Beschreibung:
Abstract Bone marrow (BM) analysis show that 20% of PAD patients harbor a block at an early stage of B cell development. The aim of this work is to study the mechanisms of this developmental block. We set up a feeder free cultivation system that in healthy donors leads to the development of lymphocyte progenitors until the stage of Immature B cells. BM derived CD34+ cells are expanded in cytokine cocktail for 2 weeks. From day 14 to day 49 cells are cultivated in cytokine-free medium and analyzed once a week by flow cytometry and RNA is collected for PCR assays. BM derived CD34+ cells from 15 PAD patients and 2 Btk-deficient patients were tested in vitro for their capability to develop into IgM+ Immature B cells. CD34+ cells from patients showing a normal B cell development in vivo (6/17) developed in vitro until the stage of Immature B cells. Among PAD patients presenting a block at early B cell stage, 5/11, including the Btk-deficient patients, could not reach the Immature B cell stage, while 6/11 could develop in vitro until the stage of IgM+ B cells. Among this last group, reaching Immature B cell stage in vitro but not in vivo, 2/6 patients showed a rapid in vitro development of Immature B cells at day 14, but presented a progressive exhaustion of the common lymphoid progenitors (CLP) compartment. In patients with a block in B cell development (in vivo and in vitro), PCR analysis revealed adelayed expression of transcription factors E2A, CD79a and PAX5, reflecting an impaired lineage commitment. Combining BM phenotyping and in vitro modeling we identified four patients’ groups characterized by (1) an intrinsic B cell defect, (2) a defect in the BM microenvironment impairing early stages of B cell development, (3) a defect in repopulation of CLP and (4) normal B cell development.