Journal: PLoS ONE

Article Title: Neutrophil Gelatinase-Associated Lipocalin Increases HLA-G + /FoxP3 + T-Regulatory Cell Population in an In Vitro Model of PBMC

doi: 10.1371/journal.pone.0089497

Figure Lengend Snippet: A) Flow cytometry analysis of surface HLA-G on CD4 + and CD8 + cells. PBMCs were treated with increasing concentrations of NGAL:Enterobactin:Iron (40 ng/ml, 80 ng/ml, 160 ng/ml, and 320 ng/ml). A dose-dependent rise in the percentage of CD4 + HLA-G + cells was evident. The percentage of CD8 + HLA-G + cells did not show any marked changes. Data are representative of 8 independent experiments. The number in each quadrant represents the percentage of the total population. B) Data are expressed as percentages of CD4 + HLA-G + cells. Means ± SD; n = 8. * p<0.05.

Article Snippet: Detection of CD4+ CD25+ FoxP3+ regulatory T cells on PBMC was performed with the Treg Detection Kit, CD4/CD25/FoxP3 (Miltenyi, Bergish Gladbach, Germany) as described in the manufacturer's protocol.

Techniques: Flow Cytometry

Journal: PLoS ONE

Article Title: Neutrophil Gelatinase-Associated Lipocalin Increases HLA-G + /FoxP3 + T-Regulatory Cell Population in an In Vitro Model of PBMC

doi: 10.1371/journal.pone.0089497

Figure Lengend Snippet: Flow cytometry analysis of HLA-G expression on CD4 + cells. Iron treatment (without NGAL) did not increase the percentage of HLA-G + cells. Treatment with the NGAL:enterobactin complex (without iron) reduced HLA-G expression in contrast to stimulation with NGAL:Enterobactin:Iron. Incubation with anti-NGAL antibody reduced the percentage of HLAG + cells. Data are representative of 8 independent experiments. The number in each quadrant represents the percentage of the total population. B) Data are expressed as percentages of CD4 + HLA-G + cells. Means ± SD; n = 8. * p<0.05.

Article Snippet: Detection of CD4+ CD25+ FoxP3+ regulatory T cells on PBMC was performed with the Treg Detection Kit, CD4/CD25/FoxP3 (Miltenyi, Bergish Gladbach, Germany) as described in the manufacturer's protocol.

Techniques: Flow Cytometry, Expressing, Incubation

Journal: PLoS ONE

Article Title: Neutrophil Gelatinase-Associated Lipocalin Increases HLA-G + /FoxP3 + T-Regulatory Cell Population in an In Vitro Model of PBMC

doi: 10.1371/journal.pone.0089497

Figure Lengend Snippet: A). The percentage of CD4 + /CD25 + /FoxP3 + cells in PHA-activated PBMCs after stimulation with increasing concentrations of NGAL: Enterobactin:Iron (40 ng/ml, 80 ng/ml, 160 ng/ml, and 320 ng/ml), raised in a dose dependent manner. Data are representative of 8 independent experiments. The number in each quadrant represents the percentage of CD25 + /FoxP3 + cells on gated CD4 + cells of the total population. B) Data are expressed as percentages of CD4 + /CD25 + /FoxP3 + cells. Means ± SD; n = 8. *p<0.05.

Article Snippet: Detection of CD4+ CD25+ FoxP3+ regulatory T cells on PBMC was performed with the Treg Detection Kit, CD4/CD25/FoxP3 (Miltenyi, Bergish Gladbach, Germany) as described in the manufacturer's protocol.

Techniques:

Journal: PLoS ONE

Article Title: Neutrophil Gelatinase-Associated Lipocalin Increases HLA-G + /FoxP3 + T-Regulatory Cell Population in an In Vitro Model of PBMC

doi: 10.1371/journal.pone.0089497

Figure Lengend Snippet: Flow cytometry analysis of CD25 + /FoxP3 + expression on CD4 + cells in PHA-activated PBMCs. Iron treatment (without NGAL) did not increase the percentage of CD25 + /FoxP3 + cells. Treatment with NGAL:enterobactin complex (without iron) reduced the percentage of CD25 + /FoxP3 + cells in contrast to stimulation with NGAL:Enterobactin:Iron. Incubation with anti-NGAL antibody reduced the percentage of CD25 + /FoxP3 + cells. Data are representative of 8 independent experiments. The number in each quadrant represents the percentage of CD25+foxP3 cells on gated CD4 + cells of the total population. B) Data are expressed as percentages of CD4+/CD25+/FoxP3+ cells. Means ± SD; n = 8. * p<0.05.

Article Snippet: Detection of CD4+ CD25+ FoxP3+ regulatory T cells on PBMC was performed with the Treg Detection Kit, CD4/CD25/FoxP3 (Miltenyi, Bergish Gladbach, Germany) as described in the manufacturer's protocol.

Techniques: Flow Cytometry, Expressing, Incubation

Journal: PLoS ONE

Article Title: Neutrophil Gelatinase-Associated Lipocalin Increases HLA-G + /FoxP3 + T-Regulatory Cell Population in an In Vitro Model of PBMC

doi: 10.1371/journal.pone.0089497

Figure Lengend Snippet: Flow cytometry analysis of HLA-G + /FoxP3 + expression on CD4 + cell PHA-activated PBMCs. PBMCs were treated with increasing concentrations of NGAL:Enterobactin:Iron (40 ng/ml, 80 ng/ml, 160 ng/ml, and 320 ng/ml). A dose-dependent raise in the percentage of HLA-G + /FoxP3 + cells was evident. Data are representative of 8 independent experiments. The number in each quadrant represents the percentage of HLA-G + /FoxP3 + cells on gated CD4 + cells of the total population. B) Data are expressed as percentages of CD4 + /HLA-G + /FoxP3 + cells. Means ± SD; n = 8. * p<0.05.

Article Snippet: Detection of CD4+ CD25+ FoxP3+ regulatory T cells on PBMC was performed with the Treg Detection Kit, CD4/CD25/FoxP3 (Miltenyi, Bergish Gladbach, Germany) as described in the manufacturer's protocol.

Techniques: Flow Cytometry, Expressing

Journal: European journal of immunology

Article Title: Phenotypic and functional characteristics of ATP-hydrolysing CD4 + CD39 + FOXP3 + and CD4 + CD39 + FOXP3 neg T-cell subsets in patients with cancer

doi: 10.1002/eji.201142347

Figure Lengend Snippet: (A) PBMCs from HCs were isolated, gated as shown. FOXP3 and PD-1 expression by the CD25+FOXP3+ subset and % of FOXP3+ expression by CD4+CD39+ and CD4+CD39neg T cells were examined by flow cytometry. Box plots show quartiles for 25, 50, 75 as boxes, and values for 0% and 100% as whiskers. (B) CD4+CD39+ T cells were stimulated with SEB for 6 h. Co-expression of TGF-β-associated LAP, GARP, IL-1-receptor CD121a, CTLA4 with FOXP3 was determined. Data are representative (contour plots) or means + SD (bar graphs) of 10 independent experiments each performed using cells from a different HC *p < 0.05 for A&B. All p values in this and other figures were determined using Kruskal-Wallis and two-tailed exact Wilcoxon-Mann Whitney tests.

Article Snippet: For intracellular staining of cytokines, FOXP3, and CTLA4 cells were fixed for 30 min and washed twice with permeabilization buffer using a commercially available FOXP3 staining kit (eBioscience) as previously described [ 22 ].

Techniques: Isolation, Expressing, Flow Cytometry, Two Tailed Test, MANN-WHITNEY

Journal: European journal of immunology

Article Title: Phenotypic and functional characteristics of ATP-hydrolysing CD4 + CD39 + FOXP3 + and CD4 + CD39 + FOXP3 neg T-cell subsets in patients with cancer

doi: 10.1002/eji.201142347

Figure Lengend Snippet: (A) PD-L1 and FOXP3 co-expression on unstimulated CD4+CD39+ T cells isolated from PBMCs of HCs. (B) PBMCs obtained from HCs were stimulated with SEB for 6 h in the presence of Brefeldin A and stained for intracellular IL-6, IL-2, IFN-γ and TNF-α expression in CD4+CD39+ T cells. Density plots are representative for more than 30 individual experiments each performed with cells of a different HC; (C) Subsets of CD4+CD39+ and CD4+CD39neg cells were single-cell sorted, stimulated with OKT3/anti-CD28 Ab-coated beads and cultured. Supernatants were collected after 48 h, and cytokine concentrations were measured by Luminex. The data are means ± SD for three cultures. CD4+CD39neg Tconv cells served as a positive reference for cytokine measurements.

Article Snippet: For intracellular staining of cytokines, FOXP3, and CTLA4 cells were fixed for 30 min and washed twice with permeabilization buffer using a commercially available FOXP3 staining kit (eBioscience) as previously described [ 22 ].

Techniques: Expressing, Isolation, Staining, Cell Culture, Luminex

Journal: European journal of immunology

Article Title: Phenotypic and functional characteristics of ATP-hydrolysing CD4 + CD39 + FOXP3 + and CD4 + CD39 + FOXP3 neg T-cell subsets in patients with cancer

doi: 10.1002/eji.201142347

Figure Lengend Snippet: (A) The two CD4+CD39+ subsets are distinguished by CD25 expression; (B) Mean fluorescence intensity (MFI) of CD39 expression in CD25+FOXP3+ cells and CD25negFOXP3neg cells is shown (n=3 for each subset). (C) The purity of CD4+CD39+ cells single cell-sorted into CD25+ and CD25neg cells is shown in a representative plot. (D) The utilization of exogenous ATP by T cells in Subsets 1 and 2. The data are means ± SD from 3 independent experiments with cells of different HCs (*p < 0.05). (E) Induction of CD39+FOXP3+ Treg cells in vitro from separated CD4+CD39neg Tconv cells stimulated via TCR and cultured in the presence of IL-2 (50 IU/mL) ± TGF-β (10ng/mL). (F) Bar graphs show the percentages of CD39+FOXP3+ Treg cells induced in the presence of IL-2 alone or IL-2 and TGF-β. The data are means ± SD from 5 independent experiments with cells of different HCs.

Article Snippet: For intracellular staining of cytokines, FOXP3, and CTLA4 cells were fixed for 30 min and washed twice with permeabilization buffer using a commercially available FOXP3 staining kit (eBioscience) as previously described [ 22 ].

Techniques: Expressing, Fluorescence, In Vitro, Cell Culture

Journal: European journal of immunology

Article Title: Phenotypic and functional characteristics of ATP-hydrolysing CD4 + CD39 + FOXP3 + and CD4 + CD39 + FOXP3 neg T-cell subsets in patients with cancer

doi: 10.1002/eji.201142347

Figure Lengend Snippet: Unstimulated CD4+CD39+ T cells consisted of two subpopulations, which differed in their expression of CD25, FOXP3, CTLA4 and PD-1. The cell phenotype was unchanged after a 3-day culture + IL-2 (50 IU/mL) without stimulation. After 3 day stimulation with SEB, all CD39+ T cells expressed Treg cell -associated markers CD25, FOXP3, CTLA4 and PD-1 as well as LAP. Dot plots are gated on CD4+CD39+ T cells. Bar graphs present data (means ± SD) from 4 independent experiments with cells of different HCs (* p < 0.05).

Article Snippet: For intracellular staining of cytokines, FOXP3, and CTLA4 cells were fixed for 30 min and washed twice with permeabilization buffer using a commercially available FOXP3 staining kit (eBioscience) as previously described [ 22 ].

Techniques: Expressing

Journal: European journal of immunology

Article Title: Phenotypic and functional characteristics of ATP-hydrolysing CD4 + CD39 + FOXP3 + and CD4 + CD39 + FOXP3 neg T-cell subsets in patients with cancer

doi: 10.1002/eji.201142347

Figure Lengend Snippet: Phenotypic and functional characteristics of the two CD4 + CD39 + T cell subsets

Article Snippet: For intracellular staining of cytokines, FOXP3, and CTLA4 cells were fixed for 30 min and washed twice with permeabilization buffer using a commercially available FOXP3 staining kit (eBioscience) as previously described [ 22 ].

Techniques: Functional Assay, Expressing