Monocytes contribute to immune responses as a source for subsets of dendritic cells and macrophages. Human blood monocytes are classified as classical, non-classical and intermediate cells. However, the particular functions of these subsets have been hard to define, with conflicting results and significant overlaps. One likely reason for these ambiguities is in the heterogeneity of these monocyte subsets regrouping cells with divergent functions. To better define monocyte populations, we have analysed expression of 17 markers by multicolour flow cytometry in samples obtained from 28 control donors. Data acquisition was tailored to detect populations present at low frequencies. Our results reveal the existence of novel monocyte subsets detected as larger CD14 + cells that were CD16 + or CD16 neg. These large monocytes differed from regular, smaller monocytes with respect to expression of various cell surface molecules, such as FcR, chemokine receptors, and adhesion molecules. Unsupervised multidimensional analysis confirmed the existence of large monocytes and revealed interindividual variations that were grouped according to unique patterns of expression of adhesion molecules CD62L, CD49d, and CD43. Distinct inflammatory responses to TLR agonists were found in small and large monocytes. Overall, refining the definition of monocyte subsets should lead to the identification of populations with specific functions. Monocytes, which are mostly precursors of some macrophage and dendritic cell populations, have been hard to divide into populations with clear-cut inflammatory and immune functions. This may be due in part to the high number and complexity of phenotypes present in monocyte populations. A classification of human blood monocyte subsets based on the expression of CD14 and CD16 cell surface receptors was proposed 1 and refined over the years 2,3. It consisted initially of two populations described as "classical" monocytes, which express CD14 but no CD16, and "nonclassical" monocytes with low CD14 and strong CD16 expression. Further analysis identified CD14 + CD16 + monocytes with expression of CCR5 and intermediate expression of receptors divergently expressed in the two other subsets (e.g. CCR2 and CX3CR1 4. This additional subset was identified as "intermediate" 5. Evidence for this third subset was confirmed in transcriptome analysis 6-10. Despite progress in phenotypic analysis, immune functions associated with monocyte subpopulations in the steady state remained ill defined. Marked functional redundancies between the sub-populations were found, and contradicting results in the literature added to the puzzling difficulties in assigning functions to specific populations 11-13. Thus, intermediate monocytes were described as the major source of pro-inflammatory cytokines upon stimulation 14,15. In contrast, non-classical monocytes were also described as the most inflammatory monocytes 3,13,16. Similarly, anti-inflammatory cytokine secretion was alternatively found high in intermediate 13 or in classical monoc...

The clinical significance of the BRAFV600E mutation in adult Langerhans cell histiocytosis (LCH), including pulmonary Langerhans cell histiocytosis (PLCH), is not well understood. Similarly, the spectrum of molecular alterations involved in adult LCH has not been fully delineated. To address these issues, we genotyped a large number of adult LCH biopsies and searched for an association of identified molecular alterations with clinical presentation and disease outcome.Biopsies from 117 adult LCH patients, 83 with PLCH (median age 36.4 years, 56 females, 38 multisystem disease, 79 single system disease, 65 current smokers) were genotyped for the BRAFV600E mutation. In 69 cases, LCH lesions were also genotyped by whole-exome sequencing (WES) or targeted gene panel next-generation sequencing (NGS). Cox models were used to estimate the association of baseline characteristics with the hazard of LCH progression.MAPK pathway alterations were detected in 59 out of 69 cases (86%) (BRAFV600E mutation: 36%, BRAFN486_P490 deletion: 28%, MAP2K1 mutations: 15%, isolated NRASQ61 mutations: 4%), while KRAS mutations were virtually absent in PLCH lesions. The BRAFV600E mutation was not associated with LCH presentation at diagnosis, including smoking status and lung function, in PLCH patients. BRAFV600E status did not influence the risk of LCH progression over time.Thus, MAPK alterations are present in most lesions from adult LCH patients, particularly in PLCH. Unlike reports in paediatric LCH, BRAFV600E genotyping did not provide additional information on disease outcome. The search for alterations involved in the MAPK pathway, including BRAF deletions, is useful for guiding targeted treatment in selected patients with refractory progressive LCH.