Journal: The Journal of Experimental Medicine

Article Title: Enhanced TLR7-dependent production of type I interferon by pDCs underlies pandemic chilblains

doi: 10.1084/jem.20231467

Figure Lengend Snippet: Enhanced TLR7 responses in patients with PC. (A and B) PBMCs from chilblain patients (PC, n = 16) or HD (controls, n = 20) were stimulated with the TLR7-selective agonist IMQ for 24 h. (A) Secreted IFN-α was evaluated in a LEGENDplex assay. (B) IFN-α production in response to IMQ is shown relative to the age of female (F) and male (M) patients or controls. Freshly isolated PBMCs were used in these experiments. (C) PBMCs from chilblain patients (PC, n = 12) or HD (controls, n = 12) were stimulated with the TLR7-selective agonist CL087 and the dual TLR7/8 agonist R848 for 24 h. Secreted IFN-α was evaluated in a LEGENDplex assay. Cryopreserved PBMCs were used in these experiments. (D) ISRE reporter cells (expressing luciferase under the control of an ISRE, THP1-Dual) were treated with supernatants from the CL087-stimulated PBMCs of chilblain patients (PC, n = 12) or HD (controls, n = 12). Luciferase activity was quantified with a luminometer, and RLA is represented as fold induction over untreated reporter cells. (E) THP1-Dual cells were treated with the supernatants of CL087-stimulated PBMCs from chilblain patients (PC, n = 12) or HD (controls, n = 12). The expression of ISG15 and SIGLEC1 was analyzed by flow cytometry with intracellular staining. Graphs show MFI as fold induction over basal levels in THP1 cells. (F) PBMCs from chilblain patients (PC, n = 15) or HD (controls, n = 16) were stimulated with viral nucleic acid surrogates for 24 h. Secreted IFN-α levels were evaluated in a LEGENDplex assay after stimulation with liposome-encapsulated poly(I:C), cGAMP (STING agonist) or poly(dA:dT) (cytosolic DNA sensors), or naked CpG-A (TLR9 agonist). Freshly isolated PBMCs were used in these experiments. (G) PBMCs from chilblain patients (PC) or HD (controls) were stimulated for 24 h with SARS-CoV-2 virus ( n = 8) or IAV ( n = 12). Secreted IFN-α was evaluated in a LEGENDplex assay. Cryopreserved PBMCs were used in these experiments. Adjusted P values in A and C–G were determined in Mann–Whitney tests with the Bonferroni correction for multiple testing. ***P < 0.001; **P < 0.01; *P < 0.05; ns = nonsignificant. IMQ, imiquimod; RLA, relative luciferase activity; MFI, mean fluorescence intensity; IAV, influenza A virus.

Article Snippet: Permeabilized cells were incubated overnight at 4°C with the following antibodies: ISG15 PE (clone 851701, Cat. IC8044P, RRID:AB_3656663, 1:200 dilution; R&D Systems) and SIGLEC1 APC (clone 7-239, Cat. 130-098-645, RRID:AB_2655547, 1:400 dilution; Miltenyi Biotec), in the presence of Human TruStain FcX (AB_2818986; BioLegend).

Techniques: Isolation, Expressing, Luciferase, Control, Activity Assay, Flow Cytometry, Staining, Virus, MANN-WHITNEY, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: Enhanced TLR7-dependent production of type I interferon by pDCs underlies pandemic chilblains

doi: 10.1084/jem.20231467

Figure Lengend Snippet: Related to . (A) PBMCs from chilblain patients (PC, n = 13) or HD (controls, n = 16) were stimulated with the TLR7-selective agonist IMQ for 24 h. Secreted cytokines were evaluated in a LEGENDplex assay. Freshly isolated PBMCs were used in these experiments. (B) THP1-Dual reporter cells were treated with various doses of recombinant IFN-α (rIFN-α2b), ranging from 10 to 1,000 IU/ml. Left panel: luciferase activity was quantified with a luminometer, and RLA is represented as fold induction over untreated reporter cells. Right panels: the expression of ISG15 and SIGLEC1 was analyzed by flow cytometry with intracellular staining. Graphs show MFI as fold induction over basal levels in THP1 cells. The dashed lines represent the mean type I IFN activity levels for supernatants from the TLR7-stimulated leukocytes of HD (black) (controls, n = 12) and chilblain patients (red) (PC, n = 12). (C) PBMCs from chilblain patients (PC, n = 15) or HD (controls, n = 16) were stimulated with viral nucleic acid surrogates for 24 h. Secreted IFN-β levels were evaluated in a LEGENDplex assay after stimulation with liposome-encapsulated poly(I:C), cGAMP (STING agonist) or poly(dA:dT) (cytosolic DNA sensors), or naked CpG-A (TLR9 agonist). Freshly isolated PBMCs were used in these experiments. (D) PBMCs from PC patients or controls were stimulated for 24 h with SARS-CoV-2 ( n = 8) or IAV ( n = 12). Secreted IFN-β levels were evaluated in a LEGENDplex assay. Cryopreserved PBMCs were used in these experiments. (E) PBMCs from PC patients (PC, n = 12) or HD (controls, n = 12) were stimulated for 24 h with HSV-1. Secreted IFN-α and IFN-β levels were evaluated in a LEGENDplex assay. (F) Distribution of lymphoid and myeloid subsets among PBMCs from PC patients (PC, n = 12) and HD (controls, n = 14) was determined by flow cytometry. Data are represented as percentages of total CD45 + cells. CM, central memory T cells; EM, effector memory T cells; EMRA, CD45RA + effector memory T cells; NK, natural killer cells; cDC CD1c + , CD1c + conventional dendritic cells; cDC CD1c − , CD1c − conventional dendritic cells. (G) Frequency of CD123 + BDCA2 + pDCs in PBMCs from PC patients ( n = 12) and controls ( n = 14), as analyzed in F, is shown on a separate graph. Bars represent the mean ± SD. The adjusted P values in A, C–E, and G were determined in Mann–Whitney tests with the Bonferroni correction for multiple testing. *P < 0.05; ns = nonsignificant. IMQ, imiquimod; RLA, relative luciferase activity; MFI, mean fluorescence intensity; IAV, influenza A virus.

Article Snippet: Permeabilized cells were incubated overnight at 4°C with the following antibodies: ISG15 PE (clone 851701, Cat. IC8044P, RRID:AB_3656663, 1:200 dilution; R&D Systems) and SIGLEC1 APC (clone 7-239, Cat. 130-098-645, RRID:AB_2655547, 1:400 dilution; Miltenyi Biotec), in the presence of Human TruStain FcX (AB_2818986; BioLegend).

Techniques: Isolation, Recombinant, Luciferase, Activity Assay, Expressing, Flow Cytometry, Staining, MANN-WHITNEY, Fluorescence, Virus