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Extended Data Figure 1 Extended Data Figure 1 a SARS-CoV2 PCR(+) (days from COVID-19 diagnosis) -14 -10 -6 -2 0 2 6 10 14 20 60 100 140 AP1 AP2 AP3 AP4 AP5 AP6 Adult AP7 COVID-19 AP8 AP9 AP10 symptom onset AP11 AP12 SARS-CoV2 PCR detection AP13 Nasal/Tracheal/Bronchi brush, AP14 blood BP1 BP2 Convalescent blood BP3 BP4 PP1 Paediatric PP2 COVID-19 PP3 PP4 PC2 Post PC6 COVID-19 PC9 PC11 PC12 PC21 b Neonate Infant Young Child Child Adolescent Adult Patient 0 1 0 1 0 6 Sample Nasal brush 0 1 0 1 0 5 Tracheal/bronchial brush 0 0 0 0 0 0 Blood 0 1 0 1 0 5 Severe Post COVID-19 Moderate severity Mild Age group Neonate Infant Young Child Child Adolescent Adult 0 30d 1y 2y 6y 12y 18y 30y 50y 70y 90y Bronchial brush Tracheal brush Nasal brush Blood c East Asian African South Asian PC2 PC3 PC1 European Extended Data Figure 2 a b c Extended DataFigure3 c a b Neuroendocrine Fraction of cells with expression Deuterosomal Basal cycling 0.025 0.050 0.075 0.100 Inflamm epi Melanocyte Squamous Secretory B/Plasma Ciliated 2 Ciliated 1 T/NK/ILC Spreaman rho Ionocyte Goblet 2 Goblet 1 Myeloid Basal 2 Basal 1 0 −0.4 Brush Mast Club 0.4 0 B/Plasma SARS-CoV-2 receptor ACE2 Correlation betweenmeanviral expressionandmeanofentryfactorsacrosscelltypes ACE2 Basal 1 NRP1 NRP1 BSG BSG Basal 2 TFRC TFRC HCoV-229E TMPRSS2 SARS-CoV, Basal cycling CTSL receptor CD209 CTSB CLEC4G Brush TMPRSS4 CLEC4M TMPRSS11D ANPEP Ciliated 1 LY6E MERS-CoV receptor DPP4 Rhinovirus-C receptor CDHR3 Ciliated 2 IFITM1 IFITM2 Influenza A (H1N1) receptor ST3GAL1 Club IFITM3 TMPRSS2 HNRNPA1 CTSL Healthy Children Protease Cell type Deuterosomal FURIN CTSB DDX3X TMPRSS4 ACE2 TOP3B Goblet 1 TMPRSS11D DPP4 FURIN Goblet 2 ST3GAL1 ST14 ST14 Restriction factors Inflamm epi ANPEP LY6E VCP IFITM1 Ionocyte IFIT2 IFITM2 IFIT3 IFITM3 Mast CDHR3 IFIT2 CD209 IFIT3 Melanocyte CLEC4G CHMP2A CLEC4M AP2A2 Myeloid CHMP2A Post-entry factors AP2M1 AP2A2 Neuroendocrine TAPT1 AP2M1 RHOA TAPT1 Secretory RHOA RAB14 RAB10 Squamous RAB14 RAB10 RAB1A TOP3B T/NK/ILC RAB1A ZCRB1 0 123 5 10152025 Lorem ipsum Fraction ofcells Mean expression in group(%) Group p <0.05 in group Ped_Post-COVID Ped_Healthy Ped_COVID+ Adult_Post-COVID Adult_COVID+ True False Extended Data Figure 4 a KRT14 KRT6A KRT13 KRT24 marker genes “Hillock” associated b Tissue Patient COVID_status Age_bin Cell_viability 10x_kit Ethnicity Sex Smoker Date_of_process residual −0.5 0.0 0.5 1.0 1.5 c Healthy nasal epithelial cells Neonate 0.3 Infant Young child Child 0.2 Adolescent 0.1 Proportion 0.0 Club Brush Basal 1 Basal 2 Goblet 1 Ionocyte Goblet 2 Ciliated 1 Ciliated 2 Secretory Squamous Melanocyte Inflamm epi Basal cycling Deuterosomal Neuroendocrine a Extended DataFigure5 a c b Gamma Type 1 1.0 0.0 0.5 1.0 0.0 0.5 Basal 1 Basal 2 Basal cycling Squamous Secretory Goblet 1 Goblet 2 all cells Club Inflamm epi Deuterosomal Ciliated 1 Expression signaturescoringofcellularresponse tointerferon Ciliated 2 Ionocyte Brush Neuroendocrine Melanocyte T naive T reg T CD4 FRMD4B+ T CD4 mem T CD8 mem T CD8 exhausted T gd NKT NK ILC B naive B mem B mem exhausted B mem activated Monocytes B cycling Plasma IGK Plasma IGL Mac (GO:0034341) response tointerferon-gamma (GO:0035455) response tointerferon-alpha Mac activated LC Mono Mono GPBAR1+ Neut cDC activated pDC fDC Mast Extended Data Figure 6 a Upper airway b Lower airway CCNO CDC20B FOXN4 CCNO CDC20B FOXN4 Deu Deu KRT4 KRT7 IF43 KRT4 KRT7 IF43 Ba-d Ba-d ISG15 IFIT1 CXCL10 ISG15 IFIT1 CXCL10 IRC IRC SAA1 S100A8 S100A9 SAA1 S100A8 S100A9 IETCs IETCs c Chua et al. dataset CCNO CDC20B FOXN4 Deu KRT4 KRT7 IF43 Ba-d ISG15 IFIT1 CXCL10 IRC SAA1 S100A8 S100A9 IETCs Extended Data Figure 7 a log10 #genes log10 #proteins 5 detected 5 detected 4.0 2.5 0 0 3.5 2.0 1.5 −5 3.0 −5 1.0 −10WNN UMAP 2 −10WNN UMAP 2 2.5 0.5 −10 −5 0 5 10 2.0 −10 −5 0 5 10 0.0 WNN UMAP 1 WNN UMAP 1 log10 #UMIs log10 #UMIs from 5 from genes 5 antibody derived tags 5 5 0 4 0 4 −5 3 −5 3 2 2 WNN UMAP 2 −10 −10WNN UMAP 2 1 1 −10 −5 0 5 10 0 −10 −5 0 5 10 0 WNN UMAP 1 WNN UMAP 1 Sequencing library batch S1 S8 S15 S22 5 5 S2 S9 S16 S23 Sex 0 Female 0 S3 S10 S17 S24 S4 S11 S18 S25 −5 Male −5 S5 S12 S19 S26 −10WNN UMAP 2 −10WNN UMAP 2 S6 S13 S20 S27 S7 S14 S21 S28 −10 −5 0 5 10 −10 −5 0 5 10 WNN UMAP 1 WNN UMAP 1 Sample ID Percentage AP1 AP3 NP16 NP24 NP41 5 mitochondrial 5 10.0 AP1−post AP4 NP17 NP26 NP44 0 0 AP10 AP5 NP18 NP32 PC11 7.5 AP11 AP5−post NP19 NP35 PC2 −5 −5 5.0 AP12 AP6 NP20 NP36 PC21 WNN UMAP 2 WNN UMAP 2 −10 2.5 −10 AP13 AP9−post NP21 NP37 PC6 AP2 NP13 NP22 NP38 PC9 −10 −5 0 5 10 0.0 −10 −5 0 5 10 WNN UMAP 1 WNN UMAP 1 AP2−post NP15 NP23 NP39 b 100,000 30,000 10,000 genes 3,000 #UMIs from 1,000 ost p AP1 AP2 AP3 AP4 AP5 AP6 AP8 AP9 PP1 PP2 PP3 PP4 PC2 PC6 PC9 AP10 AP11 AP12 AP13 NP13 NP15 NP16 NP17 NP18 NP19 NP20 NP21 NP22 NP23 NP24 NP26 NP27 NP28 NP30 NP31 NP32 NP35 NP36 NP37 NP38 NP39 NP41 NP44 PC11 PC21 AP2− AP1−post AP5−post AP6−post AP7−post AP9−post 100,000 1,000 10 #UMIs from ost p AP1 AP2 AP3 AP4 AP5 AP6 AP8 AP9 PP1 PP2 PP3 PP4 PC2 PC6 PC9 antibody derived tags AP10 AP11 AP12 AP13 NP13 NP15 NP16 NP17 NP18 NP19 NP20 NP21 NP22 NP23 NP24 NP26 NP27 NP28 NP30 NP31 NP32 NP35 NP36 NP37 NP38 NP39 NP41 NP44 PC11 PC21 AP2− AP1−post AP5−post AP6−post AP7−post AP9−post c 15,000 10,000 5,000 after filtering #cells detected 0 ost p AP1 AP2 AP3 AP4 AP5 AP6 AP8 AP9 PP1 PP2 PP3 PP4 PC2 PC6 PC9 AP10 AP11 AP12 AP13 NP13 NP15 NP16 NP17 NP18 NP19 NP20 NP21 NP22 NP23 NP24 NP26 NP27 NP28 NP30 NP31 NP32 NP35 NP36 NP37 NP38 NP39 NP41 NP44 PC11 PC21 AP2− AP1−post AP5−post AP6−post AP7−post AP9−post Extended Data Figure 8 a b CD4 naive B exhausted CD4 Tcm Percent cells B naive CD4 Tem expressed B non−switched mem CD4 CTL B switched mem 0 CD14 monocytes T regs 25 CD16 monocytes CD8 naive 50 CD16+C1 monocytes CD8 Tcm CD4 CTL CD8 Tem 75 CD4 naive CD8 CTL CD4 Tcm CD4 Tem g/d T mRNA expression CD8 CTL MAIT (column z-score) CD8 naive Cycling T and NK CD8 Tcm NKT 2.5 CD8 Tem 2.0 cDC1 NK cDC2 NK CD56hi 1.5 Cycling T and NK cells ILC 1.0 g/d T CD14 monocytes 0.5 HSPCs CD16 monocytes ILC 0.0 MAIT CD16+C1 monocytes NK pDC Protein expression NK CD56hi cDC1 (column z-score) NKT cDC2 pDC 2.5 Plasma cells B naive 2.0 B non−switched mem Plasmablasts 1.5 Platelets B switched mem RBCs B exhausted 1.0 T regs Plasmablasts 0.5 K Percentage N ILC dnT gdT Plasma cells pDC 0.0 Treg Eryth MAIT overlap (%) cDC1 cDC2 HSPCs ASDC HSPC B naive Platelet 100 Platelets CD4 CTL CD4 TEM CD8 TEM CD4 TCM CD8 TCM B memory CD8 Naive RBCs CD4 Naive CD14 Mono CD16 Mono Plasmablast 50 B intermediate NK Proliferating NK_CD56bright KIT CD8 Proliferating PF4 CD4 Proliferating CD4 HBB IGHD 0 CD34 CD19 CD27 CD14 PPBP CD8A CCR6 C1QA CD3D CCR7 NCR1 IL3RA IL2RA MKI67 GZMH Azimuth annotation FXYD7 FCER2 MS4A1 TRDV2 FOXP3 TRGV9 NCAM1 CLEC9A ENTPD1 CD45RA FCER1A CLEC4C CD45RO FCGR3A SLC4A10 TRAV1−2 c TNFRSF13B Extended Data Figure 9 a Nasal immune cells Nasal immune cells (with adult and paed PBMC samples (with adult and paed COVID19 effects considered seperately) COVID19 effects considered seperately) Residual Patient Patient Patient Age group Age group Residual Residual Age group COVID19 status COVID19 status COVID19 status Sex Sex Ethnicity Sequencing batch Sex 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 b T naive T reg T CD4 FRMD4B+ T CD4 mem T CD8 mem T CD8 exhausted T gd NKT NK ILC B naive B mem Fraction of cells B mem exhausted in group (%) B mem activated B cycling Plasma IGK Plasma IGL 10 20 30 40 50 Mac Mac activated LC Mono Mono GPBAR1+ Mean expression Neut in group cDC activated pDC fDC 0.25 0.50 0.75 1.00 Mast CR2 SDS CD4 IGKC LEF1 LST1 GNLY CHPF CCR7 CCR4 CD27 CD8A CD1C C1QA VCAN CD3D IGLC2 GZMK MCM2 MKI67 KLRF1 KRT81 FCRL5 FCRL4 FCRL3 TPSB2 CTLA4 FOXP3 IGHG1 FCER2 TBX21 FDCSP LAMP3 CD207 PROK2 LILRA4 TRDV1 NR4A3 TRGV5 MS4A1 MS4A2 CAMK1 EOMES NCAM1 MARCO CLEC4C RNASE1 FCGR3B CD40LG GPBAR1 FRMD4B COL19A1 c Extended Data Figure 10 a combined_annot b chain_pairing c 100000 1 2 3 75000 4 5 6 50000 7 8 9 25000 >=10 UMAP2 UMAP2 Total # of T cell clones 0 Nasal PBMC 1.00 Tracheal 1 2 3 0.75 UMAP1 4 UMAP1 5 CD4 CTL CD8 Tem extra VDJ 6 extra VJ 0.50 7 CD4 Tcm CD8 naive 8 CD4 Tem Cycling T and NK cells no IR 9 >=10 CD4 naive MAIT orphan VDJ 0.25 fraction of TCR’s with CD8 CTL NKT orphan VJ differenting clone size CD8 T effect T regs single pair 0.00 two full chains CD8 Tcm Nasal PBMC Tracheal d patient_id tissue AP1 AP12 NP28 AP1-post AP13 NP30 AP2 NP13 NP31 AP2-post NP15 NP32 AP3 NP16 NP35 AP4 NP17 NP36 AP5 NP18 NP37 Nasal AP5-post NP19 NP38 PBMC AP6 NP20 NP39 Tracheal AP6-post NP21 NP41 AP7-post NP22 NP44 AP8 NP23 PC11 clonotype_network2 clonotype_network2 AP9 NP24 PP1 AP9-post NP26 PP2 AP10 NP27 PP3 AP11 PP4 clonotype_network1 clonotype_network1 e receptor_subtype combined_annot chain_pairing ambiguous B exhausted extra VDJ IGH B naive extra VJ IGH+IGK B non-switched mem no IR IGK+IGL B switched mem orphan VDJ UMAP2 ambiguous UMAP2 UMAP2 Plasma cells orphan VJ no IR Plasmablasts single pair two full chains UMAP1 UMAP1 UMAP1 patient_id f g AP1 AP13 NP30 1 AP1-post NP13 NP31 2 AP2 NP15 NP32 30000 3 AP2-post NP16 NP35 4 AP3 NP17 NP36 5 AP4 NP18 NP37 20000 6 AP5 NP19 NP38 7 AP5-post NP20 NP39 8 AP6 NP21 NP41 9 AP7-post NP22 NP44 10000 >=10 AP8 NP23 PC11 clonotype_network2 AP9 NP24 PP1 AP9-post NP26 PP2 0 AP10 NP27 PP3 AP11 NP28 PP4 AP12 Nasal PBMC Tracheal clonotype_network1 1.00 1 2 origin 3 h 0.75 4 5 6 0.50 7 8 9 0.25 >=10 Nasal PBMC Tracheal 0.00 clonotype_network2 Nasal PBMC Tracheal clonotype_network1 Extended Data Table 1 SARS-CoV-2 negative children SARS-CoV-2 positive children SARS-CoV-2
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