Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by granulomatous lesions containing pathological CD207+ dendritic cells (DCs) with persistent mitogen-activated protein kinase (MAPK) pathway activation. Standard of care chemotherapy strategies is inadequate for most patients with multisystem disease, and optimal strategies for relapsed and refractory disease are not defined. The mechanisms underlying development of inflammation in LCH lesions, the role of inflammation in pathogenesis and potential for immunotherapy are unknown. Analysis of the immune infiltrate in LCH lesions identified the most prominent immune cells as T lymphocytes. Both CD8+ and CD4+ T cells exhibited 'exhausted' phenotypes with high expression of the immune checkpoint receptors. LCH DCs showed robust expression of ligands to checkpoint receptors. Intra-lesional CD8+ T cells showed blunted expression of Tc1/Tc2 cytokines and impaired effector function. In contrast, intra-lesional regulatory T cells (Tregs) demonstrated intact suppressive activity. Treatment of BRAFV600ECD11c LCH mice with anti-PD-1 or MAPK inhibitor reduced lesion size, but with distinct responses: whereas MAPK inhibitor treatment resulted in reduction of the myeloid compartment, anti-PD-1 treatment was associated with reduction in the lymphoid compartment. Notably, combined treatment with MAPK inhibitor and anti-PD-1 significantly decreased both CD8+ T cell and myeloid LCH cells in a synergistic fashion. These results are consistent with a model that MAPK hyperactivation in myeloid LCH cells drives recruitment of functionally exhausted T cells within the LCH microenvironment and highlight combined MAPK and checkpoint inhibition as a potential therapeutic strategy.

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The majority of children with high-risk LCH have BRAFV600E mutation. BRAFV600E alleles are detectable in myeloid mononuclear cells at diagnosis but it is not known if the cellular distribution of mutation evolves over time. Here, the profiles of 16 high risk patients were analyzed. Two received conventional salvage chemotherapy, 4 patients on inhibitors were tracked at intervals for 3-6 years and 10 patients, also given inhibitors, were analyzed once more than 2 years after diagnosis. In contrast to patients responding to salvage chemotherapy, who completely cleared BRAFV600E within six months, children who received inhibitors maintained high BRAFV600E alleles in their blood. At diagnosis, mutation was detected predominantly in monocytes and myeloid dendritic cells. With time, mutation switched to the T cell compartment, which accounted for most of the mutational burden in PBMC more than two years from diagnosis (median 85.4%; range 44.5-100%). The highest level of mutation occurred in naïve CD4+ T cells (median 51.2%; range 3.8-93.5%). This study reveals an unexpected lineage-switch of BRAFV600E mutation in high risk LCH, which may influence monitoring strategies for the potential withdrawal of inhibitor treatment and has new implications for the pathogenesis of neurodegeneration, which occurred in 4 patients.