The role of pro-apoptotic BH3-only proteins in survival and differentiation of lymphocytes

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Mitochondrial apoptosis is a key regulatory mechanism during differentiation and life of haematopoietic cells. Mitochondrial apoptosis is governed by the Bcl-2-family of proteins; within this family the group of eight BH3-only proteins are initiators of apoptosis, which by their expression levels and possibly their activation regulate apoptosis. In immune cells, Bim is the most prominent and important BH3-only protein but a number of other BH3-only proteins (especially Puma, Bmf and Noxa) appear to play an incompletely understood role. We have learned much about mitochondrial apoptosis in immune cells especially from gene-deficient mouse models. However, further progress is slow in particular because of the limitations in obtaining the necessary cellular material. We have established a cell model where mouse haematopoietic progenitor cells can be expanded in vitro and differentiated into myeloid cells as well as into lymphocytes in vitro and in vivo. We have established such progenitor cells from mice double deficient in BH3-only proteins (Bim/Puma, Bim/Bmf, Bim/Noxa) and have found intriguing apoptosis defects in some situations. We here propose to use these cells, and to establish additional cell lines carrying triple-deficiencies for BH3-only proteins, in order to analyse the contribution of BH3-only proteins to development, maintenance and apoptosis sensitivity especially in B and T lymphocytes. In vitro, progenitors as well as "B cells" and "T cells" of the various genotypes will be tested for their apoptotic response (B cells can from the progenitors be differentiated in vitro to IgM-positive cells, T cells into DN3/4 stage "thymocytes"). Apoptosis sensitivity will further be tested ex vivo from mice injected with progenitor cells (by injecting irradiated mice with progenitors, fully mature lymphocytes can be obtained). The expression of BH3-only proteins during differentiation towards B and T cells in vitro will further be measured. Through the assessment of complex formation with anti-apoptotic Bcl-2 proteins, open questions of the molecular function of BH3-only proteins will be addressed. Lastly, by measuring competitive productivity of differentiating, gene-deficient cells during injection of two genetically different sets of progenitor cells into mice (such as Bim- plus Bim/Noxa-deficient) we will assess potential survival advantages upon loss of BH3-only proteins during differentiation of myeloid and lymphoid cells in vivo. We believe that with this approach we will be able to obtain substantial new information on BH3-only proteins and their roles in differentiation and apoptosis sensitivity of mouse haematopoietic cells.