Figure 4
Figure 4. DC-derived f-actin dynamics play a role in the maturation of interactions with resting NK cells and DC survival on NK cell activation. (A) Mature DCs treated with siRNA control or siRNA targeting WASP were lysed and protein extracts were run on SDS-page gels. Vinculin (as a loading control) and WASP were detected by Western blot and protein levels were quantified by ImageJ. (B) Immature or mature control and ΔWASP DCs were stained with fluorochrome conjugated anti–HLA-ABC, -CD86, -CD83 and –HLA-DR antibodies and compared with unstained DCs by flow cytometry. For microscopy images, mature control or ΔWASP DCs were fixed and MHC class I molecules (red) and nuclear DNA (blue) were stained. (C) Mature control DCs, mature ΔWASP DCs and resting NK cells were prestained with vital dyes and cocultured for 1, 20, or 120 minutes. The percentage of NK cell-conjugated DCs was determined by flow cytometry. (D) NK cells were cultured alone or with mature control or ΔWASP DCs for 6 hours, in the presence of BFA. IFN-γ production by NK cells, as well as degranulation (CD107a staining) were assessed by ICS in flow cytometry. NK cells were gated as live, individual, CD3−CD56+ cells. (E) Mature control DCs, mature ΔWASP DCs and resting NK cells were prestained with vital dyes and cocultured for 6 hours, in the presence of BFA, and IFN-γ production by DC-conjugated NK cells was assessed by ICS in flow cytometry, above the high autofluorescence of nonconjugated DCs. (F) Mature control and ΔWASP DCs were cultured with resting NK cells for 6 hours, and the percentage of surviving DCs (% DCs of live cells) was assessed by flow cytometry, using the Fixable Aqua Live/Dead reagent (left graph). Mature control and ΔWASP DCs were prestained with vital dyes and cultured alone or with IL-2 activated NK cells, for 6 hours. The percentage of killed DCs (% killed DCs) was assessed by flow cytometry, using the TO-PRO-3 reagent (right graph). Medians ± interquartile ranges were plotted in the graphs, after subtracting the values of spontaneous lysis of DC single cell cultures. (G) Mature control and ΔWASP DCs were cultured alone for 6 hours, and the percentage of surviving DCs (% DCs of live cells) was assessed by flow cytometry, using the Fixable Aqua Live/Dead reagent. Plots are representative of at least 3 independent experiments. Values on graph bars represent medians from at least 3 independent experiments with duplicates. Error bars indicate interquartile ranges. Original magnifications are 100× for all the microscopy images. *P < .05. P values from Mann-Whitney test, nonparametric and bi-caudal. Scale bars are 10 μm.

DC-derived f-actin dynamics play a role in the maturation of interactions with resting NK cells and DC survival on NK cell activation. (A) Mature DCs treated with siRNA control or siRNA targeting WASP were lysed and protein extracts were run on SDS-page gels. Vinculin (as a loading control) and WASP were detected by Western blot and protein levels were quantified by ImageJ. (B) Immature or mature control and ΔWASP DCs were stained with fluorochrome conjugated anti–HLA-ABC, -CD86, -CD83 and –HLA-DR antibodies and compared with unstained DCs by flow cytometry. For microscopy images, mature control or ΔWASP DCs were fixed and MHC class I molecules (red) and nuclear DNA (blue) were stained. (C) Mature control DCs, mature ΔWASP DCs and resting NK cells were prestained with vital dyes and cocultured for 1, 20, or 120 minutes. The percentage of NK cell-conjugated DCs was determined by flow cytometry. (D) NK cells were cultured alone or with mature control or ΔWASP DCs for 6 hours, in the presence of BFA. IFN-γ production by NK cells, as well as degranulation (CD107a staining) were assessed by ICS in flow cytometry. NK cells were gated as live, individual, CD3−CD56+ cells. (E) Mature control DCs, mature ΔWASP DCs and resting NK cells were prestained with vital dyes and cocultured for 6 hours, in the presence of BFA, and IFN-γ production by DC-conjugated NK cells was assessed by ICS in flow cytometry, above the high autofluorescence of nonconjugated DCs. (F) Mature control and ΔWASP DCs were cultured with resting NK cells for 6 hours, and the percentage of surviving DCs (% DCs of live cells) was assessed by flow cytometry, using the Fixable Aqua Live/Dead reagent (left graph). Mature control and ΔWASP DCs were prestained with vital dyes and cultured alone or with IL-2 activated NK cells, for 6 hours. The percentage of killed DCs (% killed DCs) was assessed by flow cytometry, using the TO-PRO-3 reagent (right graph). Medians ± interquartile ranges were plotted in the graphs, after subtracting the values of spontaneous lysis of DC single cell cultures. (G) Mature control and ΔWASP DCs were cultured alone for 6 hours, and the percentage of surviving DCs (% DCs of live cells) was assessed by flow cytometry, using the Fixable Aqua Live/Dead reagent. Plots are representative of at least 3 independent experiments. Values on graph bars represent medians from at least 3 independent experiments with duplicates. Error bars indicate interquartile ranges. Original magnifications are 100× for all the microscopy images. *P < .05. P values from Mann-Whitney test, nonparametric and bi-caudal. Scale bars are 10 μm.

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