Impaired induction of IFNs via TBK1-mediated pathways in the patient’s SV40-fibroblasts. (A) TBK1 mRNA levels (left panel) were measured by RT-qPCR in fibroblasts (SV40-fibroblasts) from healthy controls (C1, C2), P1, and two other TBK1-deficient patients (WT/G159A, W619*/W619*), and an immunoblot analysis of endogenous TBK1 protein levels (right panel) was performed with antibodies against the N-terminus and C-terminus of TBK1. The results shown are representative of three independent experiments. (B) SV40-fibroblasts from healthy controls (C1, C2), P1, two other TBK1 patients (WT/G159A, W619*/W619*), and a TLR3^−/− HSE patient were left unstimulated (NS) or were stimulated with poly (I:C) alone, Lipofectamine alone (Lipo), or both (poly (I:C)+Lipo), for 6 h. The relative expression levels of IFNB1, IFNL1, IFIT1, and IL6 were measured by RT-qPCR. The results shown are from three independent experiments. (C)IFNB mRNA levels were measured by RT-qPCR in SV40-F from healthy controls (n = 3), P1, and HSE patients with TBK1-deficient (WT/G159A) and AR IFNAR1 deficiencies. Cells were either left uninfected (NS) or infected with HSV-1 (KOS strain, MOI = 1) for 24 h. Data represent the means of three independent experiments. (D) SV40-F from healthy controls (Ctrls, n = 3), P1, and HSE patients with TBK1-deficient (WT/G159A) and AR IFNAR1 deficiencies were either left untreated or pretreated with IFN-β for 24 h, followed by infection with HSV-1 (MOI = 0.001). Viral replication was assessed at the indicated time points post-infection using the TCID₅₀ virus titration method. Data represent means ± SEM from three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test; P values at 48 h after infection compare P1’s cells to controls. ***P < 0.0001. Source data are available for this figure: SourceData F3.