Figure 2.
Figure 2. CLEC-1 triggering on human moDCs prevents downstream Th17 activation. (A) Human moDCs were stimulated with pb anti–CLEC-1 or IgG1 isotype control mAb for 5 minutes. CLEC-1 and binding partners were immunoprecipitated in low-stringent conditions (D6 clone) and were revealed by western blot using anti-phosphotyrosine mAb (4G10). Representative image of western blot with arrows indicating bands with changes in phosphorylation intensity between isotype control and anti–hCLEC-1 mAb stimulation, and IgG HC and LC chains of immunoprecipitating antibody (at the expected size of 50 and 25 kDa, respectively). M line represents molecular-weight size markers. Data are representative of 3 independent experiments. (B) Human moDCs were incubated with or without (−) pb anti–hCLEC-1 or IgG1 isotype control mAbs, and were alternatively stimulated simultaneously with TLR 4-L (LPS) or zymosan for 24 hours, and CD80, CD86, CD83, and HLA-DR were evaluated by flow cytometry (overlays are representative of 8 independent experiments). (C) Tumor necrosis factor-α, IL-12p70, IL-6, IL-23, and IL-10 were assessed by ELISA in supernatants (histograms represent mean ± SEM of 8 independent experiments). (D) Human moDCs were stimulated with pb anti–hCLEC-1 or IgG1 Iso control mAbs for 20 minutes and with or without zymosan. Representative images of western blot revealing phosphorylation of IκBα (PSer32/36) or the degradation of total IκBα at the expected size of 40 and 39 kDa, respectively. Data are representative of 3 independent experiments. M line represents molecular-weight size markers. (E) Following 24 hours of CLEC-1 triggering, human moDCs were extensively washed and subjected to MLR with allogeneic T cells for 5 days. (i) T-cell proliferation was assessed (CFSE dilution) by flow cytometry in allogeneic T cells, and (ii) IL-17 and IFN-γ production was evaluated by ELISA in supernatants. Data were expressed in histograms as mean ± SEM of 8 independent experiments. **P < .01; ***P < .001. IgG HC, IgG heavy chain; IgG LC, IgG light chain; Iso, isotype; UT, untreated.

CLEC-1 triggering on human moDCs prevents downstream Th17 activation. (A) Human moDCs were stimulated with pb anti–CLEC-1 or IgG1 isotype control mAb for 5 minutes. CLEC-1 and binding partners were immunoprecipitated in low-stringent conditions (D6 clone) and were revealed by western blot using anti-phosphotyrosine mAb (4G10). Representative image of western blot with arrows indicating bands with changes in phosphorylation intensity between isotype control and anti–hCLEC-1 mAb stimulation, and IgG HC and LC chains of immunoprecipitating antibody (at the expected size of 50 and 25 kDa, respectively). M line represents molecular-weight size markers. Data are representative of 3 independent experiments. (B) Human moDCs were incubated with or without (−) pb anti–hCLEC-1 or IgG1 isotype control mAbs, and were alternatively stimulated simultaneously with TLR 4-L (LPS) or zymosan for 24 hours, and CD80, CD86, CD83, and HLA-DR were evaluated by flow cytometry (overlays are representative of 8 independent experiments). (C) Tumor necrosis factor-α, IL-12p70, IL-6, IL-23, and IL-10 were assessed by ELISA in supernatants (histograms represent mean ± SEM of 8 independent experiments). (D) Human moDCs were stimulated with pb anti–hCLEC-1 or IgG1 Iso control mAbs for 20 minutes and with or without zymosan. Representative images of western blot revealing phosphorylation of IκBα (PSer32/36) or the degradation of total IκBα at the expected size of 40 and 39 kDa, respectively. Data are representative of 3 independent experiments. M line represents molecular-weight size markers. (E) Following 24 hours of CLEC-1 triggering, human moDCs were extensively washed and subjected to MLR with allogeneic T cells for 5 days. (i) T-cell proliferation was assessed (CFSE dilution) by flow cytometry in allogeneic T cells, and (ii) IL-17 and IFN-γ production was evaluated by ELISA in supernatants. Data were expressed in histograms as mean ± SEM of 8 independent experiments. **P < .01; ***P < .001. IgG HC, IgG heavy chain; IgG LC, IgG light chain; Iso, isotype; UT, untreated.

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