Fig 2: Validation of the Severe COVID-19 Innate Immune Signature(A) Spectral flow pipeline.(B) Fraction of B cells, CD4+ T cells, CD8+ T cells, and NK cells among total cells in individual samples (circles) in each group.(C) Fraction of neutrophils in individual samples in each group.(D) Neutrophil subsets identified by CD101 and CD10 expression in each group (pooled data).(E) Fraction of CD10LowCD101- neutrophils among total neutrophils in individual samples within each group.(F) Fraction of CXCR4+ neutrophils among CD10LowCD101– neutrophils as in (E).(G) Spearman correlation of time spent in the ICU and non-classical monocyte fraction among total monocytes. Patients with a bacterial infection are shown as red dots. Mean time spent in the ICU was 5.46 days for patients with a low (=5%) and 8.83 days in those with a high (>5%) CD14lowCD16High monocyte fraction.(H) Fraction of classical monocytes among white blood cells in each individual sample in each group.(I) Fraction of non-classical monocytes among total monocytes as in (H).(J) Monocyte subset partition in each group (pooled data), with the severe group split in two groups based on mean time spent in the ICU. Top panels: monocyte subsets identified by CD14 and CD16 expression. Bottom panels: HLA-DR and CD169 or CD11b and CD141 expression in the classical monocyte subset.(K) Fraction of monocytes expressing CD169 among classical monocytes as in (H).(L) Fraction of monocytes expressing CD141 among classical monocytes as in (H).Kruskal-Wallis test, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: Validation of the Innate Immune Signature of Severe COVID-19, Related to Figure 6 and Table S6A-B Non-supervised UMAP representation generated by pooling data from all the patient samples; cell identification (A) surface marker expression (B); C. Bar plots representing the percentage of CD10LowCD16Low neutrophils among all neutrophils in individual patients from each group in the validation cohort (n = 90). D. Spearman correlation between CD169 (SIGLEC-1) mean fluorescence intensity (MFI) and days spent by severe patients in ICU. E. Spearman correlation between CD169 (SIGLEC-1) mean fluorescence intensity (MFI) and plasma IFNa concentration; yellow, mild COVID-19 patients; red, severe COVID-19 patients. F.G. Bar plots representing the percentage of HLA-DRLow classical monocytes, B cells, CD4+ and CD8+ T cells and NK cells (F) and neutrophils among CD45+ cells, CD10LowCD101- neutrophils among all neutrophils and CD10LowCXCR4+ neutrophils among CD10LowCD101- neutrophils (G) in individual patients from each group in the validation cohort (n = 90). Kruskal-Wallis test, *p < 0.05, **p < 0.01, ***p < 0.001; ns, non-significant.

Fig 4: Changes in Innate Immune Cell Phenotype Are Detected in Patients with Moderate COVID-19, Related to Figure 7 and Tables S6 and S7A. Bar plots representing the percentage of B cells, CD4+ T cells, CD8+ T cells, NK cells, total monocytes, CD169+, HLA-DRLow and CD141+ classical monocyte subsets, total neutrophils among CD45+ cells, and CD10LowCD101- and CD10LowCD16Low neutrophil subset among all neutrophils in individual patients from each group, with the moderate category (6 patients) highlighted. B. Plasma concentration of IFNa in moderate COVID-19 patients compared to the three other groups. C. ROC analysis showing performance of a diagnostic test using percentage of non-classical monocytes among total monocytes to distinguish controls and mild COVID patients from moderate and severe COVID patients; D. Monocyte subset analysis in the peripheral blood of 2 severe patients, before (left panels) and after (right panels) treatment with the indicated anti-IL-6 antibodies; E. Percentage of HLA-DRlow classical monocytes among classical monocytes in a cohort of 22 patients and 17 controls grouped into 4 clinical categories; F. Correlation between the percentage of HLA-DRlow classical monocytes and non-classical monocytes; G. Percentage of CD16low neutrophils among neutrophils in control and COVID-19 patients of the learning cohort described in Figure 7. H. ROC curves evaluating the discriminating significance of calprotectin plasma level (yellow), nonclassical monocyte fraction (red), CD16low circulating neutrophils (blue) and IFNa2a plasma level (white) between controls/mild patients and moderate/severe patients. AUC, Area Under the Curve; Mann Whitney test; I. Heatmap of blood and bronchoalveolar lavage fluid scRNaseq cells integrated defining the 5 regions of cell populations; J. Pathway analysis (Cytoscape and ClueGo) of DEGs expressed at a higher level in bronchoalveolar monocytes/macrophages from mild versus severe patients. K. UMAP analysis of neutrophils with S100A8 (left panel) and S100A9 (right panel) gene expression level projection (low expression = gray dots; high expression = dark blue dots). Kruskal-Wallis test, *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 5: Surface protein abundance changes are observed in leukocyte subsets of patients with critical COVID-19.(A) Volcano plot of log fold change between COVID-19+ and healthy controls (x axis) versus -log10(P value) (y axis) in cM cells. Proteins that are significantly (FDR < 0.05) differentially abundant and have a log2(fold change) > 0.5 are highlighted. (B) Normalized (Norm.) LAIR1 and SIGLEC1 surface expression (exp.; y axis) at day 0 across eight cell types separated by case control status, severity, and presence of anti–IFN-a2 antibodies (x axis). Box plots show median and 25th and 75th percentile. (C) Normalized LAIR1 surface expression (y axis) in cMs over the course of disease for healthy controls, COVID-19- controls, and COVID-19+ cases. COVID-19- controls and COVID-19+ cases are separate by severity and the presence of anti–IFN-a2 antibodies. Insets show significant results of LMM testing for changes over time. (D) The bar plot shows correlation between the surface expression of 52 statistically significantly proteins to ISG-I score in cMs at day 0. Proteins are colored by their log2(fold change) expression between COVID-19+ cases and healthy controls. Red indicates higher expression in COVID-19+ cases. Blue indicates lower expression in COVID-19+ cases. (E) Scatter plot of normalized LAIR1 expression (y axis) versus the ISG-I score (x axis) for COVID-19+ cases colored by severity and anti–IFN-a2 status. *P < 0.05, **P < 0.01, and ***P < 0.001.