Journal: Immunity

Article Title: Deep spatial profiling of human COVID-19 brains reveals neuroinflammation with distinct microanatomical microglia-T-cell interactions

doi: 10.1016/j.immuni.2021.06.002

Figure Lengend Snippet: The molecular and cellular census of the CNS shows neuroinflammatory alterations in response to SARS-CoV-2 infection (A) Segmentation of IMC images into cellular masks was performed on the entire dataset by supervised machine learning. Single-cell data extracted was clustered with PhenoGraph and visualized on a t-SNE map. (B) Heatmap visualization of cluster marker expression. Normalized median marker expression after subtraction of background is shown. Clusters were annotated according to their expression pattern and spatial distribution; key expression features are indicated. A corresponding Z- score-normalized heatmap is shown in Figure S2 B. (C) Heatmap of myeloid cluster heterogeneity. Median marker intensity, distance-to-vessel, and microglia nodule index (see Figure 4 ) of myeloid clusters were determined in the extension cohort and are visualized in a hierarchically clustered column-normalized heatmap. (D) t-SNE visualization of the brain immune map based on the immune cell clusters identified in (B). (E) Immune cell cluster composition of COVID-19 and control patients is shown by stacked bar charts displaying mean counts per group. (F) Brain immune landscape of COVID-19 (blue) and control patients (black) is shown as in (D). See also and .

Article Snippet: Heatmap visualization of correlations between clinical and high-dimensional data of patients in F was conducted in R studio using corrplot and RColorBrewer packages.

Techniques: Infection, Marker, Expressing, Control

Journal: Immunity

Article Title: Deep spatial profiling of human COVID-19 brains reveals neuroinflammation with distinct microanatomical microglia-T-cell interactions

doi: 10.1016/j.immuni.2021.06.002

Figure Lengend Snippet: Spatial profiling of the brain immune response in COVID-19 indicates localized and orchestrated adaptive immune infiltration (A) High-dimensional annotated cell clusters were compared across patients and controls. Immune cell clusters with significantly different abundances in COVID-19 and control patients are shown by scattered dot plots with bar graphs indicating means ± SEM; each dot represents one patient. (B) Spatial interactions between each pair of cell types in patients with COVID-19 were analyzed by permutation-based neighborhood analysis. The percentages of images with significant neighborhood interactions are displayed as a hierarchically clustered heatmap, ranging from −0.4 (avoidance) to +1 (interaction). Rows represent the neighborhood of a cell phenotype of interest. Columns indicate the enrichment or depletion of a cell in other neighborhoods. (C) Cluster c19 spatial neighborhood interactions were determined in COVID-19 and control patients. Columns indicate significant enrichment or depletion of c19 cells in the vicinity of cells from clusters c1–c34. (D) Distance of cluster c19 cells (blue dots) to the nearest collagen + or CD34 + vessel was determined in COVID-19 brain sections and compared with a random distribution. The 25th, 50th, and 75th percentiles are depicted. (E) CD8 T cell activation in the brains of patients with COVID-19. Cluster c19 cells were analyzed for markers of T cell activation, differentiation, exhaustion, and function. Absolute cell counts were compared among patient groups and visualized by boxplots; dots represent samples. (F) CD8 T cells isolated from medulla, olfactory bulb, cortex, and regional lymph node of a deceased COVID-19 patient were analyzed by suspension-mode mass cytometry. CD8 T cell heterogeneity is shown on a t-SNE map; expression of indicated exhaustion markers is indicated by heatmap coloring. Frequencies are illustrated by bar graphs. See also Figure S4 .

Article Snippet: Heatmap visualization of correlations between clinical and high-dimensional data of patients in F was conducted in R studio using corrplot and RColorBrewer packages.

Techniques: Control, Activation Assay, Isolation, Suspension, Mass Cytometry, Expressing

Journal: Immunity

Article Title: Deep spatial profiling of human COVID-19 brains reveals neuroinflammation with distinct microanatomical microglia-T-cell interactions

doi: 10.1016/j.immuni.2021.06.002

Figure Lengend Snippet: Immune cell activation in anatomical compartments indicates pervasive inflammatory effect of microglial nodules on T cell activation and immune checkpoint expression (A) Cluster c19 CD8 T cells were assessed across perivascular, juxtavascular, parenchymal, and nodule compartments for the fraction of PD-1 + , CD39 + , PD-1 + CD39 + , Tim3 + , and Eomes + cells. (B) Spatial heatmap of HLA-DR signal intensities in segmented cells in a representative IMC image. Scale bar: 100 μm. (C) Fluorescent IHC for Iba1 (green), HLA-DR (yellow), CD8a (red), and DAPI (blue) of a microglia nodule. Image shows a three-dimensional (3D) Z stack. Scale bars: 10 μm, 3 μm, and 1 μm. White arrows indicate HLA-DR expression at CD8 + cell contact sites. (D) Spatial heatmap of PD-1 signal intensities as in (B) (E) Cluster c19 CD8 T cells were analyzed in different anatomical compartments depending on presence or absence of microglial nodules. Fraction of PD-1 + , CD39 + , PD-1 + CD39 + , Eomes + , and HLA-DR + cells is shown. (F) Spatial heatmap of PD-L1 signal intensities as in (B) (G and H) Fraction of Iba1 + PD-L1 + cells (G) and of PD-L1-expressing CD45 + Iba1 + cells (H) was compared across perivascular, juxtavascular, parenchymal, and nodule compartments. (I) Confocal immunofluorescence analysis of Iba1 (green), PD-L1 (violet), CD8a (red), and DAPI (blue) of a microglia nodule. Image shows a Z stack. The scale bar: 10 μm. Boxplots with dots display the median with interquartile range (IQR) and upper and lower whiskers. See also Figure S5 .

Article Snippet: Heatmap visualization of correlations between clinical and high-dimensional data of patients in F was conducted in R studio using corrplot and RColorBrewer packages.

Techniques: Activation Assay, Expressing, Immunofluorescence

Journal: Immunity

Article Title: Deep spatial profiling of human COVID-19 brains reveals neuroinflammation with distinct microanatomical microglia-T-cell interactions

doi: 10.1016/j.immuni.2021.06.002

Figure Lengend Snippet: COVID-19 patients show disease-specific alterations in the central nervous system that correlate with blood chemistry (A) IMC image with expression of SARS-CoV-spike protein (pink), collagen (light blue), ACE2 (yellow), CD34 (blue), GFAP (green), Iba1 (red), HLA-DR (orange), and histone H3 (blue), and an overlay graph depicts an olfactory bulb section from a patient with COVID-19. For each marker the whole image (left) and two areas of interest (right) are shown. Scale bars: 100 μm and 20 μm. (B) The immunohistochemical reaction for SARS-CoV-spike protein (brown) and counterstaining with hematoxylin (blue) is shown at multiple magnifications in the medulla section of a patient with COVID-19. The arrowhead points to a SARS-CoV-spike-protein-positive endothelial cell. The asterisk indicates positive signal in the blood within the vessel lumen. Scale bars: 500 μm, 50 μm, or 20 μm. (C) Violin plot visualizing the distance to the closest vessel (in μm) for all SARS-CoV + cells. Each dot represents one cell; dotted lines indicate median and IQR. (D) ACE-2 (left) and collagen (right) expression by endothelial cell cluster c8 was compared among patient groups and localizations. Fraction of positive cells is depicted per patient. Box and whiskers plot displays median and IQR. (E) A total of nine different tests for viral protein or RNA were performed in the brains of patients with COVID-19. Results are shown in the heatmap (positive, red; negative, blue; gray; test was not performed). (F) Spearman correlations are visualized between clinical parameters, neuroinflammatory features, and immune clusters from the deep spatial analysis. The heatmap coloring indicates the correlation coefficient; significance levels are indicated by asterisks, and boxes indicate an adjusted FDR < 0.05. See also Figure S7 .

Article Snippet: Heatmap visualization of correlations between clinical and high-dimensional data of patients in F was conducted in R studio using corrplot and RColorBrewer packages.

Techniques: Expressing, Marker, Immunohistochemical staining