Figure 4. NK cells induce apoptosis of DCs treated with SEA.
(A) NK cell expression of CD107a after 5 h culture alone (NK only) or at a 1:1 ratio with immature DCs or DCs treated for 24 h with LPS, Poly(I:C) or SEA. Plots show analysis of CD107a and CD56 gated on live singlet NK cells; representative from one donor of 6 tested. (B) Total percentage of CD107a positive NK cells after 5 h culture alone (NK only), with immature DCs, or DCs treated with LPS, Poly(I:C) or SEA; points of the same shape/colour show measurements from the same donor. Bars show mean (± standard deviation) of data pooled from 6 donors. (C) Annexin V staining of DCs cultured alone (top) or with autologous NK cells at a 1:1 ratio (bottom) for 5 h. Histograms from one representative donor, showing the proportion of annexin V negative (left) and positive populations (right) of DCs. (D) Difference in annexin V staining of DCs cultured alone (circles) compared to DCs cultured with autologous NK cells at a 1:1 ratio for 5 h (squares). Connected data points show paired measurements from 6 independent donors. (E) Specific lysis of 221 target cells, immature DCs or DCs treated for 24 h with LPS, or SEA by autologous NK cells at 1:1, 5:1 and 10:1 NK:DC (E:T) ratios, measured by release of 35S over 5 h. Plots shows data from one representative donor of three; mean of values measured in triplicate. (F) Percent of DCs expressing CD86 after treatment for 24 h with LPS, Poly(I:C) or SEA in the presence of 50 ng/ml TNF-α and 20 ng/ml IL-1β as maturation factors. (G) The proportion of NK cells stained positive for CD107a after 5 hours culture with immature DCs or DCs which had previously been treated for 24 h LPS, Poly(I:C) or SEA in the presence of maturation factors TNF- α and IL-1β. (H) The difference in annexin V staining of immature DCs or DCs treated with LPS, Poly(I:C) or SEA in the presence of maturation factors TNF- α and IL-1β cultured alone (circles) compared to DCs cultured with autologous NK cells at a 1:1 ratio for 5 h (squares). Connected data points show paired measurements from 5 independent donors. (I) IL-4 and IFN-γ production by CD4+ T cells stimulated with PMA and ionomycin after 13 d culture with immature DCs or LPS/omega-1treated DCs in the presence of staphylococcal enterotoxin B; ratio of IL-4 positive over IFN-γ positive CD4+ T cells. (J) Total percentage of CD107a positive NK cells after 5 h culture alone (NK only), with immature DCs, or DCs previously treated with LPS or omega-1; points of the same shape/colour show measurements from the same donor. Bars show mean (± standard deviation) of data pooled from 5 donors. In all plots circles represent data points from individual donors and bars show mean (± standard deviation) of 3-6 independent donors. *p<0.05, ** p<0.01, ***p<0.001; (B, G, J) analysed by repeated measures 1-way ANOVA with Tukey’s multiple comparisons. (F) Analysed by 1-way ANOVA with Dunnett’s multiple comparisons. (D, H) Analysed by repeated measures 2-way ANOVA with Sidak’s multiple comparisons.