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Blood, Vol. 112, Issue 9, 3713-3722, November 1, 2008
Plasmacytoid dendritic cells efficiently cross-prime naive T cells in vivo after TLR activation
Blood Mouriès et al.
112: 3713
Supplemental materials for: Mouries et al
Files in this Data Supplement:
- Figure S1. Phenotypic and functional characterization of DC subsets (JPG, 107 KB)
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(A) DCs subsets were gated as shown in Fig. 1 and expression of surface markers (thick line) was analyzed for pDCs (top panel) and cDCs (bottom panel) from naïve spleen. Filled histograms represent control isotype for each marker. (B) Mice were i.v. injected either with PBS (top row), CpG in DOTAP (100 µg, second row) or R848 (10 µg, third row). Three hours later, pDCs gated among total splenocytes were analyzed for co-stimulatory and MHC molecules expression (thick line). Filled histograms represent control isotype for each marker. Specific fluorescent mean (marker fluorescent mean – control isotype fluorescent mean) is indicated in each case. (C) Production of IFN-α, IL-6, and IL-12 was assayed by ELISA after 24 h of culture of purified DC subsets in the presence of medium alone (CM) or CpG (10 µg.mL−1) or R848 (1 µg.mL−1), as indicated. Results are expressed as mean ± SD for duplicate wells. One representative experiment of 3 is depicted in each case.
- Figure S2. Purity of isolated pDCs (JPG, 87 KB)
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(A–B) Purified pDCs were stained with anti-CD11c, BST-2 (PDCA-1), CD8α, and CD11b mAbs to determine the percentage of cDC contamination. (A) One representative sample is shown. (B) Percentage of each cDC subset in 12 independent sortings. (C) Purified pDCs were stained with anti-CD11c, BST-2 (PDCA-1), and CD45RA mAbs to determine the percentage of CD11c− BST-2+ cells following activation. Three individual mice are shown in each condition of activation.
- Figure S3. TLR-7 activation induces cross-presentation by pDCs (JPG, 38.9 KB)
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Various concentrations of purified pDCs were cultured for 24 h with 105 OVA-specific B3Z cells in the presence of OVA257–264 peptide (1 µg.mL−1) or OVA (3 mg.mL−1), as indicated, and either medium alone (○, CM), Loxoribine (●, left panel − 100 µM) or PolyU (●, right panel – 10 µg.mL−1). Stimulation of B3Z was measured as the release of IL-2 into supernatants as assayed by CTLL proliferation monitored by 3H-labeled thymidine incorporation. Results are expressed as mean cpm ± SD for duplicate wells and are representative of 3 experiments.
- Figure S4. Influence of DC activation on capture ability (JPG, 57.8 KB)
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(A) Spleen cells from mice injected with either PBS, 10 µg of R848 or 100 µg CpG in DOTAP were incubated for one hour in the presence of labeled Ags at 37°C in culture medium (CM) or at 4°C in CM containing 0.1% azide. DCs subsets were analyzed by flow cytometry as in Fig. 1. Fluorescent mean was determined for each Ag in each condition of activation. Capture by DCs isolated from PBS-injected mice was considered as 100%, and percentage of increase was determined for R848 or CpG activated DC subsets. Cumulative data from 4 independent experiments are plotted. Statistical analysis showed non significant difference between activated and non-activated DCs. (B) Spleen cells were incubated for one hour in the presence of beads coated or not with OVA protein at 37°C in culture medium (CM) or at 4°C in CM containing 0.1% azide. DC subsets were analyzed by flow cytometry as in Fig. 1. The percentage of cells gated in M1 that have captured beads is plotted for each subset. Results represent cumulative data from 4 independent experiments and are expressed as mean percentages ± SD. ns non significant, *** p<0.001.
- Figure S5. Internalization of OVA by pDCs following i.v. injection (JPG, 63.1 KB)
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Purified pDCs isolated from mice injected with PBS or 3 or 9 mg of OVA-A488 (green) were stained with anti–BST-2 (120G8 – red) and CD11c (dark blue) mAbs and Hoechst 44432 (light blue) and then analyzed by fluorescent microscopy (original magnification ×630). Several cells from one representative experiment out of 3 are shown.
- Figure S6. Induction of cross-presentation by CpG types A, B, and C (JPG, 32.9 KB)
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129sv mice were injected i.v. with PBS or OVA (9 mg) and either 10 µg of R848, or 50 µg of CpG type A (2216), B (1826), or C (2395) in DOTAP. Two hours later, splenic DC subsets were purified by magnetic sorting, and their capacity to cross-prime OT-I T cells in vitro was assessed. Purified pDCs or cDCs were cultured with 2.5 × 105 CFSE-labeled OT-I T cells and OT-I T-cell proliferation was analyzed by flow cytometry 72 h later. Proliferating T cells were assessed by CFSE dye dilution as described in Fig 5. Percentages of proliferating T cells are plotted. Cumulative results from two independent experiments are shown.
- Figure S7. In vitro cytotoxic assay following cross-priming of primary CD8+ T cells (JPG, 28.6 KB)
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Naïve 129sv mice were immunized by two consecutive i.v. injections (at days 0 and 7) of pDCs or cDCs purified from mice injected with either PBS or OVA (9 mg) alone or with 10 µg of R848. Splenocytes were isolated from immunized mice 7 days after the last injection and restimulated in vitro for 5 days with OVA257–264 peptide (1 µg.mL−1) in the presence of syngeneic irradiated naïve spleen cells. The cytotoxic activity was determined during a 5-hour in vitro 51Cr-release assay. Briefly, 51Cr–EL-4 (H-2b) tumor cells loaded with 50 µM OVA257–264 peptide were used as target cells. Various effector-to-target ratios were used and all assays were done in duplicate. The 51Cr released in each well was counted by using a MicroBeta Trilux liquid scintillation Counter (Wallac, Turku, Finland). The percentage of specific lysis was calculated as 100 × (experimental release spontaneous release)/ (maximal release spontaneous release). Maximum release was obtained by adding 10% Triton X-405 to target cells, and spontaneous release was determined by incubating target cells in medium alone. The plotted percentages of specific lysis presented were obtained for a 90:1 effector-to-target ratio after subtraction of unspecific lysis obtained with unspulsed target cells. ns non significant, *** p<0.001.
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