Introduction: Moyamoya Disease (MMD) is a rare cerebrovascular disease characterized by the narrowing of the intracranial arteries and the formation of compensatory collateral vessels. Previous research suggests a role of elevated immune response and abnormal extracellular matrix-related gene expression in the progression of the disease. In this study we characterized the immune composition and cell type–specific transcriptomes of peripheral blood mononuclear cells (PBMCs) from MMD patients using single cell RNA sequencing (scRNA-seq) and flow cytometry. Methods: Whole blood samples were collected from bilateral MMD patients (n=3), and healthy controls (n=2). PBMCs were isolated within two hours and processed for 10x scRNA-seq (Chromium Single Cell 3′ Gene Expression v3); libraries were sequenced on a NovaSeq 6000 (PE150). We identified immune-cell heterogeneity and cell type–specific differentially expressed genes (DEGs) and performed pathway enrichment with Ingenuity Pathway Analysis (IPA). An 11-color flow-cytometry panel validated key populations, and selected subsets were sorted by FACS for qPCR (MMD n=5; non-MMD n=5; healthy n=4). Results: Our results showed a distinct immune cell landscape in MMD patients, including CD4+ NKT (5.78% vs 3.22%), CD8+ T (9.21% vs 7.11%), and CD8+ NKT (9.59% vs 15.74%) for MMD vs healthy. DEG analyses revealed that CD8+ NKT cells, naïve CD4+ T cells, and classical monocytes are more transcriptionally active in MMD. Pathway analysis shows predicted changes at the immune-vascular interface, as well as inhibition in mTOR, interleukin, and CXCR4 signaling. Flow cytometry with additional patient samples showed a significant increase in circulating CD8+ NKT cells in MMD (% of total NKT; CD8+ NKT p=0.018) compared to healthy control. Notably, ligand–receptor interaction analysis suggested that CD8+ NKT cells are major targets of intercellular communication, receiving signals from multiple immune cell types. Among these, the CD94/NKG2A receptor appears as a prominent inhibitory signal in this interaction. Conclusions: This study highlights MMD-specific immune cell types, cell type-specific transcriptomics and intercellular signaling networks, suggesting involvement of immune-vascular crosstalk in MMD. Ongoing studies utilize FACS sorting of CD8+ NKT cells and qPCR to validate candidate genes. These results demonstrate a potential role of peripheral immune dysregulation in MMD vascular pathology.
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