Journal: bioRxiv

Article Title: Metabolic and functional impairment of CD8 + T cells from the lungs of influenza-infected obese mice

doi: 10.1101/2020.04.19.047282

Figure Lengend Snippet: Lungs were harvested, digested, and homogenized into single cell suspension. Total CD8 + T cells from day 10-post influenza infected mice were isolated using negative selection magnetic bead separation. Cells were plated at 300,000 cells per well, and extracellular flux analysis was performed using the Agilent Seahorse XF e 96 flux analyzer. (A) Oxygen consumption rates (OCR), a surrogate of glycolysis, and (B) Extracellular Acidification Rates (ECAR), a surrogate of mitochondrial respiration, were measured in response to the Mitochondrial Stress Test for CD8 + T cells from Lean (n=11) and Obese (n=12) mice. (C) Basal OCR and (D) Basal ECAR were determined as the last point before oligomycin injection. Data represents mean ± SEM and were normalized to cell number. Wilcoxon rank sum test was used to compare groups. *p<0.05, **p<0.01.

Article Snippet: CD8 + T cells were isolated from the single cell suspension of mouse lungs at day 10 post PR8 infection using magnetic bead negative selection (Stemcell, Vancoover, Canada) in EasySep buffer (PBS + 2% FBS + 1mM EDTA).

Techniques: Suspension, Infection, Isolation, Selection, Injection

Journal: bioRxiv

Article Title: Metabolic and functional impairment of CD8 + T cells from the lungs of influenza-infected obese mice

doi: 10.1101/2020.04.19.047282

Figure Lengend Snippet: Lungs were harvested, digested, and homogenized into single cell suspension. Total CD8 + T cells from day 10-post influenza infected mice were isolated using negative selection magnetic bead separation. Cells were plated at 300,000 cells per well, and extracellular flux analysis was performed using the Agilent Seahorse XF e 96 flux analyzer and Mitochondrial Stress test for lean (n=11) and obese (n=12) mice. (A) Maximal respiration was measured as peak OCR following injection of 1.5μM FCCP. (B) Spare Respiratory Capacity was calculated as the difference in Max OCR minus Basal OCR. (C) ATP production was calculated as the difference between Basal OCR and the minimum OCR following injection of 0.5 μM oligomycin. (D) Glycolytic potential (OCR vs. ECAR) before and after injection of oligomycin. Data represents mean ± SEM and were normalized to cell number. Wilcoxon rank sum test was used to compare groups. *p<0.05.

Article Snippet: CD8 + T cells were isolated from the single cell suspension of mouse lungs at day 10 post PR8 infection using magnetic bead negative selection (Stemcell, Vancoover, Canada) in EasySep buffer (PBS + 2% FBS + 1mM EDTA).

Techniques: Suspension, Infection, Isolation, Selection, Injection

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

Journal: Foods

Article Title: Immunomodulatory Role of BLG-Derived Peptides Based on Simulated Gastrointestinal Digestion and DC-T Cell from Mice Allergic to Cow’s Milk

doi: 10.3390/foods11101450

Figure Lengend Snippet: Peptide BLG 40–60 showing the promote maturation ability of LP cells and directed differentiation of activated CD4 + T lymphocytes towards a Th1 phenotype, but not the proliferation, in the peptide–LP cell–MLN cell-co-culture assay. ( A ) The intensity of CFSE staining after stimulation. ( B ) The histogram results of CD80, CD86, TIM-4, CD28, and CD40. Each row corresponds to a different gate result, and each column corresponds to a different peptide treatment group. ( C ) Bar graphs showing the MFI levels summarized from ( B ). ( D ) Bar graphs showing the cytokines levels of IFN-γ, IL-4, IL-5, IL-13, IL-10, IL-17e, IL-12p70, and MIP-3α. ( E ) Bar graphs showing the IFN-γ/IL-4 ratio summarized from ( D ). The results are represented as the mean ± SD. (The data were calculated in three replicates for flow cytometry assay, and two replicates for cytokine level. The same letters indicate the lack of significance, as determined by two-way analysis of variance (ANOVA) with Duncan’s post-test ( p < 0.05).) LP cell, intestinal lamina propria cell. Th, T helper lymphocyte. MLN, mesenteric lymph node. CFSE, 5-(and 6)-carboxyfluorescein diacetate, succinimidyl ester. TIM-4, T cell immunoglobulin and mucin domain (TIM)-4. MFI, median fluorescence intensity. IFN-γ, interferon γ. IL, interleukin.

Article Snippet: MLN-derived CD4 + T cells were sorted by a CD4-positive sorting magnetic bead kit (anti-mouse CD4 magnetic particles-DM, 551539, BD Bioscience, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions.

Techniques: Co-culture Assay, Staining, Flow Cytometry, Fluorescence

Journal: Foods

Article Title: Immunomodulatory Role of BLG-Derived Peptides Based on Simulated Gastrointestinal Digestion and DC-T Cell from Mice Allergic to Cow’s Milk

doi: 10.3390/foods11101450

Figure Lengend Snippet: Peptides BLG 1–14 , BLG 40–60 , BLG 82–101 , and BLG 123–139 directed differentiation of activated MLN-CD4 + T lymphocytes towards a Th1 phenotype in the peptide–MLN cell-co-culture assay. ( A ) The histogram results of CD4 + IFN-γ + , CD4 + IL-4 + , CD4 + IL-17e + , and CD4 + CD28 + , and the contour plot of CD4 + CD25 + Foxp3 + after peptide stimulation. Each row corresponds to a different gate result, and each column corresponds to a different peptide treatment group. ( B ) Bar graphs showing the MFI levels summarized from ( A ). ( C ) Bar graphs showing the CD4 + IFN-γ + /CD4 + IL-4 + ratio summarized from ( B ). The results are represented as the mean ± SD ( n = 3, the data were calculated in three replicates, the same letters indicate the lack of significance, as determined by two-way analysis of variance (ANOVA) with Duncan’s post-test ( p < 0.05)). MLN, mesenteric lymph node. Th, T helper lymphocyte. MFI, median fluorescence intensity.

Article Snippet: MLN-derived CD4 + T cells were sorted by a CD4-positive sorting magnetic bead kit (anti-mouse CD4 magnetic particles-DM, 551539, BD Bioscience, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions.

Techniques: Co-culture Assay, Fluorescence