Additional file 1 of Aged microglia promote peripheral T cell infiltration by reprogramming the microenvironment of neurogenic niches
Creators: name: Zhang, Xiaotao
name: Wang, Rui
name: Chen, Haoran
name: Jin, Chenghao
name: Jin, Ziyang
name: Lu, Jianan
name: Xu, Liang
name: Lu, Yunrong
name: Zhang, Jianmin
name: Shi, Ligen
Publisher: name: figshare
Year: 2022
Subjects: subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Neuroscience
subject: Physiology
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Mental Health
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Supplementary Fig. 1. Functional implication of infiltrated T cells. (a) Schematic illustration of original processing steps by Dulken et al. Single-cell RNA sequencing data of the SVZ from three young (3 months old) and three old (28–29 months old) mice were used for downstream analysis. (b–c) t-SNE projection of whole cells in the SVZ from young and aged mice (B). CD45+ cells were extracted for sub-clustering analysis (c). (d) Heatmap showing the expression levels of top five marker genes in each cell type. The most significantly upregulated genes are shown in yellow and downregulated genes are shown in Tibetan blue. (e) Feature plot depicting the expression levels of T cell markers. (f) Number of CD8+ T cells per coronal section in five young (6–8 weeks old) and five old (18 months old) female mice. **P = 0.0079, Mann–Whitney test, two sided. Data are shown as mean ± s.e.m. (g) Functional enrichment analysis was performed using Metascape on the T cells’ DEGs in the old SVZ. The bar plot shows the significance of the enriched terms. (h) Sankey diagram depicting the interaction between T cells and resident cells in the SVZ based on the cytokines released by T cells. The proportional flow represents the number of gene pairs. Gene pairs are listed in Supplementary Table 3. Supplementary Fig. 2. Single-cell transcriptomic analysis of CD8+ T cells. (a) Schematic illustration of original processing steps by Kimmel et al. Single-cell RNA sequencing data of the spleen from four young (7–8 months old) and three old (22–23 months old) mice were used for downstream analysis. (b) t-SNE projection of whole cells in the spleen from young and aged mice (left). CD8+ T cells were extracted for subclustering analysis (right). (c) Expression profiles of Cd44 (encoding the CD44 antigen) and Sell (encoding the CD62L antigen) in CD8+ T cells are shown using the UMAP visualization approach. (d) Flow cytometry gating strategy for CD45+CD3+CD8a+ CD44+ CCR2+ memory T cells. (e) Protein expression of CCR2 by splenic CD8+ memory T cells of five young (6–8 weeks old) and five aged (18 months old) female mice. **P = 0.0054, two-tailed Student’s t test. Data are shown as mean ± s.e.m. (f) Quantification of CCL3 fluorescence in IBA1+ cells in five young (6–8 weeks old) and five aged (18 months old) female mice. ****P &lt; 0.0001, two-tailed Student’s t test. Data are shown as mean ± s.e.m. (g) Left: Representative confocal microscopic images of young and old SVZs stained for CCL4 and IBA-1. Nuclei are labeled with DAPI. Scale bars: 200 μm or 25 μm. Right: Quantification of CCL4 fluorescence in IBA1+ cells in four young (6–8 weeks old) and four aged (18 months old) male mice. ***P = 0.0005, two-tailed Student’s t test. Data are shown as mean ± s.e.m. Supplementary Fig. 3. Single-cell transcriptomic analysis of brain endothelial cells. (a) Representative confocal microscopic images of young and old mouse brains stained for CD31 and CCL3. Nuclei are labeled with DAPI. Scale bars: 50 μm. (b) Schematic illustration of original processing steps by Dulken et al. Single-cell RNA sequencing data of the SVZ from three young (3 months old) and three old (28–29 months old) mice were used for downstream analysis. (c) t-SNE projection of whole cells in the SVZ from young and aged mice (left). Endothelial cells were extracted for subclustering analysis. (d) Plots showing the expression of representative well-known arterial (Fbln5), capillaries (Rgcc), and venous (Nr2f2) marker genes on the pseudotime trajectory. (e) Cellular trajectory of all endothelial cell subclusters (E0–E6) generated by Monocle. (f) Ternary diagram showing the marker genes of arteries, capillaries, and veins. Red nodes represent arterial marker genes, green nodes represent capillary marker genes, and blue nodes represent venous marker genes. (g) Expression profiles of Cldn5 and Ocln in both young and aged BECs are shown using the t-SNE visualization approach. Supplementary Fig. 4. Single cell transcriptomic analysis of micrglia. (a) Schematic illustration of original processing steps by Dulken et al. Single-cell RNA sequence data of SVZ from three young (3 months old) and three old (28–29 months old) mice were used for downstream analysis. (b) T-SNE projection of whole cells in SVZ from young and aged mice (left). Microglia were extracted for sub-clustering analysis. (c) volcano plot depicting the DEGs in microglia cluster 2 compared to cluster 0. DEGs were colored (red for upregulated DEGs and green for downregulated DEGs). (d) Functional enrichment analysis was performed using Metascape on the upregulated DEGs in (c). The significantly overrepresented (P &lt; 0.01) terms were grouped into color-coded clusters based on their membership similarities and rendered as a network plot. Each node represents an enriched term, and one representative term is shown for each cluster. Terms with a similarity &gt; 0.3 are connected by edges. (e) The relationship between the enriched terms in (d) and the involved genes is depicted. Circos plot shows genes related to inflammatory response and cytokine production were explored for their involvement in five functional sub-categories. (f) Quantification of TNF-α fluorescence in IBA1+ cells in five young (6–8 weeks old) and five aged (18 months old) female mice. ****P &lt; 0.0001, two-tailed Student’s t test. Data are shown as mean ± s.e.m. (g) Design foe brain slice – CD8+ T cells coculture experiment for flow cytometry analysis.