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Background: Systemic mastocytosis (SM) is a haematological disease characterised by organ infiltration by neoplastic mast cells. Almost all SM patients have a mutation in the gene encoding the tyrosine kinase receptor KIT causing a D816V substitution and autoactivation of the receptor. Mast cells and CD34(+) haematopoietic progenitors can carry the mutation: however, in which progenitor cell subset the mutation arises is unknown. We aimed to investigate the distribution of the D816V mutation in single mast cells and single haematopoietic stem and progenitor cells. Methods: Fluorescence-activated single-cell index sorting and KIT D816V mutation assessment were applied to analyse mast cells and >10,000 CD34(+) bone marrow progenitors across 10 haematopoietic progenitor subsets. In vitro assays verified cell-forming potential. Findings: We found that in SM 60-99% of the mast cells harboured the KIT D816V mutation. Despite increased frequencies of mast cells in SM patients compared with control subjects, the haematopoietic progenitor subset frequencies were comparable. Nevertheless, the mutation could be detected throughout the haematopoietic landscape of SM patients, from haematopoietic stem cells to more lineage-primed progenitors. In addition, we demonstrate that Fc epsilon RI+ bone marrow progenitors exhibit mast cell-forming potential, and we describe aberrant CD45RA expression on SM mast cells for the first time. Interpretation: The KIT D816V mutation arises in early haematopoietic stem and progenitor cells and the mutation frequency is approaching 100% in mature mast cells, which express the aberrant marker CD45RA.

Mast cells are tissue-resident immune cells that are well known for their involvement in asthma and allergy. They originate in the fetal yolk sac, or in the bone marrow. From the bone marrow, they are released as immature progenitors that are transported via the blood circulation to the peripheral tissue, where they mature. Human mast cells accumulate at certain sites in the airways of individuals with asthma, and in a mouse model of allergic airway inflammation, an influx of mast cell progenitors (MCp) to the lung has been demonstrated. We quantified MCp frequency by flow cytometry to determine if there is a correlation between MCp frequency and reduced lung function among allergic asthmatics and controls. We found that individuals with reduced lung function had an increased MCp frequency in the blood circulation. Additionally, women had a higher MCp frequency than men. In the second study, we identified a bone marrow counterpart to the human blood MCp. These two MCp populations had similar mast cell-related gene expression, morphology and proliferation capacity. However, the blood MCp expressed higher levels of the integrin β7 receptor, which in mice has been shown to be required for MCp transmigration into peripheral tissues. Mast cells can be activated by antigen crosslinking of IgE bound to FcεRI. Both human and mouse MCp express FcεRI, but it is unknown whether they can be activated via this receptor. In the third study, we demonstrated that human and mouse MCp can become activated in vitro via FcεRI by measuring tyrosine kinases phosphorylation by flow cytometry. Furthermore, we showed the activation in vivo by measuring IL-13 production in MCp in an allergic airway inflammation model. In the final study, we investigated the chemokine receptor expression on mouse MCp. We found that MCp in the peritoneal cavity express CCR5 whereas bone marrow MCp express CCR1. To summarize, my thesis has provided novel insights into the significance of circulating human MC