Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Toll‐Like Receptor 9 Deficiency Breaks Tolerance to RNA‐Associated Antigens and Up‐Regulates Toll‐Like Receptor 7 Protein in Sle1 Mice

doi: 10.1002/art.40535

Figure Lengend Snippet: Regulation of antibody production by Toll‐like receptor 9 (TLR‐9) in Sle1 mice. A and B , B cell (B220+) activation ( A ) and proliferation ( B ) in splenocytes from young (8–10‐week‐old) Sle1 and Sle1 TLR‐9 −/− mice that were left untreated (media), stimulated with R848 (0.01 μg/ml), or stimulated with lipopolysaccharide (LPS; 1 μg/ml). B cell activation was measured by flow cytometry after 24 hours of stimulation and is shown as the percentage of CD69+ cells. B cell proliferation was measured according to the 5,6‐carboxyfluorescein succinimidyl ester dilution after 72 hours of stimulation. C , TLR‐7 expression, measured by intracellular flow cytometry, in mouse splenic B220+ B cells. TLR‐7–deficient Sle1 mice ( Sle1 TLR‐7 −/− ) (n = 4) and fluorescence minus one (FMO) samples were used as negative controls. MFI = median fluorescence intensity. D , Levels of IgG subtypes, measured by Luminex, in Sle1 and Sle1 TLR‐9 −/− mouse culture supernatants collected after 96 hours of incubation. Cultures were left untreated, stimulated with R848, or stimulated with LPS. E , Expression of surface IgG (sIgG) (IgG1/IgG2a/IgG2b/IgG3) on freshly isolated B220+CD19+ splenocytes from Sle1 and Sle1 TLR‐9 −/− mice. F and G , TLR‐7 expression and frequencies of splenic CD138+ plasma/plasmablasts ( F ) and CD11b+ dendritic cells (DCs) ( G ) in Sle1 and Sle1 TLR‐9 −/− mice. In A , B , and D , bars show the mean ± SEM from 2–3 independent experiments (n = 9–15 mice per group). Data were assessed by multiple t ‐tests, and statistical significance was corrected using the Holm‐Sidak method. In C , E , F , and G , data are from 1 representative experiment with 8–10‐week‐old mice (n = 8 Sle1 and 5 Sle1 TLR‐9 −/− mice). Circles represent individual mice; horizontal lines and error bars show the mean ± SEM. Significance was determined by Student's t ‐test. * = P < 0.05; ** = P < 0.01; *** = P < 0.001; **** = P < 0.0001.

Article Snippet: For intracellular staining with anti–TLR‐7, the BD Cytofix/Cytoperm fixation/permeabilization solution kit was used according to the recommendations of the manufacturer.

Techniques: Activation Assay, Flow Cytometry, Expressing, Fluorescence, Luminex, Incubation, Isolation

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Toll‐Like Receptor 9 Deficiency Breaks Tolerance to RNA‐Associated Antigens and Up‐Regulates Toll‐Like Receptor 7 Protein in Sle1 Mice

doi: 10.1002/art.40535

Figure Lengend Snippet: Infiltration of dendritic cells (DCs) with increased Toll‐like receptor 7 (TLR‐7) protein expression into Sle1 TLR‐9 −/− mouse kidneys. A , Analysis of renal CD45+CD11b+Gr‐1 −/low subsets using antibodies to CD11c, major histocompatibility complex class II (MHCII), F4/80, and CD11b. The magenta boxed area includes the CD11c+MHCII− precursor population. The black boxed area includes the CD11c+MHCII+ cells. CD11c+MHCII+ cells were further subdivided into F4/80+ macrophages (Mϕ; green encircled area) and F4/80 −/low DCs (blue encircled area). B , Cumulative frequencies of renal CD11b+Gr‐1− subsets identified in A in Sle1 and Sle1 TLR‐9 −/− mice. Bars show the mean ± SEM. Data are from 3 independent cohorts of 4.5–6.5‐month‐old mice with a total of 9–16 mice per group. C , Proliferation of CD4+TCRvα2+ Sle1 OT‐II cells exposed to ovalbumin‐pulsed F4/80+ macrophages or F4/80 −/low DCs, with or without prior stimulation with R848. Circles represent cells sorted from an individual mouse (n = 6), except for Sle1 F4/80 −/low DCs that were pooled to a total of 3 samples due to low cell numbers. D , Analysis of TLR‐7 protein expression, using flow cytometry, in the CD11b+Gr‐1− subsets identified in A in Sle1 and Sle1 TLR‐9 −/− mice. Symbols represent individual samples; horizontal lines and error bars show the mean ± SEM. E , Correlation of TLR‐7 expression in Sle1 and Sle1 TLR‐9 −/− mice with percentages of renal macrophage and DC infiltration. Data in D and E are from 3 independent experiments with a total of 8 mice (ages 5.7–6.4 months) per group. Each experiment was conducted with a TLR‐7 −/− and a fluorescence minus one (FMO) control (a total of 3 each). Parametric data were assessed by one‐way analysis of variance (with Bonferroni adjustment for multiple comparisons) or Student's t ‐test, and nonparametric data were assessed by Kruskal‐Wallis test (with Dunn's multiple comparison test) or Mann‐Whitney test. Correlations were determined using Spearman's rank correlation for nonparametric data and Pearson's correlation for parametric data. * = P < 0.05; ** = P < 0.01. NS = not significant; MFI = median fluorescence intensity.

Article Snippet: For intracellular staining with anti–TLR‐7, the BD Cytofix/Cytoperm fixation/permeabilization solution kit was used according to the recommendations of the manufacturer.

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

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Toll‐Like Receptor 9 Deficiency Breaks Tolerance to RNA‐Associated Antigens and Up‐Regulates Toll‐Like Receptor 7 Protein in Sle1 Mice

doi: 10.1002/art.40535

Figure Lengend Snippet: Schematic overview of the proposed mechanisms leading to severe disease in the absence of Toll‐like receptor 9 (TLR‐9). B cells from prediseased Sle1 TLR‐9 −/− mice are primed to produce more IgG‐switched antibodies, most likely due to the presence of increased numbers of CD11b+ dendritic cells (DCs) with increased TLR‐7 protein expression. In the absence of TLR‐9, the autoantibody repertoire shifts toward RNA/RNA‐associated antigens (Ags), which leads to the generation of RNA‐containing immune complexes (ICs). These complexes act as TLR‐7 ligands through Fc receptor (FcR) and B cell receptor (BCR)–mediated uptake and induce TLR‐7 protein expression through a positive feedback loop at the systemic and organ levels. A simultaneous increase in the levels of TLR‐7 and its ligands leads to ongoing inflammation and tissue destruction through various mechanisms, including T cell proliferation and inflammatory cytokine generation. Anti‐snRNP = anti–small nuclear RNP; pDCs = plasmacytoid DCs; PMNs = polymorphonuclear leukocytes; Mϕ = macrophages.

Article Snippet: For intracellular staining with anti–TLR‐7, the BD Cytofix/Cytoperm fixation/permeabilization solution kit was used according to the recommendations of the manufacturer.

Techniques: Expressing

Journal: Oncoimmunology

Article Title: Development of immune memory to glial brain tumors after tumor regression induced by immunotherapeutic Toll-like receptor 7/8 activation

doi: 10.4161/onci.19068

Figure Lengend Snippet: Figure 4. Lack of detection of TLR 7 and TLR 8 expression in both rat glioma CNS-1 cells and murine glioma GL261 cells. However, in rat spleen tissue, used as positive control, expression of both TLR7 and TLR8 can be detected.

Article Snippet: Oligonucleotide primers used for PCR amplification: Primers for the PCR amplification were obtained by Real Time Primers LLC, according to successful approach for TLR-7, or as customized primers for TLR-8 obtained from Real Time Primers LLC.

Techniques: Expressing, Positive Control