We investigated the mechanism by which B lymphocyte stimulator (BLyS)/BAFF, a tumor necrosis factor superfamily ligand, promotes B-cell survival and resistance to atrophy. BLyS stimulation activates 2 independent signaling pathways, Akt/mTOR and Pim 2, associated with cell growth and survival. BLyS blocks the cell volume loss (atrophy) that freshly isolated B cells normally undergo when maintained in vitro while concurrently increasing glycolytic activity and overall metabolism. This atrophy resistance requires Akt/mTOR. We used a genetic approach to resolve the contributions of Akt/mTOR and Pim kinase pathways to BLyS-mediated survival. Pim 2-deficient B cells are readily protected from death by BLyS stimulation, but this protection is completely abrogated by treatment with the mTOR inhibitor rapamycin. Furthermore, rapamycin treatment in vivo significantly reduces both follicular and marginal zone B cells in Pim-deficient but not healthy hosts. BLyS-dependent survival requires the antiapoptotic protein Mcl-1. Mcl-1 protein levels rise and fall in response to BLyS addition and withdrawal, respectively, and conditional deletion of the Mcl-1 gene renders B cells refractory to BLyS-mediated protection. Because BlyS is required for the normal homeostasis of all B cells, these data suggest a therapeutic strategy simultaneously inhibiting mTOR and Pim 2 could target pathogenic B cells.

CBA/N mice carry an X-linked immunodeficiency (xid) due to a point mutation in the Bruton's tyrosine kinase (btk) gene. xid mice have a smaller peripheral B cell pool than normal animals, lack CD5+ B cells (B1), and are hyporesponsive to mitogenic anti-Igs and thymus-independent type 2 Ags. The proto-oncogene bcl-2 affects B cell homeostasis by suppressing programmed cell death. We hypothesized that reduced bcl-2 expression could enhance programmed cell death in xid B cells, directly causing poor peripheral B cell survival and indirectly affecting Ag responsiveness. We measured and compared levels of endogenous Bcl-2 protein and spontaneous apoptosis in xid and normal B cells, and determined the effect of a human bcl-2/Ig minigene on B cell survival and Ag responsiveness in bcl-2 transgenics. The amount of endogenous Bcl-2 was reduced fivefold in freshly isolated xid B cells compared with that in normal cells, but was equal in xid and normal T cells. Attrition by spontaneous apoptosis was significantly higher in cultured xid B cells. Expression of the bcl-2 transgene suppressed apoptosis equally in normal and xid B cells, prolonged in vitro survival, and markedly expanded in vivo the follicular B cell population normally reduced in xid mice. However, most xid defects persisted; xid/bcl-2 mice remained deficient in B1 cells and hyporesponsive to anti-Igs, thymus-independent type 1 Ags, and thymus-independent type 2 Ags. The data suggest that signal transduction pathways using Btk independently regulate B cell survival and Ag responsiveness.

Normal B cells responsive to thymus independent-type 1 Ags (TI-1) are resistant to low doses of ionizing radiation in vivo (200-300 cGy), compared with TI-1 responsive B cells of mice with the CBA/N X-linked immunodeficiency (xid). This difference in radiosensitivity is an intrinsic B cell property; normal B cells adoptively transferred into xid mice remain TI-1-responsive after irradiation in situ. Because irradiation induces programmed cell death (PCD) in lymphocytes, we determined whether PCD were regulated differently in normal and xid B cells. B cells isolated immediately after irradiation from normal or xid donors when cultured without stimulators became apoptotic with the same kinetics and to the same extent, showing that apoptosis was induced equally in both populations. Apoptosis could be suppressed and mitogenesis could be induced frequently, however, if irradiated B cells were cultured with B cell activators. When activators using separate signal transduction pathways were compared, a hierarchy of efficiency at effecting apoptosis rescue was observed, and activators used singly without effect could synergize to protect. xid B cells were more resistant to rescue than normal B cells unless PMA was used as a stimulant. Although the mechanism of activator-induced rescue was not established, selective overexpression of a bcl-2 transgene rendered xid B cells radioresistant. The data suggest that a signal(s) delivered to irradiated B cells in the in vivo microenvironment suppresses apoptosis and that xid B cells and a radiosensitive subpopulation of normal B cells are refractory to this signal(s).