Journal: Cell reports

Article Title: TGF-β in the microenvironment induces a physiologically occurring immune-suppressive senescent state

doi: 10.1016/j.celrep.2023.112129

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Article Snippet: The antibodies used were: CD45 (Cat #: MCD4501, Invitrogen); Ly-6G (Cat #: 60031PB.1, StemCell Technologies Inc); Ly6C (Cat #: 17-5932-82, Life Technologies), CD11b (Cat #: 53-0112-82, Life Technologies); F4/80 (Cat #: 17-4801-80, Life Technologies), B220 (Cat #: 17-0452-82, Life Technologies), CD3 (Cat #: 11-0031-82, Life Technologies), CD8 (Cat #: 100712, BioLegend), CD4 (Cat #: 48-0041-82, Life Technologies), NK1.1 (Cat #: 108706, BioLegend), FoxP3 (Cat #: 17-4801-80, Life Technologies).

Techniques: Sequencing, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Transfection, SYBR Green Assay, Viability Assay, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Derivative Assay, Real-time Polymerase Chain Reaction, Software

Journal: Immunology

Article Title: Hepatitis C virus core protein reduces CD 8 + T‐cell proliferation, perforin production and degranulation but increases STAT 5 activation

doi: 10.1111/imm.12882

Figure Lengend Snippet: Hepatitis C virus (HCV) core protein reduces CD8+ T‐cell proliferation and viability. (a) A scatter plot graph shows the proliferation of CFSE‐labelled CD8+ T‐cells cultured alone (Medium) or pre‐incubated with HCV core for increasing periods of time (0, 24, 48 and 72 hr) followed by a 5‐day stimulation with anti‐CD3/CD28 (n = 3, repeated measures analysis of variance with Bonferroni's multiple comparison test). *P < .05, **P < 0·01. (b) A second scatter plot graph indicates the viability of stimulated CD8+ T‐cells incubated with HCV core in a time–course experiment, as determined in studies using phytohaemagglutinin as a stimulant (n = 4; relative change in MTT absorbance). To confirm effects on proliferation, CFSE‐labelled CD8+ T‐cells were pre‐incubated with or without HCV core (5 µg/ml) for 72 hr followed by 5 days of stimulation with anti‐CD3/28 (0·0625 µg/ml). (c) A representative histogram shows the proliferation profile of CFSE‐labelled CD8+ T‐cells. The marker indicates the decreasing fluorescence intensity traces of proliferating (i.e. CFSE low) cells. (d) A scatter plot summarizes the proliferation of CD8+ T‐cells pre‐incubated with medium or HCV core for 72 hr followed by stimulation with anti‐CD3/28 for 72 hr (mean ± SD, two‐tailed paired Student's t‐test, n = 5).

Article Snippet: Cells Human peripheral blood mononuclear cells were isolated from the blood of healthy HCV − donors using Lymphoprep (StemCell Technologies, Vancouver, BC, Canada) density gradient centrifugation, followed by isolation of CD8 + T‐cells using CD8 + T‐cell Positive Magnetic Selection Kit I or II (StemCell Technologies).

Techniques: Virus, Cell Culture, Incubation, Comparison, Marker, Fluorescence, Two Tailed Test

Journal: Immunology

Article Title: Hepatitis C virus core protein reduces CD 8 + T‐cell proliferation, perforin production and degranulation but increases STAT 5 activation

doi: 10.1111/imm.12882

Figure Lengend Snippet: Incubation with hepatitis C virus (HCV) core protein decreases interleukin‐7 (IL‐7) ‐mediated Bcl‐2 production in CD8+ T‐cells. CD8+ T‐cells incubated with or without HCV core protein (5 µg/ml) for 72 hr were treated with IL‐7 (1 ng/ml) for 48 hr. Cells were then stained with anti‐human Bcl‐2 antibody before analysis on a flow cytometer. (a) Representative histograms, with marker indicating Bcl‐2hi cells. (b) Average proportion of Bcl‐2hi cells ± SD (two‐tailed paired Student's t‐test, n = 8).

Article Snippet: Cells Human peripheral blood mononuclear cells were isolated from the blood of healthy HCV − donors using Lymphoprep (StemCell Technologies, Vancouver, BC, Canada) density gradient centrifugation, followed by isolation of CD8 + T‐cells using CD8 + T‐cell Positive Magnetic Selection Kit I or II (StemCell Technologies).

Techniques: Incubation, Virus, Staining, Flow Cytometry, Marker, Two Tailed Test

Journal: Immunology

Article Title: Hepatitis C virus core protein reduces CD 8 + T‐cell proliferation, perforin production and degranulation but increases STAT 5 activation

doi: 10.1111/imm.12882

Figure Lengend Snippet: Incubation with hepatitis C virus (HCV) core protein increases signal transducer and activator of transcription 5 (STAT5) activation in CD8+ T‐cells. CD8+ T‐cells were incubated with or without HCV core protein (5 µg/ml) for 72 hr before treatment with interleukin‐7 (IL‐7; 1 ng/ml) for 15 min. Cells were then labelled with anti‐pSTAT5 antibody and analysed on a flow cytometer. (a) Representative histogram depicting effect of HCV core on STAT5 activation. (b) Mean MFI ± SD of pSTAT5 production (i.e. activated STAT5; two‐tailed paired Student's t‐test, n = 6).

Article Snippet: Cells Human peripheral blood mononuclear cells were isolated from the blood of healthy HCV − donors using Lymphoprep (StemCell Technologies, Vancouver, BC, Canada) density gradient centrifugation, followed by isolation of CD8 + T‐cells using CD8 + T‐cell Positive Magnetic Selection Kit I or II (StemCell Technologies).

Techniques: Incubation, Virus, Activation Assay, Flow Cytometry, Two Tailed Test

Journal: Immunology

Article Title: Hepatitis C virus core protein reduces CD 8 + T‐cell proliferation, perforin production and degranulation but increases STAT 5 activation

doi: 10.1111/imm.12882

Figure Lengend Snippet: Hepatitis C virus (HCV) core protein reduces perforin production of CD8+ T‐cells. CD8+ T‐cells were incubated with or without HCV core protein (5 µg/ml) for 72 hr before stimulation with anti‐CD3/28 (1 µg/ml) for 48 hr, and then labelled with anti‐human perforin antibody and analysed by flow cytometry, or stimulated for a further 72 hr before collection of supernatants for quantification of perforin release. (a) A representative histogram is shown with marker indicating perforin+ cells. (b) Average proportion of perforin+ cells ± SD (two‐tailed paired Student's t‐test, n = 9). (c) Perforin release from stimulated CD8+ T‐cells as measured from supernatants by Milliplex Bead Array (one‐tailed paired Student's t‐test, n = 5). ULOD, upper limit of detection.

Article Snippet: Cells Human peripheral blood mononuclear cells were isolated from the blood of healthy HCV − donors using Lymphoprep (StemCell Technologies, Vancouver, BC, Canada) density gradient centrifugation, followed by isolation of CD8 + T‐cells using CD8 + T‐cell Positive Magnetic Selection Kit I or II (StemCell Technologies).

Techniques: Virus, Incubation, Flow Cytometry, Marker, Two Tailed Test, One-tailed Test

Journal: Immunology

Article Title: Hepatitis C virus core protein reduces CD 8 + T‐cell proliferation, perforin production and degranulation but increases STAT 5 activation

doi: 10.1111/imm.12882

Figure Lengend Snippet: Incubation with hepatitis C virus (HCV) core protein reduces degranulation (as indicated by CD107a expression) of CD8+ T‐cells. CD8+ T‐cells incubated with or without HCV core protein (5 µg/ml) for 72 hr were stimulated with anti‐CD3/28 stimulation (1 µg/ml) for 48 hr before analysis by flow cytometry. For the last 6 hr of stimulation, anti‐human CD107a antibody and BD GolgiPlug (containing monensin) was added. (a) A representative histogram with marker indicating CD107a+ cells. (b) Average proportion of CD107a+ cells ± SD (two‐tailed paired Student's t‐test, n = 5).

Article Snippet: Cells Human peripheral blood mononuclear cells were isolated from the blood of healthy HCV − donors using Lymphoprep (StemCell Technologies, Vancouver, BC, Canada) density gradient centrifugation, followed by isolation of CD8 + T‐cells using CD8 + T‐cell Positive Magnetic Selection Kit I or II (StemCell Technologies).

Techniques: Incubation, Virus, Expressing, Flow Cytometry, Marker, Two Tailed Test

Journal: Immunology

Article Title: Hepatitis C virus core protein reduces CD 8 + T‐cell proliferation, perforin production and degranulation but increases STAT 5 activation

doi: 10.1111/imm.12882

Figure Lengend Snippet: Incubation with hepatitis C virus (HCV) core protein does not affect proportion of IFN‐γ + CD8+ T‐cells. CD8+ T‐cells incubated with or without HCV core protein (5 µg/ml) for 72 hr were stimulated with anti‐CD3/28 (1 µg/ml) for 24 hr before labelling with anti‐human interferon‐ γ (IFN ‐ γ) antibody and analysis by flow cytometry. Cells were treated with BD GolgiPlug (containing Brefeldin‐A) for the last 5 hr of stimulation. (a) A representative histogram, with analysis marker indicating IFN‐γ + cells. (b) Average proportion of IFN‐γ + cells ± SD (two‐tailed paired Student's t‐test, n = 7).

Article Snippet: Cells Human peripheral blood mononuclear cells were isolated from the blood of healthy HCV − donors using Lymphoprep (StemCell Technologies, Vancouver, BC, Canada) density gradient centrifugation, followed by isolation of CD8 + T‐cells using CD8 + T‐cell Positive Magnetic Selection Kit I or II (StemCell Technologies).

Techniques: Incubation, Virus, Flow Cytometry, Marker, Two Tailed Test

Journal: Immunology

Article Title: Hepatitis C virus core protein reduces CD 8 + T‐cell proliferation, perforin production and degranulation but increases STAT 5 activation

doi: 10.1111/imm.12882

Figure Lengend Snippet: Cells treated with hepatitis C virus (HCV) ‐infected serum produce less perforin upon stimulation. CD8+ T‐cells were incubated with serum from HCV + or healthy HCV − serum for 72 hr before stimulation with anti‐CD3/28 for 48 hr. Cells were then labelled with anti‐human perforin antibody and analysed by flow cytometry. Average proportion of perforin+ cells ± SD (two‐tailed paired Student's t‐test, n = 6).

Article Snippet: Cells Human peripheral blood mononuclear cells were isolated from the blood of healthy HCV − donors using Lymphoprep (StemCell Technologies, Vancouver, BC, Canada) density gradient centrifugation, followed by isolation of CD8 + T‐cells using CD8 + T‐cell Positive Magnetic Selection Kit I or II (StemCell Technologies).

Techniques: Virus, Infection, Incubation, Flow Cytometry, Two Tailed Test