Journal: PLoS ONE

Article Title: Recruitment of Cbl-b to B Cell Antigen Receptor Couples Antigen Recognition to Toll-Like Receptor 9 Activation in Late Endosomes

doi: 10.1371/journal.pone.0089792

Figure Lengend Snippet: (A) Representative confocal microscopic images of splenocytes from mice with indicated genotypes. For experiments, cells were stimulated through the BCR (green) for 30 minutes then fixed and stained for TLR9 (blue) and Lamp-1 (red)(n = 3). (B) Quantitation of experiments shown in (A) for fraction of cells demonstrating significant co-localization of TLR9 with Lamp-1 (n = 3, 30 cells/exp) (Cbl-b −/− vs. Cblb C373A or WT, p<0.001). (C) Quantitation of fractions of cells in (A) demonstrating significant co-localization between BCR and TLR9 (n = 3, 30 cells/exp). (D) In vitro assay of T-bet induction in response to ODN 1826 or control ODN targeted through the BCR (n = 3, p<0.01).

Article Snippet: Antibodies used for visualization were Lamp-1 (1D4B) (BD Biosciences), TLR9 (26C593.2) (Imgenex), Cathepsin L (CPLH-3G10) and Cbl-b (H-121 and H-454) (Santa Cruz Biotechnology).

Techniques: Staining, Quantitation Assay, In Vitro

Journal: Genetic Vaccines and Therapy

Article Title: A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

doi: 10.1186/1479-0556-6-3

Figure Lengend Snippet: Phenotypic characterization of monocyte-derived macrophages and DCs . (A) Expression of markers CD11b (Mac-1), CD86 and HLA-DR on the surface of macrophages, and (B) CD11c, CD86 and HLA-DR on the surface of DCs was evaluated by flow cytometry (all markers are indicated by solid lines). Dotted-line histograms indicate isotype control mAb. These results are representative of seven independent experiments. (C) Expression of CD1c, CD123 (IL-3 receptor) and BDCA-4 on the surface of DCs. (D) IFN-alpha production by monocyte-derived DC (mo-DC) and plasmacytoid DC (pDC). These results are representative of three independent experiments. (E) Intracellular expression of TLR9 by macrophages and DCs analysed by confocal microscopy.

Article Snippet: The purified mAb TLR9 (26C593 clone) was obtained from Imgenex (San Diego, CA, USA), and the biotinylated anti-mouse IgG was obtained from Bioscience (Toronto, Canada).

Techniques: Derivative Assay, Expressing, Flow Cytometry, Confocal Microscopy

Journal: The Journal of Clinical Investigation

Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

doi: 10.1172/JCI127542

Figure Lengend Snippet: WT and Tlr9–/– mice were subjected to CLP. PLF was collected at 18 hours after CLP. (A) Percentages of indicated immune cells were measured by flow cytometry. (B) TLR9 expression in indicated peritoneal cells. MFI for TLR9 expression was measured by flow cytometry. (C–G) Peritoneal cell counts of (C) B cells, (D) macrophages, (E) DCs, (F) neutrophils, and (G) T cells in control and after CLP. Data are shown as mean ± SD from 2 separate experiments. Symbols represent individual mice. *P < 0.05; **P < 0.01; ***P < 0.001, unpaired, 2-tailed Student’s t tests.

Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

Techniques: Flow Cytometry, Expressing

Journal: The Journal of Clinical Investigation

Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

doi: 10.1172/JCI127542

Figure Lengend Snippet: (A–D) WT and Tlr9–/– mice were subjected to CLP. Plasma and PLF were collected at 18 hours after CLP. (A) Peritoneal B-1 cell numbers. (B) Plasma IgM levels. (C) Peritoneal IgM levels. (D) Peritoneal GM-CSF levels. (E–G) Tlr9–/– mice were treated with CD19 neutralizing antibodies (10 mg/mouse) or control IgG (10 mg/mouse) at 24 hours before CLP. PLF was collected at 18 hours after CLP. (E) Seven-day survival after CLP. Data are from 2 separate experiments. n = 10. *P < 0.05, log-rank test. (F) Bacterial clearance in PLF. Data are from 2 separate experiments. Symbols represent individual mice. *P < 0.05, nonparametric Mann-Whitney U test. (G) Peritoneal IgM levels. For A–D and G, data are shown as mean ± SD from 2 separate experiments. Symbols represent individual mice. *P < 0.05; **P < 0.01; ***P < 0.001, unpaired, 2-tailed Student’s t test.

Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

Techniques: MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

doi: 10.1172/JCI127542

Figure Lengend Snippet: (A) WT and Tlr9–/– mice were subjected to CLP. PLF was collected at 18 hours after CLP. Peritoneal CXCL13 levels were assessed using ELISA. (B–F) WT and Tlr9–/– mice were treated with CXCL13 neutralizing antibodies (10 mg/mouse) or control IgG (10 mg/mouse) immediately after CLP. PLF and plasma were collected at 18 hours after CLP. (B) Peritoneal B-1 cell numbers. (C) Peritoneal IgM levels. (D) Bacterial load in PLF. (E) Plasma IL-6 levels. (F) Seven-day survival. For A–C and E, data are shown as mean ± SD. Symbols represent individual mice. *P < 0.05; **P < 0.01, unpaired, 2-tailed Student’s t tests. For D, symbols represent individual mice. *P < 0.05, nonparametric Mann-Whitney U test. For F, n = 13–19/group as indicated. *P < 0.05 versus Tlr9–/– IgG, log-rank test. (G–K) WT mice were treated with recombinant CXCL13 (10 mg/mouse) or PBS immediately after CLP. (G) Peritoneal B-1 cell number. (H) Peritoneal IgM levels. (I) Bacterial load in PLF. (J) Plasma IL-6 levels. (K) Seven-day survival. For G, H, and J, data are shown as mean ± SD. Symbols represent individual mice. *P < 0.05, unpaired, 2-tailed Student’s t test. For I, symbols represent individual mice. *P < 0.05, nonparametric Mann-Whitney U test. For K, n = 10/group. *P < 0.05, log-rank test.

Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Recombinant

Journal: The Journal of Clinical Investigation

Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

doi: 10.1172/JCI127542

Figure Lengend Snippet: (A–C) WT and Tlr9–/– mice were subjected to CLP. PLF and mesenteric adipose tissue were collected at18 hours after CLP. (A and B) Cxcl13 expression in peritoneal cells (A) and mesenteric adipose tissue (B) were assessed using quantitative PCR. Data are shown as mean ± SD from 2 separate experiments. Symbols represent individual mice. *P < 0.05, 2-tailed Student’s t test. (C) Immunofluorescence staining of CXCL13 (red), CD45 (green), CD11c (white), and nucleus (blue) in mesenteric adipose tissue. Scale bar: 5 μm. (D and E) Cxcl13 expression in mouse FRCs. WT and Tlr9–/– FRCs were isolated from mesenteric adipose tissues and expanded ex vivo. FRCs were stimulated with indicated TLR ligands (ODN1585, 5 μM; ODN1826, 5 μM; ODN2395, 5 μM, LPS, 1 μg/mL; PIC, 20 μg/mL) for 18 hours. Cxcl13 expression was assessed using quantitative PCR. Data are shown as mean ± SD from 1 representative experiment. Experiments were performed 3 times. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA with Bonferroni’s post hoc analysis.

Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Isolation, Ex Vivo

Journal: The Journal of Clinical Investigation

Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

doi: 10.1172/JCI127542

Figure Lengend Snippet: (A) Schematic timeline of experimental set and analysis for adoptive transfer studies. (B and C) Mice were subjected to CLP. WT or Tlr9–/– FRCs (2 × 105/ mouse) were injected i.p. at 1 hour after CLP. (B) Bacterial load in PLF at 18 hours after CLP. (C) Circulating IL-6 levels at 18 hours after CLP. Data are shown as mean ± SD from 2 separate experiments. *P < 0.05, unpaired, 2-tailed Student’s t test. (D and E) Seven-day survival. (D) Mice were subjected to CLP. PBS, WT, or Tlr9–/– FRCs (2 × 105/ mouse) were injected i.p. 1 hour after CLP. n = 13 in PBS group; n = 19 in WT FRC group; n = 22 in Tlr9–/– FRC group. (E) Mice were subjected to CLP. PBS, WT, or Tlr9–/– FRCs (2 × 105/ mouse) were injected i.p. at 12 hours after CLP. n = 10 in PBS group; n = 14 in WT FRC group; n = 15 in Tlr9–/– FRC group. *P < 0.05 vs. PBS, log-rank test. Data are from 3 separate experiments for D and 2 separate experiments for E. Statistical differences were determined using the log-rank test.

Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

Techniques: Adoptive Transfer Assay, Injection

Journal: The Journal of Clinical Investigation

Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

doi: 10.1172/JCI127542

Figure Lengend Snippet: Human FRCs were isolated from adipose tissue and expanded ex vivo. (A) TLR9 expression in human FRCs. TLR9 expression was assessed using flow cytometry. Numbers indicate percentage of FRCs (CD45–CD31–PDPN+). (B) Chemokine expression in human FRCs. FRCs were stimulated with indicated TLR ligands (ODN2216, 5 μM; LPS, 1 μg/mL; PIC, 20 μg/mL) for 18 hours. Indicated chemokine expression was assessed using quantitative PCR. Data are shown as mean ± SD from 1 representative experiment. Experiments were performed 3 times. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA with Bonferroni’s post hoc analysis.

Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

Techniques: Isolation, Ex Vivo, Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction

Journal: The Journal of Clinical Investigation

Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

doi: 10.1172/JCI127542

Figure Lengend Snippet: Activation of TLR9 signaling in FRCs suppresses the production of chemokines, which are essential for recruiting B cells, T cells, macrophages, and neutrophils into the peritoneal cavity as well as driving the formation of FALCs.

Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

Techniques: Activation Assay