Journal: Journal of Extracellular Vesicles

Article Title: Extracellular vesicles derived from nasopharyngeal carcinoma induce the emergence of mature regulatory dendritic cells using a galectin‐9 dependent mechanism

doi: 10.1002/jev2.12390

Figure Lengend Snippet: Characterization of moDCs pre‐cultured with recombinant Gal9S after maturation. (A) Expression level of surface markers CD11c, CD14, DC‐SIGN, HLA‐DR (A.a‐d) , CD40, CD80, CD83 and CD86 (B.a‐d) in mature (mDC) immature (iDC), tolerogenic (tDC) and co‐cultured with Gal9S (DCGal9S) blocked or not with 1g3 (DCGal9S + 1g3), isotype IgG1K (DCGal9S + Iso), lactose (DCGal9S + lactose) moDCs after maturation. The histograms are representative of four independent experiments for conditions iDC (except for CD80 where n = 2), mDC, tDC and DCGal9S, three independent experiments for DCGal9S + 1g3 and one experiment for DCGal9S + Iso and DCGal9S + lactose. The histograms are expressed as the median fluorescence ratio normalized to mDC; MEAN ± SEM. Statistical differences were analysed by one‐way ANOVA or Kruskall Wallis after outlier identification. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (C) Cytokine secretion of moDCs after maturation. Dosage of cytokines secretion by ELISA in the culture supernatant of moDCs. (C.a‐b) Secretion of the effector cytokines IL‐12 and IL‐6 (C.c‐d) Secretion of immunoregulatory cytokines TGF‐ß and IL‐10. Results expressed in cytokines concentrations (pg/mL) from four independent experiments for conditions iDC, mDC, tDC and DCGal9S, three independent experiments for DCGal9S + 1g3 and 1 experiment for DCGal9S + Iso and DCGal9S + lactose, MEAN ± SEM. Statistical differences between conditions were analysed by one‐way ANOVA or Kruskall Wallis after outlier identification. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: C15SEVs (5 μg/mL) and recombinant Galectine‐9S (Gal9S, 3 μg/mL) (Galpharma, Japan) blocked or not with anti‐Gal9 antibody (1g3, 3 μg/mL) (Biotem, Apprieu, France), isotype mouse IgG1K (3 μg/mL) (Biolegend, San Diego, CA, USA) or lactose (5 mM) (Fluka, St. Louis, MO, USA) were added to PBMC (100 000 cells/wells) in round bottom 96‐well plate (Corning Incorporated, NY, USA) for 48 or 72 h. We realized a pre‐incubation of C15SEVs or recombinant Gal9S with 1g3 / isotype / lactose during at least 2 h before adding them to culture with PBMC.

Techniques: Cell Culture, Recombinant, Expressing, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular vesicles derived from nasopharyngeal carcinoma induce the emergence of mature regulatory dendritic cells using a galectin‐9 dependent mechanism

doi: 10.1002/jev2.12390

Figure Lengend Snippet: Immunosuppressive properties of recombinant Galectin‐9S and suppressive function of DCGal9S reverse by the use of an antibody neutralizing galectin‐9. (A) Proliferation assay of PBMCs co‐culture with recombinant galectin‐9S (3 µg/mL) ± an antibody neutralizing galectin‐9 (1g3) (3 µg/mL) or isotype IgG1K (3 µg/mL) or lactose (5 mM) and with anti‐gal9 antibody alone (1g3) after 48 h of culture. Results are expressed in proliferation index normalized on activated PBMCs, MEAN ± SEM. Results are representatives of three independents experiments for the condition 1g3 and Gal9S + lactose, four independent experiments for the condition Gal9S + Iso and seven independents’ experiments for the conditions Gal9S and Gal9S + 1g3. Statistical differences between activated PBMCs and other conditions were analysed by one‐way ANOVA with **** p < 0.0001 consider to be significant. (B) Suppression assay of CD3+ T cells co‐cultured for 48 h (B.a), 72 h (B.b) or 96 h (B.c) with DCs that were pre‐cultured with Galectin‐9S (3 µg/mL) ± an antibody neutralizing galectin‐9 (1g3) (3 µg/mL) or IgG1K isotype (3 µg/mL) or lactose (5 mM). Results are expressed in proliferation index normalized on mDC/CD3TL proliferation, MEAN ± SEM, n = 4 (iDC, mDC, tDC et DCGal9S), n = 3 (DCGal9S + 1g3) at 48 and 72 h. n = 3 (iDC, mDC, tDC et DCGal9S, DCGal9S + 1g3) at 96 h. n = 1 for the others conditions. Statistical differences between conditions were analysed by one‐way ANOVA or kruskal Wallis after outlier identification with ** p < 0.01, *** p < 0.001, **** p < 0.0001 consider to be significant.

Article Snippet: C15SEVs (5 μg/mL) and recombinant Galectine‐9S (Gal9S, 3 μg/mL) (Galpharma, Japan) blocked or not with anti‐Gal9 antibody (1g3, 3 μg/mL) (Biotem, Apprieu, France), isotype mouse IgG1K (3 μg/mL) (Biolegend, San Diego, CA, USA) or lactose (5 mM) (Fluka, St. Louis, MO, USA) were added to PBMC (100 000 cells/wells) in round bottom 96‐well plate (Corning Incorporated, NY, USA) for 48 or 72 h. We realized a pre‐incubation of C15SEVs or recombinant Gal9S with 1g3 / isotype / lactose during at least 2 h before adding them to culture with PBMC.

Techniques: Recombinant, Proliferation Assay, Co-Culture Assay, Suppression Assay, Cell Culture

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular vesicles derived from nasopharyngeal carcinoma induce the emergence of mature regulatory dendritic cells using a galectin‐9 dependent mechanism

doi: 10.1002/jev2.12390

Figure Lengend Snippet: Impact of blocking SEVs Galectin‐9 on their suppressive function (a) Proliferation assay of PBMCs co‐culture with C15SEVs (5 µg/mL) ± an antibody neutralizing galectin‐9 (1g3) (3 µg/mL) or isotype IgG1K (3 µg/mL) or lactose (5 mM) after 72 h of culture. Results are expressed in proliferation index normalized on activated PBMCs, MEAN ± SEM. Results are representatives of three independents experiments. Statistical differences between conditions were analysed by one‐way ANOVA, ** p < 0.01, **** p < 0.0001. (b) Suppression assay of CD3+ T cells co‐cultured for 96 h with DCs that were pre‐cultured with C15SEVs (5 µg/mL) ± an antibody neutralizing galectin‐9 (1g3) (3 µg/mL) or isotype IgG1K (3 µg/mL) or lactose (5 mM). Results are expressed in proliferation index normalized on mDC/CD3TL proliferation on three independents experiments, MEAN ± SEM. Statistical differences between conditions were analysed by one‐way ANOVA after outlier identification.

Article Snippet: C15SEVs (5 μg/mL) and recombinant Galectine‐9S (Gal9S, 3 μg/mL) (Galpharma, Japan) blocked or not with anti‐Gal9 antibody (1g3, 3 μg/mL) (Biotem, Apprieu, France), isotype mouse IgG1K (3 μg/mL) (Biolegend, San Diego, CA, USA) or lactose (5 mM) (Fluka, St. Louis, MO, USA) were added to PBMC (100 000 cells/wells) in round bottom 96‐well plate (Corning Incorporated, NY, USA) for 48 or 72 h. We realized a pre‐incubation of C15SEVs or recombinant Gal9S with 1g3 / isotype / lactose during at least 2 h before adding them to culture with PBMC.

Techniques: Blocking Assay, Proliferation Assay, Co-Culture Assay, Suppression Assay, Cell Culture