The belx gene has been shown to regulate programmed cell death in-vitro. We PCD when exposed to glucocorticoids, radiation, or cross-linking of T-cell receptor/CD3 molecules (2-5). Most DP cells die via PCD in vivo, either due to negative selection or to neglect, the lack of positive selection (6-8). A minority of DP thymocytes undergo positive selection and mature to the single-positive (SP) stage, where they express either CD4 or CD8 but not both.The protooncogene bc12 was the first gene studied in the context of PCD regulation during lymphoid development. Bc12 was originally cloned at the chromosomal breakpoint of t(14;18)-bearing B-cell lymphomas (9, 10). Bcl2 protects some, but not all, factor-dependent cell lines from PCD in vitro, and deregulated bc12 expression prolongs the survival of B cells in transgenic mice (11-13). In the thymus, Bcl2 expression is predominantly restricted to mature SP thymocytes found in the medulla and is largely absent from DP thymocytes that reside in the cortex. Bcl2 expression is up-regulated in DP thymocytes after positive selection and may assist them in differentiating to SP cells (14).Forced expression of bc12 in DP thymocytes of transgenic mice enhances the resistance of these cells to 'y-radiation and glucocorticoid-induced PCD but does not prevent the physiological elimination of DP thymocytes by negative selection (15, 16). Elimination of bc12 by gene targeting results in progressive apoptosis of B and T lymphocytes beginning at 3 to 4 weeks of age (17,18). In addition, bc12--SP thymocytes and mature peripheral T lymphocytes demonstrate increased sensitivity to PCD by -y-irradiation and dexamethasone. However, T-cell development occurs normally in these animals, andThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. MATERIALS AND METHODSGeneration ofAnti-Bclx Monoclonal Antibodies and Immunoanalyses of Lymphocytes. Recombinant Bclx proteins were produced in Escherichia coli and used to generate murine monoclonal antibodies. Several monoclonal antibodies were identified that reacted with murine Bclxl and BcIxS, in addition to human Bclx proteins. For immunoblotting, single-cell suspensions of lymphocytes from lymph nodes (LN) or thymuses of 6-week-old CD1 mice were lysed in 1% Nonidet P-40/1% deoxycholate/0.1% SDS with protease inhibitors. The protein concentration was determined by bicinchoninic acid assay (BCA, Pierce), and 25 ,ug of protein per sample was separated in SDS/15% polyacrylamide gels. Proteins were blotted onto nitrocellulose and probed with monoclonal antibodies directed against either Bclx (2A1) or Bc12 (3F11, PharMingen) proteins. Reactive proteins were detected by enhanced chemiluminescence (ECL, Amersham).Gene Targeting of bclx. To target the bclx gene in ES cells, a murine clone of the bclx gene was isolated from a 129SV genomic library (Stratagene). Targeting constructs (20) were e...

SummaryWe have assessed during B cell development, the regulation and function of bcl-x, a member of the bcl-2 family of apoptosis regulatory genes. Here we show that Bcl-xr, a product of bd-x, is expressed in pre-B cells but downregulated at the immature and mature stages of B cell development. Bcl-xr but not Bcl-2 is rapidly induced in peripheral B cells upon surface immunoglobulin M (IgM) cross-linking, CD40 signaling, or LPS stimulation. Transgenic mice that overexpressed Bcl-xt within the B cell lineage exhibited marked accumulation of peripheral B cells in lymphoid organs and enhanced survival of developing and mature B cells. B cell survival was further increased by simultaneous expression of bcl-xL and bcl-2 transgenes. These studies demonstrate that Bcl-2 and Bcl-x t are regulated differentially during B cell development and activation of mature B cells. Induction of Bcl-XL after signaling through surface IgM and CD40 appears to provide mature B cells with an additional protective mechanism against apoptotic signals associated with antigen-induced activation and proliferation.

To study effects of Bcl-x(L) in the pancreatic beta-cell, two transgenic lines were produced using different forms of the rat insulin promoter. Bcl-x(L) expression in beta-cells was increased 2- to 3-fold in founder (Fd) 1 and over 10-fold in Fd 2 compared with littermate controls. After exposure to thapsigargin (10 microM for 48 h), losses of cell viability in islets of Fd 1 and Fd 2 Bcl-x(L) transgenic mice were significantly lower than in islets of wild-type mice. Unexpectedly, severe glucose intolerance was observed in Fd 2 but not Fd 1 Bcl-x(L) mice. Pancreatic insulin content and islet morphology were not different from control in either transgenic line. However, Fd 2 Bcl-x(L) islets had impaired insulin secretory and intracellular free Ca(2+) ([Ca(2+)](i)) responses to glucose and KCl. Furthermore, insulin and [Ca(2+)](i) responses to pyruvate methyl ester (PME) were similarly reduced as glucose in Fd 2 Bcl-x(L) islets. Consistent with a mitochondrial defect, glucose oxidation, but not glycolysis, was significantly lower in Fd 2 Bcl-x(L) islets than in wild-type islets. Glucose-, PME-, and alpha-ketoisocaproate-induced hyperpolarization of mitochondrial membrane potential, NAD(P)H, and ATP production were also significantly reduced in Fd 2 Bcl-x(L) islets. Thus, although Bcl-x(L) promotes beta-cell survival, high levels of expression of Bcl-x(L) result in reduced glucose-induced insulin secretion and hyperglycemia due to a defect in mitochondrial nutrient metabolism and signaling for insulin secretion.

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Both rapid B-cell proliferation and programmed cell death (PCD) occur during the differentiation and selection of B cells within the germinal center. To help elucidate the role of Bcl-x in B-cell antigen selection and PCD within the germinal center, we examined its expression in defined B-cell populations and by immunochemistry of tonsil tissue. Purified B-cell fractions enriched for centrocytes express high amounts of Bcl-x and relatively low amounts of Bcl-2, whereas fractions enriched for centroblasts lack significant levels of both proteins. Consistent with this observation, immunocytochemistry localized Bcl-x within cells scattered throughout the germinal center. Stimulation of tonsil B cells with either CD40 or Staphylococcus aureus Cowan increase bcl-x mRNA and protein levels. Treatment of a cell line with a germinal center phenotype (RAMOS) or the tonsillar B-cell centroblast fraction with CD40 rapidly increased Bcl-x levels and partially rescued B cells from PCD. These data suggest that Bcl-x rather than Bcl-2 may rescue centrocytes during selection in the germinal center.

Transgenic mice that overexpress the anti-apoptotic gene bcl-x L under the control of the keratin 14 promoter have significantly shorter hair than non-transgenic littermates. The deficit in hair length correlated with a decrease in the duration of anagen, the growth phase of the hair cycle. A prolongation in telogen, the resting phase of the hair cycle, was also observed in adult animals. In the developing hair bulb, bcl-x L transgene expression was observed exclusively in the outer root sheath (ORS) cells. Bcl-x L expression enhanced the survival of ORS cells treated with apoptotic stimuli. The results suggest that preventing the apoptotic death of ORS cells during anagen leads to a more rapid termination of progenitor cell commitment/proliferation, while the increased survival of ORS cells during telogen delays the initiation of a new hair cycle. ORS cells produce fibroblast growth factor-5 (FGF-5), which acts in a paracrine fashion to terminate precursor cell division during anagen. The short hair phenotype of bcl-x L transgenic mice was substantially reversed in FGF-5-deficient mice. Thus, the production of growth inhibitory factors by ORS cells may provide a mechanism through which the hair-growth cycle is regulated by cell survival.

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The development of TCR-gamma delta cells during thymic ontogeny has been studied using fetal thymic organ cultures of normal and transgenic (Tg) mice. The expression of the cell-surface markers--heat stable Ag (HSA), MEL-14, CD5, CD25 (IL-2R), and CD44 (Pgp-1)--correlated with TCR-gamma delta maturation. As the fetal thymus developed, there was an increase in HSA-, CD5dull, and CD44+ cells for each TCR-gamma delta cell subset. Moreover, the expression of recombination activating genes-1 and -2 (RAG-1 and RAG-2) also correlated with TCR-gamma delta maturation as only HSA+ TCR-gamma delta cells transcribed these genes. Cyclosporin A inhibited the development of the TCR-gamma delta thymocytes if it was introduced early during thymic ontogeny by arresting the differentiation of TCR-gamma delta thymocytes at the HSA+ stage. Immature HSA+ TCR-gamma delta thymocytes isolated from both TCR-gamma delta Tg and normal mice did not respond to nominal Ag or anti-TCR mAb unless exogenous IL-2 was added to the cultures. In contrast, HSA- TCR-gamma delta cells from Tg and normal mice responded to TCR/ligand interactions in the absence of additional IL-2. Finally, the development of functionally mature TCR-gamma delta cells could be induced in vitro. Interaction of the HSA+ Tg+ TCR-gamma delta cells with anti-TCR-gamma delta mAb or Ag-bearing thymic stromal cells resulted in RAG-1 and RAG-2 down-regulation. These data strongly suggest that TCR-gamma delta HSA+, RAG+ thymocytes differentiate into a more mature stage under the pressure of positive selection and that TCR-gamma delta cell development is regulated in a manner similar to TCR-alpha beta cells. In addition, the ability of Cyclosporin A to inhibit TCR-gamma delta cell development combined with the findings that Ag-bearing stromal cells can induce Tg TCR-gamma delta cell development suggests that maturation and selection of TCR-gamma delta cells depends on receptor-mediated physiologic stimuli delivered during thymic development.