Detailed knowledge of human B-cell development is crucial for the proper interpretation of inborn errors of immunity and malignant diseases. It is of interest to understand the kinetics of protein expression changes during development, but also to properly interpret the major and possibly alternative developmental trajectories. We have investigated human samples from healthy individuals with the aim of describing all B-cell developmental trajectories. We validated a 30-parameter mass cytometry panel and demonstrated the utility of "vaevictis" visualization of B-cell developmental stages. We used the trajectory inference tool "tviblindi" to exhaustively describe all trajectories leading to all developmental ends discovered in the data. Focusing on Natural Effector B cells, we demonstrated the dynamics of expression of nuclear factors (PAX-5, TdT, Ki-67, Bcl-2), cytokine and chemokine receptors (CD127, CXCR4, CXCR5) in relation to the canonical B-cell developmental stage markers. We observed branching of the memory development, where follicular memory formation was marked by CD73 expression. Lastly, we performed an analysis of two example cases of abnormal B-cell development caused by mutations in RAG-1 and Wiskott-Aldrich syndrome gene in patients with primary immunodeficiency. In conclusion, we developed, validated, and presented a comprehensive set of tools for the investigation of B-cell development in the bone marrow compartment.

Center for Chronic Immunodeficiency University Medical Center Freiburg Faculty of Medicine University of Freiburg Freiburg Germany

CLIP Department of Paediatric Haematology and Oncology 2nd Faculty of Medicine Charles University Prague Czech Republic

Data Mining and Modeling for Biomedicine Center for Inflammation Research VIB UGent Ghent Belgium

Department of Applied Mathematics Computer Science and Statistics Ghent University Ghent Belgium

Department of Internal Medicine Erasmus MC University Medical Center Rotterdam Rotterdam the Netherlands

Department of Paediatric Haematology and Oncology University Hospital Motol Prague Czech Republic

Department of Pediatrics Laboratory for Pediatric Immmunology Leiden University Medical Center Leiden the Netherlands

Department of Rheumatology and Clinical Immunology Freiburg University Medical Center University of Freiburg Freiburg Germany

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