Fig. 4.

MyD88 is required for NK cell activation induced by Pf-RBC. (A) Splenocytes from Ly5.1⁺ WT mice were mixed with splenocytes from either Ly5.1^- MyD88^-/- mice (Top), Ly5.1^- IL-18R^-/- mice (Middle), or Ly5.1^- WT mice (Bottom) at a 1/1 ratio. Mixed splenocytes were then stimulated for 20-24 h in the presence of IL-12 with 3D7-RBC, RBC, or PMA/ionomycin. Two parameter flow cytometry contour plots, gated on NK cells, are shown. The frequencies of IFN-γ⁺ NK cells expressing Ly5.1⁺ or Ly5.1^- are indicated in the upper and lower quadrant of each plot, respectively. Similar results were obtained in three experiments. (B) The Ly5.1⁺ mouse NK cell line Ky.2 was cultured in the absence (0) or presence of freshly isolated splenocytes from Ly5.1^- WT or deficient mice and stimulated with 3D7-RBC for 20-24 h in the presence of IL-12. The percentages of Ly5.1⁺ Ky.2 cells producing IFN-γ are indicated. Results are expressed as means ± SEM of three experiments. Statistical analyses were performed by using a one-tailed Wilcoxon signed rank test. When cocultured with uninfected RBC, the percentage of IFN-γ⁺ Ky.2 cells was 1.9 ± 1.5 in the absence of splenocytes (n = 4), 4.4 ± 1.2 with WT splenocytes (n = 5), 2.9 ± 1.9 with MyD88 knockout (KO) splenocytes (n = 5), and 6.8 ± 2.0 with IL-18R knockout splenocytes (n = 3).