Type I IFN receptor and its respective signaling through IRF9 is required for NK cell IFN-γ production during HSV-2 infection. (A) WT B6 mice were infected with 10⁴ pfu HSV-2 intravaginally (ivag). On day 2 p.i., vaginal tracts were processed and examined for NKp46, CD3, and IFN-γ expression. (B) WT B6 mice were depleted of NK cells using anti-NK1.1 antibody or IL-15 using anti–IL-15 antibody and then infected with 10⁴ pfu HSV-2 ivag. Day 1–3 p.i. vaginal lavages were examined for IFN-γ levels (n = 5). (C) Peripheral blood was examined for NK cells in mice given anti–IL-15 antibody (n = 5). (D) WT and Ifnar^−/− mice were infected with 10⁴ pfu HSV-2 ivag, and vaginal lavages were examined for IFN-γ production on days 1–3 p.i. (n = 3; repeated twice with similar results). (E) WT B6 mice were administered anti-IFNAR antibody or the respective isotype-matched control Ig on days −1 through 2 i.p. and then infected with 10⁴ pfu HSV-2 ivag. Their vaginal lavages were examined for IFN-γ content (n = 4; repeated once with similar results). (F) WT and Irf9^−/− mice were infected with 10⁴ pfu HSV-2 ivag, and vaginal lavages were examined for IFN-γ levels on days 1–3 p.i. (n = 3; repeated once with similar results). (G) WT and Irf9^−/− mice were infected with 10⁴ pfu HSV-2 ivag. At day 3 p.i., the vaginal mucosa was examined for CD3-NK1.1⁺ NK cells (n = 3; repeated once with similar results). (H) WT, Ifnar^−/−, and Irf9^−/− mice were infected with 10⁴ pfu HSV-2 ivag and followed for survival (n = 5; repeated once with similar results). (I) WT mice were administered anti-IFNAR antibody or the respective isotype control Ig on days −1 through 2 and infected with 10⁴ pfu HSV-2 ivag. Mice were followed for survival (n = 4). (J) After infection with HSV-2, vaginal lavages were collected from WT, Ifnar^−/−, and Irf9^−/− mice and assessed for HSV-2 level via plaque assay (n = 5). (K) WT and Ifnb^−/− mice were infected with 10⁴ pfu HSV-2 ivag. Day 1-3 vaginal washes were collected and examined for IFN-γ amount (n = 4). Data in B, D–F, and K are displayed as mean ± SEM and were analyzed using two-way ANOVA: n.s., not significant; ***, P < 0.001; ****, P < 0.0001. Data in C and G are displayed as mean ± SEM and were analyzed using an unpaired Student’s t test and a Mann–Whitney test (for nonparametric data), respectively: n.s., not significant; **, P < 0.01. Data in J are displayed as mean ± SEM and were analyzed using one-way ANOVA. Data in H and I were analyzed using a log-rank test: *, P < 0.05; **, P < 0.01.