by Alexa Fluor 555-conjugated goat anti-rabbit IgG. The cells were stained with biotinylated anti-TLR9 mAb followed by Alexa Fluor 488-conjugated streptavidin, and were observed by confocal microscopy. The micrographs shown in (A) are representative of 3 experiments. Pixels with positive signals for both probes are shown in white. Scale bars, 5 μm. (B) Statistical analysis of microscopy data pooled from 3 experiments. Each symbol represents a cell, and 15 cells were analyzed for each condition. The coefficient value represents the fraction of green in compartments containing red. n.s. : not significant. The significance of differences was determined by Mann-Whitney U-test with Bonferroni correction following Kruskal-Wallis H-test.

Figure S9. Dasatinib and PP2, but not chloroquine, suppress CpG-triggered global tyrosine phosphorylation CAL-1 cells were stimulated with ODN2216 plus DOTAP in the absence or presence of PP3, PP2, chloroquine (CQ), dasatinib, imatinib (Ima) or nilotinib (Nilo) for 24 h. (A) The concentration of IFN-α in the supernatants was measured in duplicate by ELISA, and were normalized to the maximum value obtained without the inhibitors. The data are shown as means +/- SE of 3 experiments. *P < 0.05; **P < 0.01; ***P < 0.001. The significance of differences was determined by paired two-tailed t-test. The mean and range of absolute concentrations of IFN-α obtained without the inhibitors are 1411 pg/mL (857-2426 pg/mL). (B) Cell lysates were fractionated by SDS-PAGE followed by immunoblotting with anti-phosphotyrosine mAb or anti-β-actin mAb. The data are representative of 3 experiments.

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