Newly generated immature B cells are selected to enter the peripheral mature B-cell pool only if they do not bind (or bind limited amount of) self-antigen. We previously suggested that this selection relies on basal extracellular signal-regulated kinase (Erk) activation mediated by tonic B-cell antigen receptor (BCR) signaling and that this signal can be replaced by an active rat sarcoma (Ras), which are small GTPase proteins. In this study we compared the activity of Ras and Erk in nonautoreactive and autoreactive immature B cells and investigated whether activation of Ras can break tolerance. Our results demonstrate lower levels of active Erk and Ras in autoreactive immature B cells, although this is evident only when these cells display medium/high avidity for self-antigen. Basal activation of Erk in immature B cells is proportional to surface IgM and dependent on sarcoma family kinases, whereas it is independent of B-cell activating factor, IFN, and Tolllike receptor signaling. Ectopic expression of the constitutively active mutant Ras form N-RasD12 in autoreactive cells raises active Erk, halts receptor editing via PI3 kinase, and promotes differentiation via Erk, breaking central tolerance. Moreover, when B cells coexpress autoreactive and nonautoreactive BCRs, N-RasD12 leads also to a break in peripheral tolerance with the production of autoantibodies. Our findings indicate that in immature B cells, basal activation of Ras and Erk are controlled by tonic BCR signaling, and that positive changes in Ras activity can lead to a break in both central and peripheral B-cell tolerance.Src | BAFF

B cell selection mechanisms in the bone marrow control the nature of the primary B cell repertoire, generating diverse specificities while reducing autoreactivity. Our studies aim to characterize the mechanisms that drive the positive selection of immature B cells in the bone marrow. It has been shown that the B cell receptor (BCR) is able to signal in the absence of ligand (tonic signal) and this signal is required for B cell development. We hypothesize that alterations in tonic signaling could cause aberrant positive selection of autoreactive immature B cells. We have previously shown that low levels of active Ras, Erk, and Akt correlate with levels of BCR tonic signal and that constitutive activation of Ras allows for differentiation of autoreactive immature B cells in vitro. We are currently testing the individual contributions of the Erk and PI3K pathways in this selection process. By using retroviral transduction of primary bone marrow B cells in vitro, we show that Erk activation can overcome a lack of tonic signaling to induce differentiation of nonautoreactive or slightly autoreactive B cells. We have also found that the Dual Specificity Phosphatase 5 (DUSP5), a novel phosphatase, may play a role in Erk regulation and the selection of immature B cells. In order to study these pathways in vivo we generated mouse models in which constitutively active forms of MEK or PI3K are specifically expressed in autoreactive B cells to determine if activation of these molecules is sufficient to bypass central tolerance. Preliminary data suggests that in vivo activation of PI3K can drive development of autoreactive B cells and allow some escape to the periphery.