The suppression of DC activation by peritoneal fluid from patients with beningn conditions is fully mediated by IL-10 unlike the suppressive activity of OC-associated ascites.

<p>Monocyte-derived DC were stimulated overnight with 3μg/ml R848 in the presence or absence of (A) 10% malignant ascites from OC patients or (B) 10% peritoneal fluid from benign ovarian tumours and IL-10 neutralizing antibody (5μg/ml) as indicated. CD86 expression levels were assessed by flow cytometry, and cytokine levels were measured in cell culture supernatants by flow cytomix analysis (A: IL-6 and TNFα) or sandwich ELISA (A: IL-12p40 and B: TNFα, IL-6 and IL-12p40); for malignant samples n = 6 (6 independent experiments; monocyte-derived DC from 5 different healthy donors, cultured with 1 (n = 4), or 2 (n = 1) different ascites samples); for benign samples n = 9 (9 independent experiments; monocyte-derived DC from 5 different healthy donors, cultured with 1 (n = 3), or 3 (n = 2) different peritoneal fluids); One-way ANOVA (Friedman test with Dunn post test): * = p<0.05, ns = not significant.</p

Neutralisation of IL-10 releases the impairment of DC activation in response to TLR stimulation in the presence of OC-associated ascites.

<p>(A, B) Monocyte derived DC were cultured in the presence of 3μg/ml R848, 10% ascites and neutralizing antibodies (5μg/ml) as indicated. Neutralizing antibodies were added to cultures alone or in combination. (A) CD86 expression levels were measured by flow-cytometry, and (B) cytokine levels were measured in cell culture supernatants by flow cytomix analysis (IL-6, TNFα) or sandwich ELISA (IL-12p40). n = 9–13 (13 independent experiments; monocyte-derived DC from 8 different healthy donors, cultured with 1 (n = 4), 2 (n = 3) or 3 (n = 1) different ascites samples. IL-6 neutralizing antibody was only used in 9 out of 13 experiments). One-way ANOVA (Friedman test with Dunn post test): *** = p<0.001. Please note that the quantification of IL-6 in samples containing IL-6 neutralizing antibody (αIL-6 and all AB) is compromised. (C, D) For allogeneic MLR, monocyte-derived DC from healthy donors were cultured overnight in complete medium only, or stimulated with 3μg/ml R848 in the presence or absence of 10% ascites fluid (AF) and/or IL-10 neutralizing antibody (5μg/ml) as indicated. Subsequently, such pre-treated DC were washed and co-cultured with CFSE-stained CD4⁺ naïve T cells from another healthy donor at a 1:200 ratio for 6 days. Naïve T cells from the same donor were used consistently for all experiments. The proliferation of T cells was determined by CFSE dilution after 6 days. (C) A representative example of flow-cytometry analysis is shown. (D) Cumulative data from 4 independent experiments showing proliferation in percent of CD3⁺ cells that have undergone at least one round of division. n = 4 (4 independent experiments; monocyte-derived DC from 3 different healthy donors, cultured with ascites from two (n = 1) or one (n = 2) OC patient).</p

Up-regulations of CD86 and induction of cytokines in response to TLR stimulation in the presence or absence of OC-associated ascites.

<p>(A) Monocyte-derived DC were stimulated overnight with 3μg/ml R848, 1μg/ml LPS, 100μg/ml polyI:C in the presence of 0%, 10% or 25% of ascites from patients with malignant OC. The mean fluorescence intensity (MFI) of the surface marker CD86 was assessed by flow cytometry. (B) Monocyte-derived DC were cultured overnight as described above and cytokines were measured in culture supernatants by flow cytomix analysis or sandwich ELISA (IL-12p40). In total, 12 independent experiments were performed (n = 12) with DC from individual healthy volunteers cultured with ascites from 4, 3, 2 or 1 OC patients (n = 1, 1, 1 and 3 healthy volunteers, respectively). One-way ANOVA was used for statistical analysis (Friedman test with Dunn post test): * = p<0.05; ** = p<0.01; *** = p<0.001; ns = not significant.</p

Depletion of IL-10 from OC-associated ascites does not release the suppression of TLR-mediated DC activation.

<p>Monocyte-derived DC were stimulated with 3μg/ml R848 in the presence or absence of 10% ascites from ovarian carcinoma patients which had previously been depleted of IL-10 (IL10 ΔAF) or which had undergone mock depletion (mock ΔAF). Alternatively, DC cultures were treated with R848 and untreated ascites (AF) in the presence of αIL-10 neutralizing antibody or isotype control antibody (5μg/ml); n = 3 (3 independent experiments; monocyte-derived DC from 1 healthy donor, cultured with three ascites samples from three individual ovarian carcinoma patients). IL-6 levels were measured in cell culture supernatants by sandwich ELISA.</p

Up-regulations of CD86 and induction of cytokines in response to TLR stimulation in the presence or absence of peritoneal fluid from patients with benign conditions.

<p>Monocyte-derived DC were stimulated overnight with 3μg/ml R848, 1μg/ml LPS or 100μg/ml polyI:C in the presence of 0%, 10% or 25% peritoneal fluid obtained from patients suffering from benign ovarian conditions. The following day, (A) the MFI of surface marker CD86 was assessed by flow cytometry and (B) cytokines were measured in culture supernatants by flow cytomix analysis or sandwich ELISA (IL-12p40). In total, 12 experiments (n = 12) were performed with DC from 8 different healthy volunteers cultured with ascites from 2 (n = 4) or 1 (n = 4) patient with benign ovarian conditions. One-way ANOVA was used for statistical analysis (Friedman test with Dunn post test); * = p<0.05; ** = p<0.01; *** = p<0.001; ns = not significant.</p

Levels of various immunomodulatory factors in OC-associated ascites and their correlation with the immunosuppressive effects observed in DC activation assays.

<p>(A) Protein levels of various factors in ascites samples collected from OC patients were measured by sandwich ELISA. Eight malignant ascites samples used throughout the study are shown. Each symbol represents one patient sample throughout the graphs (n = 8). (B) Levels of IL-10 in ascites samples are correlated to the suppression of TLR-mediated up-regulation of CD86 and production of IL-6, IL-12 and TNFα. Suppression is expressed in per cent reduction of surface marker and cytokine levels when 10% ascites was added to the cell culture as compared to no ascites present; n = 26 (26 independent experiments conducted with monocyte-derived DC from 9 different healthy controls, cultured with ascites from two (n = 4), three (n = 2) or four (n = 3) different OC patients. The Pearson coefficient was calculated to assess statistical significance of correlations.</p