Journal: Frontiers in Immunology

Article Title: Shifting gears: Study of immune system parameters of male habitual marathon runners

doi: 10.3389/fimmu.2022.1009065

Figure Lengend Snippet: Gating strategy for isolation of CD3+CD4+CD25+ T cells and CD3+CD4+CD25- T cells from PBMCs. Characteristic dot plots showing purity after cell sorting using BD FACS Aria III Cell Sorter.

Article Snippet: CD3+CD4+CD25+ and CD3+CD4+CD25- T cells were isolated from PBMCs using the BD FACS Aria III Cell Sorter.

Techniques: Isolation, FACS

Journal: Theranostics

Article Title: Inhibition of Notch1 induces population and suppressive activity of regulatory T cell in inflammatory arthritis

doi: 10.7150/thno.26093

Figure Lengend Snippet: Administration of γ-secretase inhibitors induces Treg population in CIA-induced DBA1/J mice and cytokines are altered by γ-secretase inhibitors in human RA patients' synoviocytes. (A) Proportions of CD4 + CD25 + Foxp3 + cells among splenocytes of vehicle-, L-685,458-, or RO4929097-treated mice at 46 days after primary immunization evaluated by flow cytometry. Numbers indicate percentages of CD25 + Foxp3 + cells among CD4 + cells. (B) Statistical analysis of CD4 + CD25 + Foxp3 + cells in splenocytes. Values are mean ± SEM. ** P < 0.01. (C) Quantitative real-time PCR analyses of relative mRNA expression levels in RA patients' synoviocytes treated with 5 μM DAPT or 5 μM L-685,458 for 24 h. Value are mean ± SEM (n=3). * P < 0.05, ** P < 0.01, *** P < 0.001 versus controls.

Article Snippet: Non-regulatory CD4 + CD25 - T responder (Tresp) cells were labeled with 1 μM of carboxy fluorescein diacetate succinimidyl ester (CFSE: eBioscience).

Techniques: Flow Cytometry, Real-time Polymerase Chain Reaction, Expressing

Journal: Theranostics

Article Title: Inhibition of Notch1 induces population and suppressive activity of regulatory T cell in inflammatory arthritis

doi: 10.7150/thno.26093

Figure Lengend Snippet: Treg population is increased in CIA-induced NAS mice. (A) Proportions of CD4 + CD25 + Foxp3 + cells among splenocytes of NAS CIA or WT CIA mice at 28 days after primary immunization evaluated by flow cytometry. Numbers indicate percentages of CD25 + Foxp3 + cells among CD4 + cells. (B) Statistical analysis of CD4 + CD25 + Foxp3 + cells. Values are mean ± SEM. *** P < 0.001. (C) At 28 days after primary immunization, both CD4 and CD8 T cells from splenic lymphocytes of NAS or WT CIA mice were analysed by flow cytometry. (D) Statistical data shown as bar graphs. Values are mean ± SEM. *** P < 0.001.

Article Snippet: Non-regulatory CD4 + CD25 - T responder (Tresp) cells were labeled with 1 μM of carboxy fluorescein diacetate succinimidyl ester (CFSE: eBioscience).

Techniques: Flow Cytometry

Journal: Theranostics

Article Title: Inhibition of Notch1 induces population and suppressive activity of regulatory T cell in inflammatory arthritis

doi: 10.7150/thno.26093

Figure Lengend Snippet: (A, C) Notch inhibition increases Treg population in the CAIA model. Proportions of CD4 + Foxp3 + Treg cells among splenocytes of indicated mice at 9 days after αCII-IA induction were evaluated by flow cytometry. Numbers indicate percentages of CD4 + Foxp3 + cells. Values are mean ± SEM. ** P < 0.01. (B, D) Proportions of T effector cells among splenocytes. Numbers indicate percentages of CD62L low CD44 high cells. *P < 0.05 versus control. (E) Ratios between effector T cells and regulatory T cells (Teff/Treg). Values are mean ± SEM. * P < 0.05 versus control (WT/Vehicle). (F) Suppressive effects of Treg cells were anlayzed by CFSE-based suppression assay. Values are mean ± SEM. ## P < 0.01 versus WT mice; * P < 0.05 versus NAS mice. (G, H) Synovial tissues of CAIA were used for immunohistochemical staining analysis. Consecutive sections of synovial tissues from WT CAIA and NAS CAIA mice were DAB-stained for CD3 and Foxp3. Quantitative analysis is shown in the bar graph. Values are mean ± SEM. * P < 0.05. Scale bar: 25 μm.

Article Snippet: Non-regulatory CD4 + CD25 - T responder (Tresp) cells were labeled with 1 μM of carboxy fluorescein diacetate succinimidyl ester (CFSE: eBioscience).

Techniques: Inhibition, Flow Cytometry, Control, Suppression Assay, Immunohistochemical staining, Staining

Journal: Diabetes

Article Title: Rapamycin Monotherapy in Patients With Type 1 Diabetes Modifies CD4 + CD25 + FOXP3 + Regulatory T-Cells

doi: 10.2337/db08-0138

Figure Lengend Snippet: Percentages and phenotype of circulating CD4 + T-cells in type 1 diabetic patients before and during rapamycin treatment. PBMCs isolated from type 1 diabetic patients before (PRE) and during (RAPA) rapamycin treatment were stained with the indicated mAbs and analyzed by FACS. Representative plots of samples collected and analyzed from patient Hsr-064-ITA-rp04, before and during rapamycin therapy, are shown on the left . Numbers indicate how many cells express each marker. Graphs including analyses performed in all patients (each distinguishable by a specific symbol, see ) are shown on the right , and the solid line represents the average level of type 1 diabetes PRE and type 1 diabetes RAPA samples. Statistical analysis is shown in each graph. A : Percentages of total CD4 + T-cells within PBMCs. B : Percentages of CD25 bright T-cells within the CD4 + T-cell compartment. C : Percentages of CD127 − FOXP3 + , CTLA-4 + , CD45RO + , HLA-DR + , and CD62L + cells within the CD4 + CD25 bright T-cells. FOXP3 mean fluorescence intensity of CD4 + CD25 bright CD127 − FOXP3 + cells is shown for both PRE and RAPA samples.

Article Snippet: FACS-sorted CD4 + CD25 − T-cells were plated at 1 × 10 4 cells per well (in 200 μl X-vivo 15 [Cambrex-Biowhittaker] supplemented with 10% human serum AB [Sigma], 100 units/ml penicillin, and 100 units/ml streptomycin) in 96 round-bottom plates and stimulated with anti CD3/CD28-coupled beads (one bead per six cells) (Invitrogen-Dynal, Oslo, Norway).

Techniques: Isolation, Staining, Marker, Fluorescence

Journal: Diabetes

Article Title: Rapamycin Monotherapy in Patients With Type 1 Diabetes Modifies CD4 + CD25 + FOXP3 + Regulatory T-Cells

doi: 10.2337/db08-0138

Figure Lengend Snippet: In vitro suppressive function of FACS-sorted CD4 + CD25 bright CD127 − and CD4 + CD25 bright T-cells. FACS-sorted 10 4 effector CD4 + CD25 − T-cells (Eff.) were activated polyclonally with magnetic beads coated with anti-CD3+CD28 mAbs alone or in the presence of equal amounts of FACS-sorted autologous CD4 + CD25 bright CD127 − nTregs ( left ) or CD4 + CD25 bright nTregs ( right ). Cell proliferation was assessed in all cultures at day 4 after addition of [ 3 H]thymidine for the last 18 h of culture. Percentage of suppression is indicated. One representative experiment of four is shown.

Article Snippet: FACS-sorted CD4 + CD25 − T-cells were plated at 1 × 10 4 cells per well (in 200 μl X-vivo 15 [Cambrex-Biowhittaker] supplemented with 10% human serum AB [Sigma], 100 units/ml penicillin, and 100 units/ml streptomycin) in 96 round-bottom plates and stimulated with anti CD3/CD28-coupled beads (one bead per six cells) (Invitrogen-Dynal, Oslo, Norway).

Techniques: In Vitro, Magnetic Beads

Journal: Diabetes

Article Title: Rapamycin Monotherapy in Patients With Type 1 Diabetes Modifies CD4 + CD25 + FOXP3 + Regulatory T-Cells

doi: 10.2337/db08-0138

Figure Lengend Snippet: Cytokine production profile of FACS-sorted CD4 + CD25 − T-cells. FACS-sorted CD4 + CD25 − T-cells isolated from PBMCs of type 1 diabetic patients before (PRE) and during (RAPA) rapamycin treatment and from normal donors (ND) were activated polyclonally (5 × 10 4 cells/ml) with beads coated with anti-CD3 + CD28 mAbs, and supernatants were collected 72 h after activation. The indicated cytokines were evaluated by cytokine-bead array. Each patient is identifiable by a specific symbol, and each star represents a different normal donor. The solid line represents average levels of cytokines produced by type 1 diabetes PRE, type 1 diabetes RAPA, and ND CD4 + CD25 − T-cells.

Article Snippet: FACS-sorted CD4 + CD25 − T-cells were plated at 1 × 10 4 cells per well (in 200 μl X-vivo 15 [Cambrex-Biowhittaker] supplemented with 10% human serum AB [Sigma], 100 units/ml penicillin, and 100 units/ml streptomycin) in 96 round-bottom plates and stimulated with anti CD3/CD28-coupled beads (one bead per six cells) (Invitrogen-Dynal, Oslo, Norway).

Techniques: Isolation, Activation Assay, Produced

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:

Article Title: Functional Foxp3 + CD4 + CD25 (Bright+) "Natural" Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4 + and CD8 + Effector T Cells during Ocular Herpes Infection ▿

doi: 10.1128/JVI.00294-07

Figure Lengend Snippet: Number of immune cells isolated from individual conjunctivas of rabbits with no ocular inflammatory disease

Article Snippet: All protein-loading controls were confirmed by stripping and reprobing the same blots with a mAb specific for the rabbit housekeeping protein β-actin. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Western blot analysis of CD4, CD25, CTLA4, GITR, and Foxp3 proteins from CD4 + CD25 + T cells versus CD4 + CD25 − T cells isolated from normal rabbit conjunctiva. (A) CD4 (lanes: 1, positive control CCRF-CEM cell extract; 2, 13 μg CD4 + CD25 + cell lysate; 3, 13 μg CD4 + CD25 − cell lysates; 4, 27 μg CD4 + CD25 + ; 5, 27 μg CD4 + CD25 − ), CD25 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 − CD25 − ), CTLA4 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 + CD25 − ), GITR (lanes: 1, CCRF-CEM; 2, 7 μg CD4 + CD25 + ; 3, 7 μg CD4 + CD25 − ) (arrowheads indicate GITR monomer and dimer), and Foxp3 (lanes: 1, 2.5 μg human PBMC; 2, 5 μg human PBMC; 3, 7.5 μg human PBMC; 4, 6 μg CD4 + CD25 − ; 5, 12 μg CD4 + CD25 − ; 6, 18 μg CD4 + CD25 − ; 7, 6 μg CD4 + CD25 + ; 8, 12 μg CD4 + CD25 + ; 9, 18 μg CD4 + CD25 +) . (B) Results from three experiments normalized to β-actin.

Techniques: Isolation

Journal:

Article Title: Functional Foxp3 + CD4 + CD25 (Bright+) "Natural" Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4 + and CD8 + Effector T Cells during Ocular Herpes Infection ▿

doi: 10.1128/JVI.00294-07

Figure Lengend Snippet: Percentages of CD4 + CD25 + and CD4 + CD25 − T cells detected within CD45 + ICL populations a

Article Snippet: All protein-loading controls were confirmed by stripping and reprobing the same blots with a mAb specific for the rabbit housekeeping protein β-actin. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Western blot analysis of CD4, CD25, CTLA4, GITR, and Foxp3 proteins from CD4 + CD25 + T cells versus CD4 + CD25 − T cells isolated from normal rabbit conjunctiva. (A) CD4 (lanes: 1, positive control CCRF-CEM cell extract; 2, 13 μg CD4 + CD25 + cell lysate; 3, 13 μg CD4 + CD25 − cell lysates; 4, 27 μg CD4 + CD25 + ; 5, 27 μg CD4 + CD25 − ), CD25 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 − CD25 − ), CTLA4 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 + CD25 − ), GITR (lanes: 1, CCRF-CEM; 2, 7 μg CD4 + CD25 + ; 3, 7 μg CD4 + CD25 − ) (arrowheads indicate GITR monomer and dimer), and Foxp3 (lanes: 1, 2.5 μg human PBMC; 2, 5 μg human PBMC; 3, 7.5 μg human PBMC; 4, 6 μg CD4 + CD25 − ; 5, 12 μg CD4 + CD25 − ; 6, 18 μg CD4 + CD25 − ; 7, 6 μg CD4 + CD25 + ; 8, 12 μg CD4 + CD25 + ; 9, 18 μg CD4 + CD25 +) . (B) Results from three experiments normalized to β-actin.

Techniques: Control

Journal:

Article Title: Functional Foxp3 + CD4 + CD25 (Bright+) "Natural" Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4 + and CD8 + Effector T Cells during Ocular Herpes Infection ▿

doi: 10.1128/JVI.00294-07

Figure Lengend Snippet: CD4+ CD25+ T cells are a predominant subpopulation of rabbit intraconjunctival CD45+ leukocytes. Fresh conjunctiva, PBMC, and spleen were harvested from normal rabbits. Immunomagnetic enrichment was used to sort out CD45+ leukocytes. The sorted CD45+ leukocyte population was stained with rabbit-specific antibody to CD4 and CD25 molecules (left three graphs) or with isotype control mAbs. Shown are representative experiments from a total of five experiments.

Article Snippet: All protein-loading controls were confirmed by stripping and reprobing the same blots with a mAb specific for the rabbit housekeeping protein β-actin. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Western blot analysis of CD4, CD25, CTLA4, GITR, and Foxp3 proteins from CD4 + CD25 + T cells versus CD4 + CD25 − T cells isolated from normal rabbit conjunctiva. (A) CD4 (lanes: 1, positive control CCRF-CEM cell extract; 2, 13 μg CD4 + CD25 + cell lysate; 3, 13 μg CD4 + CD25 − cell lysates; 4, 27 μg CD4 + CD25 + ; 5, 27 μg CD4 + CD25 − ), CD25 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 − CD25 − ), CTLA4 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 + CD25 − ), GITR (lanes: 1, CCRF-CEM; 2, 7 μg CD4 + CD25 + ; 3, 7 μg CD4 + CD25 − ) (arrowheads indicate GITR monomer and dimer), and Foxp3 (lanes: 1, 2.5 μg human PBMC; 2, 5 μg human PBMC; 3, 7.5 μg human PBMC; 4, 6 μg CD4 + CD25 − ; 5, 12 μg CD4 + CD25 − ; 6, 18 μg CD4 + CD25 − ; 7, 6 μg CD4 + CD25 + ; 8, 12 μg CD4 + CD25 + ; 9, 18 μg CD4 + CD25 +) . (B) Results from three experiments normalized to β-actin.

Techniques: Staining, Control

Journal:

Article Title: Functional Foxp3 + CD4 + CD25 (Bright+) "Natural" Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4 + and CD8 + Effector T Cells during Ocular Herpes Infection ▿

doi: 10.1128/JVI.00294-07

Figure Lengend Snippet: Western blot analysis of CD4, CD25, CTLA4, GITR, and Foxp3 proteins from CD4+ CD25+ T cells versus CD4+ CD25− T cells isolated from normal rabbit conjunctiva. (A) CD4 (lanes: 1, positive control CCRF-CEM cell extract; 2, 13 μg CD4+ CD25+ cell lysate; 3, 13 μg CD4+ CD25− cell lysates; 4, 27 μg CD4+ CD25+; 5, 27 μg CD4+ CD25−), CD25 (lanes: 1, 7 μg CD4+ CD25+; 2, 7 μg CD4+ CD25−; 3, 14 μg CD4+ CD25+; 4, 14 μg CD4− CD25−), CTLA4 (lanes: 1, 7 μg CD4+ CD25+; 2, 7 μg CD4+ CD25−; 3, 14 μg CD4+ CD25+; 4, 14 μg CD4+ CD25−), GITR (lanes: 1, CCRF-CEM; 2, 7 μg CD4+ CD25+; 3, 7 μg CD4+ CD25−) (arrowheads indicate GITR monomer and dimer), and Foxp3 (lanes: 1, 2.5 μg human PBMC; 2, 5 μg human PBMC; 3, 7.5 μg human PBMC; 4, 6 μg CD4+ CD25−; 5, 12 μg CD4+ CD25−; 6, 18 μg CD4+ CD25−; 7, 6 μg CD4+ CD25+; 8, 12 μg CD4+ CD25+; 9, 18 μg CD4+ CD25+). (B) Results from three experiments normalized to β-actin.

Article Snippet: All protein-loading controls were confirmed by stripping and reprobing the same blots with a mAb specific for the rabbit housekeeping protein β-actin. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Western blot analysis of CD4, CD25, CTLA4, GITR, and Foxp3 proteins from CD4 + CD25 + T cells versus CD4 + CD25 − T cells isolated from normal rabbit conjunctiva. (A) CD4 (lanes: 1, positive control CCRF-CEM cell extract; 2, 13 μg CD4 + CD25 + cell lysate; 3, 13 μg CD4 + CD25 − cell lysates; 4, 27 μg CD4 + CD25 + ; 5, 27 μg CD4 + CD25 − ), CD25 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 − CD25 − ), CTLA4 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 + CD25 − ), GITR (lanes: 1, CCRF-CEM; 2, 7 μg CD4 + CD25 + ; 3, 7 μg CD4 + CD25 − ) (arrowheads indicate GITR monomer and dimer), and Foxp3 (lanes: 1, 2.5 μg human PBMC; 2, 5 μg human PBMC; 3, 7.5 μg human PBMC; 4, 6 μg CD4 + CD25 − ; 5, 12 μg CD4 + CD25 − ; 6, 18 μg CD4 + CD25 − ; 7, 6 μg CD4 + CD25 + ; 8, 12 μg CD4 + CD25 + ; 9, 18 μg CD4 + CD25 +) . (B) Results from three experiments normalized to β-actin.

Techniques: Western Blot, Isolation, Positive Control

Journal:

Article Title: Functional Foxp3 + CD4 + CD25 (Bright+) "Natural" Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4 + and CD8 + Effector T Cells during Ocular Herpes Infection ▿

doi: 10.1128/JVI.00294-07

Figure Lengend Snippet: Normal conjunctiva CD4+ CD25+ T cells exhibit phenotypic characteristics of nTreg cells. Upper and lower fresh conjunctivas were collected from normal rabbits, and CD4+ CD25+ T cells were analyzed by FACS either as unsorted conjunctival cells or as sorted CD45+ leukocytes. (A) The percentage of CD4+ CD25(Bright+) and CD4+ CD25(low) T-cell subsets before sorting of cells (left) and within the CD45+ T-cell-enriched population (right). (B) Expression of transcription factor Foxp3 in conjunctiva CD4+ CD25(Bright+) T cells in two independent experiments. (C) Detection of cell surface GITR and intracytoplasmic CTLA4 proteins in CD4+ CD25(Bright+) T cells by FACS. CTLA4 staining was performed after cell membrane permeabilization. Shown is a representative experiment of a total of five experiments. (D) Relative mean florescent intensities in CD4+ CD25(Bright+) versus CD4+ CD25(low) cells.

Article Snippet: All protein-loading controls were confirmed by stripping and reprobing the same blots with a mAb specific for the rabbit housekeeping protein β-actin. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Western blot analysis of CD4, CD25, CTLA4, GITR, and Foxp3 proteins from CD4 + CD25 + T cells versus CD4 + CD25 − T cells isolated from normal rabbit conjunctiva. (A) CD4 (lanes: 1, positive control CCRF-CEM cell extract; 2, 13 μg CD4 + CD25 + cell lysate; 3, 13 μg CD4 + CD25 − cell lysates; 4, 27 μg CD4 + CD25 + ; 5, 27 μg CD4 + CD25 − ), CD25 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 − CD25 − ), CTLA4 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 + CD25 − ), GITR (lanes: 1, CCRF-CEM; 2, 7 μg CD4 + CD25 + ; 3, 7 μg CD4 + CD25 − ) (arrowheads indicate GITR monomer and dimer), and Foxp3 (lanes: 1, 2.5 μg human PBMC; 2, 5 μg human PBMC; 3, 7.5 μg human PBMC; 4, 6 μg CD4 + CD25 − ; 5, 12 μg CD4 + CD25 − ; 6, 18 μg CD4 + CD25 − ; 7, 6 μg CD4 + CD25 + ; 8, 12 μg CD4 + CD25 + ; 9, 18 μg CD4 + CD25 +) . (B) Results from three experiments normalized to β-actin.

Techniques: Expressing, Staining, Membrane

Journal:

Article Title: Functional Foxp3 + CD4 + CD25 (Bright+) "Natural" Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4 + and CD8 + Effector T Cells during Ocular Herpes Infection ▿

doi: 10.1128/JVI.00294-07

Figure Lengend Snippet: CD4+ CD25+ cell distribution in superior and inferior conjunctiva. (A) Schematic location of samples tested in this study. a and f, limbal conjunctiva; b and g, bulbar conjunctiva, c and h, fornix conjunctiva; d and i, palpebral conjunctiva; e and j, conjunctiva near the lid margin. Sections from different regions of superior and inferior conjunctiva, indicated by the letters a to j shown in A, were stained with mAbs specific for rabbit CD4 or anti-CD25 molecules. (B) Maximum-intensity projection image of scanning of conjunctiva stained for anti-CD4 (upper two lines) or anti-CD25 (lower two lines). Labels a thorough j are matched to Fig. ​Fig.2A.2A. The scale bar shows 200 mm. (C) Image reconstructed from scanned images of (a) limbal conjunctiva, (b) fornix conjunctiva, and (c) lid margin conjunctiva.

Article Snippet: All protein-loading controls were confirmed by stripping and reprobing the same blots with a mAb specific for the rabbit housekeeping protein β-actin. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Western blot analysis of CD4, CD25, CTLA4, GITR, and Foxp3 proteins from CD4 + CD25 + T cells versus CD4 + CD25 − T cells isolated from normal rabbit conjunctiva. (A) CD4 (lanes: 1, positive control CCRF-CEM cell extract; 2, 13 μg CD4 + CD25 + cell lysate; 3, 13 μg CD4 + CD25 − cell lysates; 4, 27 μg CD4 + CD25 + ; 5, 27 μg CD4 + CD25 − ), CD25 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 − CD25 − ), CTLA4 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 + CD25 − ), GITR (lanes: 1, CCRF-CEM; 2, 7 μg CD4 + CD25 + ; 3, 7 μg CD4 + CD25 − ) (arrowheads indicate GITR monomer and dimer), and Foxp3 (lanes: 1, 2.5 μg human PBMC; 2, 5 μg human PBMC; 3, 7.5 μg human PBMC; 4, 6 μg CD4 + CD25 − ; 5, 12 μg CD4 + CD25 − ; 6, 18 μg CD4 + CD25 − ; 7, 6 μg CD4 + CD25 + ; 8, 12 μg CD4 + CD25 + ; 9, 18 μg CD4 + CD25 +) . (B) Results from three experiments normalized to β-actin.

Techniques: Staining

Journal:

Article Title: Functional Foxp3 + CD4 + CD25 (Bright+) "Natural" Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4 + and CD8 + Effector T Cells during Ocular Herpes Infection ▿

doi: 10.1128/JVI.00294-07

Figure Lengend Snippet: CD4+ CD25+ cell distribution in superior and inferior conjunctivas. (A) Schematic location of samples tested in this study. Images were reconstructed from scanned images of (a) limbal conjunctiva, (b) fornix conjunctiva, and (c) lid margin conjunctiva. Blue and green indicate nuclei of conjunctival cells and CD4+ cells, respectively. (B) accumulation of the CD4+ CD25+ cells at the superior palpebral conjunctiva. (C) Maximum-intensity projection image of x-y scanning of CD4+ cells at the lid margin (region e). (D) x-z reconstructed image from x-y scanning image. CD4+ cells accumulate at the superficial conjunctiva. Scale bar, 200 mm.

Article Snippet: All protein-loading controls were confirmed by stripping and reprobing the same blots with a mAb specific for the rabbit housekeeping protein β-actin. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Western blot analysis of CD4, CD25, CTLA4, GITR, and Foxp3 proteins from CD4 + CD25 + T cells versus CD4 + CD25 − T cells isolated from normal rabbit conjunctiva. (A) CD4 (lanes: 1, positive control CCRF-CEM cell extract; 2, 13 μg CD4 + CD25 + cell lysate; 3, 13 μg CD4 + CD25 − cell lysates; 4, 27 μg CD4 + CD25 + ; 5, 27 μg CD4 + CD25 − ), CD25 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 − CD25 − ), CTLA4 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 + CD25 − ), GITR (lanes: 1, CCRF-CEM; 2, 7 μg CD4 + CD25 + ; 3, 7 μg CD4 + CD25 − ) (arrowheads indicate GITR monomer and dimer), and Foxp3 (lanes: 1, 2.5 μg human PBMC; 2, 5 μg human PBMC; 3, 7.5 μg human PBMC; 4, 6 μg CD4 + CD25 − ; 5, 12 μg CD4 + CD25 − ; 6, 18 μg CD4 + CD25 − ; 7, 6 μg CD4 + CD25 + ; 8, 12 μg CD4 + CD25 + ; 9, 18 μg CD4 + CD25 +) . (B) Results from three experiments normalized to β-actin.

Techniques:

Journal:

Article Title: Functional Foxp3 + CD4 + CD25 (Bright+) "Natural" Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4 + and CD8 + Effector T Cells during Ocular Herpes Infection ▿

doi: 10.1128/JVI.00294-07

Figure Lengend Snippet: Normal conjunctiva CD4+ CD25(Bright+) T cells exhibit functional properties of nTreg cells. (A) Effect of addition of naïve conjunctiva-derived CD4+ CD25(Bright+) T cells on proliferative responses of CD4+ Teff cells from ocularly infected rabbits to whole HSV-1 recall antigens. Proliferation assays of HSV-1-specific CD4+ CD25− Teff cells were performed by stimulating a constant number (1 × 105 cells) of immune HSV-specific CD4+ CD25− Teff cells from either the conjunctiva (top) or PBMC (bottom) of HSV-1-infected rabbits in the absence or presence of increasing numbers of conjunctiva-derived CD4+ CD25(Bright+) T cells. As a control, the open bars show CD4+ CD25+ instead of CD4+ CD25− cells used as responder cells. Data are the differences between the mean counts per minute for CD4+ CD25−-stimulated Teff cells minus the mean counts per minute for unstimulated CD4+ CD25− Teff cells. (B) T-cell proliferation assays were performed by stimulating CD4+ CD25− Teff cells (left panels) or CD8+ Teff cells (right panels) derived from immune conjunctiva (5 × 104 cells) with either heat-inactivated HSV (multiplicity of infection of 0.3) (circles) or an anti-rabbit CD3 mAb (triangles) in the absence or in the presence of increasing numbers of conjunctiva-derived CD4+ CD25(Bright+) T cells (closed symbols) or CD4+ CD25− T cells (open symbols). The ratio of CD4+ CD25+ T cells to CD4+ CD25− and CD8+ CD25− Teff cells is indicated on the x axis in each graph. These panels are representative data from a total of three separate experiments. Values are means ± standard deviations of triplicate experiments per condition (*, P < 0.05).

Article Snippet: All protein-loading controls were confirmed by stripping and reprobing the same blots with a mAb specific for the rabbit housekeeping protein β-actin. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Western blot analysis of CD4, CD25, CTLA4, GITR, and Foxp3 proteins from CD4 + CD25 + T cells versus CD4 + CD25 − T cells isolated from normal rabbit conjunctiva. (A) CD4 (lanes: 1, positive control CCRF-CEM cell extract; 2, 13 μg CD4 + CD25 + cell lysate; 3, 13 μg CD4 + CD25 − cell lysates; 4, 27 μg CD4 + CD25 + ; 5, 27 μg CD4 + CD25 − ), CD25 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 − CD25 − ), CTLA4 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 + CD25 − ), GITR (lanes: 1, CCRF-CEM; 2, 7 μg CD4 + CD25 + ; 3, 7 μg CD4 + CD25 − ) (arrowheads indicate GITR monomer and dimer), and Foxp3 (lanes: 1, 2.5 μg human PBMC; 2, 5 μg human PBMC; 3, 7.5 μg human PBMC; 4, 6 μg CD4 + CD25 − ; 5, 12 μg CD4 + CD25 − ; 6, 18 μg CD4 + CD25 − ; 7, 6 μg CD4 + CD25 + ; 8, 12 μg CD4 + CD25 + ; 9, 18 μg CD4 + CD25 +) . (B) Results from three experiments normalized to β-actin.

Techniques: Functional Assay, Derivative Assay, Infection, Control

Journal:

Article Title: Functional Foxp3 + CD4 + CD25 (Bright+) "Natural" Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4 + and CD8 + Effector T Cells during Ocular Herpes Infection ▿

doi: 10.1128/JVI.00294-07

Figure Lengend Snippet: Suppressive activity of conjunctival CD4+ CD25+ Treg cells is cell-cell contact dependent but independent of IL-10 and TGF-β1. (A) CD4+ CD25+ and CD4 CD25− T cells were isolated from naïve rabbit conjunctiva. The cells were lysed in RLT buffer by scraping, immediately frozen in liquid nitrogen, and then thawed on ice. The RNAs were eluted from the silica column, and the product was analyzed to verify both the quantity and quality of the RNA as described in Materials and Methods. The RNA was obtained from CD4+ CD25+ and CD4 CD25 rabbit T cells and was subjected to PCR using primers specific for IL-10, TGF-β1, GAPDH, and β-actin. Lanes: −, CD4+ CD25 rabbit cell RNAs; +, CD4+ CD25+ cell RNAs; B, control buffer. (B) Western blot analysis of IL-10 and TGF-β1 proteins from CD4+ CD25+ T cells versus CD4+ CD25− T cells isolated from normal rabbit conjunctiva. Data for TGF-β1 (lanes: 1, 13 μg CD4+ CD25+ cell lysate; 2, 13 μg CD4+ CD25− cell lysates) and IL-10 (lanes: 1, positive control CCRF-CEM cell extract; 2, 13 μg CD4+ CD25+ cell lysate; 3, 13 μg CD4+ CD25− cell lysates) are shown. (C) Immune PBMC (white columns) or immune conjunctiva (black columns)-infiltrating CD4 CD25− T cells induced during an ocular infection with HSV-1 (McKrae) were depleted of CD8+ cells and cultured in the presence of normal conjunctiva CD4+ CD25+ regulatory T cells alone (no mAbs) or with anti-IL-10, anti-TGF-β1, or isotype control IgG. (D) Immune conjunctiva-infiltrating CD4 CD25− Teff cells induced during an ocular infection with HSV-1 (McKrae) were cultured in the presence of normal conjunctiva CD4+ CD25+ T cells but separated through a microporous membrane (no cell-cell contact) and not separated together in one well (cell-cell contact). Data are the means of data from triplicate wells.

Article Snippet: All protein-loading controls were confirmed by stripping and reprobing the same blots with a mAb specific for the rabbit housekeeping protein β-actin. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Western blot analysis of CD4, CD25, CTLA4, GITR, and Foxp3 proteins from CD4 + CD25 + T cells versus CD4 + CD25 − T cells isolated from normal rabbit conjunctiva. (A) CD4 (lanes: 1, positive control CCRF-CEM cell extract; 2, 13 μg CD4 + CD25 + cell lysate; 3, 13 μg CD4 + CD25 − cell lysates; 4, 27 μg CD4 + CD25 + ; 5, 27 μg CD4 + CD25 − ), CD25 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 − CD25 − ), CTLA4 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 + CD25 − ), GITR (lanes: 1, CCRF-CEM; 2, 7 μg CD4 + CD25 + ; 3, 7 μg CD4 + CD25 − ) (arrowheads indicate GITR monomer and dimer), and Foxp3 (lanes: 1, 2.5 μg human PBMC; 2, 5 μg human PBMC; 3, 7.5 μg human PBMC; 4, 6 μg CD4 + CD25 − ; 5, 12 μg CD4 + CD25 − ; 6, 18 μg CD4 + CD25 − ; 7, 6 μg CD4 + CD25 + ; 8, 12 μg CD4 + CD25 + ; 9, 18 μg CD4 + CD25 +) . (B) Results from three experiments normalized to β-actin.

Techniques: Activity Assay, Isolation, Control, Western Blot, Positive Control, Infection, Cell Culture, Membrane

Journal:

Article Title: Functional Foxp3 + CD4 + CD25 (Bright+) "Natural" Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4 + and CD8 + Effector T Cells during Ocular Herpes Infection ▿

doi: 10.1128/JVI.00294-07

Figure Lengend Snippet: Relative quantitation of IL-10 from rabbit conjunctiva CD4 + CD25 + and CD4 + CD25 − T cells by real-time PCR a

Article Snippet: All protein-loading controls were confirmed by stripping and reprobing the same blots with a mAb specific for the rabbit housekeeping protein β-actin. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Western blot analysis of CD4, CD25, CTLA4, GITR, and Foxp3 proteins from CD4 + CD25 + T cells versus CD4 + CD25 − T cells isolated from normal rabbit conjunctiva. (A) CD4 (lanes: 1, positive control CCRF-CEM cell extract; 2, 13 μg CD4 + CD25 + cell lysate; 3, 13 μg CD4 + CD25 − cell lysates; 4, 27 μg CD4 + CD25 + ; 5, 27 μg CD4 + CD25 − ), CD25 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 − CD25 − ), CTLA4 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 + CD25 − ), GITR (lanes: 1, CCRF-CEM; 2, 7 μg CD4 + CD25 + ; 3, 7 μg CD4 + CD25 − ) (arrowheads indicate GITR monomer and dimer), and Foxp3 (lanes: 1, 2.5 μg human PBMC; 2, 5 μg human PBMC; 3, 7.5 μg human PBMC; 4, 6 μg CD4 + CD25 − ; 5, 12 μg CD4 + CD25 − ; 6, 18 μg CD4 + CD25 − ; 7, 6 μg CD4 + CD25 + ; 8, 12 μg CD4 + CD25 + ; 9, 18 μg CD4 + CD25 +) . (B) Results from three experiments normalized to β-actin.

Techniques: Quantitation Assay, Real-time Polymerase Chain Reaction

Journal:

Article Title: Functional Foxp3 + CD4 + CD25 (Bright+) "Natural" Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4 + and CD8 + Effector T Cells during Ocular Herpes Infection ▿

doi: 10.1128/JVI.00294-07

Figure Lengend Snippet: Relative quantitation of TGF-β1 from rabbit conjunctiva CD4 + CD25 + and CD4 + CD25 − T cells by real-time PCR a

Article Snippet: All protein-loading controls were confirmed by stripping and reprobing the same blots with a mAb specific for the rabbit housekeeping protein β-actin. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Western blot analysis of CD4, CD25, CTLA4, GITR, and Foxp3 proteins from CD4 + CD25 + T cells versus CD4 + CD25 − T cells isolated from normal rabbit conjunctiva. (A) CD4 (lanes: 1, positive control CCRF-CEM cell extract; 2, 13 μg CD4 + CD25 + cell lysate; 3, 13 μg CD4 + CD25 − cell lysates; 4, 27 μg CD4 + CD25 + ; 5, 27 μg CD4 + CD25 − ), CD25 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 − CD25 − ), CTLA4 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 + CD25 − ), GITR (lanes: 1, CCRF-CEM; 2, 7 μg CD4 + CD25 + ; 3, 7 μg CD4 + CD25 − ) (arrowheads indicate GITR monomer and dimer), and Foxp3 (lanes: 1, 2.5 μg human PBMC; 2, 5 μg human PBMC; 3, 7.5 μg human PBMC; 4, 6 μg CD4 + CD25 − ; 5, 12 μg CD4 + CD25 − ; 6, 18 μg CD4 + CD25 − ; 7, 6 μg CD4 + CD25 + ; 8, 12 μg CD4 + CD25 + ; 9, 18 μg CD4 + CD25 +) . (B) Results from three experiments normalized to β-actin.

Techniques: Quantitation Assay, Real-time Polymerase Chain Reaction

Journal:

Article Title: Functional Foxp3 + CD4 + CD25 (Bright+) "Natural" Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4 + and CD8 + Effector T Cells during Ocular Herpes Infection ▿

doi: 10.1128/JVI.00294-07

Figure Lengend Snippet: Increased CD4+ CD25+ nTreg cells and their suppressor activity in immune conjunctiva of rabbits with ocular herpes infection. (A) Relative frequency of CD4+ CD25+ T cells isolated from the immune conjunctiva, spleen, and PB of HSV-1-infected rabbits and uninfected controls. Rabbits were either infected ocularly with 2 × 105 PFU of HSV-1 (left panels) or left uninfected (right panels). Eleven days postinfection, conjunctiva, spleen, and blood were collected. The percentages of Foxp3+ CD4+ CD25+ T cells in total CD4+ cells isolated from the conjunctivas of normal rabbits (n = 15) and infected rabbits (n = 17) were determined by using FACS. *, P < 0.05. (B) Conjunctiva CD4+ CD25+ T cells from HSV-1-infected rabbits possess higher regulatory activity. A constant number of immune conjunctiva CD4+ CD25− Teff cells (2 × 105 cells/well) were cocultured with an increasing number of CD4+ CD25+ T cells derived from either normal or immune conjunctiva. As control, a constant number of immune conjunctiva CD4+ CD25− Teff cells (2 × 105 cells/well) were cocultured with an increasing number of CD4+ CD25− T cells derived from normal conjunctiva. After 4 days of culture, T-cell proliferation was measured using an additional overnight [3H]thymidine (1 μCi/well) uptake assay. Results from a representative experiment of three are shown. (C) CD4+ CD25+ Treg cells sorted from immune conjunctiva-mediated suppression were cell-cell contact dependent but independent of IL-10 and TGF-β1. Immune conjunctiva-infiltrating CD4+ CD25− Teff cells induced during an ocular HSV-1 infection were depleted of CD8+ cells and cultured alone or in the presence of immune or normal conjunctiva-derived CD4+ CD25+ regulatory T cells. In some experiments, immune conjunctiva-infiltrating CD4+ CD25− Teff cells were incubated with CD4+ CD25+ T cells that were also from immune conjunctiva and separated through a microporous membrane (no cell-cell contact), and neutralizing antibodies against either IL-10 or TGF-β1 were added to the culture wells. Data are the means of duplicate wells.

Article Snippet: All protein-loading controls were confirmed by stripping and reprobing the same blots with a mAb specific for the rabbit housekeeping protein β-actin. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Western blot analysis of CD4, CD25, CTLA4, GITR, and Foxp3 proteins from CD4 + CD25 + T cells versus CD4 + CD25 − T cells isolated from normal rabbit conjunctiva. (A) CD4 (lanes: 1, positive control CCRF-CEM cell extract; 2, 13 μg CD4 + CD25 + cell lysate; 3, 13 μg CD4 + CD25 − cell lysates; 4, 27 μg CD4 + CD25 + ; 5, 27 μg CD4 + CD25 − ), CD25 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 − CD25 − ), CTLA4 (lanes: 1, 7 μg CD4 + CD25 + ; 2, 7 μg CD4 + CD25 − ; 3, 14 μg CD4 + CD25 + ; 4, 14 μg CD4 + CD25 − ), GITR (lanes: 1, CCRF-CEM; 2, 7 μg CD4 + CD25 + ; 3, 7 μg CD4 + CD25 − ) (arrowheads indicate GITR monomer and dimer), and Foxp3 (lanes: 1, 2.5 μg human PBMC; 2, 5 μg human PBMC; 3, 7.5 μg human PBMC; 4, 6 μg CD4 + CD25 − ; 5, 12 μg CD4 + CD25 − ; 6, 18 μg CD4 + CD25 − ; 7, 6 μg CD4 + CD25 + ; 8, 12 μg CD4 + CD25 + ; 9, 18 μg CD4 + CD25 +) . (B) Results from three experiments normalized to β-actin.

Techniques: Activity Assay, Infection, Isolation, Derivative Assay, Control, Cell Culture, Incubation, Membrane