Journal: eLife

Article Title: Short-range interactions between fibrocytes and CD8 + T cells in COPD bronchial inflammatory response

doi: 10.7554/eLife.85875

Figure Lengend Snippet: ( A ) CD8 (brown) staining of a representative bronchus. ( B ) CD45 (red) and fibroblast-specific protein 1 (FSP1, green) stainings of the same bronchus. ( C, D ) The left panels show a higher magnification image (indicated by the boxed regions) of the images in A and B . The lamina propria is shown in light blue. ( C ) Middle panel: image for CD8 staining obtained after color deconvolution. Right panel: image obtained after segmentation by a binary threshold followed by a watershed transformation to the segmented image. CD8 + T cells are shown in magenta. ( D ) Middle panels: images for CD45 (top) and FSP1 (bottom) stainings and images obtained after segmentation by a binary threshold. Right panel: segmented image obtained after the combination of CD45 and FSP1 segmented images to select cells dual positive for FSP1 and CD45 double staining, followed by a watershed transformation to separate potential neighboring cells. CD45 + FSP1 + cells are shown in yellow. ( E ) Top panels: dilatation of each CD8 positive particle with an area greater than 64 µm 2 . Bottom panels: combination of these modified images with segmented image for dual CD45 FSP1 positive staining ( B ) to automatically select overlapping dilated CD8 + T cells with CD45 + FSP1 + cells. The white arrow indicates interacting cells. ( F ) Top panels: a CD8 distance map is built from the binary image produced from CD8 staining. Bottom panels: each area corresponding to a FSP1 + CD45 + cell was reported on the CD8 distance map (blue outlines), and the minimal gray value in each area is measured and converted to a distance, allowing to measure the minimal distance between the CD45 + FSP1 + cell and neighboring CD8 + T cells.

Article Snippet: Cells were stained with anti-CD8-PerCP-Vio700 or anti-CD4-PerCP-Vio700, anti-CD45-RA-FITC, and then fixed, permeabilized using the IntraPrep Permeabilization Reagent Kit (Beckman Coulter) and stained with anti-Granzyme-APC, anti-TNF-α-PE, anti-IFN-γ-APC, anti-IL-17- PE-Cy7, anti-IL-10-PE or isotype controls (Miltenyi Biotech, Paris, France).

Techniques: Staining, Transformation Assay, Double Staining, Modification, Produced

Journal: eLife

Article Title: Short-range interactions between fibrocytes and CD8 + T cells in COPD bronchial inflammatory response

doi: 10.7554/eLife.85875

Figure Lengend Snippet: ( A, B ) Representative stainings of CD8 (brown, A ), CD45 (red, B ), and FSP1 (green, B ) in distal bronchial tissue specimens from a control subject (left) and a COPD patient (right). The yellow arrowheads indicate fibrocytes, defined as CD45 + FSP1 + cells. ( C ) Quantification of CD8 + T cells and fibrocyte densities (normalized by the sub-epithelial area) in one specimen/patient (n=20 control subjects, n=12 patients with COPD). ( D ) Merged segmented images for CD8 and CD45-FSP1 staining, showing CD8 + T cells and CD45 + FSP1 + cells, respectively, in magenta and yellow. The white arrows indicate interacting cells, detected by dilatation of CD8 positive particles. ( E ) Table showing the correspondence between dilatations in pixels and µm. ( F ) Quantification of interacting cell densities (normalized by the sub-epithelial area) in one specimen/patient, using the different dilatations sizes ( E ). ( G ) Distance maps built from the binary image produced from CD8 staining, with FSP1 + CD45 + cells (blue outlines). ( H ) Quantification of the mean minimal distances between fibrocyte and CD8 + T cells in one specimen/patient. ( I ) Cluster analysis performed by Delaunay triangulation on segmented images for CD8 and CD45-FSP1 staining, followed by the application of a threshold value (40 μm) above which connections are not kept. CD8 + T cells and fibrocytes appear, respectively, with green and red dots, connections are shown in blue. ( J ) First row: densities of clusters containing exclusively CD8 + T cells (‘CD8 clusters’), fibrocytes (‘Fib clusters’), and both cell types (‘CD8-Fib clusters’) normalized by the sub-epithelial area in one specimen/patient. Second row: mean number of cells by cluster. ( C, F, H, J ) The medians are represented as horizontal lines, n=20 specimens from control subjects, n=12 specimens from patients with COPD. *p<0.05, **p<0.01; ***p<0.001. unpaired t-tests or Mann-Whitney tests.

Article Snippet: Cells were stained with anti-CD8-PerCP-Vio700 or anti-CD4-PerCP-Vio700, anti-CD45-RA-FITC, and then fixed, permeabilized using the IntraPrep Permeabilization Reagent Kit (Beckman Coulter) and stained with anti-Granzyme-APC, anti-TNF-α-PE, anti-IFN-γ-APC, anti-IL-17- PE-Cy7, anti-IL-10-PE or isotype controls (Miltenyi Biotech, Paris, France).

Techniques: Staining, Produced, MANN-WHITNEY

Journal: eLife

Article Title: Short-range interactions between fibrocytes and CD8 + T cells in COPD bronchial inflammatory response

doi: 10.7554/eLife.85875

Figure Lengend Snippet: ( A ) Binary images for CD8 (left panel) and double CD45-FSP1 (right panel) stainings were obtained after segmentation. ( B ) Images after Delaunay triangulation, were performed on the centers of mass of CD8 + T cells and fibrocytes and on the points defining area edges. CD8 + T cells and fibrocytes appear, respectively, with red and green dots. The right panel is a higher magnification of the peribronchial area (indicated by the boxed purple region on the left panel). The connections including the points defining area edges are shown in red, all the other connections are shown in blue. ( C ) Left panel: image with Delaunay triangulation after elimination of the connections including the points defining area edges. The lamina propria is shown in blue. Right panel: image shown on the left panel, after applying a threshold value (40 µm) above which connections are not kept.

Article Snippet: Cells were stained with anti-CD8-PerCP-Vio700 or anti-CD4-PerCP-Vio700, anti-CD45-RA-FITC, and then fixed, permeabilized using the IntraPrep Permeabilization Reagent Kit (Beckman Coulter) and stained with anti-Granzyme-APC, anti-TNF-α-PE, anti-IFN-γ-APC, anti-IL-17- PE-Cy7, anti-IL-10-PE or isotype controls (Miltenyi Biotech, Paris, France).

Techniques:

Journal: eLife

Article Title: Short-range interactions between fibrocytes and CD8 + T cells in COPD bronchial inflammatory response

doi: 10.7554/eLife.85875

Figure Lengend Snippet: Relationships between the density of CD45 + FSP1 + cells ( A ), the density of interacting cells ( B ), the mean minimal distance between fibrocytes and CD8 + T cells ( C ), the density of mixed cell clusters ( D ) and the FEV 1 /FVC ratio measured in control subjects (open circles) and chronic obstructive pulmonary disease (COPD) patients (black circles). The correlation coefficient (R) and significance level (p value) were obtained by using nonparametric Spearman analysis. n=20 specimens from control subjects, n=12 specimens from patients with COPD.

Article Snippet: Cells were stained with anti-CD8-PerCP-Vio700 or anti-CD4-PerCP-Vio700, anti-CD45-RA-FITC, and then fixed, permeabilized using the IntraPrep Permeabilization Reagent Kit (Beckman Coulter) and stained with anti-Granzyme-APC, anti-TNF-α-PE, anti-IFN-γ-APC, anti-IL-17- PE-Cy7, anti-IL-10-PE or isotype controls (Miltenyi Biotech, Paris, France).

Techniques:

Journal: eLife

Article Title: Short-range interactions between fibrocytes and CD8 + T cells in COPD bronchial inflammatory response

doi: 10.7554/eLife.85875

Figure Lengend Snippet: Prior to co-culture, CD8 + T cells have been activated. ( A, B, C, D ) Experiment design. Pure CD8 + T cells were characterized by flow cytometry for CD8 expression ( A ) before being were cultured either alone ( B ) or with fibrocytes ( D ). Fibrocytes were either cultured alone ( C ) or with CD8 + T cells ( D ). ( E, F, G, H ) Dot plots represent representative CD8-PerCP-Cy5-5 fluorescence (y-axis) versus granularity (Side Scatter, SSC) (x-axis) of cells removed either without trypsinization ( E, G ) or with trypsinization ( F, H ) after 6 days in culture/co-culture. ( I, J, K, L ) Dot plots represent representative Collagen Type I (Coll)-FITC fluorescence (y-axis) versus CD45-APC fluorescence (x-axis) of cells removed either without trypsinization ( I, K ) or with trypsinization ( J, L ) after 6 days in culture/co-culture. For conditions with CD8 + T cells only ( I ), fibrocytes only ( J ), and adherent cells removed from co-culture ( K ), the cytographs were generated by gating the total cell population. For non-adherent cells removed from co-culture ( L ), the cytograph was generated by gating the CD8 low population (pink).

Article Snippet: Cells were stained with anti-CD8-PerCP-Vio700 or anti-CD4-PerCP-Vio700, anti-CD45-RA-FITC, and then fixed, permeabilized using the IntraPrep Permeabilization Reagent Kit (Beckman Coulter) and stained with anti-Granzyme-APC, anti-TNF-α-PE, anti-IFN-γ-APC, anti-IL-17- PE-Cy7, anti-IL-10-PE or isotype controls (Miltenyi Biotech, Paris, France).

Techniques: Co-Culture Assay, Flow Cytometry, Expressing, Cell Culture, Fluorescence, Generated

Journal: eLife

Article Title: Short-range interactions between fibrocytes and CD8 + T cells in COPD bronchial inflammatory response

doi: 10.7554/eLife.85875

Figure Lengend Snippet:

Article Snippet: Cells were stained with anti-CD8-PerCP-Vio700 or anti-CD4-PerCP-Vio700, anti-CD45-RA-FITC, and then fixed, permeabilized using the IntraPrep Permeabilization Reagent Kit (Beckman Coulter) and stained with anti-Granzyme-APC, anti-TNF-α-PE, anti-IFN-γ-APC, anti-IL-17- PE-Cy7, anti-IL-10-PE or isotype controls (Miltenyi Biotech, Paris, France).

Techniques: Blocking Assay, Recombinant

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal

doi: 10.1073/pnas.1506943112

Figure Lengend Snippet: Robust human BC CML engraftment in hematopoietic mouse tissues. (A) Graphical summary of results and hypothesized effector function of CD44 mAb therapy. (B) Representative FACS plots showing human BC CML (BC) engraftment in peripheral blood (PB), BM, spleen, and myeloid sarcoma 8–10 wk posttransplant compared with no transplant control (No Tp Ctrl). Mice were transplanted with CD34+ BC11, BC12, or BC19 patient cells. Human cells were gated as live human CD45+. (C) Representative FACS plots showing BC CML (BC) progenitor cells (Lin−CD45+CD34+CD38+) in BM from mice transplanted with BC patient BC11, BC12, and BC19. Prog, Lin−CD45+CD34+CD38+ progenitor cells; Stem, Lin−CD45+CD34+CD38− stem cells. (D) Pooled data from six separate experiments using three different BC CML patient samples (BC11, BC12, BC19) showing human engraftment level (live CD45+ cells) in peripheral blood (PB), BM, spleen, and myeloid sarcomas from control mAb-treated mice. Graphs show mean ± SEM.

Article Snippet: The presence of human cells was analyzed with human-specific antibodies: anti-lineage markers (Invitrogen) (CD2-PE-Cy5.5, CD3-PE-Cy5.5, CD4-PE-Cy5.5, CD8-PE-Cy5.5, CD14-PE-Cy5.5, CD19-PE-Cy5.5, CD20-PE-Cy5.5, CD56-PE-Cy5.5), anti–CD34-APC (BD Biosciences), anti–CD38-PECy7 (BD Biosciences), anti–CD45-RA FITC (Invitrogen), and anti–CD44-APC (BD Biosciences).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal

doi: 10.1073/pnas.1506943112

Figure Lengend Snippet: Disruption of BC LSC ligand interactions with CD44 and BCR-ABL inhibition. (A) Human BC CML LSC engraftment in peripheral blood, spleen, and BM from mice transplanted with patient BC11 (magenta; n ≥ 5 per treatment group), BC12 (blue; n ≥ 5 per treatment group), and BC19 (green; n ≥ 4 per treatment group). Combination therapy significantly reduced BC LSCs in the hematopoietic tissues of all three patient models. Graphs depict mean frequency BC LSCs (Live, Lin−, CD45+CD34+CD38+) out of live cells ± SEM and values for individual mice. Pooled data are from five separate experiments. (B) Confocal fluorescence microscopic analysis of femur sections stained for CD34, CD38, and CD44. Mice treated with CD44 mAb alone or in combination with dasatinib showed a decrease in CD38 and CD44-positive cells. (C) Confocal fluorescence microscopic analysis revealed colocalization of CD34, CD38, and CD44 expression in the control group (C + V) and dasatinib-treated group (C + D). Dashed line, colocalization cutoff value of 0.5. (D) Frequency of human BC CML cells (Lin−CD45+) in peripheral blood, spleen, and BM from secondary recipient mice transplanted with human CD34+ BM cells from primary patient BC11 engrafted mice after treatment (n ≥ 4 per treatment group). Graphs show mean ± SEM and values for individual mice. (E) qRT-PCR gene expression of CD44, CD44v3, β-catenin, BCR-ABL1, and OPN in human CD34+ BC CML cells isolated from the BM of secondary recipient mice. Graphs show transcript levels normalized to control group.

Article Snippet: The presence of human cells was analyzed with human-specific antibodies: anti-lineage markers (Invitrogen) (CD2-PE-Cy5.5, CD3-PE-Cy5.5, CD4-PE-Cy5.5, CD8-PE-Cy5.5, CD14-PE-Cy5.5, CD19-PE-Cy5.5, CD20-PE-Cy5.5, CD56-PE-Cy5.5), anti–CD34-APC (BD Biosciences), anti–CD38-PECy7 (BD Biosciences), anti–CD45-RA FITC (Invitrogen), and anti–CD44-APC (BD Biosciences).

Techniques: Inhibition, Fluorescence, Staining, Expressing, Quantitative RT-PCR, Isolation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal

doi: 10.1073/pnas.1506943112

Figure Lengend Snippet: Targeted CD44 and BCR-ABL1 inhibition reduces human BC LSC survival in hematopoietic tissues. (A) Human BC CML LSC cell engraftment in mice transplanted with patient BC12 (n ≥ 5 per treatment group). (B) Human BC CML LSC engraftment in mice transplanted with patient BC19 (n ≥ 4 per treatment group). (C) Human BC CML LSC engraftment in mice transplanted with patient BC11 (n ≥ 5 per treatment group). (D) Myeloid sarcoma formation in primary (Left) and secondary (Right) recipient mice transplanted with patient BC11 (n ≥ 5 per treatment group). (E) No mice within any of the four treatment groups showed any dramatic increase or decrease in body weight during the treatment period (Left). All four treatment groups show variability in the activity level throughout the treatment (Middle). However, none of the mice showed signs of low activity or being hunched down, a sign of low overall health. Mice in the control groups treated with control IgG + vehicle or control IgG + dasatanib both showed decreased signs of grooming during the course of treatment (Right). Mice treated with CD44 mAb and vehicle, however, had constant good grooming throughout the treatment. Treatment groups, control IgG + vehicle (C + V), control IgG + dasatinib (C + D), CD44 mAb + vehicle (44 + V), and CD44 mAb + dasatinib (44 + D). Graphs (A–D) depict mean frequency human BC CML LSCs (Live, Lin−, CD45+CD34+CD38+) out of live cells ± SEM. Myeloid sarcoma graphs show number of tumors per mouse. Treatment groups, control IgG + vehicle (C + V), control IgG + dasatinib (C + D), CD44 mAb + vehicle (44 + V), and CD44 mAb + dasatinib (44 + D).

Article Snippet: The presence of human cells was analyzed with human-specific antibodies: anti-lineage markers (Invitrogen) (CD2-PE-Cy5.5, CD3-PE-Cy5.5, CD4-PE-Cy5.5, CD8-PE-Cy5.5, CD14-PE-Cy5.5, CD19-PE-Cy5.5, CD20-PE-Cy5.5, CD56-PE-Cy5.5), anti–CD34-APC (BD Biosciences), anti–CD38-PECy7 (BD Biosciences), anti–CD45-RA FITC (Invitrogen), and anti–CD44-APC (BD Biosciences).

Techniques: Inhibition, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal

doi: 10.1073/pnas.1506943112

Figure Lengend Snippet: Targeted CD44 and BCR-ABL1 inhibition reduces human BC LSC survival in hematopoietic tissues. (A and B) Representative dot plots of human BC CML cell (Live, Lin−CD45+) and progenitor cell (Live, Lin−CD45+CD34+CD38+) engraftment in BM from BC11, BC12, and BC19 transplanted mice after 14 d of therapy with control IgG + vehicle (C + V), control IgG + dasatinib (C + D), CD44 mAb + vehicle (44 + V), and CD44 mAb + dasatinib (44 + D). (C) In vitro cytotoxicity assay shows no direct cytotoxic effect of CD44 mAb compared with control IgG in CD34+ cells from cord blood (Left), CP CML (Middle), or BC CML (Right). (D) In vitro treatment with CD44 mAb shows no change in differentiation compared with control IgG in CD34+ cells from cord blood (Left), CP CML (Middle), or BC CML (Right).

Article Snippet: The presence of human cells was analyzed with human-specific antibodies: anti-lineage markers (Invitrogen) (CD2-PE-Cy5.5, CD3-PE-Cy5.5, CD4-PE-Cy5.5, CD8-PE-Cy5.5, CD14-PE-Cy5.5, CD19-PE-Cy5.5, CD20-PE-Cy5.5, CD56-PE-Cy5.5), anti–CD34-APC (BD Biosciences), anti–CD38-PECy7 (BD Biosciences), anti–CD45-RA FITC (Invitrogen), and anti–CD44-APC (BD Biosciences).

Techniques: Inhibition, In Vitro, Cytotoxicity Assay

Journal: BMC Pulmonary Medicine

Article Title: Epigenetics modifications and Subclinical Atherosclerosis in Obstructive Sleep Apnea: The EPIOSA study

doi: 10.1186/1471-2466-14-114

Figure Lengend Snippet: Subset of T cell population to be analyzed

Article Snippet: CD45 RA , HI100 , APC , 45RAA2-100 T IMMUNOSTEP.

Techniques: Fluorescence