Transcriptomic profiling of circulating immune cells from LCH patients using scRNA-seq. (A) Dot plot showing the cfBRAF^V600E load distribution of PB samples used for scRNA-seq (n = 14). Shapes represent the clinical classification, and colors represent the mutational classification. (B) tSNE plot visualization of 11 cell clusters identified from a total of 2727 single-cell transcriptome profiles, pooled from LCH patients and healthy controls. The Lin⁻HLA-DR⁺ population (green box) and the subsets within it including CD14⁺ monocyte (pink box), CD14⁻CD11C⁺BDCA2⁻ subset containing cDC and CD16⁺⁺ monocyte (yellow box), and CD14⁻BDCA2⁺CD11c^– pDCs (blue box) are sorted for single-cell RNA sequencing (Figure 1C). (C) Violin plot overview of the expression of curated feature genes in each immune cell cluster. (D) tSNE plots of circulating hematopoietic cell clusters overlaid with mutational (i) and clinical classification (ii). (E) Dot plots showing the average expression level and percentage of cells expressing top 5 differential expression genes (DEGs) for CD14⁺ Mo, CD16⁺⁺ Mo, iMo, and cDC2/DC3 of each mutational group compared with normal control. DEGs are detected using function FindMarkers in Seurat (Wilcoxon-rank-sum test, with P value adjusted for multiple testing using Benjamini-Hochberg correction). Top 5 DEGs with log fold change >1, adjusted P < .01, and expressed by more than 50% cells in each group are selected for visualization.