HLA-DQB2 expression is identified on blood BRAF-V600E⁺CD1c⁺mDCs in patients with high-risk (HR) LCH. (A) Genomic DNA was isolated from peripheral blood specimens from LCH patients (N = 11; n = 3 for high-risk multisystem, n = 3 for low-risk [LR] multisystem, n = 5 for low-risk single lesion) with BRAFV600E⁺ lesions and healthy donors (N = 11). The percentage of circulating cells with BRAFV600E allele was determined by qPCR.³  As expected from previous studies, BRAFV600E expression was detected at low levels in PBMCs from patients with BRAFV600E⁺ lesions and active high-risk LCH, and was absent from PBMCs from patients with low-risk LCH and healthy donors (HD).³  Technical duplicates were used in this experiment. LCH patients’ PBMC samples used in the study are listed in supplemental Table 1b. Blue dot, Patient LCH 0019; red dot, patient LCH 0020; green dot, patient LCH 0021. (B) RNA from peripheral blood specimens from the same set of LCH patients as in panel A was extracted and cDNA was amplified, and then the HLA-DQB2 expression was determined by qPCR (normalized to GAPDH mRNA expression). HLA-DQB2 expression was specifically detected in PBMCs from the same patients with detectable BRAFV600E⁺ PBMCs. Technical duplicates were used in this experiment. Blue dot, Patient LCH 0019; red dot, patient LCH 0020; green dot, patient LCH 0021). (C) Representative dot plots showing identification of CD1c⁺ mDCs (green gate) within HLA-DR⁺ cells from PBMCs of an HR LCH patient. Overlay histograms show HLA-DQB2 expression in LCH lesion CD1c⁺ mDCs (green) compared with control (gray). HLA-DQB2 expression was detectable on some CD1c⁺ mDCs. Representative dot plots showing identification of CD1c⁺ mDCs (green gate) from PBMCs of a healthy donor were illustrated in supplemental Figure 8. No HLA-DQB2 expression was detectable on CD1c⁺ mDCs from PBMCs of a healthy donor. (D) Genomic DNA from unsorted and sorted cells from PBMCs of high-risk LCH patients (N = 3) with BRAFV600E⁺ lesions was isolated and amplified, and the percentage of cells with BRAFV600E allele was determined by qPCR. As demonstrated in previous studies, many lineages have the potential to carry the BRAFV600E mutation.³  Technical duplicates were used in this experiment. Blue dot, Patient LCH 0019; red dot, patient LCH 0020; green dot, patient LCH 0021. (E) Genomic DNA from CD1c⁺ mDCs (HLA-DQB2^- and HLA-DQB2⁺) from PBMCs of high-risk LCH patients (N = 3) was isolated and amplified, and the percentage of cells with BRAFV600E allele was determined by qPCR. BRAFV600E was highly enriched in the HLA-DQB2⁺CD1c⁺ mDC population. Technical duplicates were used in this experiment. Blue dot, Patient LCH 0019; red dot, patient LCH 0020; green dot, patient LCH 0021.