Article Figures & Data
- Figure 1
Study design and transcriptome profiling of PBMCs from controls and patients who had a stroke. (A) Schematic picture showing the overall study design; PBMCs were pulled from controls (n=9) and patients who had an acute ischaemic stroke (n=10), and single-cell RNA sequencing was performed. (B) Cell clusters were identified with UMAP projection of 101 481 cells from controls and patients who had a stroke. (C,E) Selected canonical cell markers were used to identify cell clusters. cDC, classical dendritic cell; MK, megakaryocyte; NK, natural killer; PBMC, peripheral blood mononuclear cell; pDC, plasmacytoid dendritic cell; UMAP, uniform manifold approximation and projection.
- Figure 2
Differences in NK cell population between controls and patients who had a stroke. (A) Top dot plot shows the total PBMC counts of each subject used in scRNA-seq. The bar plot shows the percentage of cell clusters in each subject. The average percentage of each cell cluster analysed using scRNA-seq (B) and flow cytometry (C) is shown in green for controls and orange for patients who had a stroke. From both scRNA-seq and flow cytometry, it is demonstrated that NK cell cluster is increased in patients who had a stroke. (D) Representative flow cytometry plot shows increased NK cell population in patients who had a stroke. (E) DEGs (adjusted p<0.05) in NK cell clusters of patients who had a stroke compared with controls are demonstrated using a heatmap. (F) Ingenuity pathway analysis was performed with all DEGs in the NK cell cluster, demonstrating enhanced NK cell signalling. (G) Pathway analysis was performed with upregulated DEGs (orange) and downregulated DEGs (blue) of the NK cell cluster. Upregulated DEGs are involved in the activation of the NK cell-related pathway. cDC, classical dendritic cell; DEG, differentially expressed gene; MK, megakaryocyte; NK, natural killer; PBMC, peripheral blood mononuclear cell; pDC, plasmacytoid dendritic cell; scRNA-seq, single-cell RNA sequencing; PKR, Protein Kinase R; STAT3, Signal Transducer and Activator of Transcription 3; MAPK, Mitogen-Activated Protein Kinase.
- Figure 3
Changes of monocytes subclusters in patients who had a stroke. (A) Two CD14+ monocytes clusters were classified into 14 subclusters; CD14+ monocyte cluster A is classified into nine subclusters (0, 2–4 and 6–10), and CD14+ monocyte cluster B is classified into five subclusters (1, 9 and 11–13). (B) Based on the gene expression characteristics, cluster A is named dendritic cell-related CD14+ monocyte cluster (subclusters 0, 2–4, 7 and 8 in a circle in peach) and cluster B is named NK cell-related CD14+ monocyte cluster (subclusters 1, 9 and 11–13 in a circle in cyan). Besides, there is one plasma cell-related cluster (5), one erythrocyte progenitor cell-related cluster (6) and one megakaryocyte-related cluster (10). (C) The violin plot shows specifically expressed genes of each subcluster using classification. (D) The proportion of each subcluster was compared between controls and patients who had a stroke. (E) Individual variations of each subcluster in controls and patients who had a stroke are shown. Horizontal lines represent the average value for the proportion of each subcluster. *Significant differences (p<0.05) between controls and patients who had a stroke. NK, natural killer; UMAP, uniform manifold approximation and projection.
- Table 1
Characteristics of patients who had a stroke
Subject characteristics All patients (n=10) Demographic characteristics Age (years), mean±SD 68.8±13.1 Sex, female, n (%) 4 (40) Race Caucasian (100%) Hispanic 0 Risk factors, n (%) Hypertension 7 (70) Diabetes 0 Hyperlipidaemia 2 (20) Previous ischaemic stroke/transient ischaemic attack 0 Smoking (current and former within 5 years) 1 (10) Alcohol (two or fewer drinks/day) 6 (60) Medication, n (%) Antiplatelet 3 (30) Anticoagulant 0 Lipid-lowering 4 (30) Blood pressure medication 5 (50) NIHSS, median (IQR 25–75) 1.5 (0–4) Time intervals (min), median (IQR 25–75) Last known well to triage 111.5 (40.0–698.0) Last known well to blood draw 297.5 (278.8–869.3) Stroke location, n (%) Left cerebrum 3 (30) Left cerebrum and cortical cerebrum 2 (20) Left cerebrum and deep cerebrum 2 (20) Right cerebrum 1 (10) None 2 (20) Vascular territory, n (%) Left ACA 2 (20) Left MCA 6 (60) Right ACA and right PCA 1 (10) Acute treatment with intravenous tPA, n (%) Intravenous tPA 4 (40) None 6 (60) Days of hospital stay, median (IQR 25–75) 2.5 (1.0–4.3) Last known well indicates the time before hospital arrival at which the patient was last known to be without the signs and symptoms of the current stroke.
ACA, anterior cerebral artery; MCA, middle cerebral artery; NIHSS, National Institutes of Health Stroke Scale; PCA, posterior cerebral artery; tPA, tissue plasminogen activator.
- Table 2
Differentially expressed genes in natural killer cell cluster
Gene name Average log2 fold change Adjusted P value GIMAP7 0.444456 0.000791 CX3CR1 0.370523 1.54E-10 H3F3B −0.25528 0.008194 EIF3H −0.27161 0.000524 RPL7 −0.27724 0.008482 BTG1 −0.29421 1.80E-08 CD74 −0.32504 5.67E-05 HLA.DRB1 −0.3366 0.000357 CD69 −0.35405 0.004314 NFKBIA −0.35952 0.006542 TSC22D3 −0.36775 1.30E-05 CD3D −0.41581 0.000151 TNFAIP3 −0.42138 0.004851 DUSP1 −0.42501 3.43E-05 LTB −0.53388 7.39E-10 ZFP36 −0.55201 2.21E-13 IL7R −0.69229 1.54E-17 DUSP2 −0.76309 3.16E-20 CXCR4 −0.79897 3.37E-29 Log2 fold change, positive values indicate that the gene is more highly expressed in the patient who had a stroke compared with controls; adjusted p values are calculated based on Bonferroni correction.
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