BRAFV600E⁺cells localize to LCH lesion CD1c⁺mDCs, CD1a⁺CD207⁻, CD1a⁺CD207^low, and CD1a⁺CD207^highsubpopulations. (A) Dot plots showing identification of 3 LCH subpopulations within HLA-DR⁺ and CD1a⁺ fractions from 12 LCH lesions: CD1a⁺CD207⁻ (yellow gate), CD1a⁺CD207^low (red gate), and CD1a⁺CD207^high cells (green gate). (B) Flow cytometry of LCH lesion specimens with representative dot plots showing identification of 3 subpopulations within HLA-DR⁺, CD45⁺, CD207⁻CD1a^−, and LIN⁻ fractions from LCH lesions: CD14⁺ DCs, CD1c⁺ mDCs, and CD11c⁺CD1c⁻ cells. (C) PCA of global transcriptome data from LCH lesion subpopulations (LCH lesion CD3⁺ lymphocytes, CD1a⁺CD207⁻ cells, CD1a⁺CD207^low cells and CD1a⁺CD207^high cells) demonstrates clustering of all the CD1a⁺ populations and also clustering of the CD3⁺ populations. All samples used in the study are listed in supplemental Table 3a. (D) Lesions from LCH patients (N = 8) (supplemental Table 3b) were FACS-purified according to gating strategies shown in Figure 3A-B and supplemental Figure 4. Genomic DNA was extracted and amplified, and then the BRAFV600E allele was quantified by quantitative PCR (qPCR) as described in Berres et al.³ BRAFV600E was highly enriched in all CD1a⁺ populations, and was also detected in CD1c⁺ mDCs, but not in other LCH lesion populations. Technical duplicates were used in this experiment. CD207 and CD1a expression in LCH lesion subpopulations was further determined and shown in supplemental Figure 3. PC1, first principal component; PC2, second principal component.