Single cell RNA sequencing. (A) UMAP plot of single-cell gene expression in BMMNCs of HLA alloimmunized (n = 8) and nonalloimmunized (n = 12) patients with SAA. Cells are colored by type (HSPC; ProB; erythroblast, neutrophil at different differentiation stages, monocyte at different differentiation stages, CD8⁺ T cell, CD4⁺ T cell, NK cell, B/plasma, DC, stromal cell, and platelet). Frequency (% in BMMNCs) of cell populations in HLA alloimmunized SAA (n = 8) and nonalloimmunized (n = 12) patients with SAA, by 2-sided unpaired Mann-Whitney test. No statistical significance is observed. (B) A bar chart showing enriched gene sets by Gene Set Enrichment Analysis (GSEA). Enriched plots of differentially expressed genes for Hallmark complement, Hallmark interferon gamma response, Hallmark TNF-α signaling via NFkB, and Hallmark complement in BMMNCs of HLA alloimmunized compared with nonalloimmunized patients with SAA. GSEA based on the Kolmogorov-Smirnov test. (C) A network of upregulated genes involved in interferon gamma response, TNF-α signaling via NFkB, and complement pathway in those with postive HLA class I Ab (n = 8) compared to nonalloimmunized (n = 12) patients with SAA. B/Plasma, B cell and plasma cell; DC, dendritic cell; HSPC, hematopoietic stem and progenitor cells; IFN-γ, interferon gamma; NES, normalized enrichment score; NK, natural killer; ProB, B-cell progenitors.