Fig 2: Cohort Definition and Single-Cell Multi-omics Analysis Strategy(A) Pipeline for control and COVID-19 blood samples of the two cohorts (see also Table S1). Whole blood samples were subjected to red blood cell (RBC) lysis and processed for CyTOF mass cytometry (two antibody panels), multi-color flow cytometry (MCFC), or scRNA-seq (BD Rhapsody). PBMCs were isolated by density centrifugation and processed directly or after frozen storage, labeled with cell hashing antibodies and loaded on droplet-based (10x) or microwell-based (BD Rhapsody) scRNA-seq platforms. Box (bottom left): number of subjects in each cohort. Boxes (on the right): number of samples analyzed with each technique.(B) Number of samples per technique summarized across cohorts, divided by disease severity according to WHO ordinal scale and by the time after onset of first symptoms (early: days 0–10, late: >day 11).(C) UMAP of CD45+ leukocytes, down-sampled to 70,000 cells, from mass cytometry using antibody panel 2 (30 markers, Table S2). Cells are colored according to donor origin (blue, age-matched controls; gray, FLI; yellow, mild COVID-19; red, severe COVID-19) and major lineage subtypes.(D) Box and whisker (10–90 percentile) plots of major cell lineage composition in whole blood from FLI (n = 8), COVID-19 patients with mild (n = 8) or severe disease (n = 9), age-matched controls measured by mass cytometry (ctrl CyTOF, n = 9) or by flow cytometry (ctrl flow, n = 19) (Kverneland et al., 2016). Kruskal-Wallis and Dunn’s multiple comparison test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.a., not available.See also Figure S1 and Table S3.

Fig 3: 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 4: Expansion of EpCAM+/NCAM+/TROP2− human foetal liver HHyPs in the renal capsule. a 2D t-SNE visualisation of single cells isolated from foetal and adult human liver coloured by TROP-2 gene expression. Expression is Log10(TPM). b Schematic of hepatic and BEC lineage restriction of HHyPs based on NCAM/TROP-2 expression. c Gating scheme for the isolation of distinct foetal human liver populations based on expression of CD235a, CD45, EpCAM, NCAM and TROP2. d Hematoxylin and eosin (H&E) staining in tissue cross-sections of explant region for 4-week post renal capsule transplantation. Scale bars represent 250 μm. e Immunofluorescence (IF) staining of FAH (red) in explant region of CD235a−/CD45−/EpCAM+/NCAM+/TROP2− HHyPs 4-week post transplantation. Slides counterstained in DAPI (cyan). Scale bars represent 75 μm. t-SNE t-distributed stochastic neighbour embedding, TPM transcripts per million

Fig 5: Impaired T-cell antigen-specific recognition of HCC827-GR6 cells despite elevated STING. A, TCRaß expression on J76 cells transduced with or without LMP2B TCR. B, HLA-A*11:01 Tetramer and CD45 expression on J76 cells transduced with or without LMP2B TCR. C, ELISA of human IFN? levels in CM derived from HCC827 cells with J76 cells transduced with LMP2B TCR for 72 hours and pretreated ±10 µg/mL peptides (SSCSSCPLSK) for 3 hours. Each data represents one experiment. D, ELISA of human IFN? levels in CM derived from HCC827 or HCC827GR6 cells with J76 cells transduced with LMP2B TCR for 72 hours and pretreated ± 0.1, 1, 10µg/mL peptides (SSCSSCPLSK) for 3 hours. Mean ± SEM of n = 2 biological replicates is shown (unpaired two-tailed Student t test; left). ELISA of human IL2 levels in CM derived from HCC827 or HCC827GR6 cells with J76 cells transduced with LMP2B TCR for 72 hours and pretreated ±1 µg/mL peptides (SSCSSCPLSK) for 3 hours. Mean ± SEM of n = 3 biological replicates shown (unpaired two-tailed Student t test; right). *, P < 0.05; **, P < 0.005.