IL-18 and IL-18R are both required for NK cell IFN-γ production during HSV-2 infection. (A) WT and Ifnar^−/− mice were infected with 10⁴ pfu HSV-2 ivag, and day 1–3 vaginal lavages were examined for IL-18. Data were normalized to Il18^−/− data (n = 5; repeated once with similar results). (B) WT, Il18^−/−, and Il18r1^−/− B6 mice were infected with 10⁴ pfu HSV-2 ivag, and on day 1–3 p.i. vaginal lavages were examined for IFN-γ content (n = 5; repeated once with similar results). (C and D) Vaginal tissue was isolated on day 3 p.i. and examined for CD45⁺CD3⁻NK1.1⁺ cells in Il18^−/− (C; n = 3) and Il18r1^−/− (D; n = 3) mice. (E) WT and Il18r1^−/− NK cells were isolated from the spleen and adoptively transferred into Rag2^−/−Il2rg^−/− mice i.v. 24 h p.i. The mice were infected with 10⁴ pfu HSV-2 ivag. On days 1–3 p.i., vaginal lavages were collected and examined for IFN-γ (n = 6). (F) NK cells were isolated from WT, Ifnar^−/−, and Il18r^−/− spleens and stimulated with 50 ng/ml PMA and 500 ng/ml ionomycin for 24 h ex vivo. Supernatants were collected and assayed for IFN-γ (n = 3). Data in A, B, and E are displayed as mean ± SEM and were analyzed using two-way ANOVA: *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Data in C and D are displayed as mean ± SEM and were analyzed using an unpaired Student’s t test: n.s., not significant. Data in F are displayed as mean ± SEM and were analyzed using one-way ANOVA: n.s., not significant.