cl097 tlr7 agonist  (InvivoGen)

Journal: The Journal of Clinical Investigation

Article Title: Divergent TIR signaling domains in TLR7 and TLR9 control opposing effects on systemic autoimmunity

doi: 10.1172/JCI189566

Figure Lengend Snippet: ( A ) Amino-acid sequences of the transmembrane-TIR junctions. ( B ) surface and intracellular staining of TLR7 in CD23 hi CD21 lo FO, CD23 lo CD21 hi MZ, CD11c + CD11b + ABCs B cells of 5-week-old Tlr7 +/Y or Tlr 779/Y (all Tlr 9 –/– ) male MRL/lpr mice. Data were analyzed by Wilcoxon matched-pairs signed rank test. ( C ) TLR7 endosomal localization was evaluated by confocal microscopy in sorted splenic MZ B cells and in ABCs of 6–7week old female Tlr7 +/+ and Tlr 779/779 MRL/lpr mice. Left and right panels show representative 1,000x magnification images and 3D reconstructed images of TLR7+ endosomes. Colored spheres indicate EEA1, LAMP-1, and TLR7 spot counting. The percentage of TLR7 + EEA1 + or LAMP-1 + endosomes in Tlr7 +/+ or Tlr 779/779 MZ B cells or ABCs (EEA1 staining, MZ B cells, Tlr7 +/+ n = 10 cells, Tlr 779/779 n = 13 cells; LAMP-1 staining, MZ B cells Tlr7 +/+ n = 20 cells, Tlr 779/779 n = 11 cells; ABC Tlr7 +/+ n = 15 cells, Tlr 779/779 n = 14 cells; from 2 mice).The mean volume of the reconstructed TLR7 spots (MZ B cells, Tlr7 +/+ n = 30 cells, Tlr 779/779 n = 24 cells; ABC B cells, Tlr7 +/+ n = 15 cells, Tlr 779/779 n = 14 cells, from 2 mice) Data were analyzed by 1-way ANOVA with Sidak’s multiple comparisons test. ( D ) Splenocytes from 5–6-week-old Tlr7 +/Y Tlr9 –/– or Tlr 779/Y Tlr9 –/– male MRL/lpr mice were stimulated with different doses of TLR7 agonist (CL097; μg/ml) for 120 min. Quantification of the NF-κB nuclear localization score in FO CD21 int or MZ CD21 hi B cells (upper and lower panels). Data points indicate the mean score quantified for n = 6 mice per genotype and bars indicate the SEM of 2 experiments pooled. Data were analyzed by multiple paired t test. For statistics, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Bead-purified B cells (4–5 million per conditions) were resuspended in B cell media (RPMI 1640 with 10% Fetalplex, Glutamax, Penicillin/Streptomycin, HEPES and 50M of 2-mercaptoethanol) at 10 x 10 6 cells/ml, warmed at 37°C for 45 minutes and stimulated for 4 hours with TLR7 agonist CL097 (Invivogen) at 5 μg/ml or TLR9 agonist CpG at 10 μg/ml or left unstimulated as indicated in the figure legends.

Techniques: Staining, Confocal Microscopy

Journal: The Journal of Clinical Investigation

Article Title: Divergent TIR signaling domains in TLR7 and TLR9 control opposing effects on systemic autoimmunity

doi: 10.1172/JCI189566

Figure Lengend Snippet: Transcriptome analysis of bead-purified B cells from 5–7week-old Tlr7 +/Y Tlr9 –/– or Tlr 779/Y Tlr9 –/– male MRL/lpr mice that were stimulated with TLR7 agonist (CL097; 5 μg/ml) for 4 hours. ( A ) Number of differentially expressed genes (DEGs) identified using the limma R package (log 2 FC > 0.5 and FDR-corrected P < 0.05), between stimulated versus unstimulated samples (first 2 columns) or between the 2 genotypes, with or without stimulation (last 2 columns). ( B ) Upstream regulators that are predicted to be significantly activated upon CL097 stimulation by the IPA software in Tlr7 +/Y (Y-axis) or Tlr 779/Y (X-axis) B cells. XY plot shows the predicted z-score. ( C ) Bubble plot shows the top 15 reactome pathways enriched in Tlr 779/Y (stim) (pink bubbles) and Tlr7 +/Y (stim) (dark blue bubbles) regulated genes (from the Tlr 779/Y (stim) vs Tlr7 +/Y (stim) comparison). X-axis shows the –log 10 FDR for the enriched terms displayed on Y-axis. Bubble size shows the genes in the pathway that are also differentially expressed in Tlr 779/Y versus Tlr7 +/Y . Enrichment was calculated using Fisher’s exact test (with all expressed genes as background) followed by Storey’s Q value FDR correction. ( D ) Volcano plot representing the DEGs between Tlr7 +/Y and Tlr 779/Y stimulated B cells. X-axis shows the log 2 fold change value and Y-axis shows –log10 (FDR). The dotted lines separate the significant (FDR < 0.05) and nonsignificant (FDR > 0.05) genes and indicate the log 2 FC –0.5 and 0.5 cut-offs. The significant DEGs (log 2 FC > 0.5, and FDR-corrected P value of < 0.05) were annotated based on the reported functions of their corresponding proteins in B cell activation (green dots), cell death (yellow dots), TLR-mediated inflammation (red dots), negative regulation of TLR-mediated inflammation and cell activation (blue dots), or if the genes were IFN-induced genes (pink dots).

Article Snippet: Bead-purified B cells (4–5 million per conditions) were resuspended in B cell media (RPMI 1640 with 10% Fetalplex, Glutamax, Penicillin/Streptomycin, HEPES and 50M of 2-mercaptoethanol) at 10 x 10 6 cells/ml, warmed at 37°C for 45 minutes and stimulated for 4 hours with TLR7 agonist CL097 (Invivogen) at 5 μg/ml or TLR9 agonist CpG at 10 μg/ml or left unstimulated as indicated in the figure legends.

Techniques: Purification, Software, Comparison, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: Divergent TIR signaling domains in TLR7 and TLR9 control opposing effects on systemic autoimmunity

doi: 10.1172/JCI189566

Figure Lengend Snippet: 16–18-week-old Tlr7 +/+ , Tlr 779/779 or Tlr7 –/– (all Tlr9 –/– ) MRL/lpr mice were assessed for disease endpoints. Disease endpoints were also assessed in age-matched WT Tlr7 +/+ and Tlr9 +/+ MRL/lpr mice as a reference but were not included in the statistical analysis ( B – E ). ( A ) Schematic of the different mouse genotypes that are compared. The groups were labeled Tlr7 +/+ , Tlr 779/779 or Tlr7 –/– if both males and females were included. ( B and C ) Spleen and lymph node weights were measured in mice of the indicated sex and genotypes. ( D ) Kidney pathology was assessed in mice of the indicated sex and genotypes. For B–D , reference group female n = 12, male n = 11; experimental group female Tlr7 +/+ n = 19, Tlr 779/779 n = 21, Tlr7 –/– n = 25; male Tlr7 +/Y n = 20, Tlr 779/Y n = 24, Tlr7 –/Y n = 14. ( E ) Quantification of anti-RNA (reference group n = 9; experimental group Tlr7 +/+ n = 6, Tlr 779/779 n = 13, all females) and anti-Smith autoantibodies (reference group n = 18; experimental group Tlr7 +/+ n = 16, Tlr 779/779 n = 19, males and females). ( F – H ) Splenic B and T cell subsets were assessed by flow-cytometry in MRL/lpr mice of the indicated genotypes. ( F ) Percent of CD19 + cells among live splenocytes, and CD11b + CD11c + ABCs among live B cells (CD19 + : Tlr7 +/+ n = 14, Tlr 779/779 n = 25, Tlr7 –/– n = 24; ABC: Tlr7 +/+ n = 12, Tlr 779/779 n = 17, Tlr 7 –/– n = 24). ( G ) Percent of TCR – CD44 hi CD138 + plasmablasts among live splenocytes in mice of the indicated genotypes ( Tlr7 +/+ n = 9, Tlr 779/779 n = 25, Tlr7 –/– n = 24). ( H ) Percent of naive (CD62L hi CD44 lo ), activated (CD62L hi CD44 hi ), and memory (CD62L lo CD44 hi ) T cells among live TCR + CD4 + splenocytes ( Tlr7 +/+ n = 14, Tlr 779/779 n = 25, Tlr7 –/– n = 24). For all panels, data points indicate individual mice and bars indicate the mean ± SEM For statistics, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, using a 1-way ANOVA with Tukey’s multiple comparisons test for all panels except E ; Mann-Whitney test for panel E .

Article Snippet: Bead-purified B cells (4–5 million per conditions) were resuspended in B cell media (RPMI 1640 with 10% Fetalplex, Glutamax, Penicillin/Streptomycin, HEPES and 50M of 2-mercaptoethanol) at 10 x 10 6 cells/ml, warmed at 37°C for 45 minutes and stimulated for 4 hours with TLR7 agonist CL097 (Invivogen) at 5 μg/ml or TLR9 agonist CpG at 10 μg/ml or left unstimulated as indicated in the figure legends.

Techniques: Labeling, Flow Cytometry, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Divergent TIR signaling domains in TLR7 and TLR9 control opposing effects on systemic autoimmunity

doi: 10.1172/JCI189566

Figure Lengend Snippet: ( A ) Amino-acid sequences of the transmembrane-TIR junctions of TLR999, TLR777, and the TLR997 mutant. The TLR molecules are described based on the source of their 3 functional domains: endosomal domain–transmembrane domain–signaling TIR domain. ( B ) intracellular staining of TLR9 in FO, MZ, CD11c + CD11b + ABCs, CD11b + , and CD11c + B cells of 5–7-week-old Tlr9 +/+ , Tlr9 +/– , or Tlr 997 (all Tlr7 –/Y ) male MRL/lpr mice. Data points indicate individual mice ( n = 6 per genotype and bars indicate the mean ± SEM of 2 pooled experiments, except for Tlr9 +/+ , n = 2 mice). ( C ) TLR9 endosomal localization in flow-sorted splenic CD21 hi MZ (left column) and CD11c + CD11b + ABC (right column) B cells of 6–7 week old Tlr9 +/– or Tlr 997 (all Tlr7 –/– ) female MRL/lpr mice was evaluated by confocal microscopy. Images were acquired at 1,000x magnification. Representative images of TLR9 + endosomes were made with 3D reconstruction (left panels). Colored spheres indicate LAMP1 and TLR9 spot counting generated from confocal images (right panels). The percentage of TLR9 + LAMP-1 + endosomes and the mean volume of the reconstructed TLR9 spots were measured using the Imaris software in Tlr9 +/– or Tlr 997 MZ and ABC B cells. (MZ B cells, Tlr9 +/– n = 12 cells, Tlr 997 n = 10 cells; ABC B cells, Tlr9 +/– n = 20 cells, Tlr 997 n = 11 cells, from 2 mice per genotype). ( D ) Splenocytes from 5–7-week-old Tlr9 +/– Tlr7 –/Y or Tlr 997 Tlr7 –/Y male MRL/lpr mice were stimulated with different doses of TLR9 agonist (CpG, μg/ml) for 120 min. Quantification of the NF-κB nuclear localization score in FO CD21 int or MZ CD21 hi B cells (upper and lower panels). Data points indicate the mean score quantified for n = 6 mice per genotype and bars indicate the SEM of 2 experiments pooled. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data between the 2 genotypes were analyzed by multiple paired t test.

Article Snippet: Bead-purified B cells (4–5 million per conditions) were resuspended in B cell media (RPMI 1640 with 10% Fetalplex, Glutamax, Penicillin/Streptomycin, HEPES and 50M of 2-mercaptoethanol) at 10 x 10 6 cells/ml, warmed at 37°C for 45 minutes and stimulated for 4 hours with TLR7 agonist CL097 (Invivogen) at 5 μg/ml or TLR9 agonist CpG at 10 μg/ml or left unstimulated as indicated in the figure legends.

Techniques: Mutagenesis, Functional Assay, Staining, Confocal Microscopy, Generated, Software

Journal: The Journal of Clinical Investigation

Article Title: Divergent TIR signaling domains in TLR7 and TLR9 control opposing effects on systemic autoimmunity

doi: 10.1172/JCI189566

Figure Lengend Snippet: Transcriptome analysis of bead-purified B cells from 5-week-old Tlr9 +/– Tlr7 –/– or Tlr 997 Tlr7 –/– female MRL/lpr mice that were stimulated with TLR9 agonist (CpG, 10 μg/ml) for 4 hours. ( A ) Number of differentially expressed genes (DEGs) identified using the limma R package (log 2 FC > 0.5, and FDR-corrected P value of < 0.05). ( B ) Bubble plots show the top 15 reactome pathways enriched in Tlr 997 (purple bubbles) and Tlr9 +/– (salmon bubbles) regulated genes from the Tlr 997 (stim) vs Tlr9 +/– (stim) comparison. Bubble size reflects the number of genes in the pathway that are also differentially expressed in Tlr 997 vs Tlr9 +/– . Enrichment was calculated using Fisher’s exact test (with all expressed genes as background) followed by Storey’s Q value FDR correction. ( C ) Volcano plot representing the DEGs between Tlr 997 and Tlr9 +/– CpG-stimulated B cells. The significant DEGs (log 2 FC > 0.5) were annotated based on the reported functions of their corresponding proteins in B cell activation (green dots), cell death (yellow dots), TLR-mediated inflammation (red dots), negative regulation of TLR-mediated inflammation and/or B cell activation (blue dots), or if the genes were IFN-induced genes (pink dots). ( D ) Diagram depicting how proteins encoded by the curated DEGs in C could promote or regulate NF-κB, IRF, MAPK, IFN type 1 or 2 signaling pathways. In the cartoons of the chimeric TLR, TLR9-driven domains are in red and TLR7-derived domain in blue. ( E ) A TLR9-induced gene set signature for B cells of Tlr9 +/+ BALB/c mice was generated. It comprises 1,724 upregulated genes (log 2 FC > 1 and FDR P value < 0.05) after a 4-hour in vitro CpG stimulation compared with unstimulated cells. Enrichment of this TLR9-induced gene set was assessed before and after TLR9 stimulation in Tlr9 +/– B cells (left panel) and in Tlr 997 (middle panel) B cells and between Tlr9 +/– and Tlr 997 stimulated B cells (right panel). The P value was calculated using the rankSumTestWithCorrelation function from the R limma package.

Article Snippet: Bead-purified B cells (4–5 million per conditions) were resuspended in B cell media (RPMI 1640 with 10% Fetalplex, Glutamax, Penicillin/Streptomycin, HEPES and 50M of 2-mercaptoethanol) at 10 x 10 6 cells/ml, warmed at 37°C for 45 minutes and stimulated for 4 hours with TLR7 agonist CL097 (Invivogen) at 5 μg/ml or TLR9 agonist CpG at 10 μg/ml or left unstimulated as indicated in the figure legends.

Techniques: Purification, Comparison, Activation Assay, Protein-Protein interactions, Derivative Assay, Generated, In Vitro

Journal: The Journal of Clinical Investigation

Article Title: Divergent TIR signaling domains in TLR7 and TLR9 control opposing effects on systemic autoimmunity

doi: 10.1172/JCI189566

Figure Lengend Snippet: B cells from 5–7-week-old Tlr9 +/– or Tlr 997 (all Tlr7 –/– ) MRL/lpr mice were labeled with violet proliferation dye (VPD) and cultured for 1, 2, or 3 days with CpG (1 μg/ml). ( A ) Representative flow cytometry plots gated on live B cells. ( B ) Quantification of BLIMP1 hi CD138 + plasmablasts (PB) among live B cells. One-way ANOVA with Sidak’s multiple comparisons test was used to compare both genotypes. ( C ) BLIMP1 MFI in live B cells. Symbols indicate individual mice and error bars represent SEM. An unpaired t test was used to compare both genotypes at day 1. ( D ) Percentage of live-dead dye positive (LD + ) and LD + VPD lo cells (which correspond to post-proliferative dead cells). For panels E – H , due to batch effects that led to differences in the overall B cell proliferation profiles, results from experiments 1 and 2 (shown in E – H ) and experiments 3 and 4 (shown in ) were analyzed separately. ( E ) Percentage of live B cells that divided. ( F ) The FlowJo Proliferation Platform analysis was used to determine the division index. ( G ) Cell divisions were gated based on each proliferation peak of live B cells. Division 0 corresponds to undivided cells. Y-axis shows the percentage of total live B cells within each division number at day 3 . ( H ) The percentage of PB for each division number was plotted. For panels E – H , symbols indicate mean and error bars are the SEM from n = 4 mice per genotype derived from 2 experiments. For E and F , 1-way ANOVA with Sidak’s multiple comparisons test was used. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Bead-purified B cells (4–5 million per conditions) were resuspended in B cell media (RPMI 1640 with 10% Fetalplex, Glutamax, Penicillin/Streptomycin, HEPES and 50M of 2-mercaptoethanol) at 10 x 10 6 cells/ml, warmed at 37°C for 45 minutes and stimulated for 4 hours with TLR7 agonist CL097 (Invivogen) at 5 μg/ml or TLR9 agonist CpG at 10 μg/ml or left unstimulated as indicated in the figure legends.

Techniques: Labeling, Cell Culture, Flow Cytometry, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Divergent TIR signaling domains in TLR7 and TLR9 control opposing effects on systemic autoimmunity

doi: 10.1172/JCI189566

Figure Lengend Snippet: 18–21-week-old Tlr9 +/+ , Tlr9 +/– or Tlr 997/997 (referred to as Tlr 997 ) (all Tlr7 –/– ) MRL/lpr mice were assessed for disease endpoints. Disease endpoints were also assessed in 18–20-week-old WT Tlr7 +/+ and Tlr9 +/+ MRL/lpr mice as a reference but were not included in the statistical analysis ( B and C ). ( A ) Schematic of the different mouse genotypes that are compared. ( B ) Spleen weights were measured in mice of the indicated gender and genotypes. ( C ) Kidney pathology was assessed in mice of the indicated gender and genotypes. (For B and C , female Tlr9 +/+ n = 17, Tlr9 +/– n = 28, Tlr 997 n = 22, Tlr9 +/+ Tlr7 +/+ n = 8; male Tlr9 +/+ n = 16, Tlr9 +/– n = 24, Tlr 997 n = 11, Tlr9 +/+ Tlr7 +/Y n = 2 or 3) ( D ) Splenic B cell subsets were assessed by flow cytometry in mice of the indicated genotypes. Percent of CD19 + cells among live splenocytes, and percent of CD23 lo CD21 hi MZ, CD11b + CD11c + ABCs and CD11b + cells among live B cells ( Tlr9 +/+ n = 26, Tlr9 +/– n = 36, Tlr 997 n = 25). For all panels, data points indicate individual mice and bars indicate the mean ± SEM. For statistics, * P < 0.05, ** P < 0.01, **** P < 0.001, **** P < 0.0001, 1-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Bead-purified B cells (4–5 million per conditions) were resuspended in B cell media (RPMI 1640 with 10% Fetalplex, Glutamax, Penicillin/Streptomycin, HEPES and 50M of 2-mercaptoethanol) at 10 x 10 6 cells/ml, warmed at 37°C for 45 minutes and stimulated for 4 hours with TLR7 agonist CL097 (Invivogen) at 5 μg/ml or TLR9 agonist CpG at 10 μg/ml or left unstimulated as indicated in the figure legends.

Techniques: Flow Cytometry

Journal: Pharmacological research

Article Title: The host cell factor DDX3 mediates sex dimorphism in the IFNα response of plasmacytoid dendritic cells upon TLR activation.

doi: 10.1016/j.phrs.2025.107764

Figure Lengend Snippet: Fig. 5. Transfection with recombinant DDX3X enhances the IFNα response without affecting TNF production upon TLR7 activation in pDCs. A. Schema of the transfection procedure created with BioRender.com. Isolated pDCs were transfected either with recombinant DDX3X or with TUBA1A via cell squeezing, rested for 4 hour, and stimulated with TLR7 agonist CL097 (1 µg/mL) in the presence of Brefeldin A. B. Mean Fluorescence Intensity (MFI) of DDX3 in pDCs after transfection with recombinant DDX3X or TUBA1A (control) are expressed as scatter plots of individual donors. C. MFI of IFNα and TNF in pDCs after transfection with re combinant DDX3X or TUBA1A (control) in the context of TLR7 activation (CL097 (1 µg/mL). D. Percentage of IFNα+ and TNF+ pDCs after transfection with re combinant DDX3X or TUBA1A (control) in the context of TLR7 activation (CL097 (1 µg/mL). Lines connect the corresponding conditions of one donor. Wilcoxon- signed rank test was used to assess statistical differences between the groups, ns = not significant ***p < 0.001, ****p ≤0.0001.

Article Snippet: For the stimulation experiments, 5 × 104 pDCs of each donor were treated with the indicated concentration of the DDX3 inhibitor RK-33 (stock 1 mM in DMSO, Biotrend Chemikalien GmbH, purity of powder >99 %) and stimulated either with 1 μg/mL TLR7/8 agonist CL097 (stock 1 mg/mL in water, InvivoGen) or with 1 μM TLR9 agonist CpG-A Fig. 3.

Techniques: Transfection, Recombinant, Activation Assay, Isolation, Fluorescence, Control

Journal: iScience

Article Title: Reduction of IFN-I responses by plasmacytoid dendritic cells in a longitudinal trans men cohort

doi: 10.1016/j.isci.2023.108209

Figure Lengend Snippet: TLR7/8 stimulation of PBMCs induced distinct transcriptomic changes in pDCs (A) Workflow of the scRNA-seq experiment, shown for one donor. Peripheral blood mononuclear cells (PBMCs) from time points before (TP0) and under (TP3) testosterone injections were isolated and stimulated with the TLR7/8 ligand CL097 for 2 h or left unstimulated before FACS-isolation of pDCs and subsequent scRNA-seq analysis (n = 3). Created with BioRender.com . (B) Heatmap depicting enrichment (AUC values) of immune cell subset marker gene sets (ref. ) across all pDC clusters. Both unstimulated and stimulated pDC clusters displayed high AUC values for pDC marker genes, confirming pDC identity. Stimulated pDC clusters exhibited high AUC values for the set of ISGs, indicating effects of CL097 stimulation. (C) Uniform manifold approximation and projection (UMAP) colored by condition. Unstimulated and stimulated pDCs from both assessed time points each form a separate group. (D) UMAP colored by unsupervised clusters determined using Louvain method after constructing a shared nearest neighbor (SNN) graph based on k-nearest neighbors. Five clusters were associated with unstimulated pDCs and six clusters with stimulated pDCs; cluster 11 was equally distributed between both groups. (E) Bar plots showing the percentage of cells in each pDC cluster at different stimulation conditions and time points. Cluster frequencies did not change significantly after testosterone administration (Wilcoxon signed-rank tests).

Article Snippet: Freshly isolated PBMCs (2.5 x 10 6 for cytokine production experiments by flow cytometry, 5 x 10 5 for cytokine quantification by ELISA and ISG qPCR) were stimulated with 1 μg/mL of the synthetic TLR7/8 agonist CL097 (catalog no. tlrl-c97, InvivoGen, San Diego, CA, USA) for 17 hours at 37°C, 5% CO 2 in RPMI Medium (catalog no. 21875-034, Thermo Fisher, Waltham, MA, USA) + 10% (v/v) FBS (Sigma Aldrich) + Penicillin-Streptomycin (catalog no. P4333-100ML, Sigma Aldrich).

Techniques: Isolation, Marker

Journal: iScience

Article Title: Reduction of IFN-I responses by plasmacytoid dendritic cells in a longitudinal trans men cohort

doi: 10.1016/j.isci.2023.108209

Figure Lengend Snippet: Heterogeneity of IFN-I-related transcriptomic responses upon testosterone injections in pDCs of trans men (A) Enrichment of genes associated with the GO term “IFN-I signaling pathway” visualized as AUC values on UMAP and violin plot. Upregulation of respective genes upon CL097 stimulation was significant (p = 2.2e-16, Wilcoxon signed-rank test). (B) Venn diagrams of upregulated genes upon CL097 stimulation at TP0 and TP3 in the three donors used for scRNA-seq analysis. Gene counts were largely overlapping between the two time points. (C) Volcano plots of differentially expressed genes (DEGs) for the three individual donors comparing TP3 to TP0 in TLR7/8-stimulated pDCs. IFN-I genes belonged to genes with the strongest differential expression, exhibiting downregulation in donor 1 and 2, and upregulation in donor 3 comparing TP0 to TP3. p value threshold: <0.05; Log2FC threshold: >0.25. (D) Top 5 GO terms associated with DEGs (TP3 CL097 vs. TP0 CL097) from each donor and enrichment of these GO terms across all donors, reflecting differential expression of IFN-I-related genes and inter-donor heterogeneity. (E) Heatmap displaying enrichment of gene sets (AUC values) associated with IFN-I responses, displayed for each donor.

Article Snippet: Freshly isolated PBMCs (2.5 x 10 6 for cytokine production experiments by flow cytometry, 5 x 10 5 for cytokine quantification by ELISA and ISG qPCR) were stimulated with 1 μg/mL of the synthetic TLR7/8 agonist CL097 (catalog no. tlrl-c97, InvivoGen, San Diego, CA, USA) for 17 hours at 37°C, 5% CO 2 in RPMI Medium (catalog no. 21875-034, Thermo Fisher, Waltham, MA, USA) + 10% (v/v) FBS (Sigma Aldrich) + Penicillin-Streptomycin (catalog no. P4333-100ML, Sigma Aldrich).

Techniques: Expressing

Journal: iScience

Article Title: Reduction of IFN-I responses by plasmacytoid dendritic cells in a longitudinal trans men cohort

doi: 10.1016/j.isci.2023.108209

Figure Lengend Snippet: IFN-I production by pDCs decreased under testosterone injections (A) Frequency of IFNα2+ pDCs over time under testosterone injections of one donor from the trans men cohort. (B) Upon TLR7/8 stimulation of PBMCs, frequencies of IFNα2-producing pDCs in n = 6 trans men decreased significantly over time under testosterone administration (R 2 = 0.76, p = 0.0009). Statistical significance was assessed by extra sum-of-squares F test comparing a linear regression model with a shared slope and individual intercepts to a respective model with a hypothetical slope of 0. (C) IFNα+ pDC frequencies upon TLR7/8 stimulation of PBMCs in an age-matched control cohort of cis women (n = 21) and cis men (n = 17) and in the trans men cohort, displayed for each time point. Cis women controls vs. cis men controls: p = 0.003; cis women controls vs. trans men TP3: p = 0.007; Mann-Whitney test, mean with SD displayed as error bars. ∗p < 0.05, ∗∗p < 0.01. (D) IFNα and (E) IFNβ levels in supernatant of TLR7/8-stimulated PBMCs showed a decreasing trend over time under testosterone injections (IFNα and IFNβ: p = 0.073, two-way ANOVA followed by Fisher’s least significant difference test, n = 5). Blue dots represent CL097 stimulation, and green dots represent the unstimulated condition. Mean with SD indicated as error bars.

Article Snippet: Freshly isolated PBMCs (2.5 x 10 6 for cytokine production experiments by flow cytometry, 5 x 10 5 for cytokine quantification by ELISA and ISG qPCR) were stimulated with 1 μg/mL of the synthetic TLR7/8 agonist CL097 (catalog no. tlrl-c97, InvivoGen, San Diego, CA, USA) for 17 hours at 37°C, 5% CO 2 in RPMI Medium (catalog no. 21875-034, Thermo Fisher, Waltham, MA, USA) + 10% (v/v) FBS (Sigma Aldrich) + Penicillin-Streptomycin (catalog no. P4333-100ML, Sigma Aldrich).

Techniques: Control, MANN-WHITNEY

Journal: iScience

Article Title: Reduction of IFN-I responses by plasmacytoid dendritic cells in a longitudinal trans men cohort

doi: 10.1016/j.isci.2023.108209

Figure Lengend Snippet: Expression of interferon-stimulated genes in PBMCs is reduced under testosterone administration (A) Relative interferon-stimulated gene (ISG) expression levels of PBMCs upon TLR7/8 stimulation (blue) and without stimulation (green). CL097 stimulation induced significant upregulation of ISG expression (TP0: p < 0.0001, TP3: p = 0.0027, two-way ANOVA followed by Šidák multiple comparison test, n = 5). TLR7/8-mediated ISG expression was significantly reduced at TP3 compared to TP0 (p = 0.001, two-way ANOVA followed by Šidák multiple comparison test). Mean with SD indicated as error bars. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (B) Heatmap of mean relative expression values of 15 ISGs from TP0 and TP3, depicted from the same dataset used in (A). ISG expression was generally reduced under testosterone injections. (C) Linear regression of relative ISG expression values upon CL097 stimulation of PBMCs from TP0 and TP3 (R 2 = 0.75, Slope = 0.6).

Article Snippet: Freshly isolated PBMCs (2.5 x 10 6 for cytokine production experiments by flow cytometry, 5 x 10 5 for cytokine quantification by ELISA and ISG qPCR) were stimulated with 1 μg/mL of the synthetic TLR7/8 agonist CL097 (catalog no. tlrl-c97, InvivoGen, San Diego, CA, USA) for 17 hours at 37°C, 5% CO 2 in RPMI Medium (catalog no. 21875-034, Thermo Fisher, Waltham, MA, USA) + 10% (v/v) FBS (Sigma Aldrich) + Penicillin-Streptomycin (catalog no. P4333-100ML, Sigma Aldrich).

Techniques: Expressing, Comparison

Journal: iScience

Article Title: Reduction of IFN-I responses by plasmacytoid dendritic cells in a longitudinal trans men cohort

doi: 10.1016/j.isci.2023.108209

Figure Lengend Snippet:

Article Snippet: Freshly isolated PBMCs (2.5 x 10 6 for cytokine production experiments by flow cytometry, 5 x 10 5 for cytokine quantification by ELISA and ISG qPCR) were stimulated with 1 μg/mL of the synthetic TLR7/8 agonist CL097 (catalog no. tlrl-c97, InvivoGen, San Diego, CA, USA) for 17 hours at 37°C, 5% CO 2 in RPMI Medium (catalog no. 21875-034, Thermo Fisher, Waltham, MA, USA) + 10% (v/v) FBS (Sigma Aldrich) + Penicillin-Streptomycin (catalog no. P4333-100ML, Sigma Aldrich).

Techniques: Control, Recombinant, Enzyme-linked Immunosorbent Assay, Software