Journal: BioMed Research International

Article Title: Chronic Heat Stress Inhibits Immune Responses to H5N1 Vaccination through Regulating CD4 + CD25 + Foxp3 + Tregs

doi: 10.1155/2013/160859

Figure Lengend Snippet: CHS regulates Tregs prevalence. Splenic T cells from each group ( n = 3) were isolated on the indicated days postprimary immunization and then analyzed by flow cytometry. (a) Percentages of CD25 + and Foxp3 + cells in CD4 + T cells were calculated at the indicated time points. Results shown are pooled from three independent repeats. (b) Intracellular staining of IL-10 + CD4 + T cells on day 21 was performed (stimulated with a viral-specific antigen for 6 h in vitro before being analyzed by flow cytometry). Representative plots showing the percentages of positive T cells are shown on the left. Data from all experiments are summarized in bar graphs to the right. (c) The expression levels of TGF- β in the CD4 + cells of CHS groups were analyzed by real-time PCR at indicated time points. Data are presented as relative changes in gene expression (fold) normalized to the TN groups and are one representative from three independent experiments.

Article Snippet: CD4 + CD25 + Tregs were isolated and purified using the MagCellect Mouse CD4 + CD25 + T Cell Isolation Kit according to the manufacturer's protocol (R&D Systems, Inc., Minneapolis, USA).

Techniques: Isolation, Flow Cytometry, Staining, In Vitro, Expressing, Real-time Polymerase Chain Reaction, Gene Expression

Journal: BioMed Research International

Article Title: Chronic Heat Stress Inhibits Immune Responses to H5N1 Vaccination through Regulating CD4 + CD25 + Foxp3 + Tregs

doi: 10.1155/2013/160859

Figure Lengend Snippet: Adoptive transfer of CD4 + CD25 + Tregs reduces the vaccine efficacy under TN conditions. 10 6 CD4 + CD25 + Tregs from CHS+Ag group and TN+Ag group were adoptively transferred into TN+Ag mice on day 21 in conjunction with a 50 PFU H5N1 virus challenge. The mortalities of the two transferred groups ( n = 6) were observed for 14 days.

Article Snippet: CD4 + CD25 + Tregs were isolated and purified using the MagCellect Mouse CD4 + CD25 + T Cell Isolation Kit according to the manufacturer's protocol (R&D Systems, Inc., Minneapolis, USA).

Techniques: Adoptive Transfer Assay, Virus

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: Splenic cells were isolated on day 6 after viral infection and purified by using MagCellect Mouse CD4 + CD25 + , CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer∼s protocol.

Techniques: Staining, Infection, Virus, Purification, Quantitative RT-PCR, 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: Splenic cells were isolated on day 6 after viral infection and purified by using MagCellect Mouse CD4 + CD25 + , CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer∼s protocol.

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: Splenic cells were isolated on day 6 after viral infection and purified by using MagCellect Mouse CD4 + CD25 + , CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer∼s protocol.

Techniques: Virus, Isolation, Infection, 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: Splenic cells were isolated on day 6 after viral infection and purified by using MagCellect Mouse CD4 + CD25 + , CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer∼s protocol.

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: Splenic cells were isolated on day 6 after viral infection and purified by using MagCellect Mouse CD4 + CD25 + , CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer∼s protocol.

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

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: Splenic cells were isolated on day 6 after viral infection and purified by using MagCellect Mouse CD4 + CD25 + , CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer∼s protocol.

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