Journal: International Journal of Molecular Sciences

Article Title: Myelin Oligodendrocyte Glycoprotein (MOG)35–55 Mannan Conjugate Induces Human T-Cell Tolerance and Can Be Used as a Personalized Therapy for Multiple Sclerosis

doi: 10.3390/ijms25116092

Figure Lengend Snippet: Antibodies used for phenotypic analysis.

Article Snippet: CD45-RA , Becton Dickinson , HI100 , PE.

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: ( 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