Journal: Infection and Immunity

Article Title: Fimbriated Salmonella enterica Serovar Typhimurium Abates Initial Inflammatory Responses by Macrophages

doi: 10.1128/iai.70.8.4273-4281.2002

Figure Lengend Snippet: FIG. 4. IL-12 is not stimulated following the infection of RAW 264.7 macrophages with Salmonella-CFA/I. RAW 264.7 cells were infected as described in the legend to Fig. 1 but at Salmonella-to-macrophage ratios between 0.1:1 and 100:1. Infected cells were cultured for 24 h, and supernatants were analyzed for the presence of IL-10 (A), IL-12 p40 (B), and IL-12 p70 (C) by ELISA. Data are the means SEM of values from four experiments. Statistical differences between cytokines induced by infection with the Salmonella vector versus the Salmonella-CFA/I strain are represented by (P 0.001) and (P 0.009).

Article Snippet: The following primary (coating) and secondary antibodies (Abs), respectively, were used in 50- l volumes: for IL-1 , 3.0 g of hamster anti-mouse IL-1 MAb (clone ALF-161; BD Pharmingen) per ml and 2.5 g of goat anti-mouse IL-1 (R&D Systems, Minneapolis, Minn.) per ml; for IL-1 , 4.0 g of rat anti-mouse IL-1 (clone 30311.11; R&D Systems) per ml and 0.2 g of biotinylated goat anti-IL-1 (R&D Systems) per ml; for TNF- , 10 g of rat antimouse TNF- (clone G281-2626; BD Pharmingen) per ml and 0.25 g of bio- tinylated rat anti-mouse TNF- (clone MP6-XT3; BD Pharmingen) per ml; for IL-6, 2.0 g of rat anti-mouse IL-6 (clone MP5-20F3; BD Pharmingen) per ml and 0.5 g of biotinylated rat anti-IL-6 MAb (clone MP5-32C11; BD Pharmingen) per ml; for IL-10, 2.0 g of rat anti-mouse IL-10 (clone JES5-2A5; BD Pharmingen) per ml and 0.3 g of biotinylated rat anti-IL-10 MAb (clone SXC-1; BD Pharmingen) per ml; for IL-12 p70, 2.0 g of rat anti-mouse IL-12 p70 (clone R2-9A5; American Type Culture Collection) per ml; and 1.0 g of biotinylated rat anti-mouse IL-12 p40 (clone C17.8; BD Pharmingen) per ml; and for IL-12 p40, 6.0 g of rat anti-mouse IL-12p40 (clone C15.6; BD Pharmingen) per ml and 1.0 g of biotinylated rat anti-mouse IL-12 p40 per ml.

Techniques: Infection, Cell Culture, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Infection and Immunity

Article Title: Fimbriated Salmonella enterica Serovar Typhimurium Abates Initial Inflammatory Responses by Macrophages

doi: 10.1128/iai.70.8.4273-4281.2002

Figure Lengend Snippet: FIG. 5. IL-12 is not stimulated following the infection of peritoneal macrophages with Salmonella-CFA/I. Macrophages were infected as described in the legend to Fig. 1 but with Salmonella-to-macrophage ratios between 0.1:1 and 100:1. Infected cells were cultured for 24 h, and supernatants were analyzed for the presence of IL-10 (A), IL-12 p40 (B), and IL-12 p70 (C) by ELISA. Data are the means SEM of values from four experiments. Statistical differences are represented by (P 0.001), (P 0.016), and (P 0.025).

Article Snippet: The following primary (coating) and secondary antibodies (Abs), respectively, were used in 50- l volumes: for IL-1 , 3.0 g of hamster anti-mouse IL-1 MAb (clone ALF-161; BD Pharmingen) per ml and 2.5 g of goat anti-mouse IL-1 (R&D Systems, Minneapolis, Minn.) per ml; for IL-1 , 4.0 g of rat anti-mouse IL-1 (clone 30311.11; R&D Systems) per ml and 0.2 g of biotinylated goat anti-IL-1 (R&D Systems) per ml; for TNF- , 10 g of rat antimouse TNF- (clone G281-2626; BD Pharmingen) per ml and 0.25 g of bio- tinylated rat anti-mouse TNF- (clone MP6-XT3; BD Pharmingen) per ml; for IL-6, 2.0 g of rat anti-mouse IL-6 (clone MP5-20F3; BD Pharmingen) per ml and 0.5 g of biotinylated rat anti-IL-6 MAb (clone MP5-32C11; BD Pharmingen) per ml; for IL-10, 2.0 g of rat anti-mouse IL-10 (clone JES5-2A5; BD Pharmingen) per ml and 0.3 g of biotinylated rat anti-IL-10 MAb (clone SXC-1; BD Pharmingen) per ml; for IL-12 p70, 2.0 g of rat anti-mouse IL-12 p70 (clone R2-9A5; American Type Culture Collection) per ml; and 1.0 g of biotinylated rat anti-mouse IL-12 p40 (clone C17.8; BD Pharmingen) per ml; and for IL-12 p40, 6.0 g of rat anti-mouse IL-12p40 (clone C15.6; BD Pharmingen) per ml and 1.0 g of biotinylated rat anti-mouse IL-12 p40 per ml.

Techniques: Infection, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Targeting Xcr1 on Dendritic Cells Rapidly Induce Th1-Associated Immune Responses That Contribute to Protection Against Influenza Infection

doi: 10.3389/fimmu.2022.752714

Figure Lengend Snippet: Xcl1-OVA induces Th1 polarization of DO11.10 cells in vitro and in vivo . (A) CD4 + cells were purified from spleen of DO11.10 TCR transgenic mice and incubated with BMDCs as APC in the presence of indicated amounts of Xcl1, fliC, or NIP-OVA protein for 72 h. Incorporation of radioactive thymidine was analyzed after 48 h. (B–D) CD4 + from DO11.10 transgenic mice were incubated with BMDC in the presence of Xcl1-, fliC-, or NIP-OVA proteins (0.5 μg/ml) for 72 h. (B) CD4 + DO11.10 + cells were evaluated for expression of T-bet by flow cytometry. MFI for T-bet expression is summarized in the right graph. (C) Ratio of T-bet/GATA3 MFI for CD4 + DO11.10 + cells in mice from (B) and Supplementary Figure 3A . (D) Concentrations of IFNγ, IL-12, TNFα, and IL-13 in supernatants. (E, F) 1 × 10 6 naïve DO11.10 cells were transferred to BALB/c mice that were subsequently injected i.v. with purified Xcl1-OVA, fliC-OVA, or αNIP-OVA proteins (5 μg). Spleens were harvested 72 h later and the percentage of (E) CD4 + DO11.10 + and (F) T-bet + DO11.10 + cells determined by flow cytometry. Data are representative of one (A) or three (B–D) independent experiments with n = 3 samples per group. (E, F) Data from one experiment with n = 3 mice per group. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple-comparison corrections. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For the early time-point blockade, 500 μg of anti-IL12 antibody (clone R2-9A5) (Bio X Cell, Lebanon, NH, USA) was injected i.p 1 and 2 days after vaccination, while for the later time inhibition the anti-IL12 antibody was injected on days 6 and 7.

Techniques: In Vitro, In Vivo, Purification, Transgenic Assay, Incubation, Expressing, Flow Cytometry, Injection, Comparison

Journal: Frontiers in Immunology

Article Title: Targeting Xcr1 on Dendritic Cells Rapidly Induce Th1-Associated Immune Responses That Contribute to Protection Against Influenza Infection

doi: 10.3389/fimmu.2022.752714

Figure Lengend Snippet: Xcl1-OVA induced Th1 polarization is IL12 dependent. (A, B) CD4 + cells from DO11.10 mice were incubated with BMDCs and Xcl1-OVA, fliC-OVA, or αNIP-OVA proteins (0.5 μg/ml), and either anti-IL-12 or isotype-matched mAbs (10 μg/ml) for 72 h. (A) DO11.10 cells were evaluated for expression of T-bet by flow cytometry, and (B) supernatants tested for secretion of IFNγ by ELISA. (C–E) 1 × 10 6 naïve DO11.10 cells were transferred i.v. to BALB/c mice that were subsequently immunized i.d. with 25 μg DNA encoding Xcl1-OVA. On days 1 and 2 after immunization, mice were injected i.p. with anti-IL12 or isotype-matched mAb (0.5 mg). Skin draining LNs and spleens were harvested after 1 week, and LN analyzed for (C) percentage of T-bet + DO11.10 + cells and (D) MFI of T-bet expression in T-bet + DO11.10 + cells. (E) Secretion of IFNγ from splenocytes stimulated for 24 h with the DO11.110 peptide. Data representative of two (A–D) or pooled from two (E) independent experiments with n = 3 samples per group (A, B) , n = 3–4 mice per group (C, D) or n = 7–8 mice per group (E) . Statistical analysis performed using the parametric t-test. *p < 0.05, ***p < 0.001.

Article Snippet: For the early time-point blockade, 500 μg of anti-IL12 antibody (clone R2-9A5) (Bio X Cell, Lebanon, NH, USA) was injected i.p 1 and 2 days after vaccination, while for the later time inhibition the anti-IL12 antibody was injected on days 6 and 7.

Techniques: Incubation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Injection

Journal: Frontiers in Immunology

Article Title: Targeting Xcr1 on Dendritic Cells Rapidly Induce Th1-Associated Immune Responses That Contribute to Protection Against Influenza Infection

doi: 10.3389/fimmu.2022.752714

Figure Lengend Snippet: Rapid Th1 induction is essential for induction of an IgG2a dominated response and improves protection induced by a mixed fliC-/Xcl1-HA vaccine. (A, B) DNA immunization of BALB/c mice with 25 μg DNA encoding Xcl1-HA, fliC-HA, a mixture of Xcl1-HA and fliC-HA, or Xcl1-HA and fliC-HA delivered on opposite flanks. (A) Serum titers of IgG1, IgG2a, and IgG2b were determined 2 weeks after vaccination. (B) IgG2a/IgG1 ratio of the serum samples presented in (A) . (C, D) Injection of either anti-IL-12 or isotype-matched mAbs on days 1 and 2 or 6 and 7 after DNA immunization with a mix of Xcl1-HA and fliC-HA plasmids. (C) Serum samples were harvested at the indicated time points and evaluated for the presence of HA-specific IgG1 or IgG2a. (D) Mice in (C) were challenged with 5xLD50 PR8 virus 12 weeks after immunization and weight loss monitored. Data representative of one experiment with n = 6–8 mice per group (A, B) , representative of two independent experiments with n = 4 mice per group (C) or pooled from two independent experiments with n = 8 mice per group (D) . Statistical analysis performed by non-parametric one-way ANOVA with Dunn’s multiple-comparison corrections (A, B) , or two-way ANOVA with Tukey’s multiple-comparison test (C, D) . *p < 0.05, ***p < 0.001.

Article Snippet: For the early time-point blockade, 500 μg of anti-IL12 antibody (clone R2-9A5) (Bio X Cell, Lebanon, NH, USA) was injected i.p 1 and 2 days after vaccination, while for the later time inhibition the anti-IL12 antibody was injected on days 6 and 7.

Techniques: Injection, Virus, Comparison