Extended Data Fig. 7: Single-cell transcriptomics in CD45⁺ cells from GMH cases reveal activation of proinflammatory transcriptomic profiles in monocytes, HLA⁺ myeloid cells, vasculature-associated microglia (VAM), neutrophils, and other immune cell types.

(a-b) Quantifications of the number of genes, RNA molecules, and percentage of mitochondrial genes per cell, as well as predicted doublets from scRNA-seq in age-matched CTRL and GMH cases. Each dot represents one cell. (c) UMAP feature plots of marker gene expressions that define each subtype of CD45⁺ cells. (d) UMAP feature plots of neutrophil transcripts ELANE, AZU1, and DEFA4 in CD45⁺ cells from CTRL or GMH cases. (e) Confocal images show higher abundance of ELANE⁺;CD16⁺ cells in the GE of GMH cases compared to age-matched controls. This experiment was repeated in same number of independent biological replicates for control and GMH cases as in Fig. 5e. (f) Gene burden analyses of the relative abundance of DEGs in all immune cell types. Data are from 2 independent biological samples in each condition (Control, GMH) and represent mean ± SEM. Each data point represents one pairwise comparision between a control and a GMH sample. (g-q) Volcano plots show differentially expressed genes from GMH vs CTRL cases in all CD45⁺ cell subtypes, including homeostatic microglia (MG), white matter-associated microglia (MG), cell cycle microglia (MG), vasculature-associated microglia (MG), monocytes, HLA⁺ myeloid cells, neuron-associated microglia (MG), T lymphocytes, and neutrophils. Adjusted P-values and fold changes were calculated from pseudo-bulked scRNA-seq data using DESeq2. By default in DESeq2, P-values attained by the Wald test are corrected for multiple testing using the Benjamini and Hochberg method. Genes shown were filtered to be below adjusted p-value of 0.05 and above fold change of 1.2 between CTRL and GMH comparisons. Vertical dashed lines indicate the boundary of significance in fold change. (s-t) Heatmap of GO terms in CD45⁺ cell subtypes highly associated with the GE vasculature, including monocytes, vasculature-associated microglia (MG), HLA⁺ myeloid cells, and neutrophils, which are upregulated (s) or downregulated (t) in GMH cases compared to age-matched controls. Data in all panels except e are from 2 independent biological samples in each condition (Control, GMH).