EmTIP, a T-Cell Immunomodulatory Protein Secreted by the Tapeworm Echinococcus multilocularis Is Important for Early Metacestode Development
Figure 4
Elevated secretion of IFN-γ by CD4+ T-cells activated in the presence of EmTIP in vitro.
(A) Freshly isolated CD4+CD25− T-cells (2×105/ml) were stimulated with α-CD3 (0.1 µg/ml) and α-CD28 (5 µg/ml) in the presence of E/S products of E. multilocularis primary cells (PCE/S) or metacestode vesicles (MVE/S). Three days later, the T-cell supernatants were collected and probed for IFN-γ and IL-10 concentrations by Elisa. (B) Freshly isolated CD4+CD25− T-cells (2×105/ml) were stimulated with α-CD3 (0.1 µg/ml) and α-CD28 (5 µg/ml) in medium supplemented with normalized supernatants of Mock-, control- (pSecTag2), Emtip- (pSecTag2-Emtip) or Emtip_myc (pSecTag2-Emtip_myc)- transfected HEK-293T cells. Three days later, the T-cell supernatants were collected and probed for IFN-γ and IL-10 concentrations by Elisa. (A, B) Individual results are displayed. Bars represent the mean from 4–8 independent biological replicates assessed within 3–4 independent experiments. (C) Freshly isolated CD4+CD25− T-cells (2×105/ml) were stimulated with α-CD3 (0.1 µg/ml) and α-CD28 (5 µg/ml) in the presence of conditioned medium of primary cells (PCE/S) or metacestode vesicles (MVE/S) supplemented with total rabbit IgG or purified anti-EmTIP (anti-TIP) antibody at 30 µg/ml. Three days later, the T-cell supernatants were collected and probed for IFN-γ and IL-10 concentrations by Elisa. Individual results are displayed. Bars represent the mean from experiments with 4 biological replicates. (D) Freshly isolated CD4+CD25− T-cells (2×105/ml) were stimulated with α-CD3 (0.1 µg/ml) and α-CD28 (5 µg/ml) in medium supplemented with normalized supernatants of parasite larvae (PCE/S or MVE/S) or transfected HEK-293T cells (Mock, Control or rEmTIP). At day 3 of culture, 5 µg/ml Brefeldin A was added for an additional 6 hours. Then the cells were harvested and the intracellular IFN-γ levels were determined by flow cytometry. Numbers in upper gates indicate the percentage of IFN-γ+ CD4+ cells whereas the lower gate shows the percentage of IFN-γ+ CD4− cells. One representative experiment of 3 conducted with similar results is shown (Left) and data from all three experiments are summarized as bar graphs (Right). * (p<0.05), ** (p<0.01) (D) Bars represent mean ± standard deviations from three independent experiments with 3–7 biological replicates.