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Poly(I:C)-Treated Human Langerhans Cells Promote the Differentiation of CD4+ T Cells Producing IFN-γ and IL-10  Laetitia Furio, Hermine Billard, Jenny.

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Presentation on theme: "Poly(I:C)-Treated Human Langerhans Cells Promote the Differentiation of CD4+ T Cells Producing IFN-γ and IL-10  Laetitia Furio, Hermine Billard, Jenny."— Presentation transcript:

1 Poly(I:C)-Treated Human Langerhans Cells Promote the Differentiation of CD4+ T Cells Producing IFN-γ and IL-10  Laetitia Furio, Hermine Billard, Jenny Valladeau, Josette Péguet-Navarro, Odile Berthier-Vergnes  Journal of Investigative Dermatology  Volume 129, Issue 8, Pages (August 2009) DOI: /jid Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Poly(I:C) strongly up-regulates co-stimulatory molecules on human epidermal LCs. Freshly isolated LCs were treated or not with poly(I:C) (10μg ml−1), CpG (10μg ml−1), LPS (100ng/ml), or CD40 ligation+IFN-γ for 2 days in serum-free medium. Double staining was performed using anti-langerin and the indicated mAbs. (a) Data from a representative experiment showing flow cytometry profile of LCs treated or not with poly(I:C), CpG, or LPS. (b) Mean MFI ratio of treated to untreated LCs: results are the mean±SD of five experiments carried out with different donors. Statistical significance was assessed using Student's t-test: *P<0.05; **P<0.01; ***P<0.001. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Poly(I:C) enhances LC survival upon in vitro culture. Freshly isolated LCs were treated or not with poly(I:C) (5, 10, or 50μg ml−1) for 2, 3, or 4 days. The recovered cells were analyzed for apoptosis using annexin V and propidium iodide staining. (a) Apoptotic cells are located in the lower right and necrotic cells in the upper right quadrants. Numbers represent the percentage of cells in each quadrant. A representative experiment of three is shown. (b) Mean percentage±SD of viable, apoptotic, and necrotic LCs after a 2-day culture in the presence, or not, of poly(I:C) at 10μg ml−1. The results are from the three above-mentioned experiments and from another one stopped at day 2. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Poly(I:C) enhances human LC allostimulatory function. Freshly isolated LCs were treated or not with poly(I:C) (10μg ml−1), CpG (10μg ml−1), LPS (100ng ml−1), or CD40 ligation+IFN-γ for 48hours. Cells were then recovered, and graded numbers of viable cells were added to bulk allogeneic T cells. After 5 days, T-cell proliferation was assessed by the addition of [3H]thymidine. The results are the mean cpm of triplicate wells and representative of three donors. T cells alone gave less than 100c.p.m. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Poly(I:C) induces LCs to secrete CXCL-10, IL-6, and IL-12 p40, but not IL-10 and IL-12-p70. Freshly isolated LCs were treated or not with poly(I:C) (10μg ml−1), CpG (10μg ml−1), LPS (100ng ml−1), or CD40 ligation+IFN-γ for 48hours. Cytokines were measured in culture supernatants as described in Materials and Methods. The results are the mean±SD of experiments carried out with three donors. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Bafilomycin inhibits poly(I:C)-mediated LC survival and phenotypic activation. LCs were treated or not with 50nM bafilomycin before poly(I:C) activation and analyzed after 2 days for survival (a) and phenotype (b and c). (a) LCs were double stained with Annexin V-FITC and propidium iodide. Numbers represent the percentage of cells in each quadrant. (b) For phenotype analysis, viable cells (Annexin V− and PI− cells) were first gated according to FSC and SSC parameters, and analysis was carried out in gate R1. (c) Histograms represent the mean fluorescence intensity for CD80 and CD86 on viable LCs after treatment, or not, with poly(I:C) in the presence (gray) or absence (white) of bafilomycin. The results are representative of two independent experiments. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Poly(I:C) promotes LC-induced Th1 differentiation with concomitant IL-10 production. Allogeneic naive CD4+ T cells were stimulated one or two times with LCs treated or not with poly(I:C) at 25μg ml−1. At the end of each round of stimulation, PMA and ionomycin were added. (a) After 24hours, supernatants were collected and analyzed for Th1/Th2 cytokines. The cytokine concentration is expressed in pg ml−1. The results are from three independent experiments carried out with different donors. (b) After the second round of LC stimulation, brefeldin A was added at the same time as PMA and ionomycin and intra-cellular staining was performed after 5 hours. The percentage of positive cells is indicated in the quadrants. The results are from Exp 1 and representative of the other two experiments. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 7 LC-induced IFN-γ production by CD4+ T cells is independent of bioactive IL-12 and IL-23. Allogeneic naive CD4+ T cells were co-cultured with LCs or MoDCs pre-treated or not with poly(I:C) at 25μg ml−1, in the presence, or not, of anti-IL-12 and IL-23 neutralizing mAb (anti-p40). After 6 days, PMA and ionomycin were added, and cell supernatants were collected 24hours later. IFN-γ production was quantified using the human Th1/Th2 cytokine kit. The results are from experiments carried out with two different donors. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

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