Additional file 1 of Single cell analysis identified IFN signaling activation contributes to the pathogenesis of pediatric steroid-sensitive nephrotic syndrome

Supplementary Material 1. Supplementary Fig. 1. Quality control and baseline data of each enrolled sample. (A). Principal component analysis before and after processing in Harmony. (B). t-SNE projections among different groups. (C). Quality control of the scRNA-seq data. (D). t-SNE projections from all enrolled samples. t-SNE in the control group (left), STS Pre group (middle), and STS Post group (right). (E). Boxplot comparing the proportion of plasmacytoid dendritic cells(pDCs) across the groups. The STS Pre vs. CT and STS Post vs. CT sample comparisons show exact P values determined by the Wilcoxon rank-sum test. Pre- vs. post-STS scores were calculated via the paired two-sample Wilcoxon signed-sum test. (F). The baseline information of patients and healthy controls enrolled in the scRNA-seq cohort. Supplementary Fig. 2. Focused analysis of T cells and pDCs. (A). UMAP embedding of T lymphocytes from all profiled samples in different groups. UMAP in the control group (left), STS Pre group (middle), and STS Post group (right). (B). Boxplot comparing the proportions of CRIP + CD4 + T cells, NK T cells, and TRGC2 + CD8 + T cells across the groups. The exact P values determined by the Wilcoxon rank-sum test are shown for the STS Pre vs. CT and STS Post vs. CT comparisons. Differences between STS Pre and STS Post were evaluated by the paired two-sample Wilcoxon signed-sum test. (C). Enriched pathways from Gene Ontology Biological Process Enrichment Analysis for TRGC + CD8 + T cells. (D). Enriched pathways identified by Gene Ontology Biological Process enrichment analysis in NEAT + T cells. (E). Heatmap representing the enrichment of MSigDB Hallmark gene sets for each T lymphocyte subtype across groups. (F). Pseudotime trajectory analysis of CD4 + T lymphocyte subtypes. (G). Heatmap represents DEGs within pDCs across groups. (H). Heatmap representing the enrichment of MSigDB Hallmark gene sets in the MSigDB of each group within pDCs. Supplementary Fig. 3. Focused analysis of B cells and myeloid cells. (A). Heatmap representing the enrichment of Hallmark gene sets in the MSigDB for each cell type within B lymphocytes across groups. (B). Boxplot comparing the proportions of myeloid cells across the groups. The exact P values determined by the Wilcoxon rank-sum test are shown for the STS Pre vs. CT and STS Post vs. CT comparisons. Differences between STS Pre and STS Post were evaluated by the paired two-sample Wilcoxon signed-sum test. (C). Enriched pathways from Gene Ontology Biological Process Enrichment Analysis for CD16 + monocytes. (D). Heatmap representing the enrichment of MSigDB Hallmark gene sets in each monocyte cell type across groups. Supplementary Fig. 4. The characteristics of IFN-related genes involved in pathogenesis. (A). Heatmap showing the differentially expressed genes (DEGs) in classical dendritic cells(cDCs) across groups. (B). Heatmap showing the genes differentially expressed in mast cells across groups. (C). Heatmap representing the enrichment of MSigDB Hallmark gene sets in the mast cells across groups. (D). Heatmap representing the enrichment of MSigDB Hallmark gene sets in the cDC across groups. (E). Heatmap representing the enrichment of Hallmark gene sets in the MSigDB for each cell type within Natural killer (NK) cells across groups. (F). Venn plot of the overlapping genes downregulated in the STS Pre group among B lymphocytes, T lymphocytes, monocytes, NK cells, cDCs and pDCs. (G). Receiver operating characteristic (ROC) curve and area under the curve (AUC) of overlapping genes expressed at lower levels before treatment in all cell types. (H). T-SNE analysis of CXCR4 expression in the three groups. (I). The relative expression of CXCR4 across groups was determined through qPCR. Statistical significance is denoted as P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), or P < 0.0001 (****). Supplementary Fig. 5. Analysis of transcription factors (TFs) in INS. (A). Heatmap showing TFs activation across the subcluster of T lymphocytes. (B). Heatmap showing TFs activation across the subclusters of monocytes, cDCs, neutrophils and mast cells. (C). Heatmap showing TFs activation across the subclusters of B lymphocytes. (D). Heatmap showing TFs activation across the subcluster among ΝΚ cells. (E). Heatmap showing the expression level of IFNs (IFNA1, IFNA2, IFNB1, IFNG, IFNL1, IFNL2, and IFNL3). in each cell type across groups. Supplementary Fig. 6. (A). Surface expression data of TACI and BCMA on naïve B cells, unswitched memory B cells, switched memory B cells and plasma cells determined via flow cytometry. The samples were obtained from INS patients. (B). Surface expression data of TACI and BCMA on naïve B cells, unswitched memory B cells, switched memory B cells and plasma cells determined via flow cytometry. The samples were obtained from healthy donors. (C). The proportion of naïve B cells in the serum of NS patients compared to that of healthy individuals. ns, p > 0.05. (D). The proportion of unswitched memory B cells in the serum of NS patients compared to that of healthy individuals. ns, p > 0.05. (E). The proportion of switched memory B cells in the serum of NS patients compared to that in the serum of healthy individuals. ns, p > 0.05. (F). The proportion of plasma cells in the serum of NS patients compared to that in the serum of healthy individuals. ns, p > 0.05. Supplementary Fig. 7. Supplementary analysis from an extra INS cohort (GEO233277) also validates the activation of IFN. (A). UMAP dimensionality reduction embedding from GEO datasets. (B). Heatmap showing the expression levels of the markers across each cell type using scRNAseq from the GEO datasets. The color intensity indicates the marker of interest. (C). Violin plot of the ISGs across groups using scRNAseq from the GEO datasets. Significance was evaluated with the Wilcoxon rank-sum test. (D). ISG scores among cell subtypes across groups using scRNAseq from the GEO datasets. Significance was evaluated with the Wilcoxon rank-sum test. Statistical significance is denoted as P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), or P < 0.0001 (****). (E). Incoming signaling patterns of APRIL and BAFF across groups using scRNAseq from the GEO datasets. (F). Outgoing signaling patterns of APRIL and BAFF across groups using scRNAseq from the GEO datasets. (G). Heatmap showing the expression level of all kinds of IFNs in each cell type across groups using scRNAseq from the GEO datasets. Supplementary Fig. 8. The potential mechanism underlying the pathogenesis of pediatric idiopathic nephrotic syndrome. (A). IFN-γ activation, mainly generated by T cells, stimulates the upregulation of BAFF expression among monocytes, dendritic cells, and neutrophils. Subsequently, the activation of BAFF interacts with its receptors on B cells, especially BCMA and TACI, facilitating B cell maturation and leading to autoantibody release. BAFF: B-cell activating factor; BCMA: B-cell maturation antigen; TACI: transmembrane activator and cyclophilin ligand interactor.