Effect of BCG plus ESAT-6 and HspX on DC-mediated IFN-γ release and CD69 expression by NK cells.

<p>Culture supernatants of DCs treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075684#pone-0075684-g001" target="_blank">Figure 1</a> were incubated without (filled bars) or with (open bars) 20 µg/ml IL-12-blocking antibody and then added to purified NK cells. After 24 hrs, IFN-γ release was measured by ELISA (A and C) and CD69 expression was analyzed by flow cytometry in CD56⁺ cells (B). (A) Results are the mean+SD of three experiments. Statistical analysis: NK cells stimulated with supernatants from BCG-treated DCs vs supernatants of BCG plus HspX and/or ESAT-6-treated DCs; ns P>0,05, *P<0.05. (B) Panels illustrate one representative experiment and the bar graph shows the MFI mean value+SD of three experiments expressed as fold change over control (MFI FC). Filled histograms represent the control; open histograms represent the treated cells. Statistical analysis: NK cells stimulated with supernatants from BCG-treated DCs vs supernatants of BCG/HspX/ESAT-6-treated DCs, *P<0.05; (C) Results are the mean+SD of three experiments. Statistical analysis: IL-12-blocking antibody-treated supernatants vs untreated supernatants; ns P>0,05, *P<0.05.</p

Effect of HspX and ESAT-6 on BCG-elicited DC maturation.

<p>DCs were treated with Mtb as a positive control and with BCG alone or combined with HspX and ESAT6. Cells were collected after 24(A) Histograms illustrate CD83, CD86 and HLA-DR surface expression in CD1a⁺ cells and the MFI. Filled histograms represent the control, open histograms indicate treated cells. One of four different experiments is presented. (B) Bar graphs show the CD83, CD86 and HLA-DR MFI value of the four experiments expressed as fold change (MFI FC) over control (CTRL). Statistical analysis: CTRL vs BCG alone; BCG alone vs BCG plus HspX and ESAT6; ns P>0.05, *P<0.05.</p

TLR2 is involved in IL-12-dependent IFN-γ secretion by CD4⁺ cells co-cultured with ESAT-6, HspX and BCG-treated DCs.

<p>DCs cultured in the absence (filled bars) or presence (open bars) of 5 µg/ml TLR2-blocking antibody were treated for 24 hrs with Mtb, BCG alone or combined with HspX and ESAT6, 10 µg/ml Pam3CSK4 (Pam3) or 100 ng/ml LPS. (A) Supernatants were collected and IL-12 release was analyzed by ELISA. Results are the mean value+SD of four experiments. Statistical analysis: antibody-treated vs antibody-untreated cells, ns P>0.05, *P<0.05, ***P<0.001. (B) DCs were co-cultured with autologous CD4⁺ T lymphocytes. After 7 days, culture supernatants were collected and analyzed by ELISA for IFN-γ release. Results are the mean+SD of three experiments. Statistical analysis: antibody-treated vs antibody-untreated cells, ns P>0.05, *P<0.05.</p

ESAT-6 and HspX enable BCG-stimulated DCs to elicit IFN-γ secretion and to enhance CD69 expression in CD4⁺ T lymphocytes.

<p>DCs were stimulated for 24-6 and/or HspX, and then co-cultured with autologous CD4⁺ T lymphocytes for 7 days. (A) Evaluation of IFN-γ and IL-17AF secretion in culture supernatants by ELISA. Results are expressed as the mean+SD of seven independent experiments. Statistical analysis: DCs treated with BCG alone vs BCG plus HspX and/or ESAT-6; ns P>0.05, *P<0.05. (B) Evaluation of IFN-γ and IL-17AF-production detected by intracellular staining and analyzed by FACS in Annexin V^−/CD4⁺/CD1a⁻ cells. Zebra plots illustrate one representative experiment and the percentage of positive cells (left); bar graphs show the mean+SD of three independent experiments (right). Statistical analysis: DCs treated with BCG alone vs BCG plus HspX and/or ESAT-6; ns P>0.05, *P<0.05, **P<0.01. (C) Flow cytometric analysis of CD69 expression in Annexin V^−/CD4⁺/CD1a⁻ cells. Panels illustrating one representative experiment with the MFI are shown on the left. Filled histograms represent the control, open histograms represent treated cells. Bar graphs representing the MFI mean value+SD of three experiments expressed as fold change over control (MFI FC) are shown on the right. Statistical analysis: DCs treated with BCG vs BCG/HspX/ESAT-6; **P<0.01.</p

The ability of BCG, ESAT-6 and HspX-treated DCs to elicit IFN-γ secretion by CD4⁺ cell is mediated by IL-12.

<p>DCs pre-incubated (open bars) or not (filled bars) with 20 µg/ml IL-12p70 blocking antibody were stimulated for 24 hrs with Mtb or with BCG alone or with ESAT-6 and HspX and then co-cultured for 7 days with autologous CD4⁺ T lymphocytes. IFN-γ and IL-17AF secretion was analyzed by ELISA. Results are expressed as the mean+SD of five experiments. Statistical analysis: antibody-treated vs antibody-untreated cells; ns P>0.05, *P<0.05.</p

Effect of HspX and ESAT-6 on BCG-induced cytokine secretion by DCs.

<p>Monocytes were treated (5 days) with 50 ng/ml GM-CSF and 20 ng/ml IL-4 to obtain immature DCs, that were subsequently cultured (24 hrs) in the absence (CTRL) or presence of 50 µg/ml BCG, alone or combined with 10 µg/ml HspX and/or 10 µg/ml ESAT-6. DCs were also cultured with 50 µg/ml Mtb as a positive control. Release of the indicated cytokines in culture supernatants was evaluated by ELISA. Results are expressed as the mean value+SD of seven independent experiments. Statistical analysis: DCs treated with BCG alone vs BCG plus HspX and ESAT-6 added alone or in combination; ns P>0.05, *P<0.05, **P<0.01, ***P<0.001.</p