Journal: PLoS Pathogens

Article Title: Differential expression of interferon-lambda receptor 1 splice variants determines the magnitude of the antiviral response induced by interferon-lambda 3 in human immune cells

doi: 10.1371/journal.ppat.1008515

Figure Lengend Snippet: A) Normalized expression values for each subunit of the IFN-λR as determined by RT-qPCR for normal human bronchial epithelial cells (NHBE), primary hepatocytes (hep) or immune cells purified from healthy human donor blood. Graph shows mean + SEM from 3–7 different donors for each population. Results were normalized to the geomean of HPRT1 and RPL13A reference genes. B-H) IFN-λ3 binding was quantified via flow cytometry as described in the Materials and Methods. B) Dose curves of adding 1, 2 or 5 μg/ml IFN-λ3 to epithelial cells or total human PBMCs. 0 μg/ml IFN-λ3 refers to adding the secondary antibody alone. Graph shows mean +/- SD for 3–7 different donors. C) Binding percentages as detected by flow cytometry for IFN-λ3 or a similarly His-tagged control protein (OBCAM) where means +/- SD are shown. D-H) Quantified IFN-λ3 (5 μg/ml) binding percentages to each cell type where each dot represents a different healthy individual and background binding of the secondary antibody alone was subtracted. In D), statistical significance results were identical when comparing either NHBE or hep to all other immune cell subsets. All comparisons are shown in . H) Pearson correlation coefficients (r) calculated when comparing IFN-λ3 binding to different immune cell subsets. All comparisons are shown in . *, P<0.05, ***, P<0.001, ****, P<0.0001, two-way (C) or one-way (D, G) ANOVA with Tukey’s multiple comparisons test, paired t-test (E-F).

Article Snippet: Stimuli tested in this study include: IFN-λ3 (R&D Systems), IFN-α2b (INTRON ® A, Merck), recombinant IFN-λR1 and IL-10RB (R&D Systems), R848 (Invivogen), anti-CD3 (clone HIT3a) and anti-CD28 (clone CD28.2) were from Biolegend.

Techniques: Expressing, Quantitative RT-PCR, Purification, Binding Assay, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Differential expression of interferon-lambda receptor 1 splice variants determines the magnitude of the antiviral response induced by interferon-lambda 3 in human immune cells

doi: 10.1371/journal.ppat.1008515

Figure Lengend Snippet: A-B) RT-qPCR quantification of ISG15, IFIT1, and OAS1 induced by IFN-λ3 (100 ng/ml) purified human immune cell subsets (A) or normal human bronchial epithelial cells (NHBEs) (B). All cell types were cultured with or without IFN-λ3 for 24 hrs, except neutrophils (5 hrs). Graphs show fold induction relative to unstimulated cells after normalization to the geomean of HPRT1 and RPL13A reference genes. Bars represent mean +/- SEM from 3–8 different donors. n.s., not significant, *, P<0.05. ***, P<0.001, ****, P<0.0001, one-way ANOVA, Tukey’s multiple comparisons test (A), paired t-test (B).

Article Snippet: Stimuli tested in this study include: IFN-λ3 (R&D Systems), IFN-α2b (INTRON ® A, Merck), recombinant IFN-λR1 and IL-10RB (R&D Systems), R848 (Invivogen), anti-CD3 (clone HIT3a) and anti-CD28 (clone CD28.2) were from Biolegend.

Techniques: Quantitative RT-PCR, Purification, Cell Culture

Journal: PLoS Pathogens

Article Title: Differential expression of interferon-lambda receptor 1 splice variants determines the magnitude of the antiviral response induced by interferon-lambda 3 in human immune cells

doi: 10.1371/journal.ppat.1008515

Figure Lengend Snippet: A) Normalized expression values for each subunit of the IFN-λR as determined by RT-qPCR for DG75 or BEAS-2B cell lines. Results were normalized to the geomean of HPRT1 and RPL13A reference genes. B) IFN-λ3 flow cytometry binding assay results for 0, 1, or 5 μg/ml His-tagged IFN-λ3 added to DG75 or BEAS-2B cells. C) RT-qPCR quantification of ISG15 and IFIT1 mRNA in DG75 or BEAS-2B cells induced by IFN-λ3 (100 ng/ml) as compared to unstimulated cells after 24 hrs incubation. Bar graphs (A, C) show means + SEM from 2 independent experiments and flow cytometry histograms (B) are representative of 2 independent binding assays.

Article Snippet: Stimuli tested in this study include: IFN-λ3 (R&D Systems), IFN-α2b (INTRON ® A, Merck), recombinant IFN-λR1 and IL-10RB (R&D Systems), R848 (Invivogen), anti-CD3 (clone HIT3a) and anti-CD28 (clone CD28.2) were from Biolegend.

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Binding Assay, Incubation

Journal: PLoS Pathogens

Article Title: Differential expression of interferon-lambda receptor 1 splice variants determines the magnitude of the antiviral response induced by interferon-lambda 3 in human immune cells

doi: 10.1371/journal.ppat.1008515

Figure Lengend Snippet: A) Normalized expression values for each variant of IFNLR1 (membrane, full length ( mIFNLR1 ), or small, soluble ( sIFNLR1 )) as determined by RT-qPCR for normal human bronchial epithelial cells (NHBE), hepatocytes (hep) or immune cells purified from healthy human donor blood, or cell lines (BEAS-2B or A549 lung epithelial or DG75 B cell). Means + SEM are shown from 3–6 different donors (primary cells) or 2–3 independent experiments (cell lines). Results were normalized to the geomean of HPRT1 and RPL13A reference genes. B-C) Fold induction of 3 ISGs (ISG15, IFIT1, IFI44) by IFN-λ3 (10 ng/ml) treatment of total PBMCs (B) or Huh7.5 hepatocytes (C) relative to unstimulated cells with or without simultaneous addition of recombinant sIFN-λR1 (100 ng/ml (PBMC), 1000 ng/ml (Huh7.5)). Each dot represents a different individual or experiment. D-E) Quantification of binding of recombinant sIFN-λR1 or control IL-10RB to individual cell subsets within total PBMCs (D), or to A549 or Huh7.5 cell lines (E). Binding was detected by anti-Fc PE antibody. Histograms are representative of results from 4 different PBMC donors (bar graph right panel D), or from 2–3 independent experiments (E). F) Imaging flow cytometry visualization of binding of sIFN-λR1 (500 ng/ml (A549, Huh7.5), 1 μg/ml (THP-1, Jurkat)) to 4 cell lines. The median PE intensity is shown below each representative image from 6,000–12,000 total cells acquired from 2–3 independent experiments. G-H) IFN-λ3 binding to cell subsets within PBMCs where IFN-λ3 (2 μg/ml) was added with or without sIFN-λR1 (0.5–5 μg/ml +/- boiling) or IL-10RB (5 μg/ml). Percent IFN-λ3 bound and fold increase in median PE fluorescence are representative from 2–4 donors (G) or plotted as means + SD from 2–4 different donors (H). I) The presence of a soluble IFNLR1 variant within primates and lower mammals. Gray indicates no annotation and no transcript detectable in deposited RNA sequencing data, and purple indicates both annotation and detection of RNA transcript in multiple cell types from deposited RNA sequencing data. *, P<0.05, paired t-tests (B-C).

Article Snippet: Stimuli tested in this study include: IFN-λ3 (R&D Systems), IFN-α2b (INTRON ® A, Merck), recombinant IFN-λR1 and IL-10RB (R&D Systems), R848 (Invivogen), anti-CD3 (clone HIT3a) and anti-CD28 (clone CD28.2) were from Biolegend.

Techniques: Expressing, Variant Assay, Membrane, Quantitative RT-PCR, Purification, Recombinant, Binding Assay, Imaging, Flow Cytometry, Fluorescence, RNA Sequencing Assay

Journal: PLoS Pathogens

Article Title: Differential expression of interferon-lambda receptor 1 splice variants determines the magnitude of the antiviral response induced by interferon-lambda 3 in human immune cells

doi: 10.1371/journal.ppat.1008515

Figure Lengend Snippet: A-C) IFN-λ3 (5 μg/ml) binding to LIVE/DEAD - CD20 + B cells (A-B, memory (CD27 + ), naïve (CD27 - )) or CD3 + CD4 + or CD8 + T cells (C) within total PBMCs that had been cultured for 3 days either unstimulated (unstim), or with the following stimuli: R848 (1 μg/ml), IFN-γ (10 ng/ml), IFN-α2 (1000 IU/ml) or anti-IgM/IgG/IgA (BCR, 10 μg/ml) and anti-CD40 (5 μg/ml). The percent IFN-λ3 + or median PE fluorescence is shown after background subtraction of secondary antibody alone with means +/- SD. C) RT-qPCR quantification of sIFNLR1 and mIFNLR1 transcripts in purified neutrophils that were left unstimulated (unstim) or stimulated with IFN-α2 (1000 IU/ml), R848 (1 μg/ml) or heat killed listeria (HK listeria) supernatant (1:100) for 5 hrs. *, P<0.05, **, P<0.01, ****, P<0.0001, one-way ANOVA (A) or two-way ANOVA (B-C), Dunnett’s multiple comparisons test between each treatment and unstimulated. Only significant comparisons are noted and each symbol represents a different individual donor.

Article Snippet: Stimuli tested in this study include: IFN-λ3 (R&D Systems), IFN-α2b (INTRON ® A, Merck), recombinant IFN-λR1 and IL-10RB (R&D Systems), R848 (Invivogen), anti-CD3 (clone HIT3a) and anti-CD28 (clone CD28.2) were from Biolegend.

Techniques: Binding Assay, Cell Culture, Fluorescence, Quantitative RT-PCR, Purification

Journal: PLoS Pathogens

Article Title: Differential expression of interferon-lambda receptor 1 splice variants determines the magnitude of the antiviral response induced by interferon-lambda 3 in human immune cells

doi: 10.1371/journal.ppat.1008515

Figure Lengend Snippet: A) IFN-λ3 (5 μg/ml) binding to LIVE/DEAD - CD4 + T cells within total PBMCs that had been cultured for 3 days in either media only (unstimulated (unstim)), or with the following stimuli: R848 (1 μg/ml), anti-CD3 (plate bound 1.5 μg/ml) and soluble anti-CD28 (1 μg/ml), IFN-γ (10 ng/ml) or IFN-α2 (1000 IU/ml). B-C) Total IFNLR1 , IL10RB (B), full length membrane ( mLR1 ) or soluble ( sLR1 ) IFNLR1 variant (C) transcript expression in CD4 + T cells sorted after 3 days of PBMC culture with or without anti-CD3/anti-CD28 stimulation (TCR). D) ISG induction in sorted CD4 + T cells treated with IFN-λ3 (100 ng/ml) for 24 hrs after sorting from PBMCs which had previously been cultured in unstimulated or anti-CD3/anti-CD28 stimulated (TCR) conditions for 3 days. Fold induction of 3 ISGs are shown. Graphs show means +/- SD (A) or SEM (B-D) with each symbol representing a different healthy individual. All RT-qPCR results are normalized to B2M reference gene. n.s., not significant, *, P<0.05, **, P<0.01, ***, P<0.001, one-way ANOVA, Dunnett’s multiple comparison test comparing to unstimulated (A), unpaired t-test (B-D).

Article Snippet: Stimuli tested in this study include: IFN-λ3 (R&D Systems), IFN-α2b (INTRON ® A, Merck), recombinant IFN-λR1 and IL-10RB (R&D Systems), R848 (Invivogen), anti-CD3 (clone HIT3a) and anti-CD28 (clone CD28.2) were from Biolegend.

Techniques: Binding Assay, Cell Culture, Membrane, Variant Assay, Expressing, Quantitative RT-PCR, Comparison

Journal: PLoS Pathogens

Article Title: Differential expression of interferon-lambda receptor 1 splice variants determines the magnitude of the antiviral response induced by interferon-lambda 3 in human immune cells

doi: 10.1371/journal.ppat.1008515

Figure Lengend Snippet: A) Relative expression of full length, membrane ( mIFNLR1) and small, soluble ( sIFNLR1 ) variant expression in purified CD4 + T cells on day 0 or day 3 after PHA stimulation (normalized to B2M reference gene). B) ISG ( ISG15 , IFIT1 , OAS1 ) expression in purified CD4 + T cells cultured with or without IFN-λ3 (100 ng/ml) for 24 hrs after 3 days of PHA stimulation. Results are shown as means +/- SD. C-D) Quantification of HIV-1 infection via p24 intracellular flow cytometry. C) Representative p24 staining from 1 healthy individual for all treatments tested. D) % HIV-1 p24 staining from 4–5 different individuals for HIV-1 infection alone compared to cells pre-treated with IFN-λ3 (100 ng/ml) or IFN-α2 (100 IU/ml). Symbols represent means from duplicate wells per individual with each symbol representing a different healthy blood donor. *, P<0.05, **, P<0.01, ***, P<0.001, paired t-tests.

Article Snippet: Stimuli tested in this study include: IFN-λ3 (R&D Systems), IFN-α2b (INTRON ® A, Merck), recombinant IFN-λR1 and IL-10RB (R&D Systems), R848 (Invivogen), anti-CD3 (clone HIT3a) and anti-CD28 (clone CD28.2) were from Biolegend.

Techniques: Expressing, Membrane, Variant Assay, Purification, Cell Culture, Infection, Flow Cytometry, Staining