Fig 2: Appearance of Immature and PD-L1+ Neutrophils in Severe COVID-19(A) Heatmap revealing differences in marker expression determined by mass cytometry (antibody panel 2, cohort 1) of main neutrophil cell cluster (1 to 10). Main individual neutrophil cell clusters are displayed in columns and marker identity is indicated in rows. MSI, marker staining intensity respective expression level. Significance level for the following comparisons: (1) controls (ctrl, n = 9) versus COVID-19 (mild and severe, n = 17, first row), (2) mild (n = 8) versus severe (n = 9, second row), (3) FLI (n = 8) versus mild COVID-19 (n = 8, third row), as well as (4) controls (ctrl, n = 9) versus FLI (n = 8) are indicated using a gray scale on top of the heatmap (see also p value scale next to the heatmap). Samples of COVID-19 patients collected between day 4 and 13 post-symptom onset (= first day of sample collection per patient). Abundance testing via generalized mixed effects models and multiple comparison adjustment using the Benjamini-Hochberg procedure and an FDR-cutoff of 5% across all clusters/subsets and between-group comparisons(B) UMAP of neutrophils, down-sampled to 70,000 cells (30 markers, Table S2). Cells are colored according to main cell clusters (1 to 10, see table). Donor origin (blue, controls; gray, FLI; yellow, mild COVID-19; red, severe COVID-19).(C) UMAP (from (B) with cells colored according to expression intensity of CD38, CD34, CD16, CD11b, CD33, CD64, CD62L, and CD45.(D) Box and whisker (10–90 percentile) plots of main neutrophil cell clusters 1 to 7, reaching proportions of over 1%, measured by mass cytometry (whole blood, cohort 1): controls (n = 9), FLI (n = 8), and COVID-19 (mild, n = 8; severe, n = 9). Abundance testing via generalized mixed effects models and multiple comparison adjustment using the Benjamini-Hochberg procedure and an FDR-cutoff of 5% across all clusters/subsets and between-group comparisons.(E) Box and whisker (10–90 percentile) plots of proportions of CD34+, CD11blo/-CD16-, CD64+, CD62L+, CD10-CD11blo/-CD16- (reported from panel 1) and PD-L1+ neutrophils (whole blood, cohort 1): controls (n = 9), FLI (n = 8), and COVID-19 (mild, n = 8; severe, n = 9). Kruskal-Wallis and Dunn’s multiple comparison tests.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.See also Figure S5 and Tables S1 and S3.

Fig 3: Multiple intercorrelations among CD10neg neutrophils, HLA-DRneg/low monocytes and immune-inflammation markers.Spearman-correlation matrix of CD16+CD66b+CD10neg neutrophils (%), CD14+HLA-DRneg/low monocytes (%) and circulating levels of MMP-9, S100A9/S100A8, IL-6, IL-1ß, TNF-a, MPO, and NGAL (levels). UA (n=11), NSTEMI (n=10), and STEMI (n=26). Each circle illustrates a significant correlation between different couples of parameters (p<0.05). The correlation coefficient is colored and sized up according to the value; square leaved blank indicates not significant correlation.

Fig 4: scRNA-seq T cell cluster and their cytotoxic gene signature in samples from COVID-19 patients or controls of cohort 2, related to “scRNA-seq data analysis of Rhapsody data (cohort 2)” in STAR Methods(A) UMAP of the T cell subset from the PBMC dataset of Schulte-Schrepping et al. (2020), including controls (n = 13), mild COVID-19 (n = 21) and severe COVID-19 (n = 29) patients.(B) Heatmap of selected marker expression of the T cell subset from the PBMC dataset of Schulte-Schrepping et al. (2020), including controls (n = 13), mild COVID-19 (n = 21) and severe COVID-19 (n = 29) patients.(C) UMAP of T cell clusters as shown in (A) with cells colored according to disease group origin: blue, controls (n = 13); yellow, mild COVID-19 acute phase (n = 21); red, severe COVID-19 acute phase (n = 29).(D) Box and whisker (10–90 percentile) plots of a selection of cohort 2 scRNA-seq T cell clusters, generated from controls (n = 13), mild COVID-19 acute phase (n = 8) and severe COVID-19 acute phase (n = 9) patient samples. Selected clusters show a TFH phenotype (cluster 6 and 7) or display FCGR3A expression and increased frequency in severe COVID-19 (cluster 10 and 13) generated from controls (n = 13), mild COVID-19 acute phase (n = 8) and severe COVID-19 acute phase (n = 9) patient samples. KW* shows the adjusted p value (Benjamini-Hochberg) of a Kruskal-Wallis test. The abundance of each cluster was compared between severity groups via adjusted Dunn’s post hoc test (Benjamini-Hochberg). When multiple samples for the sample patients were available only the earliest sample was used for visualization and statistical testing. All combinations where tested, only comparisons with healthy controls are shown (*p < 0.05, **p < 0.01, ***p < 0.001).(E) GSEA performed on the ranked gene list of the comparison severe versus mild COVID-19. The graph shows the mapping of the signature genes on the ranked gene list. The curve corresponds to the running sum of the weighted enrichment score (ES). The ranked gene list was calculated from the normalized pseudobulk expression data of severe and mild samples weeks 1 and 2 post symptoms onset across clusters 6, 7, 10, and 13.(F) Dot plot of the expression of the genes included in the “cytotoxicity” and “response to type I interferon” signatures in control, mild COVID-19, and severe COVID-19 samples in weeks 1 and 2 post symptoms onset across clusters 6, 7, 10, and 13. The dots are colored by the scaled gene expression across the groups and the size is proportional to the ratio of cells expressing the specific gene.

Fig 5: Increased degranulation and cytotoxic potential of T cells from severe COVID-19(A) Box and whisker (5–95 percentile) plots of SARS-CoV-2-specific IgG and IgA antibody levels detected in serum samples from mild (n = 15) and severe (n = 17) COVID-19 patients collected between day 10 and 14 post symptom onset. Wilcoxon test **p < 0.01.(B) Linear regression analysis of TFH cell proportions (CyTOF cluster 7) determined in samples collected from mild (n = 8) and severe (n = 11) COVID-19 (cohort 1) during day 5 and 14 post-symptom onset and SARS-CoV-2-specific IgG and IgA serum levels.(C) Box and whisker (min - max) plots summarizing the intracellular granzyme B expression (unstimulated) of CD8+ T cells from PBMCs of mild (n = 21) and severe (n = 28) COVID-19 patients as well as controls (n = 21). Kruskal-Wallis & post hoc Dunn’s multiple comparison test *p < 0.05, ***p < 0.001.(D) Box and whisker (5–95 percentile) plots summarizing the degranulation capacity of CD8+ T cells from PBMCs of mild (n = 21) and severe (n = 28) COVID-19 patients as well as controls (n = 20) defined by their increase of cell surface CD107a expression upon stimulation with anti-CD16 antibody-coated or isotype-coated beads. Multiple Mann-Whitney test *p < 0.05, ***p < 0.001.(E) Scatter plot of the degranulation capacity from PBMCs of severe COVID-19 patients (n = 12) upon stimulation with spike-protein-coated beads pre-incubated with control serum, with spike-protein-coated beads pre-incubated with COVID-19 serum or with anti-CD16 antibody-coated beads. Kruskal-Wallis and post hoc Dunn’s multiple comparison test ***p < 0.001, ****p < 0.0001.(F) Enrichment of CD137+CD69+ cells in activated CD16- (CD38+CD16-) and activated CD16+ (CD38+CD16+) over the total CD8+ T cell compartment upon restimulation of PBMC samples from mild (n = 5) and severe (n = 7) COVID-19 patients with a SARS-CoV-2 peptide pool. Enrichment was calculated by dividing the proportions of CD137+CD69+ T cells in non-activated CD16- (CD38+CD16-) and activated CD16+ (CD38+CD16+) by the proportions of total CD8+ T cells. Friedmann test & post hoc Dunn’s multiple comparison test. *p < 0.05, **p < 0.01.(G) Box and whisker (5–95 percentile) plots summarizing the normalized release of CXCL8 and CCL2 by primary lung endothelial cells co-cultured with CD8+ T cells enriched from PBMCs of mild (n = 6) and severe (n = 5) COVID-19 patients as well as non-infected controls (n = 5) upon stimulation with ConA or anti-CD16 antibody-coated beads. Wilcoxon test *p < 0.05.(H) Endothelial cell resistance upon stimulation with ConA alone (n = 5) or additional co-culture with CD8+ T cells enriched from PBMCs of mild (n = 6) and severe (n = 5) COVID-19 patients as well as non-infected controls (n = 5). Kruskal-Wallis test *p < 0.05.(I) Representative immunofluorescence staining of CD3 (green) and CD16 (red) in autopsy lung tissues of patients without lung pathology, with COVID-19, ARDS, or influenza pneumonia.(J) Quantification of CD3/CD16 double-positive T lymphocytes per mm2 in the autopsy cohorts of deceased patients without lung pathology (n = 4) compared with COVID-19 (n = 13), ARDS (n = 8) and influenza pneumonia (n = 6). The COVID-19 cohort was separated into early stage (death after 7–14 days after first symptoms), mid stage (15–30 days after symptom onset), and late stage (>35 days). One-way ANOVA; **, p < 0.01.