Figure 3. Impaired poly(I:C) responses in SV40-fibroblasts from patients heterozygous for TLR3 P554S or P680L, and rescue by WT TLR3. (A–D) Production of IFN-β, IFN-λ (A and B), and IL-6 (C and D) in SV40-fibroblasts from three healthy controls (CTL1/2/3), P2 (A and C), P3 (B and D), a TLR3 P554S/WT HSE patient, and a TLR3−/− HSE patient, 24 h after stimulation with 1, 5, or 25 µg/ml poly(I:C), or with 25 µg/ml poly(I:C) in the presence of lipofectamine (poly[I:C]+L; A and B), lipofectamine alone (L; A and B), or IL-1β (C and D), as assessed by ELISA. (E) IFNB, IFNL1, and IL6 mRNA levels in SV40-fibroblasts from two CTLs, P3, P554S/WT, and TLR3−/− patients, not stimulated (NS), or stimulated for 2 and 4 h with 25 µg/ml poly(I:C), or infected with VSV M51R at a MOI of 1 for 16 h. GUS was included for normalization. (F–I) Complementation of the impaired poly(I:C) response by introducing WT TLR3 into the patient’s fibroblasts. (F) IFNB, IFNL1 mRNA levels in SV40-fibroblasts from a healthy control (CTL) and P3, without plasmid transfection (NT) or after transfection with luciferase (Luc), Flag-tagged WT or P680L TLR3, and in fibroblasts from a TLR3−/− patient, not stimulated (NS), or stimulated for 2 h with 25 µg/ml poly(I:C), or infected with VSV M51R at a MOI of 1 for 16 h. GUS was included for normalization. (G) Production of IFN-λ, in the absence of stimulation, after 24 h of stimulation with 1, 5, or 25 µg/ml poly(I:C), and after stimulation with 25 µg/ml poly(I:C) in the presence of lipofectamine, or lipofectamine alone, as assessed by ELISA, in SV40-fibroblasts from a CTL and P3, without plasmid transfection or after transfection with Luc, Flag-tagged WT or P680L TLR3, and in fibroblasts from a TLR3−/− patient. (H) TLR3 mRNA levels were assessed by RT-qPCR. (I) TLR3 was detected on immunoblots following IP. Mean values ± SD were calculated from four (A–D and H) or two (E and F) independent experiments, with technical duplicates in each experiment. (G) Results from a single experiment with biological duplicates, representing three independent experiments. (I) Reproducible results from three independent experiments.
Figure 3.

Impaired poly(I:C) responses in SV40-fibroblasts from patients heterozygous for TLR3 P554S or P680L, and rescue by WT TLR3. (A–D) Production of IFN-β, IFN-λ (A and B), and IL-6 (C and D) in SV40-fibroblasts from three healthy controls (CTL1/2/3), P2 (A and C), P3 (B and D), a TLR3 P554S/WT HSE patient, and a TLR3−/− HSE patient, 24 h after stimulation with 1, 5, or 25 µg/ml poly(I:C), or with 25 µg/ml poly(I:C) in the presence of lipofectamine (poly[I:C]+L; A and B), lipofectamine alone (L; A and B), or IL-1β (C and D), as assessed by ELISA. (E) IFNB, IFNL1, and IL6 mRNA levels in SV40-fibroblasts from two CTLs, P3, P554S/WT, and TLR3−/− patients, not stimulated (NS), or stimulated for 2 and 4 h with 25 µg/ml poly(I:C), or infected with VSV M51R at a MOI of 1 for 16 h. GUS was included for normalization. (F–I) Complementation of the impaired poly(I:C) response by introducing WT TLR3 into the patient’s fibroblasts. (F) IFNB, IFNL1 mRNA levels in SV40-fibroblasts from a healthy control (CTL) and P3, without plasmid transfection (NT) or after transfection with luciferase (Luc), Flag-tagged WT or P680L TLR3, and in fibroblasts from a TLR3−/− patient, not stimulated (NS), or stimulated for 2 h with 25 µg/ml poly(I:C), or infected with VSV M51R at a MOI of 1 for 16 h. GUS was included for normalization. (G) Production of IFN-λ, in the absence of stimulation, after 24 h of stimulation with 1, 5, or 25 µg/ml poly(I:C), and after stimulation with 25 µg/ml poly(I:C) in the presence of lipofectamine, or lipofectamine alone, as assessed by ELISA, in SV40-fibroblasts from a CTL and P3, without plasmid transfection or after transfection with Luc, Flag-tagged WT or P680L TLR3, and in fibroblasts from a TLR3−/− patient. (H) TLR3 mRNA levels were assessed by RT-qPCR. (I) TLR3 was detected on immunoblots following IP. Mean values ± SD were calculated from four (A–D and H) or two (E and F) independent experiments, with technical duplicates in each experiment. (G) Results from a single experiment with biological duplicates, representing three independent experiments. (I) Reproducible results from three independent experiments.

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