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