Journal: International Journal of Molecular Sciences

Article Title: GARP as an Immune Regulatory Molecule in the Tumor Microenvironment of Glioblastoma Multiforme

doi: 10.3390/ijms20153676

Figure Lengend Snippet: ( a ) Soluble GARP (sGARP) cytokine suppression. CD4 + T cells were stimulated with 1 µg/mL anti-CD3 mAb, 1 µg/mL anti-CD28 mAb, and with or without 1 µg/mL sGARP for 24 h. INF-γ production was measured by intracellular staining via flow cytometry. Dot plots show one representative result of 5 independent experiments. ( b ) T98G cells suppress T cell proliferation and cytokine production. CD4 + T cells were cultured together with or without (w/o) T98G cells in the ratio of 8:1 and stimulated, as described above. Additionally, either 10 µg/mL anti-GARP Ab or no Ab were added into the culture and CD4 + T cells were stimulated as described before. IFN-γ production and proliferation (CFSE) were measured 4 days after stimulation by intracellular staining via flow cytometry. Dot plots show one representative result of 4 independent experiments. Data are displayed as mean values ± SEM, p -values relative to w/o ** p < 0.01. Dotted lines represent either unstained control (a + b IFN-γ), CFSE stained cells before stimulation or percentages normalized to the untreated control (w/o).

Article Snippet: The regulatory T cells were isolated with the CD4 + CD25 + CD127 dim/− isolation kit (Miltenyi #130-094-775, Bergisch Gladbach, Germany) according to the manufacturer’s protocol.

Techniques: Staining, Flow Cytometry, Cell Culture, Control

Journal: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: Phenotpyes of primed CD8 + Foxp3 + T cells. (A) Surface staining of CD8, CD25, GITR, CTLA4, and PD-1 on the splenic cells taken from Foxp3-GFPtg mice on day 6 after infection with H5N1 virus. The dot plots represent one of four independent experiments with similar results ( n = 5 mice). (B) Splenic CD8 + CD25 + T cells from Foxp3-GFPtg mice on day 6 after H5N1 viral infection were purified for quantitative RT-PCR. Naïve CD8 + T cells were purified from naïve Foxp3-GFPtg mice as control. Data are shown as mean + SEM and are pooled from three independent experiments. ** p < 0.01. n.s., p > 0.05, unpaired two-tailed t -test. (C) Splenic cells were isolated on day 6 from Foxp3-GFPtg mice infected by H5N1 virus and were stimulated with 5 μg/mL NP peptide (NP366–374) or no peptide. Cells were incubated for 6 h with 2 μM monensin before intracellular staining. CD8 + T cells were gated to analyze the expression of Foxp3 and IL-10. The dot plots represent one of three independent experiments with similar results ( n = 5 mice).

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: Staining, Infection, Virus, Purification, Quantitative RT-PCR, Control, Two Tailed Test, Isolation, Incubation, Expressing

Journal: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: Primed CD8 + CD25 + T cells expressed IL-10. Splenic cells were isolated on day 6 from IL-10-GFPtg mice infected with H5N1 virus and surface stained for CD8 and CD25. CD8 + T cells were separated into CD25 positive and negative cells. These cells were further divided into GFP positive or negative, with GFP serving as a marker for IL-10 positivity. The percentages of CD8 + CD25 + T cells and CD8 + CD25 − T cells that were IL-10 + or IL-10 − are shown together with results of statistical analysis. The dot plots represent one of five independent experiments with similar results ( n = 5 mice).

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: Isolation, Infection, Virus, Staining, Marker

Journal: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: CD8 + Treg cells resulted in enhanced mortality and increased virus load in the lung. (A) Splenic CD8 + CD25 + T cells were isolated on day 6 from Foxp3-GFPtg mice infected with H5N1 virus. The cells were transferred into BALB/c mice (1 × 10 5 or 5 × 10 5 CD8 + CD25 + T cells per mouse) and the mice ( n = 10 mice per group) were then immediately challenged with H5N1 virus. (B) Survival of mice was monitored from day 7 to 16 after virus infection. Log-rank test for comparisons of survival curves between control mice and CD8 + Treg cells recipient mice; ** p < 0.01. *** p < 0.001, log-rank test. (C) Lung viral load was assayed on day 6 after virus infection ( n = 5 mice per group). (D) Total RNA was extracted from lung for real-time PCR for IFN-β and Mx-1. Data are shown as mean + SEM and are pooled from three independent experiments. n.s., p > 0.05. * p < 0.05. ** p < 0.01. *** p < 0.001, unpaired two-tailed t -test.

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: Virus, Isolation, Infection, Control, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: CD8 + T-cell immunity was inhibited by CD8 Treg cells in vivo. (A) CD8 + CD25 + T cells, CD8 + CD25 − T cells, total CD8 + T cells were isolated from spleens of C57BL/6 mice on day 6 after infection with H5N1 virus and were transferred to CD8 KO mice. 5 × 10 5 CD8 + CD25 + T cells, 1 × 10 7 CD8 + CD25 − T cells, or 1 × 10 7 CD8 + T cells were transferred into each mouse ( n = 10 mice per group). (B) Mouse survival was monitored from day 4 to 16 after virus infection. Log-rank test for comparisons of survival curves between CD8 + T-cell recipient mice and CD8 + CD25 − T-cell recipient mice; ** p < 0.01 ( n = 10 mice per group), log-rank test. (C) Lung viral loads were assayed on day 6 after virus infection ( n = 5 mice per group). (D). Lung cells taken on day 8 of infection were stimulated with PMA and ionomycin for 5 h in the presence of monensin and then stained to detect intracellular IFN-γ expression in the CD4 + and CD8 + T cells ( n = 4 mice per group). (E) Statistical analysis of IFN-γ + cells among CD4 + and CD8 + T cells (%). (F) Serum total H5N1 virus-specific IgG titers, assayed by ELISA on day 8 post virus infection ( n = 4 mice per group). Mice receiving CD8 + CD25 − T cells served as CD8nonTreg-cell controls. Data are presented as means ± SEM and are representative of three independent experiments. * p < 0.05. ** p < 0.01. *** p < 0.001, unpaired two-tailed t -test.

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: In Vivo, Isolation, Infection, Virus, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: CD8 + CD25 − T-cell proliferation was inhibited by CD8 + Treg cells through IL-10 in vitro. (A) CD8 + CD25 + T cells, CD8 + CD25 − T cells, CD11c + cells were isolated on day 6 from spleens of H5N1-infected C57BL/6 mice. CFSE-stained CD8 + CD25 − T cells were stimulated to proliferate in vitro; 2 × 10 5 CD8 + CD25 − T cells and 5 × 10 4 CD11c + cells were stimulated with 10 μg/mL NP366–374 peptide in the presence of different numbers of CD8 + Treg cells for 5 days. (B) CD4 + CD25 + T cells, CD8 + CD25 + T cells, CD8 + CD25 − T cells, and CD11c + cells were isolated from spleens of C57BL/6 mice on day 6 after infection with H5N1 virus. CFSE-stained CD8 + CD25 − T cells were stimulated to proliferate in vitro; 2 × 10 5 CD8 + CD25 − T cells and 5 × 10 4 CD11c + cells were stimulated with 10 μg/mL NP366–374 peptide in the presence of different numbers of CD4 + or CD8 + Treg cells for 5 days. (C) 2 × 10 5 CD8 + CD25 − T cells and 5 × 10 4 CD11c + cells were stimulated with 10 μg/mL NP366–374 peptide in the presence of 1 × 10 5 CD8 + Treg cells in transwell plates for 5 days. (D) T-cell proliferation was done with 50 μg/mL anti-IL-10 mAb or isotype control antibodies in the wells. The number of CD8 + Treg cells used in the system (C and D) was 1 × 10 5 . (E) CD8 + CD25 + T cells and CD11c + cells from C57BL/6 mice and CD8 + CD25 − T cells from DNIL-10R mice were isolated 6 days after infection with H5N1 virus and T-cell proliferation assays were performed. (A–E) Data shown are representative of at least three independent experiments with four mice per group.

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: In Vitro, Isolation, Infection, Staining, Virus, Control

Journal: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: The antiviral activity of CD8 + CD25 − T cells was inhibited by CD8 + Treg cells in vivo through IL-10. (A) CD8 + CD25 + T cells and CD8 + CD25 − T cells from the spleens of WT mice and CD8 + CD25 − T cells from the spleens of DNIL-10R mice were isolated on day 6 after H5N1 infection and transferred into CD8 KO mice (5 × 10 5 CD8 + CD25 + T cells and 1 × 10 7 CD8 + CD25 − T cells per mouse) that were then immediately infected with H5N1 virus (day 0 of challenge). (B) Survival of mice ( n = 10 per group) was monitored from day 4 to 16 after virus infection. Log-rank test for comparisons of survival curves between DNIL-10R CD8 + CD25 − T cells recipient mice and DNIL-10R CD8 + CD25 − T cells plus WT CD8 + Treg cells recipient mice, or between DNIL-10R CD8 + CD25 − T cells plus WT CD8 + Treg cells recipient mice and WT CD8 + CD25 − T cells plus WT CD8 + Treg cells recipient mice; n.s., p > 0.05. **, p < 0.01, log-rank test. (C) Viral load in mouse lungs were assayed on day 6 after virus infection ( n = 5 mice per group). Data are presented as means ± SEM and are representative of four independent experiments. n.s., p > 0.05. * p < 0.05, unpaired two-tailed t -test.

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: Activity Assay, In Vivo, Isolation, Infection, Virus, Two Tailed Test

Journal: eneuro

Article Title: MicroRNA-155 Inhibition Activates Wnt/β-catenin Signaling to Restore Th17/Treg Balance and Protect against Acute Ischemic Stroke

doi: 10.1523/eneuro.0347-24.2024

Figure Lengend Snippet: Figure 4. Reduction of Th17/Treg cell ratio in MCAO mice by miR-155 antagomir. A, Evans blue extravasation indicating the extent of BBB disruption; B, Serum levels of proinflammatory cytokines IL-6, IL-17, and TNF-α measured by ELISA; C, Serum levels of anti-inflammatory cytokines IL-10 and TGF-β measured by ELISA; D, Flow cytometry analysis and determination of Th17 and Treg cell proportions; E, Th17/Treg cell ratio; F, Protein expression levels of Foxp3 and ROR-γt detected by Western blotting. ** indicates a p value of <0.01 compared with the Sham group; # indicates a p value of <0.05; ## indicates a p value of <0.01 compared with the MCAO + antagomir NC group; n = 10.

Article Snippet: Single-cell suspensions from fresh spleens of C57BL mice were prepared, and Treg cells were enriched using a negative selection and Treg cell isolation kit (130-092-984, Miltenyi Biotec) following the manufacturer’s instructions.

Techniques: Disruption, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Western Blot

Journal: eneuro

Article Title: MicroRNA-155 Inhibition Activates Wnt/β-catenin Signaling to Restore Th17/Treg Balance and Protect against Acute Ischemic Stroke

doi: 10.1523/eneuro.0347-24.2024

Figure Lengend Snippet: Figure 5. The effect of inhibiting the Wnt/β-catenin pathway on cerebral infarction and neuronal damage in MCAO mice. A, Protein expression of β-catenin and Wnt2b detected using Western blot; B, Evaluation of cerebral infarction in the ipsilateral hemisphere using TTC staining and measurement of infarct volume; C, H&E stain of hippocampal tissues with a scale bar of 50 μm; D, Evaluation of cell apoptosis using TUNEL assay, followed by quantitative anal- ysis; E, Measurement and analysis of Th17 and Treg cell ratio using flow cytometry; F, Measurement and analysis of Th17/Treg cell ratio using flow cytom- etry. *p < 0.05; **p < 0.01 indicates significant difference compared with the MCAO + antagomir NC + DMSO group; #p < 0.05; ##p < 0.01 indicates significant difference compared with the MCAO + miR-155 antagomir + DMSO group; n = 10.

Article Snippet: Single-cell suspensions from fresh spleens of C57BL mice were prepared, and Treg cells were enriched using a negative selection and Treg cell isolation kit (130-092-984, Miltenyi Biotec) following the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Staining, TUNEL Assay, Cytometry

Journal: eneuro

Article Title: MicroRNA-155 Inhibition Activates Wnt/β-catenin Signaling to Restore Th17/Treg Balance and Protect against Acute Ischemic Stroke

doi: 10.1523/eneuro.0347-24.2024

Figure Lengend Snippet: Figure 8. The modulation of the Wnt/β-catenin signaling pathway by miR-155 targeting affecting Th17/Treg cell balance in ischemic brain injury.

Article Snippet: Single-cell suspensions from fresh spleens of C57BL mice were prepared, and Treg cells were enriched using a negative selection and Treg cell isolation kit (130-092-984, Miltenyi Biotec) following the manufacturer’s instructions.

Techniques: