Fig. 2: High definition immunophenotyping of lung cells and identification of tissue structure. | Nature Communications

Fig. 2: High definition immunophenotyping of lung cells and identification of tissue structure.

From: Single cell spatial analysis reveals inflammatory foci of immature neutrophil and CD8 T cells in COVID-19 lungs

Fig. 2: High definition immunophenotyping of lung cells and identification of tissue structure.The alternative text for this image may have been generated using AI.

a UMAP representation of myeloid, lymphocyte and structural cell ‘mega clusters’ from all regions of interest (ROI) (k = 30) (COVID-19 and HC). See also Supplementary Fig. 7 for extended analysis steps. HC – healthy control. b, c Number of cells per mm2 of lung tissue sections in all COVID-19 samples (n = 12 patients, 30 ROIs’ in total) compared to healthy control (HC) samples (n = 2 individuals, 4 ROIs in total). Median shown, error bars are IQR. n = 524,552 cells in total for COVID-19 samples, n = 30,053 cells for HC. Statistical analysis performed after samples grouped into histopathology states (see Fig. 3d). Source data are provided in the Source Data File. d, e Immunofluorescence (IF) staining validation for Neut_CD8_ADJ cell cluster and Mono_CD31_ADJ cell clusters. Small panels are high magnification confocal images showing CD8 and CD15 staining (top small panel), and CD14 and CD31 staining (bottom small panel) on adjacent cells. Broken yellow circles show CD8 T cell (white- CD8) – neutrophil (green-CD15) couplets throughout lungs (d); and CD14-staining cells next to CD31-expressing cells (endothelium) in lung tissue (e). See also Supplementary Fig. 6 for negative controls. IF images shown are representative of lung sections from n = 3 patients; staining experiment performed once per lung sections. f Heatmap of median scaled intensity for each marker for all cell clusters in the ‘Myeloid’ mega cluster. ‘n_cells’ - average number of cells in all COVID-19 ROIs. Total cells – 171, 777. UD – undefined cluster. g, h Exemplar MCD image from 37-plex imaging mass cytometry (IMC) staining of a DAD ROI showing expression of CD8 T cells (CD8 -green), neutrophils (CD15-red) and CD8_CD15_ADJ cell clusters (green and red co- expression, making yellow). Image is one of n = 26 ROIs, some of which do not have the CD8_CD15_ADJ cell clusters—see Fig. 3b for number of ROIs showing presence of this cell cluster in all ROIs (n = 26 COVID-19; n = 4 HC). h Same MCD images as (G) but with IFN-β channel ‘open’ (white) showing IFN-β expression on Neut_CD8_ADJ (yellow). i Higher magnification of a set of 3 MCD panels - ‘none’ - Neut_CD8_ADJ (yellow) only (arrows); ‘IFN-γ’ – with ‘IFN-γ’ (white) channel opened on MCD viewer showing expression on Neut_CD8_ADJ (yellow) (arrows) and some CD8 (green); ‘GZB’ - with ‘GZB’ (cyan) channel opened and showing expression on Neut_CD8_ADJ (yellow) (arrows). CD172a panel shows confocal immunofluorescence staining (white) on CD15 and CD8 adjacent to each other. IF images shown are representative of lung sections from n = 3 patients; staining experiment performed once per lung sections. ALV – alveolitis, DAD – diffuse alveolar damage, OP -organising pneumonia. MCD images from all 26 ROIs (n = 10 ALV, n = 8 DAD and n = 8 OP) were analysed and median expression intensity for all ROIs shown in (f) and Supplementary Fig. S7. All scale bars in μm.

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