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: CD45 Alexa Fluor 350 (clone 2D1, FAB1430U, AB_3646482, 1:200 dilution; R&D Systems); HLA-DR BUV496 (clone L243, 753685, 1:5,000 dilution; BD Biosciences); CD56 BUV737 (clone TULY56, RRID:AB_2895975, 1:400 dilution; Thermo Fisher Scientific); CD11c eFluor 450 (clone 3.9, AB_11218498; Thermo Fisher Scientific, 1:400 dilution); CD123 BV510 (clone 6H6, AB_2562068, 1:800 dilution; BioLegend); CD16 BV570 (clone 3G8, AB_10915988, 1:200 dilution; BioLegend); BDCA-2 BV785 (clone 201A, AB_2572146, 1:800 dilution; BioLegend); CD14 Spark Blue 550 (clone 63D3, AB_2832724, 1:60,000 dilution; BioLegend); CD3 NovaFluor B610-70S (clone SK7, AB_3098363, 1:2,000 dilution; Thermo Fisher Scientific); CD1c PerCP-eFluor 710 (clone L161, AB_10545854, 1:2,000 dilution; Thermo Fisher Scientific); TLR7 PE (clone S18024F, AB_2910431, 1:400 dilution; BioLegend); TLR8 APC (clone S16018A, AB_2801050, 1:400 dilution; BioLegend); TLR9 BV421 (clone S16013D, AB_2801039, 1:800 dilution; BioLegend); and CD19 APC-Fire810 (clone HIB19, AB_2860770, 1:200 dilution; BioLegend), in the presence of Human TruStain FcX (AB_2818986; BioLegend), True-Stain Monocyte Blocker (Cat. 426102; BioLegend), and CellBlox Plus (C001T06F01; Thermo Fisher Scientific) to minimize nonspecific binding.

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

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-mediated I-IFN production by pDCs in chilblain patients. (A) Intracellular levels of IFN-α (upper panel), TNF (middle panel), and IL-6 (lower panel) in leukocyte subsets were evaluated by flow cytometry after the stimulation of PBMCs for 5 h with the TLR7-selective agonist CL087 and the dual TLR7/8 agonist R848 in the presence of brefeldin A. The subsets studied were as follows: CD14 + monocytes (orange), CD123 + BDCA2 + pDCs (red), CD1c + conventional dendritic cells (DC CD1c + ) (purple), CD1c − conventional dendritic cells (DC CD1c − ) (light purple), CD19 + B cells (blue), CD3 + T cells (green). Representative FACS plots from a single patient are shown. The gating strategy is shown in . (B) Intracellular levels of IFN-α (upper panel), TNF (middle panel), and IL-6 (lower panel) in leukocyte subsets were evaluated by flow cytometry following the stimulation of PBMCs from PC patients (PC, n = 12) or HD (controls, n = 12), as described in panel A. Graphs represent the percentage of cytokine-positive cells. (C) pDCs are the primary source of IFN-α produced in response to stimulation with TLR7 agonists. The upper panel shows the expression of IFN-α by total CD45 + PBMCs stimulated with CL087 and R848. The middle panel shows the expression of pDC markers, CD123 and BDCA2, by IFN-α–producing cells. The lower graph shows the percentage of CD123 + BDCA2 + pDCs among IFN-α–producing cells from the PBMCs of PC patients ( n = 12) stimulated with CL087 and R848. (D and E) Intracellular levels of TLR7 (left panel), TLR8 (middle panel), and TLR9 (right panel) in leukocyte subsets were evaluated by flow cytometry on PBMCs. The histograms in D show representative data from a single patient. The graphs in E represent the MFI in PBMCs from female PC patients (PC, n = 8) or healthy female donors (controls, n = 11). Cryopreserved PBMCs were used in A–E. The adjusted P values in B and E were obtained in Mann–Whitney tests with the Bonferroni correction for multiple testing for non-normally distributed datasets, or in Student’s t tests with the Bonferroni correction for multiple testing for normally distributed datasets. Normality was assessed with Shapiro–Wilk and Kolmogorov–Smirnov tests. **P < 0.01; *P < 0.05; ns = nonsignificant; MFI, mean fluorescence intensity.

Article Snippet: Permeabilized cells were incubated overnight at 4°C with the following antibodies: CD45 Alexa Fluor 350 (clone 2D1, FAB1430U, AB_3646482, 1:200 dilution; R&D Systems); HLA-DR BUV496 (clone L243, 753685, 1:5,000 dilution; BD Biosciences); CD56 BUV737 (clone TULY56, RRID:AB_2895975, 1:400 dilution; Thermo Fisher Scientific); CD11c eFluor 450 (clone 3.9, AB_11218498; Thermo Fisher Scientific, 1:400 dilution); CD123 BV510 (clone 6H6, AB_2562068, 1:800 dilution; BioLegend); CD16 BV570 (clone 3G8, AB_10915988, 1:200 dilution; BioLegend); BDCA-2 BV785 (clone 201A, AB_2572146, 1:800 dilution; BioLegend); CD14 Spark Blue 550 (clone 63D3, AB_2832724, 1:60,000 dilution; BioLegend); CD3 NovaFluor B610-70S (clone SK7, AB_3098363, 1:2,000 dilution; Thermo Fisher Scientific); CD1c PerCP-eFluor 710 (clone L161, AB_10545854, 1:2,000 dilution; Thermo Fisher Scientific); TLR7 PE (clone S18024F, AB_2910431, 1:400 dilution; BioLegend); TLR8 APC (clone S16018A, AB_2801050, 1:400 dilution; BioLegend); TLR9 BV421 (clone S16013D, AB_2801039, 1:800 dilution; BioLegend); and CD19 APC-Fire810 (clone HIB19, AB_2860770, 1:200 dilution; BioLegend), in the presence of Human TruStain FcX (AB_2818986; BioLegend), True-Stain Monocyte Blocker (Cat. 426102; BioLegend), and CellBlox Plus (C001T06F01; Thermo Fisher Scientific) to minimize nonspecific binding.

Techniques: Flow Cytometry, Produced, Expressing, MANN-WHITNEY, Fluorescence

Journal: Nature communications

Article Title: Immunotherapy that improves response to chemotherapy in high-grade serous ovarian cancer.

doi: 10.1038/s41467-024-54295-x

Figure Lengend Snippet: Fig. 3 | Human CD8 T cell subpopulations in HGSOC omental metastasis. A UMAP showing integrated CD8 cell clusters from the above seven patients. B Heatmap showing top upregulated genes per cluster. C Differentially expressed genes in CD8 T cells NACT versus PDS. Wilcoxon rank sum test. D Differentially enriched pathways in NACT versus PDS in CD8 cells. E Percentage of CD8 cells of total CD45 cells in 31 human omental samples (n = 8 PDS, n = 23 NACT) using flow

Article Snippet: Then the viable cell suspension was enriched for CD45+ cells using CD45 Microbeads (Miltenyi biotec, CD45 (TIL) microbeads, Cat. number: 130-118-780).

Techniques: