Journal: Nature Communications

Article Title: Viral transduction of primary human lymphoma B cells reveals mechanisms of NOTCH-mediated immune escape

doi: 10.1038/s41467-022-33739-2

Figure Lengend Snippet: a Representative IHC staining of NOTCH1 in CLL lymph node biopsies. b Graphical scheme of the mass cytometry analysis from nuclear NOTCH1 negative ( n = 3) or positive ( n = 4) specimens. Created with BioRender.com. c Multiplexed IMC image example of a NOTCH1 positive specimen with an enlarged area of the proliferative center (PC). Representative t-SNE plots from an individual patient specimens are presented for CD19, CD4, and CD8 cell populations ( n = 4). d Intensity of cellular signal per given cell was calculated using the HistoCat software. PD-L1 signal in CD19-gated cells in PC and non-PC areas ( p < 0.0001), PD-L1 ( p = 0.0079) and KI67 ( p = 0.011) in CD19-gated PC-cells from samples with a positive or negative NOTCH1 nuclear staining. e Total CD4 signal, KI67, and PD-1 in CD4-gated cells in PCs of NOTCH1 positive or negative samples. f Total CD8 signal and PD-1 in CD8-gated cells in PCs of NOTCH1 positive or negative samples (** p = 0.0044). g Graphical scheme of the in vivo experiment. CLL cells were transduced with an empty vector or NOTCH1 ΔPEST and intraperitoneally injected into male 8–10 week-old NSG mice. Autologous T cells were cultured with IL-2, α-CD3, and α-CD28 for 7 days prior to injection. Created with BioRender.com. h Engraftment of human CD19 + cells in the peritoneal cavity (left) and spleen (right) of mice injected with NOTCH1 ΔPEST ( n = 8) or an empty vector ( n = 8) transduced CLL cells. In total 16 mice were analyzed using cells from 3 independent donors. Each symbol refers to an individual patient sample. i Quantification of human autologous CD4 + /CD8 + T cells. Mean value was obtained from 3 independent experiment using different donor cells. Each symbol refers to an individual patient sample ( p = 0.007). j Ratio of human CD4 + /CD8 + T cells in the peripheral blood of a cohort of treatment-naive CLL patients with mutated ( n = 13) or wild-type NOTCH1 ( n = 34). Cohorts are shown as median ( d – f ) or mean ± SEM ( h – j ). Statistical analysis was done by paired ( h , i ) or unpaired t -tests ( d – f , j ) * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 and not significant (ns) P > 0.05.

Article Snippet: For the staining we used the following Fluidigm pathologist-verified Maxpar antibodies: Anti-CD19 (6OMP31)-142 Nd ; -Anti-Human CD4 (EPR6855)-156 Gd ; -Anti-Human CD8a (D8A8Y)-162 Dy ; -Anti-Human PD-1 (EPR4877(2))-165 Ho ; -Anti-Ki-67 (B56)-168 Er ; -Anti-Human PD-L1 (E1L3N)-150 Nd ; -Anti-Pan-Actin (D18C11)-175 Lu ; -Anti-Histone 3 (D1H2)-176 Yb Images acquired with the Hyperion Imaging System were reviewed and single ROI were exported using MCD Viewer (Fluidigm v1.0.560.6).

Techniques: Immunohistochemistry, Mass Cytometry, Software, Staining, In Vivo, Transduction, Plasmid Preparation, Injection, Cell Culture

Journal: Frontiers in Cell and Developmental Biology

Article Title: Spatial immune landscapes of SARS-CoV-2 gastrointestinal infection: macrophages contribute to local tissue inflammation and gastrointestinal symptoms

doi: 10.3389/fcell.2024.1375354

Figure Lengend Snippet: Summary of antibodies and their corresponding metal tags used for imaging mass cytometry.

Article Snippet: CD4 , 156Gd , EPR6855 , Fluidigm , 1/100 , 3156033D.

Techniques: Imaging, Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Spatial immune landscapes of SARS-CoV-2 gastrointestinal infection: macrophages contribute to local tissue inflammation and gastrointestinal symptoms

doi: 10.3389/fcell.2024.1375354

Figure Lengend Snippet: Gastrointestinal (GI) biopsies of COVID-19 patients showing histomorphologic changes. (A) In contrast to those of the healthy lamina propria, the gastrointestinal tracts of COVID-19 patients exhibited interstitial edema, acute inflammation (red arrow) and infiltration of plasma cells and lymphocytes (scale bar, 50 μm). (B) Immunofluorescence analysis of ACE2 and the SARS-CoV-2 nucleocapsid protein in the intestinal tissues of COVID-19 patients. ACE2 staining is shown in green, nucleocapsid staining is shown in red, and nuclear staining is shown in blue (scale bar, 50 μm). (C) Representative IMC images of gastrointestinal tissues from COVID-19 patients with (n = 12) or without (n = 4) detected NPs. Each image on the left was rendered with a selection of different markers (blue: nucleus, green: NP, red: ACE2, yellow: CD68 and CD3, magenta: CD20, cyan: E-cadherin, white: CD4 and CD8) (scale bar = 100 μm).

Article Snippet: CD4 , 156Gd , EPR6855 , Fluidigm , 1/100 , 3156033D.

Techniques: Immunofluorescence, Staining, Selection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Spatial immune landscapes of SARS-CoV-2 gastrointestinal infection: macrophages contribute to local tissue inflammation and gastrointestinal symptoms

doi: 10.3389/fcell.2024.1375354

Figure Lengend Snippet: Macrophage recruitment in GI tissue from COVID-19 patients. (A) Cell phenotypes were visualized with a heatmap based on biomarker expression in NP + tissues (n = 12) and NP − tissues (n = 4). (B) PhenoGraph defines complex cell phenotypes based on marker expression and enables the labeling of cell phenotype clusters on a t-SNE plot. (C) Analysis by t-distributed stochastic neighbor embedding (t-SNE) dimensionality reduction yielded t-SNE plots of gastrointestinal tissues from COVID-19 patients with or without NP detection. Single-cell t-SNE maps showing the expression of CD68, CD4, CD8, and CD20. Plots showing NP − (right) in comparison to NP + (left) samples. The color spectrum on the right of the plot indicates the mean expression levels of the markers (red, high expression; blue, low expression). (D) Comparisons of the numbers of CD68 + macrophages, CD3 + CD4 + T-cells, CD3 + CD8 + T-cells, and CD20 + B-cells in the groups with or without detected NPs (Mann–Whitney test). (E) The scatter plots show a positive correlation between CD68 + macrophages and CD3 + CD4 + T-cells (n = 16), but no significant correlation was observed with CD3 + CD8 + T-cells or CD20 + B-cells (n = 16).

Article Snippet: CD4 , 156Gd , EPR6855 , Fluidigm , 1/100 , 3156033D.

Techniques: Biomarker Assay, Expressing, Marker, Labeling, Comparison, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: Spatial immune landscapes of SARS-CoV-2 gastrointestinal infection: macrophages contribute to local tissue inflammation and gastrointestinal symptoms

doi: 10.3389/fcell.2024.1375354

Figure Lengend Snippet: Neighborhood analysis of cell–cell interactions in the GI tract of COVID-19 patients. (A, B) Unsupervised neighborhood analysis visualization of all cell-to-cell interactions based on the presence of significant adjacent (red) or avoidance (blue) interactions; white denotes the absence or nonsignificance of cell contacts (permutation test, p < 0.01). (C, D) Heatmap displaying cell–cell interactions in 4 NP − GI tract tissue samples (C) and 12 NP + tissue samples (D) from COVID-19 patients, in which the cell type in the row significantly neighbored (red) or avoided (blue) the cell type in the column. White represents a prevalence less than 10%. (E) Images from the NP − and NP + groups are displayed in the miCAT image window using user-defined color channels (blue, nucleus; green, NP; red, ACE2). Cells of interest, such as CD8 + T-cells (cluster 4, purple), CD4 + T-cells (cluster 5, gray), CD68 + macrophages (cluster 9, dark red), and CD20 + B-cells (cluster 13, turquoise), are highlighted in the image (scale bar: 100 μm).

Article Snippet: CD4 , 156Gd , EPR6855 , Fluidigm , 1/100 , 3156033D.

Techniques: