BCR-induced superoxide negatively regulates B-cell proliferation and T-cell-independent type 2 Ab responses.

Superoxide and its derivatives have been implicated as secondary messenger molecules that influence signaling cascades in non-phagocytes. B lymphocytes produce superoxide after BCR ligation. We found that these ROS regulate B-cell signaling and entry into the cell cycle. B cells from mice deficient in the gp91(phox) subunit of the NADPH oxidase complex are unable to generate ROS after BCR ligation. However, after BCR stimulation, more gp91(phox) KO B cells enter the G1 stage of the cell cycle and proliferate than WT B cells. BCR ligation leads to a more rapid decrease in p27(Kip1) levels in gp91(phox) KO B cells. Gp91(phox) KO mice display enhanced T-cell-independent type 2, but normal T-dependent Ab responses. ROS-dependent regulation of BCR-induced proliferation may help modulate the size of the humoral response to T-cell-independent type 2 Ag immunization.

Surface expression of Notch1 on thymocytes: correlation with the double-negative to double-positive transition.

Notch1 plays a critical role in regulating T lineage commitment during the differentiation of lymphoid precursors. The physiological relevance of Notch1 signaling during subsequent stages of T cell differentiation has been more controversial. This is due in part to conflicting data from studies examining the overexpression or targeted deletion of Notch1 and to difficulties in distinguishing between the activities of multiple Notch family members and their ligands, which are expressed in the thymus. We employed a polyclonal antiserum against the extracellular domain of Notch1 to study surface expression during thymopoiesis. We found high levels of Notch1 on the cell surface only on double negative (DN) stage 2 through the immature single-positive stage of thymocyte development, before the double-positive (DP) stage. The Notch signaling pathway, as read out by Deltex1 expression levels, is highly active in DN thymocytes. When an active Notch1 transgene, Notch1IC, is exogenously introduced into thymocytes of recombinase-activating gene 2-deficient mice, it promotes proliferation and development to the DP stage following anti-CD3 treatment without apparently affecting the intensity of pre-TCR signaling. In addition, a stromal cell line expressing the Notch ligand, Delta-like-1, promotes the in vitro expansion of wild-type DN3 thymocytes in vitro. Consistent with other recent reports, these data suggest a role for Notch1 during the DN to DP stage of thymocyte maturation and suggest a cellular mechanism by which Notch1IC oncogenes could contribute to thymoma development and maintenance.