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BH3 domain-only proteins (BH3-only) proteins are members of the Bcl-2 family that play crucial roles in embryogenesis and the maintenance of tissue homeostasis by triggering apoptotic cell death. The BH3-only protein Bim is critical for developmental apoptosis of lymphocytes, securing establishment of tolerance and for the termination of immune responses. Bim is believed to act in concert with other BH3-only proteins or members of the tumor necrosis factor receptor family in getting rid of unwanted cells. Bmf, a related BH3-only protein, was shown to play a role in B-cell homeostasis and to mediate cell death in response to certain apoptotic triggers, including glucocorticoid, histone deacetylase inhibitors, and overexpression of the c-Myc proto-oncogene. Here we show that Bim and Bmf have overlapping functions during mouse development and coregulate lymphocyte homeostasis and apoptosis in a nonredundant manner. Double deficiency of Bim and Bmf caused more B lymphadenopathy than loss of either BH3-only protein alone, and this was associated with autoimmune glomerulonephritis and a range of malignancies in aged mice. Thus, our results demonstrate that Bim and Bmf act in concert to prevent autoimmunity and malignant disease, strengthening the rational for the development of BH3-only protein mimicking therapeutics for the treatment of such disorders.

The BH3-only protein Bim is a critical initiator of apoptosis in hematopoietic cells. Bim is upregulated in response to growth factor withdrawal and in vitro studies have implicated the transcription factor Foxo3a as a critical inducer. To test the importance of this regulation in vivo, we generated mice with mutated Foxo-binding sites within the Bim promoters (Bim(ΔFoxo/ΔFoxo)). Contrary to Bim-deficient mice, Bim(ΔFoxo/ΔFoxo) mice had a normal hematopoietic system. Moreover, cytokine-dependent haematopoietic cells from Bim(ΔFoxo/ΔFoxo) and wt mice died at similar rates. These results indicate that regulation of Bim by Foxo transcription factors is not critical for the killing of hematopoietic cells.

Although the proapoptotic BH3-only protein, Bim, is required for deletion of autoreactive thymocytes, Bim-deficient mice do not succumb to extensive organ-specific autoimmune disease. To determine whether other BH3-only proteins safeguard tolerance in the absence of Bim, we screened mice lacking Bim as well as other BH3-only proteins. Most strains showed no additional defects; however, mice deficient for both Puma and Bim spontaneously developed autoimmunity in multiple organs, and their T cells could transfer organ-specific autoimmunity. Puma- and Bim-double-deficient mice had a striking accumulation of mature, single-positive thymocytes, suggesting an additional defect in thymic deletion was the basis for disease. Transgenic mouse models of thymocyte deletion by peripheral neoantigens confirmed that the loss of Bim and Puma allowed increased numbers of autoreactive thymocytes to escape deletion. Our data show that Puma cooperates with Bim to impose a thymic-deletion checkpoint to peripheral self-antigens and cement the notion that defects in apoptosis alone are sufficient to cause autoimmune disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Apoptosis is crucial for immune system homeostasis, including selection and survival of long-lived antibody-forming cells and memory cells. The interactions between proapoptotic and pro-survival proteins of the Bcl-2 family are critical for this process. In this report, we show that expression of the proapoptotic BH3-only Bcl-2 family member Puma was selectively up-regulated on in vitro activation with antigens or mitogens of both human and mouse B cells. Puma expression coincided in vivo, with the prosurvival Bcl-2 family member Mcl-1 within the germinal centers and its expression correlates with the germinal center like phenotype of Burkitt lymphoma. Experiments performed in Puma-deficient mice revealed that Puma is essential for apoptosis of mitogen-activated B cells in vitro and for the control of memory B-cell survival. In conclusion, using both human and murine models, our data show that Puma has a major role in the T cell- dependent B-cell immune response. These data demonstrate that Puma is a major regulator of memory B lymphocyte survival and therefore a key molecule in the control of the immune response.

For malignant growth, solid cancers must stimulate the formation of new blood vessels by producing vascular endothelial growth factor (VEGF-A), which is required for the survival of tumor-associated vessels. Novel anticancer agents that block VEGF-A signaling trigger endothelial cell (EC) apoptosis and vascular regression preferentially within tumors, but how the ECs die is not understood. In this study, we demonstrate that VEGF-A deprivation, provoked either by drug-induced tumor shrinkage or direct VEGF-A blockade, up-regulates the proapoptotic BH3 (Bcl-2 homology 3)-only Bcl-2 family member Bim in ECs. Importantly, the tumor growth inhibitory activity of a VEGF-A antagonist required Bim-induced apoptosis of ECs. These findings thus reveal the mechanism by which VEGF-A blockade induces EC apoptosis and impairs tumor growth. They also indicate that drugs mimicking BH3-only proteins may be exploited to kill tumor cells not only directly but also indirectly by ablating the tumor vasculature.

The size of the peripheral T-lymphocyte compartment is governed by complex homeostatic mechanisms that balance T-cell proliferation and death. Proliferation and survival signals are mediated in part by recurrent self-peptide/MHC-TCR interactions and signaling by the common γ chain-containing cytokine receptors, including those for IL-7 and IL-15. We have previously shown that the death receptor Fas (CD95/APO-1) regulates apoptosis in response to repeated TCR stimulation, whereas the Bcl-2 homology domain 3-only protein Bim mediates cytokine withdrawal-induced apoptosis. We therefore reasoned that these two molecules might cooperate in the regulation of homeostatic proliferation. In this study, we observe that the combined loss of Fas and Bim synergistically enhances the accumulation of T cells in lymphopenic host mice, and this is particularly pronounced for the unusual CD4(-) CD8(-) TCRαβ(+) T cells that are characteristic of Fas-deficient (Fas(lpr/lpr) ) mice. Our findings demonstrate that these CD4(-) CD8(-) TCRαβ(+) T cells arise from homeostatic proliferation of CD8(+) T cells. These studies also underscore the profound rate of baseline T-cell proliferation that likely occurs in wild-type mice even in the absence of foreign antigen, and the consequent need for its coordinated regulation by multiple death-signaling pathways., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: High concentrations of circulating glucose are believed to contribute to defective insulin secretion and beta-cell function in diabetes and at least some of this effect appears to be caused by glucose-induced beta-cell apoptosis. In mammalian cells, apoptotic cell death is controlled by the interplay of proapoptotic and antiapoptotic members of the Bcl-2 family. We investigated the apoptotic pathway induced in mouse pancreatic islet cells after exposure to high concentrations of the reducing sugars ribose and glucose as a model of beta-cell death due to long-term metabolic stress., Research Design and Methods: Islets isolated from mice lacking molecules implicated in cell death pathways were exposed to high concentrations of glucose or ribose. Apoptosis was measured by analysis of DNA fragmentation and release of mitochondrial cytochrome c., Results: Deficiency of interleukin-1 receptors or Fas did not diminish apoptosis, making involvement of inflammatory cytokine receptor or death receptor signaling in glucose-induced apoptosis unlikely. In contrast, overexpression of the prosurvival protein Bcl-2 or deficiency of the apoptosis initiating BH3-only proteins Bim or Puma, or the downstream apoptosis effector Bax, markedly reduced glucose- or ribose-induced killing of islets. Loss of other BH3-only proteins Bid or Noxa, or the Bax-related effector Bak, had no impact on glucose-induced apoptosis., Conclusions: These results implicate the Bcl-2 regulated apoptotic pathway in glucose-induced islet cell killing and indicate points in the pathway at which interventional strategies can be designed.

The anti-apoptotic molecule Bcl-2 inhibits apoptosis by preventing cytochrome c release from mitochondria. Although several studies have indicated the importance of Bcl-2 in maintaining skeletal integrity, the detailed cellular and molecular mechanisms remain elusive. Bcl-2(-/-) mice are growth-retarded and exhibit increased bone volume of the primary spongiosa, mainly due to the decreased number and dysfunction of osteoclasts. Osteoblast function is also impaired in Bcl-2(-/-) mice. Ex vivo studies on osteoblasts and osteoclasts showed that Bcl-2 promoted the differentiation, activation, and survival of both cell types. Because Bcl-2(-/-) mice die before 6 weeks of age due to renal failure and cannot be compared with adult wild type mice, we generated Bcl-2(-/-)Bim(+/-) mice, in which a single Bim allele was inactivated, and compared them with their Bcl-2(+/-)Bim(+/-) littermates. Loss of a single Bim allele restored normal osteoclast function in Bcl-2(-/-) mice but did not restore the impaired function of osteoblasts, and the mice exhibited osteopenia. These data demonstrate that Bcl-2 promotes the differentiation, activity, and survival of both osteoblasts and osteoclasts. The balance between Bcl-2 and Bim regulates osteoclast apoptosis and function, whereas other pro-apoptotic members are important for osteoblasts.

Proteins of the Bcl-2 family are critical regulators of apoptosis, but how its BH3-only members activate the essential effectors Bax and Bak remains controversial. The indirect activation model suggests that they simply must neutralize all of the prosurvival Bcl-2 family members, whereas the direct activation model proposes that Bim and Bid must activate Bax and Bak directly. As numerous in vitro studies have not resolved this issue, we have investigated Bim's activity in vivo by a genetic approach. Because the BH3 domain determines binding specificity for Bcl-2 relatives, we generated mice having the Bim BH3 domain replaced by that of Bad, Noxa, or Puma. The mutants bound the expected subsets of prosurvival relatives but lost interaction with Bax. Analysis of the mice showed that Bim's proapoptotic activity is not solely caused by its ability to engage its prosurvival relatives or solely to its binding to Bax. Thus, initiation of apoptosis in vivo appears to require features of both models.

The relative importance of the intrinsic and extrinsic apoptotic pathways in the control of haematopoietic cell homeostasis has been a matter of debate for many years. Cell death is omnipresent in this cellular compartment and ensures the removal of cells that are not properly equipped to assume their function as well as those that have assumed function but are no longer required. In this Review we focus on the roles of CD95 (also known as FAS) and BCL-2-interacting mediator of cell death (BIM), two major regulators of apoptosis in T cell homeostasis, with a particular emphasis on their cooperation in the shutdown of T cell responses.

Apoptotic death of hepatocytes, a contributor to many chronic and acute liver diseases, can be a consequence of overactivation of the immune system and is often mediated by TNFalpha. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation causes fatal hepatocyte apoptosis in mice, which is mediated by TNFalpha, but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the proapoptotic BH3-only protein activated by caspase-8 and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by c-Jun N-terminal kinase (JNK), protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases.

Background & Aims: Chronic infections by hepatotropic viruses such as hepatitis B and C are generally associated with an impaired CD8 T-cell immune response that is unable to clear the virus. The liver is increasingly recognized as an alternative site in which primary activation of CD8 T cells takes place, a property that might explain its role in inducing tolerance. However, the molecular mechanism by which intrahepatically activated T cells become tolerant is unknown. Here, we investigated the phenotype and fate of naïve CD8 T cells activated by hepatocytes in vivo., Methods: Transgenic mouse models in which the antigen is expressed in lymph nodes and/or in the liver were adoptively transferred with naïve CD8 T cells specific for the hepatic antigen., Results: Liver-activated CD8 T cells displayed poor effector functions and a unique CD25(low) CD54(low) phenotype. This phenotype was associated with increased expression of the proapoptotic protein Bim and caspase-3, demonstrating that these cells are programmed to die following intrahepatic activation. Importantly, we show that T cells deficient for Bim survived following intrahepatic activation., Conclusions: This study identifies Bim for the first time as a critical initiator of T-cell death in the liver. Thus, strategies inhibiting the up-regulation of this molecule could potentially be used to rescue CD8 T cells, clear the virus, and reverse the outcome of viral chronic infections affecting the liver.

Apoptotic death of T lymphocytes is critical for shutdown of immune responses and hemopoietic cell homeostasis. Both death receptor (Fas) activation and mitochondrial apoptosis triggered by the BH3-only protein Bim have been implicated in the killing of antigen-stimulated T cells. We examined mice lacking the gene encoding Bim (Bcl2l11) and with the inactivating lpr mutation in the gene encoding Fas (Fas), designated Bcl2l11(-/-)Fas(lpr/lpr) mice. Shutdown of an acute T cell response to herpes simplex virus involved only Bim with no contribution by Fas, whereas both pathways synergized in killing antigen-stimulated T cells in chronic infection with murine gamma-herpesvirus. Bcl2l11(-/-)Fas(lpr/lpr) mice developed remarkably enhanced and accelerated fatal lymphadenopathy and autoimmunity compared to mice lacking only one of these apoptosis inducers. These results identify critical overlapping roles for Fas and Bim in T cell death in immune response shutdown and prevention of immunopathology and thereby resolve a long-standing controversy.

Like Bcl-2, Mcl-1 is an important survival factor for many cancers, its expression contributing to chemoresistance and disease relapse. However, unlike other prosurvival Bcl-2-like proteins, Mcl-1 stability is acutely regulated. For example, the Bcl-2 homology 3 (BH3)-only protein Noxa, which preferentially binds to Mcl-1, also targets it for proteasomal degradation. In this paper, we describe the discovery and characterization of a novel BH3-like ligand derived from Bim, Bim(S)2A, which is highly selective for Mcl-1. Unlike Noxa, Bim(S)2A is unable to trigger Mcl-1 degradation, yet, like Noxa, Bim(S)2A promotes cell killing only when Bcl-x(L) is absent or neutralized. Furthermore, killing by endogenous Bim is not associated with Mcl-1 degradation. Thus, functional inactivation of Mcl-1 does not always require its elimination. Rather, it can be efficiently antagonized by a BH3-like ligand tightly engaging its binding groove, which is confirmed here with a structural study. Our data have important implications for the discovery of compounds that might kill cells whose survival depends on Mcl-1.

T cell-dependent B-cell immune responses induce germinal centers that are sites for expansion, diversification, and selection of antigen-specific B cells. During the immune response, antigen-specific B cells are removed in a process that favors the retention of cells with improved affinity for antigen, a cell death process inhibited by excess Bcl-2. In this study, we examined the role of the BH3-only protein Bim, an initiator of apoptosis in the Bcl-2-regulated pathway, in the programmed cell death accompanying an immune response. After immunization, Bim-deficient mice showed persistence of both memory B cells lacking affinity-enhancing mutations in their immunoglobulin genes and antibody-forming cells secreting low-affinity antibodies. This was accompanied by enhanced survival of both cell types in culture. We have identified for the first time the physiologic mechanisms for killing low-affinity antibody-expressing B cells in an immune response and have shown this to be dependent on the BH3-only protein Bim.

Programmed cell death (PCD) during development of the mouse retina involves activation of the mitochondrial pathway. Previous work has shown that the multidomain Bcl-2 family proteins Bax and Bak are fundamentally involved in this process. To induce mitochondrial membrane permeabilization, Bax and Bak require that prosurvival members of the family be inactivated by binding of "BH3-only" members. We showed previously that the BH3-only protein BimEL is highly expressed during postnatal retinal development but decreases dramatically thereafter. The purpose of this study was to investigate a possible role for Bim, in retinal development and degeneration, upstream of Bax and Bak. Bim-/- mice analyzed for defective retinal development exhibit an increase in retinal thickness and a delay in PCD, thereby confirming a role for Bim. We also demonstrate that in response to certain death stimuli, bim+/+ retinal explants upregulate BimEL leading to caspase activation and cell death, whereas bim-/- explants are resistant to apoptosis. Finally, we analyzed Bim expression in the retinal degeneration (rd) mouse, an in vivo model of retinal degeneration. Bim isoforms, which decrease during development, are not reexpressed during retinal degeneration and ultimately photoreceptor cells die by a caspase-independent mechanism. Thus, we conclude that in cases in which BimEL is reexpressed during pathological cell death, developmental cell death pathways are reactivated. However, the absence of BimEL expression correlates with caspase-independent death in the rd model.

Endoplasmic reticulum (ER) stress caused by misfolded proteins or cytotoxic drugs can kill cells and although activation of this pathway has been implicated in the etiology of certain degenerative disorders its mechanism remains unresolved. Bim, a proapoptotic BH3-only member of the Bcl-2 family is required for initiation of apoptosis induced by cytokine deprivation or certain stress stimuli. Its proapoptotic activity can be regulated by several transcriptional or posttranslational mechanisms, such as ERK-mediated phosphorylation, promoting its ubiquitination and proteasomal degradation. We found that Bim is essential for ER stress-induced apoptosis in a diverse range of cell types both in culture and within the whole animal. ER stress activates Bim through two novel pathways, involving protein phosphatase 2A-mediated dephosphorylation, which prevents its ubiquitination and proteasomal degradation and CHOP-C/EBPalpha-mediated direct transcriptional induction. These results define the molecular mechanisms of ER stress-induced apoptosis and identify targets for therapeutic intervention in ER stress-related diseases.

A central issue in the regulation of apoptosis by the Bcl-2 family is whether its BH3-only members initiate apoptosis by directly binding to the essential cell-death mediators Bax and Bak, or whether they can act indirectly, by engaging their pro-survival Bcl-2-like relatives. Contrary to the direct-activation model, we show that Bax and Bak can mediate apoptosis without discernable association with the putative BH3-only activators (Bim, Bid, and Puma), even in cells with no Bim or Bid and reduced Puma. Our results indicate that BH3-only proteins induce apoptosis at least primarily by engaging the multiple pro-survival relatives guarding Bax and Bak.

The adult, virgin mammary gland is a highly organized tree-like structure formed by ducts with hollowed lumen. Although lumen formation during pubertal development appears to involve apoptosis, the molecular mechanisms that regulate this process are not known. Here, we demonstrate that disruption of the BH3-only proapoptotic factor Bim in mice prevents induction of apoptosis in and clearing of the lumen in terminal end buds during puberty. However, cells that fill the presumptive luminal space are eventually cleared from the adjacent ducts by a caspase-independent death process. Within the filled Bim(-/-) ducts, epithelial cells are deprived of matrix attachment and undergo squamous differentiation prior to clearing. Similarly, we also detect squamous differentiation in vitro when immortalized mammary epithelial cells are detached from the matrix. These data provide important mechanistic information on the processes involved in sculpting the mammary gland and demonstrate that BIM is a critical regulator of apoptosis in vivo.

Cell killing is a critical pharmacological activity of imatinib to eradicate Bcr/Abl+ leukemias. We found that imatinib kills Bcr/Abl+ leukemic cells by triggering the Bcl-2-regulated apoptotic pathway. Imatinib activated several proapoptotic BH3-only proteins: bim and bmf transcription was increased, and both Bim and Bad were activated posttranslationally. Studies using RNAi and cells from gene-targeted mice revealed that Bim plays a major role in imatinib-induced apoptosis of Bcr/Abl+ leukemic cells and that the combined loss of Bim and Bad abrogates this killing. Loss of Bmf or Puma had no effect. Resistance to imatinib caused by Bcl-2 overexpression or loss of Bim (plus Bad) could be overcome by cotreatment with the BH3 mimetic ABT-737. These results demonstrate that Bim and Bad account for most, perhaps all, imatinib-induced killing of Bcr/Abl+ leukemic cells and suggest previously undescribed drug combination strategies for cancer therapy.

Perinatal hypoxic-ischemic injury is a common cause of neurologic disability mediated in part by Bcl-2 family-regulated neuronal apoptosis. The Bcl-2 protein family consists of both pro- (e.g. Bax, Bad, Bid, Bim) and anti-apoptotic (e.g. Bcl-2, Bcl-X(L)) proteins that regulate mitochondrial outer membrane integrity, cytochrome c release and caspase activation. Previous studies have implicated Bax as an important mediator of neuronal death in several models of brain injury, including neonatal hypoxia-ischemia (HI). In this study, we assessed the roles of several members of the pro-apoptotic BH3 domain-only Bcl-2 sub-family in an in vivo mouse model of neonatal HI. Seven-day old control and gene-disrupted mice underwent unilateral left carotid ligation followed by 45 min exposure to 8% oxygen and the extent of brain injury was assessed 2 days later. Following HI, mice deficient in Bad or Bim exhibited reduced activated caspase-3 and glial fibrillary acidic protein immunostaining in their brains compared to similarly treated control animals. Measurement of hippocampal area showed decreased parenchymal loss in both Bad- and Bim-deficient mice versus control animals. In contrast, loss of Bid, another BH3-only protein, provided no protection from neonatal HI brain injury. These results indicate that Bad and Bim are selectively involved in neuron death following neonatal HI and may be targets for therapeutic intervention.

Genes involved in the transforming growth factor beta (TGF-beta) signaling pathway are frequently altered in several types of cancers, and a gastric tumor suppressor RUNX3 appears to be an integral component of this pathway. We reported previously that apoptosis is notably reduced in Runx3-/- gastric epithelial cells. In the present study, we show that a proapoptotic gene Bim was transcriptionally activated by RUNX3 in the gastric cancer cell lines SNU16 and SNU719 treated with TGF-beta. The human Bim promoter contains RUNX sites, which are required for its activation. Furthermore, a dominant negative form of RUNX3 comprised of amino acids 1 to 187 increased tumorigenicity of SNU16 by inhibiting Bim expression. In Runx3-/- mouse gastric epithelium, Bim was down-regulated, and apoptosis was reduced to the same extent as that in Bim-/- gastric epithelium. We confirmed comparable expression of TGF-beta1 and TGF-beta receptors between wild-type and Runx3-/- gastric epithelia and reduction of Bim in TGF-beta1-/- stomach. These results demonstrate that RUNX3 is responsible for transcriptional up-regulation of Bim in TGF-beta-induced apoptosis.

Apoptosis is essential for the development, function and homeostasis of the immune system. Experiments with transgenic and gene knock-out mice have shown that defects in the control of apoptosis in the hematopoietic system can promote the development of autoimmunity or hematological malignancy. In contrast, excessive apoptosis of normally long-lived hemopoietic cells can lead to lymphopenia and immunodeficiency. In mammals, cell death in response to developmental cues and many cell stress signals is regulated by the opposing factions of the Bcl-2 family of proteins. In particular, the pro-apoptotic subgroup called BH3-only proteins, which includes Bim, is critical in the initiation of apoptosis in response to many death stimuli. Bim has been found to be an important regulator of the negative selection of B lymphocytes in the bone marrow and of T lymphocytes both in the thymus and the periphery. Mice lacking Bim accumulate self-reactive lymphocytes, develop autoantibodies and on certain genetic backgrounds succumb to SLE-like autoimmune disease. Abnormalities in Bim expression and the thymic deletion of auto-reactive lymphocytes have also been implicated as a component of the complex, polygenic predisposition to autoimmune diabetes seen in NOD mice. Bim is also an essential regulator of T lymphocyte apoptosis during the termination of an immune response. This chapter focuses on the role of Bim in the development and function of the immune system and its potential role in autoimmunity. Degenerative disorders due to increased apoptosis mediated by Bim are also discussed.

The BH3-only proteins of the Bcl-2 family initiate apoptosis through the activation of Bax-like relatives. Loss of individual BH3-only proteins can lead either to no phenotype, as in mice lacking Bik, or to marked cell excess, as in the hematopoietic compartment of animals lacking Bim. To investigate whether functional redundancy with Bim might obscure a significant role for Bik, we generated mice lacking both genes. The hematopoietic compartments of bik-/-bim-/- and bim-/- mice were indistinguishable. However, although testes develop normally in mice lacking either Bik or Bim, adult bik-/-bim-/- males were infertile, with reduced testicular cellularity and no spermatozoa. The testis of young bik-/-bim-/- males, like those lacking Bax, exhibited increased numbers of spermatogonia and spermatocytes, although loss of Bik plus Bim blocked spermatogenesis somewhat later than Bax deficiency. The initial excess of early germ cells suggests that spermatogenesis fails because supporting Sertoli cells are overwhelmed. Thus, Bik and Bim share, upstream of Bax, the role of eliminating supernumerary germ cells during the first wave of spermatogenesis, a process vital for normal testicular development.

Activation of NKT cells using the glycolipid alpha-galactosylceramide (alpha-GalCer) has availed many investigations into their immunoregulatory and therapeutic potential. However, it remains unclear how they respond to stimulation in vivo, which costimulatory pathways are important, and what factors (e.g., Ag availability and activation-induced cell death) limit their response. We have explored these questions in the context of an in vivo model of NKT cell dynamics spanning activation, population expansion, and subsequent contraction. Neither the B7/CD28 nor the CD40/CD40L costimulatory pathway was necessary for cytokine production by activated NKT cells, either early (2 h) or late (3 days) after initial stimulation, but both pathways were necessary for normal proliferative expansion of NKT cells in vivo. The proapoptotic Bcl-2 family member Bim was necessary for normal contraction of the NKT cell population between days 3-9 after stimulation, suggesting that the pool size is regulated by apoptotic death, similar to that of conventional T cells. Ag availability was not the limiting factor for NKT cell expansion in vivo, and a second alpha-GalCer injection induced a very blunted response, whereby cytokine production was reduced and further expansion did not occur. This appeared to be a form of anergy that was intrinsic to NKT cells and was not associated with inhibitory NK receptor signaling. Furthermore, NKT cells from mice pre-challenged with alpha-GalCer in vivo showed little cytokine production and reduced proliferation in vitro. In summary, this study significantly enhances our understanding of how NKT cells respond to primary and secondary antigenic challenge in vivo.

The proapoptotic members of the Bcl-2 family can be subdivided into members that contain several Bcl-2 homology (BH) domains and those that contain only the BH3 domain. Although it is known that BH3-only proteins and the multi-BH domain proteins, Bak and Bax, are essential for programmed cell death, the overlapping role of these two subgroups has not been examined in vivo. To investigate this, we generated Bak/Bim and Bax/Bim double deficient mice. We found that although Bax-/-Bim-/-, but not Bak-/-Bim-/-, mice display webbed hind and front paws and malocclusion of the incisors, both groups of mice present with dysregulated hematopoiesis. Combined loss of Bak and Bim or Bax and Bim causes defects in myeloid and B-lymphoid development that are more severe than those found in the single knock-out mice. Bak-/-Bim-/- mice have a complement of thymocytes that resembles those in control mice, whereas Bax-/-Bim-/- mice are more similar to Bim-/- mice. However, thymocytes isolated from Bak-/-Bim-/- or Bax-/-Bim-/- mice are markedly more resistant to apoptotic stimuli mediated by the intrinsic pathway as compared with thymocytes from single-knockout mice. These data suggest an essential overlapping role for Bak or Bax and Bim in the intrinsic apoptotic pathway.

Defective apoptosis not only promotes tumorigenesis, but also can confound chemotherapeutic response. Here we demonstrate that the proapoptotic BH3-only protein BIM is a tumor suppressor in epithelial solid tumors and also is a determinant in paclitaxel sensitivity in vivo. Furthermore, the H-ras/mitogen-activated protein kinase (MAPK) pathway conferred resistance to paclitaxel that was dependent on functional inactivation of BIM. Whereas paclitaxel induced BIM accumulation and BIM-dependent apoptosis in vitro and in tumors in vivo, the H-ras/MAPK pathway suppressed this BIM induction by phosphorylating BIM and targeting BIM for degradation in proteasomes. The proteasome inhibitor Velcade (P-341, Bortezomib) restored BIM induction, abrogated H-ras-dependent paclitaxel resistance, and promoted BIM-dependent tumor regression, suggesting the potential benefits of combinatorial chemotherapy of Velcade and paclitaxel.

Interleukin (IL)-7 receptor (R) signaling is essential for T and B lymphopoiesis by promoting proliferation, differentiation, and survival of cells. Mice lacking either IL-7 or the IL-7Ralpha chain have abnormally low numbers of immature as well as mature T and B lymphocytes. Transgenic expression of the apoptosis inhibitor Bcl-2 rescues T cell development and function in IL-7Ralpha-deficient mice, indicating that activation of a proapoptotic Bcl-2 family member causes death of immature and mature T cells. BH3-only proteins such as Bim, which are distant proapoptotic members of the Bcl-2 family, are essential initiators of programmed cell death and stress-induced apoptosis. We generated Bim/IL-7Ralpha double deficient mice and found that loss of Bim significantly increased thymocyte numbers, restored near normal numbers of mature T cells in the blood and spleen, and enhanced cytotoxic T cell responses to virus infection in IL-7Ralpha-/- mice. These results indicate that Bim cooperates with other proapoptotic proteins in the death of IL-7-deprived T cell progenitors in vivo, but is the major inducer of this pathway to apoptosis in mature T cells. This indicates that pharmacological inhibition of Bim function might be useful for boosting immune responses in immunodeficient patients.

T cell receptor/CD3 ligation induces apoptosis in semimature CD4(+)8(-)HSA+ thymocytes, and this helps establish immunological tolerance and constitutes one of the safeguards against autoimmune disease. We analyzed several knockout and transgenic mouse lines and found that T cell receptor/CD3-ligation-induced killing of semimature thymocytes occurred independently of Fas and "death receptor" signaling in general but required the proapoptotic BH3-only protein Bim and could be inhibited by Bcl-2. Loss of Apaf-1 or caspase-9, which act downstream of the Bcl-2 family protein family, provided only minor protection, indicating that the "apoptosome" functions as an amplifier rather than as an essential initiator of this death program. These results reveal the mechanisms of apoptosis in negative selection of semimature thymocytes and have implications for immunological tolerance and autoimmunity.

Impaired apoptosis is now recognized to be central to tumor development. Bcl2, activated by chromosomal translocation in human follicular lymphoma, promotes oncogenesis by inhibiting apoptosis. Bim, a distant proapoptotic relative, is emerging as a major physiologic antagonist of Bcl2. Here, we show that loss of Bim is oncogenic. Bim protein levels were elevated in the apoptosis-prone B lymphoid cells of Emicro-Myc-transgenic mice, and Bim-mutant Emicro-Myc mice had increased numbers of IgM-bearing B cells. Emicro-Myc-expressing B lymphoid cells deficient in Bim were refractory to apoptosis induced in vitro by cytokine deprivation or antigen receptor cross-linking. Thus, Bim is induced by Myc in B cells and mediates apoptosis. Remarkably, inactivation of even a single allele of Bim accelerated Myc-induced development of tumors, particularly acute B cell leukemia. None of the primary tumors from Bim(+/-) Emicro-Myc mice displayed loss of the second allele of Bim. These findings indicate that Bim is a tumor suppressor, at least in B lymphocytes, and is haploinsufficient. Whereas the p19Arf/p53 pathway is frequently mutated in tumors arising in Bim(+/+) Emicro-Myc mice, it was unaffected in most Bim-deficient tumors, indicating that Bim reduction is an effective alternative to loss of p53 function.

Lurcher (lc) mice have a semi-dominant mutation in the gene encoding the delta2 glutamate receptor (GRID2). The resulting constitutive activity of this receptor in heterozygous +/lc (grid(+/lc)) and homozygous (grid(lc/lc)) mice leads to the death of all cerebellar Purkinje cells and most afferent granule neurons. Some studies have indicated that the death of Purkinje cells occurs by apoptosis, and the secondary loss of granule neurons has been shown to require the pro-apoptotic Bcl-2 family member Bax. The BH3-only protein Bim has been shown to contribute to cytokine withdrawal-induced apoptosis of sympathetic neurons and to be responsible for the kidney degeneration in mice lacking the pro-survival protein Bcl-2. Because Bim is expressed strongly in cerebellar Purkinje cells, we have examined whether it has a role in their death in mutant Lurcher mice. Our studies show that Bim deficiency does not modify the Lurcher phenotype, ruling out an indispensable role for Bim in this neurodegenerative disease., (Copyright 2003 Wiley-Liss, Inc.)

We used mutant Fas-deficient (lpr) or Bim-deficient mice to investigate the role of the death receptor and Bcl-2-regulated apoptotic pathways in terminating a physiological T cell response to herpes simplex virus infection. In WT and lpr mice CD8+ antigen-specific T cells were deleted after viral clearance. In contrast, the immune response was not terminated in Bim-deficient mice despite viral clearance, and CD8+ antigen-specific T cells accumulated in the spleen. Thus, Bim is dispensable for viral clearance but is necessary for the death of activated T cells when immune responses are terminated. These findings have implications for the therapeutic manipulation of immune responses to infections and immunization.

During development, the stochastic process assembling the genes encoding antigen receptors invariably generates B and T lymphocytes that can recognize self-antigens. Several mechanisms have evolved to prevent the activation of these cells and the concomitant development of autoimmune disease. One such mechanism is the induction of apoptosis in developing or mature B cells by engagement of the B cell antigen receptor (BCR) in the absence of T cell help. Here we report that B lymphocytes lacking the pro-apoptotic Bcl-2 family member Bim are refractory to apoptosis induced by BCR ligation in vitro. The loss of Bim also inhibited deletion of autoreactive B cells in vivo in two transgenic systems of B cell tolerance. Bim loss prevented deletion of autoreactive B cells induced by soluble self-antigen and promoted accumulation of self-reactive B cells developing in the presence of membrane-bound self-antigen, although their numbers were considerably lower compared with antigen-free mice. Mechanistically, we determined that BCR ligation promoted interaction of Bim with Bcl-2, inhibiting its survival function. These findings demonstrate that Bim is a critical player in BCR-mediated apoptosis and in B lymphocyte deletion.

Programmed cell death of granulocytes is one of the mechanisms that limit inflammatory responses. Members of the Bcl-2 protein family are essential regulators of apoptosis induced by growth factor withdrawal or cytotoxic stress. We have used gene-targeted and transgenic mice to investigate the roles of the prosurvival molecules Bcl-2 and Bcl-w and their proapoptotic relatives Bax and Bim in spontaneous and stress-induced apoptosis of granulocytes from bone marrow or the peritoneum. Bim deficiency, like Bcl-2 overexpression, rendered granulocytes resistant to cytokine withdrawal and cytotoxic drugs, but absence of Bax alone had no protective effect. Loss of Bcl-2 or Bcl-w did not increase the sensitivity of granulocytes to any of these apoptotic stimuli, but Bcl-2 was essential for the in vitro survival of myeloid progenitors under conditions of cytokine withdrawal where cell death was mediated, in part, by Bim. Granulocyte colony-stimulating factor (G-CSF), a key survival factor for granulocytes, enhanced viability of cells lacking bcl-2, bcl-w, bax, or bim, indicating that none of these genes alone is the essential target of this cytokine's prosurvival function. Expression analysis of proapoptotic Bcl-2 family members in granulocytes revealed that the BH3-only protein Bmf is induced upon cytokine withdrawal. These results indicate that the BH3-only protein Bim and possibly also Bmf are critical initiators of spontaneous and drug-induced apoptosis of granulocytes, whereas Bcl-2, Bcl-w, and Bax act in a redundant manner in regulating granulocyte survival and death, respectively.

By transgenic expression of ovalbumin (OVA) as a model self antigen in the beta cells of the pancreas, we have shown that self tolerance can be maintained by the cross-presentation of this antigen on dendritic cells in the draining lymph nodes. Such cross-presentation causes initial activation of OVA-specific CD8 T cells, which proliferate but are ultimately deleted; a process referred to as cross-tolerance. Here, we investigated the molecular basis of cross-tolerance. Deletion of CD8 T cells was prevented by overexpression of Bcl-2, indicating that cross-tolerance was mediated by a Bcl-2 inhibitable pathway. Recently, Bim, a pro-apoptotic Bcl-2 family member whose function can be inhibited by Bcl-2, was found to play a critical role in the deletion of autoreactive thymocytes, leading us to examine its role in cross-tolerance. Bim-deficient T cells were not deleted in response to cross-presented self-antigen, strongly implicating Bim as the pro-apoptotic mediator of cross-tolerance.

At the end of the T cell response, the majority of the activated T cells die. We activated Vbeta8(+) T cells with staphylococcal enterotoxin B (SEB) in vivo and monitored the expansion and deletion of Vbeta8(+) T cells. We found that, in response to SEB, activated T cells died in vivo in the absence of Fas or TNF-R signaling but not when they overexpressed human Bcl-2. We also found that Vbeta8(+) T cells from Bim-deficient mice are resistant to SEB-induced deletion. While Bim levels did not change, endogenous Bcl-2 levels within Vbeta8(+) T cells decrease following SEB injection. Thus, the death of superantigen-stimulated T cells in vivo is mediated by Bim and may be modulated by a decrease in Bcl-2.

During lymphocyte development, the assembly of genes coding for antigen receptors occurs by the combinatorial linking of gene segments. The stochastic nature of this process gives rise to lymphocytes that can recognize self-antigens, thereby having the potential to induce autoimmune disease. Such autoreactive lymphocytes can be silenced by developmental arrest or unresponsiveness (anergy), or can be deleted from the repertoire by cell death. In the thymus, developing T lymphocytes (thymocytes) bearing a T-cell receptor (TCR)-CD3 complex that engages self-antigens are induced to undergo programmed cell death (apoptosis), but the mechanisms ensuring this 'negative selection' are unclear. We now report that thymocytes lacking the pro-apoptotic Bcl-2 family member Bim (also known as Bcl2l11) are refractory to apoptosis induced by TCR-CD3 stimulation. Moreover, in transgenic mice expressing autoreactive TCRs that provoke widespread deletion, Bim deficiency severely impaired thymocyte killing. TCR ligation upregulated Bim expression and promoted interaction of Bim with Bcl-XL, inhibiting its survival function. These findings identify Bim as an essential initiator of apoptosis in thymocyte-negative selection.