Journal: Immunity

Article Title: Lung γδ T cells mediate protective responses during neonatal influenza infection that are associated with Type-2 immunity

doi: 10.1016/j.immuni.2018.07.011

Figure Lengend Snippet: A. Estimation of IL-17A in whole lung homogenates from mock- (open) or influenza virus-(solid)- infected wild-type (black) and TCRδ−/− (red) neonates by ELISA at 1 day after infection. Samples are pooled from at least two independent experiments and data are shown as mean ± SEM (Mann-Whitney test).B. Survival rate of influenza virus-infected TCRδ−/− neonates administered with low levels of recombinant mouse IL-17A (rmIL-17A, green, n=37, 100pg/mouse) or PBS control (red, n=19) at the time of infection. Data are combined from six independent trials, which individually showed the same trend, and data are presented as mean ± SEM.C. Schema outlining wild-type and Il17a−/− γδ T cell transfers to TCRδ−/− neonates and subsequent infection.D and E. Body weight profile (D) and survival rate (E) of influenza-infected TCRδ−/− neonates receiving wild-type (black, n=15) or Il17a−/− (red, n=13) γδ T cells intranasally or no cell transfer (grey, n=10). Data are combined from four independent trials, which individually showed the same trend. Weight profile data are presented as mean ± SEM.F. Protein levels of IL-33 assessed by ELISA in influenza virus-infected wild-type (black, n=15) and TCRδ−/− lungs (red, n=9) at 1 day following infection. Samples are pooled from three independent experiments, and data are shown as mean ± SEM.G. Protein levels of IL-33 detected by ELISA in the lungs of TCRδ−/− neonates that had been infected with influenza virus and simultaneously treated with either a low level of rmIL-17A (green, n=7, 100pg/mouse) or PBS control (red, n=6). Data were collected 1 day after infection/treatment. Samples are pooled from at least two independent experiments, and data are shown as mean ± SEM.H. Survival rate of influenza virus-infected wild-type (black, n=20) and Il33−/− neonates (blue, n=17) with intranasal influenza virus infection. Data are combined from ten independent experiments and shown as mean ± SEM in weight change.I. Survival rate of influenza virus-infected TCRδ−/− neonates administered with recombinant mouse IL-33 (rmIL-33, brown, n=18, 10ng/mouse) or PBS control (red, n=18) at the time of infection. Data are combined from six individual experiments.*p<0.05, **p<0.01, n.s., not significant.

Article Snippet: Cytokines were measured in supernatants as follows: IL-17A, IL-33, and amphiregulin were assayed by Human IL-17A, IL-33, and amphiregulin DuoSet ELISA kits (R&D), and the level of IFN-γ was detected by Human Milliplex assays (Millipore) according to manufacturer instructions.

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

Journal: Immunity

Article Title: Lung γδ T cells mediate protective responses during neonatal influenza infection that are associated with Type-2 immunity

doi: 10.1016/j.immuni.2018.07.011

Figure Lengend Snippet: A. Relative expression of Il33 mRNA, as assessed by quantitative real-time PCR, in mouse lung epithelial cells (LET1s) treated with medium (white), A/x31 influenza virus (blue, 3 MOI), rmIL-17A (green, 50ng/ml), or virus+rmIL-17A (red) at 24hrs and 48hrs after treatment. Data are shown as mean ± SEM and combined from three separate experiments that independently showed the same trend.B. Immunoblot assay of IL-33 protein in stimulated LET1s for the same conditions as in (A) at 48 hrs after stimulation.C. Quantitative real-time PCR analysis of Il33 mRNA in LET1 cells treated as in A and B with and without STAT3 phosphorylation inhibitor S3I-201 (100μM in 0.05% DMSO) at 48hrs after treatment. Data are shown as mean ± SEM and combined from two separate experiments that independently showed the same trend.D. Immunoblot analysis of IL-33 and phosphorylated STAT3 (pSTAT3) in the LET1 lysates treated as before (A, B, and C). Total STAT3 (tSTAT3) and β-actin are also shown. Immunoblots were repeated twice showing similar results.*p<0.05, ****p<0.0001

Article Snippet: Cytokines were measured in supernatants as follows: IL-17A, IL-33, and amphiregulin were assayed by Human IL-17A, IL-33, and amphiregulin DuoSet ELISA kits (R&D), and the level of IFN-γ was detected by Human Milliplex assays (Millipore) according to manufacturer instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Immunity

Article Title: Lung γδ T cells mediate protective responses during neonatal influenza infection that are associated with Type-2 immunity

doi: 10.1016/j.immuni.2018.07.011

Figure Lengend Snippet: A- C. Correlation between concentrations of human IL-17A and IL-33 (A), IFN-γ and IL-33 (B) and IL-33 and Areg protein levels (C) in the nasal aspirates of influenza-infected children (< 8 years old, n=51).D. Concentration of IL-17A at Day 0 after enrollment in the nasal aspirates of children with mild (black, n=11) and severe (red, n=12) influenza disease outcomes.E. Concentration of IFN-γ at Day 0 after enrollment in the nasal aspirates of children with mild (black, n=11) and severe (red, n=12) disease outcomes.Data are presented as mean ± SEM, and in each case cytokine values (pg/ml) were log10 transformed for visualization only. *p<0.05.

Article Snippet: Cytokines were measured in supernatants as follows: IL-17A, IL-33, and amphiregulin were assayed by Human IL-17A, IL-33, and amphiregulin DuoSet ELISA kits (R&D), and the level of IFN-γ was detected by Human Milliplex assays (Millipore) according to manufacturer instructions.

Techniques: Infection, Concentration Assay, Transformation Assay

Journal: Immunity

Article Title: Lung γδ T cells mediate protective responses during neonatal influenza infection that are associated with Type-2 immunity

doi: 10.1016/j.immuni.2018.07.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cytokines were measured in supernatants as follows: IL-17A, IL-33, and amphiregulin were assayed by Human IL-17A, IL-33, and amphiregulin DuoSet ELISA kits (R&D), and the level of IFN-γ was detected by Human Milliplex assays (Millipore) according to manufacturer instructions.

Techniques: Plasmid Preparation, Recombinant, Cell Stimulation, Protease Inhibitor, Blocking Assay, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Sequencing, Software