Deletion Of Bim Research Articles

B cell apoptosis and inflammation conspire to promote lupus in mice

Abstract The pathogenesis of Systemic lupus erythematosus (SLE) is driven by autoreactive B and T cells and autoinflammation. Impaired apoptosis in T and B cells has been shown to result in autoimmune disease, and we recently demonstrated that reducing apoptosis in B cells alone, by B cell-specific deletion of Bim (B.Bim f/f) could lead to SLE with prominent features of Sjogren’s Syndrome in C57BL/6 mice. Consistently, B cell targeting therapies are successful in reducing SLE pathogenesis in mouse models, however, their clinical success requires a better understanding of the contributions of B cells and the mechanisms that drive inflammation to autoimmunity. To address the role of inflammation in the development and progression of autoimmunity we generated a novel mouse model that lacks ZFAND6 (Zfand6 −/−) and crossed with the B.Bim f/fmice. ZFAND6 is an A-20 like ZF domain-containing protein with a potential role in the negative regulation of NF-kB activation, however, its physiological function is unknown. The compound mutant mice displayed accelerated and exacerbated overlapping lupus and Sjogren’s autoimmune symptoms with a higher incidence of splenomegaly and kidney pathology than either single mutant alone including the early appearance of anti-SSA, anti-SSB, and anti-RNP autoantibodies. The compound mutant mice displayed increased spontaneous and TLR-induced TNFa and IL-6 and B cells from these mice produced much greater levels of these cytokines in response to TLR7 and TLR9. These results suggest that inappropriate B cell survival and persistent inflammation cooperate in the initiation and severity of lupus pathogenesis. Sylvester Comprehensive Cancer Center, University of Miami, Miami FL

Prognostic Value of BIM Deletion in EGFR-Mutant NSCLC Patients Treated with EGFR-TKIs: A Meta-Analysis.

Background Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is inevitable in EGFR-mutant non-small-cell lung cancer (NSCLC) patients. A germline 2903 bp deletion polymorphism of Bcl-2-like protein 11 (BIM) causes reduced expression of proapoptotic BH3-only BIM protein and blocks TKI-induced apoptosis of tumor cells. Yet the association between the deletion polymorphism and response to EGFR-TKI treatment remains inconsistent among clinical observations. Thus, we performed the present meta-analysis. Methods Eligible studies were identified by searching PubMed, Embase, and ClinicalTrials.gov databases prior to March 31, 2021. Hazard ratios (HRs) and 95% confidence intervals (CIs) of progression-free survival (PFS) and overall survival (OS) and odds ratios (ORs) and 95% CIs of objective response rate (ORR) and disease control rate (DCR) were calculated by using a random effects model. Sensitivity, metaregression, and publication bias analyses were also performed. Results A total of 20 datasets (3003 EGFR-mutant NSCLC patients receiving EGFR-TKIs from 18 studies) were included. There were 475 (15.8%) patients having the 2903-bp intron deletion of BIM and 2528 (84.2%) wild-type patients. BIM deletion predicted significantly shorter PFS (HR = 1.35, 95% CI: 1.10-1.64, P = 0.003) and a tendency toward an unfavorable OS (HR = 1.22, 95% CI: 0.99-1.50, P = 0.068). Patients with deletion polymorphism had lower ORR (OR = 0.60, 95% CI: 0.42-0.85, P = 0.004) and DCR (OR = 0.59, 95% CI: 0.38-0.90, P = 0.014) compared with those without deletion. Conclusion BIM deletion polymorphism may confer resistance to EGFR-TKIs and can be used as a biomarker to predict treatment response to EGFR-TKIs in EGFR-mutant NSCLC patients from Asian populations.

Impaired B Cell Apoptosis Results in Autoimmunity That Is Alleviated by Ablation of Btk.

While apoptosis plays a role in B-cell self-tolerance, its significance in preventing autoimmunity remains unclear. Here, we report that dysregulated B cell apoptosis leads to delayed onset autoimmune phenotype in mice. Our longitudinal studies revealed that mice with B cell-specific deletion of pro-apoptotic Bim (BBimfl/fl) have an expanded B cell compartment with a notable increase in transitional, antibody secreting and recently described double negative (DN) B cells. They develop greater hypergammaglobulinemia than mice lacking Bim in all cells and accumulate several autoantibodies characteristic of Systemic Lupus Erythematosus (SLE) and related Sjögren’s Syndrome (SS) including anti-nuclear, anti-Ro/SSA and anti-La/SSB at a level comparable to NODH2h4 autoimmune mouse model. Furthermore, lymphocytes infiltrated the tissues including submandibular glands and formed follicle-like structures populated with B cells, plasma cells and T follicular helper cells indicative of ongoing immune reaction. This autoimmunity was ameliorated upon deletion of Bruton’s tyrosine kinase (Btk) gene, which encodes a key B cell signaling protein. These studies suggest that Bim-mediated apoptosis suppresses and B cell tyrosine kinase signaling promotes B cell-mediated autoimmunity.

MBRS-08. SONIC HEDGEHOG SIGNALING PRIMES CEREBELLAR GRANULE NEURON PROGENITORS, THE CELL OF ORIGIN FOR MEDULLOBLASTOMA, FOR APOPTOSIS BY INDUCING PRO APOPTOTIC BIM

Medulloblastomas, unlike other malignant brain tumors, are typically sensitive to radiation therapy, but the mechanisms that mediate this sensitivity are unclear. Cerebellar granule neuron progenitors (CGNPs), the cell of origin for SHH-subgroup medulloblastoma, are also highly sensitive to radiation. In early life, CGNPs proliferate in response to Sonic Hedgehog (SHH) signaling, and hyperactivation of SHH signaling in CGNPs can lead to the development of SHH-subgroup medulloblastoma. We propose that SHH activation induces radiation sensitivity along with tumorigenesis. We have previously shown that the proapoptotic protein BAX is required for radiation sensitivity of both SHH-driven medulloblastomas and CGNPs in mice, and that BCL-xL supplies critical regulation of BAX, preventing spontaneous cell death. Here, we show that SHH signaling increases the radiation sensitivity of CGNPs by inducing the proapoptotic protein BIM. We found that BIM expression depends on SHH activity, and that genetic deletion of Bim decreases the radiation-sensitivity of CGNPs. Mechanistically, we show that BIM binds to anti-apoptotic proteins BCL-xL and MCL-1, where it may alter the balance of BAX and BCL-xL interactions. Consistent with our mechanistic model, human medulloblastoma patients with high BIM expression show a better prognosis. Based on these observations, we propose that SHH-induced BIM mediates the typical radiation sensitivity of SHH-driven medulloblastoma. Finding ways to enhance BIM activity may open new opportunities for targeted medulloblastoma therapy.

Loss of BIM in T cells results in BCL-2 family BH3-member compensation but incomplete cell death sensitivity normalization.

BIM is the master BH3-only BCL-2 family regulator of lymphocyte survival. To understand how long-term loss of BIM affects apoptotic resistance in T cells we studied animals with T cell-specific deletion of Bim. Unlike CD19CREBimfl/fl animals, LCKCREBimfl/fl mice have pronounced early lymphocytosis followed by normalization of lymphocyte counts over time. This normalization occurred in mature T cells, as thymocyte development and apoptotic sensitivity remained abnormal in LCKCREBimfl/fl mice. T cells from aged mice experienced normalization of their absolute cell numbers and responses against various apoptotic stimuli. mRNA expression levels of BCL-2 family proteins in CD4+ and CD8+ T cells from young and old mice revealed upregulation of several BH3-only proteins, including Puma, Noxa, and Bmf. Despite upregulation of various BH3 proteins, there were no differences in anti-apoptotic BCL-2 protein dependency in these cells. However, T cells had continued resistance to direct BIM BH3-induced mitochondrial depolarization. This study further highlights the importance of BIM in cell death maintenance in T cells and provides new insight into the dynamism underlying BH3-only regulation of T cell homeostasis versus induced cell death and suggests that CD4+ and CD8+ T cells compensate differently in response to loss of Bim.

Preclinical Activity of Novel MCL1 Inhibitor AZD5991 in Multiple Myeloma

Preclinical Activity of Novel MCL1 Inhibitor AZD5991 in Multiple Myeloma

Concise Review: Cheating Death for a Better Transplant.

Hematopoietic stem cell transplantation is routinely performed for the treatment of various malignant and non-malignant hematological diseases. Successful transplantation depends on the number and fitness of donor stem and progenitor cells, whose quality is substantially influenced by the sampling procedure, sample handling, and cryopreservation. BCL-2 proteins are central to the survival and maintenance of stem and progenitor cells under both, physiological and stress conditions. Transplantation-associated apoptosis of donor cells is mediated by the pro-apoptotic BCL-2 proteins BIM and BMF and prevented by overexpression of their antagonists, BCL-2 and BCL-XL. We have previously reported that deletion of BIM or BMF stabilizes donor stem cell numbers during transplantation and improves cellular fitness and transplantation outcomes, albeit posing a risk for lymphoma and autoimmunity in recipient mice. Short-term apoptosis inhibition in donor cells appears equally effective in improving the outcome of transplantation, but in contrast does not cause pathology. In this review, we discuss the role of BCL-2 proteins in the context of factors that negatively impact donor stem and progenitor cell fitness and viability. Finally, we suggest approaches to improve graft quality and transplantation procedures with an emphasis on the inhibition of BCL-2 regulated apoptosis. Stem Cells 2018;36:1646-1654.

Abstract 294: BCL-2 family compensation regulates T cell homeostasis and reveals a BIM:BCL-W axis in T-ALL

Abstract BIM is the master BH3-only BCL-2 family regulator of lymphocyte survival. To understand how long-term loss of BIM affects apoptotic resistance in T and B cells we examined peripheral blood counts in mice with T or B cell-specific deletion of Bim. In contrast to CD19CRE Bimfl/fl animals, LCKCRE Bimfl/fl mice had pronounced early lymphocytosis followed by lymphocyte count normalization as they aged. To explore BCL-2 family specific mechanism(s) leading to this normalization we compared cell death sensitivity and BCL-2 expression patterns of young versus old LCKCRE Bimfl/fl T cells. Young (6-12 weeks) and old (>40 weeks) LCKCRE Bimfl/fl mice had consistently abnormal thymocyte development hallmarked by a decrease in CD4+8+ double positive (DP) cells and accumulation of double negative (DN) and single positive (SP) cells as previously described in global Bim knockout mice. Thymocytes maintained cell death resistance to a number of apoptotic stimuli throughout all ages, reflecting a consistent loss of BIM. In contrast to thymocytes, mature splenic-derived LCKCRE Bimfl/fl T cells from young mice were equally sensitive to apoptotic stimuli as WT controls, indicating BCL-2 family compensation. Expression profiling in these cells revealed upregulation of other BH3-only proteins, including Noxa = Puma << Bmf, suggesting that these proteins functionally compensated for the loss of BIM. To determine if this was a T cell-intrinsic process and if the presence of other BH3-only proteins with pan-BCL-2 family binding affinities were required for lymphocyte number and apoptotic normalization, we analyzed peripheral blood from mice globally deleted in Bim, Bid, and Puma (TKO). TKO, like LCKCRE Bimfl/fl mice, developed profound early lymphocytosis followed by normalization as they aged. A subset of TKO mice developed T-ALL hallmarked by upregulation of Bmf and Bcl-w, an under characterized anti-apoptotic BCL-2 family protein, suggesting that BCL-W allowed for malignant T cell survival despite the presence of BMF. To determine if such a relationship exists in human disease, expression profiling of pediatric T-ALL patient samples revealed a significant negative correlation between BIM and BCL-W expression in developmentally immature and mature T-ALL. Our findings suggest a model in which the loss of BIM in T cells can be compensated for through upregulation of BMF, a BH3-only protein mechanistically similar to BIM. Loss of other ‘direct activating' BH3-only proteins increases the chances of malignant cell escape despite BMF upregulation, particularly when BCL-W is overexpressed. This work provides a developmental framework between BIM and BMF in T cells and for a BIM:BCL-W axis in T-ALL. Overexpression of BCL-W in BIMlow-expressing T-ALL may be an additional target responsible for early immature T-ALL therapeutic resistance. Citation Format: Lindsey M. Ludwig, Lauren E. Roach, Samuel G. Katz, Jill K. Fisher, Melissa Burns, Matthew R. Schnorenberg, Riyue Bao, Makda Zewde, Yusuke Nakamura, Alejandro Gutierrez, Loren D. Walensky, James L. LaBelle. BCL-2 family compensation regulates T cell homeostasis and reveals a BIM:BCL-W axis in T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 294.

Proapoptotic BIM Impacts B Lymphoid Homeostasis by Limiting the Survival of Mature B Cells in a Cell-Autonomous Manner.

The proapoptotic BH3-only protein BIM (Bcl2l11) plays key roles in the maintenance of multiple hematopoietic cell types. In mice, germline knockout or conditional pan-hematopoietic deletion of Bim results in marked splenomegaly and significantly increased numbers of B cells. However, it has remained unclear whether these abnormalities reflect the loss of cell-intrinsic functions of BIM within the B lymphoid lineage and, if so, which stages in the lifecycle of B cells are most impacted by the loss of BIM. Here, we show that B lymphoid-specific conditional deletion of Bim during early development (i.e., in pro-B cells using Mb1-Cre) or during the final differentiation steps (i.e., in transitional B cells using Cd23-Cre) led to a similar >2-fold expansion of the mature follicular B cell pool. Notably, while the expansion of mature B cells was quantitatively similar in conditional and germline Bim-deficient mice, the splenomegaly was significantly attenuated after B lymphoid-specific compared to global Bim deletion. In vitro, conditional loss of Bim substantially increased the survival of mature B cells that were refractory to activation by lipopolysaccharide. Finally, we also found that conditional deletion of just one Bim allele by Mb1-Cre dramatically accelerated the development of Myc-driven B cell lymphoma, in a manner that was comparable to the effect of germline Bim heterozygosity. These data indicate that, under physiological conditions, BIM regulates B cell homeostasis predominantly by limiting the life span of non-activated mature B cells, and that it can have additional effects on developing B cells under pathological conditions.

Transcription factor YY1 is essential for iNKT cell development

Invariant natural killer T (iNKT) cells develop from CD4+CD8+ double-positive (DP) thymocytes and express an invariant Vα14-Jα18 T-cell receptor (TCR) α-chain. Generation of these cells requires the prolonged survival of DP thymocytes to allow for Vα14-Jα18 gene rearrangements and strong TCR signaling to induce the expression of the iNKT lineage-specific transcription factor PLZF. Here, we report that the transcription factor Yin Yang 1 (YY1) is essential for iNKT cell formation. Thymocytes lacking YY1 displayed a block in iNKT cell development at the earliest progenitor stage. YY1-deficient thymocytes underwent normal Vα14-Jα18 gene rearrangements, but exhibited impaired cell survival. Deletion of the apoptotic protein BIM failed to rescue the defect in iNKT cell generation. Chromatin immunoprecipitation and deep-sequencing experiments demonstrated that YY1 directly binds and activates the promoter of the Plzf gene. Thus, YY1 plays essential roles in iNKT cell development by coordinately regulating cell survival and PLZF expression.

Dynein light chain regulates adaptive and innate B cell development by distinctive genetic mechanisms

Mechanistic differences in the development and function of adaptive, high-affinity antibody-producing B-2 cells and innate-like, “natural” antibody-producing B-1a cells remain poorly understood. Here we show that the multi-functional dynein light chain (DYNLL1/LC8) plays important roles in the establishment of B-1a cells in the peritoneal cavity and in the ongoing development of B-2 lymphoid cells in the bone marrow of mice. Epistasis analyses indicate that Dynll1 regulates B-1a and early B-2 cell development in a single, linear pathway with its direct transcriptional activator ASCIZ (ATMIN/ZNF822), and that the two genes also have complementary functions during late B-2 cell development. The B-2 cell defects caused by loss of DYNLL1 were associated with lower levels of the anti-apoptotic protein BCL-2, and could be supressed by deletion of pro-apoptotic BIM which is negatively regulated by both DYNLL1 and BCL-2. Defects in B cell development caused by loss of DYNLL1 could also be partially suppressed by a pre-arranged SWHEL Igm-B cell receptor transgene. In contrast to the rescue of B-2 cell numbers, the B-1a cell deficiency in Dynll1-deleted mice could not be suppressed by the loss of Bim, and was further compounded by the SWHEL transgene. Conversely, oncogenic MYC expression, which is synthetic lethal with Dynll1 deletion in B-2 cells, did not further reduce B-1a cell numbers in Dynll1-defcient mice. Finally, we found that the ASCIZ-DYNLL1 axis was also required for the early-juvenile development of aggressive MYC-driven and p53-deficient B cell lymphomas. These results identify ASCIZ and DYNLL1 as the core of a transcriptional circuit that differentially regulates the development of the B-1a and B-2 B lymphoid cell lineages and plays a critical role in lymphomagenesis.

Bim gene dosage is critical in modulating nephron progenitor survival in the absence of microRNAs during kidney development.

Low nephron endowment at birth has been associated with an increased risk for developing hypertension and chronic kidney disease. We demonstrated in an earlier study that conditional deletion of the microRNA (miRNA)-processing enzyme Dicer from nephron progenitors results in premature depletion of the progenitors and increased expression of the proapoptotic protein Bim (also known as Bcl-2L11). In this study, we generated a compound mouse model with conditional deletion of both Dicer and Bim, to determine the biologic significance of increased Bim expression in Dicer-deficient nephron progenitors. The loss of Bim partially restored the number of nephron progenitors and improved nephron formation. The number of progenitors undergoing apoptosis was significantly reduced in kidneys with loss of a single allele, or both alleles, of Bim compared to mutant kidneys. Furthermore, 2 miRNAs expressed in nephron progenitors (miR-17 and miR-106b) regulated Bim levels in vitro and in vivo Together, these data suggest that miRNA-mediated regulation of Bim controls nephron progenitor survival during nephrogenesis, as one potential means of regulating nephron endowment.-Cerqueira, D. M., Bodnar, A. J., Phua, Y. L., Freer, R., Hemker, S. L., Walensky, L. D., Hukriede, N. A., Ho, J. Bim gene dosage is critical in modulating nephron progenitor survival in the absence of microRNAs during kidney development.

Post-translational control of T cell development by the ESCRT protein CHMP5.

The acquisition of a protective vertebrate immune system hinges on the efficient generation of a diverse but self-tolerant repertoire of T cells by the thymus through mechanisms that remain incompletely resolved. Here we identified the endosomal-sorting-complex-required-for-transport (ESCRT) protein CHMP5, known to be required for the formation of multivesicular bodies, as a key sensor of thresholds for signaling via the T cell antigen receptor (TCR) that was essential for T cell development. CHMP5 enabled positive selection by promoting post-selection thymocyte survival in part through stabilization of the pro-survival protein Bcl-2. Accordingly, loss of CHMP5 in thymocyte precursor cells abolished T cell development, a phenotype that was 'rescued' by genetic deletion of the pro-apoptotic protein Bim or transgenic expression of Bcl-2. Mechanistically, positive selection resulted in the stabilization of CHMP5 by inducing its interaction with the deubiquitinase USP8. Our results thus identify CHMP5 as an essential component of the post-translational machinery required for T cell development.

Bim expression in endothelial cells and pericytes is essential for regression of the fetal ocular vasculature.

Apoptosis plays a central role in developmental and pathological angiogenesis and vessel regression. Bim is a pro-apoptotic Bcl-2 family member that plays a prominent role in both developmental and pathological ocular vessel regression, and neovascularization. Endothelial cells (EC) and pericytes (PC) each play unique roles during vascular development, maintenance and regression. We recently showed that germline deletion of Bim results in persistent hyaloid vasculature, increased retinal vascular density and prevents retinal vessel regression in response to hyperoxia. To determine whether retinal vascular regression is attributable to Bim expression in EC or PC we generated mice carrying a conditional Bim allele (BimFlox/Flox) and VE-cadherin-cre (BimEC mice) or Pdgfrb-cre (BimPC mice). BimEC and BimPC mice demonstrated attenuated hyaloid vessel regression and postnatal retinal vascular remodeling. We also observed decreased retinal vascular apoptosis and proliferation. Unlike global Bim -/- mice, mice conditionally lacking Bim in EC or PC underwent hyperoxia-mediated vessel obliteration and subsequent retinal neovascularization during oxygen-induced ischemic retinopathy similar to control littermates. Thus, understanding the cell autonomous role Bim plays in the retinal vascular homeostasis will give us new insight into how to modulate pathological retinal neovascularization and vessel regression to preserve vision.

Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival.

Natural killer (NK) cells are innate lymphoid cells with antitumor functions. Using an N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen in mice, we identified a strain with an NK cell deficiency caused by a hypomorphic mutation in the Bcl2 (B cell lymphoma 2) gene. Analysis of these mice and the conditional deletion of Bcl2 in NK cells revealed a nonredundant intrinsic requirement for BCL2 in NK cell survival. In these mice, NK cells in cycle were protected against apoptosis, and NK cell counts were restored in inflammatory conditions, suggesting a redundant role for BCL2 in proliferating NK cells. Consistent with this, cycling NK cells expressed higher MCL1 (myeloid cell leukemia 1) levels in both control and BCL2-null mice. Finally, we showed that deletion of BIM restored survival in BCL2-deficient but not MCL1-deficient NK cells. Overall, these data demonstrate an essential role for the binding of BCL2 to BIM in the survival of noncycling NK cells. They also favor a model in which MCL1 is the dominant survival protein in proliferating NK cells.

The Role of BAX/BAK-Mediated Apoptosis for the Cytotoxic Action of Anti-Myeloma Agents

Multiple myeloma is a malignancy of plasma cells. The abnormal clonal accumulation of plasma cells interferes with the blood cell production, thereby causing cytopenias and aberrant, excessive antibody production. Patients can also experience bone pain, neurological symptoms and renal failure. With the exception of bone marrow transplantation that is associated with significant morbidity and mortality, there is currently no cure for patients with this disease. Thus, novel therapies are desperately needed for treating patients especially those harboring disease with adverse prognostic markers or when they become refractory to current treatment regimens.Recently, we have screened a panel of well-established myeloma cell lines against multiple anti-myeloma agents, including standard-of-care agents such as bortezomib, lenalidomide, dexamethasone or melphalan, as well as novel targeted agents such as the BH3 mimetic compounds (e.g. venetoclax/ABT-199 to target BCL2) or HDAC inhibitors (e.g. panobinostat). Specifically, we sought to determine whether their cytotoxic action depends on the induction of BAX/BAK-mediated apoptosis. When activated, these two proteins drive permeabilization of the outer mitochondrial membrane. In their absence, apoptosis cannot proceed and we could readily establish if BAX/BAK-mediated apoptosis is critical by comparing the sensitivity of isogenic cell lines lacking these two cell death mediators to their wild-type parental counterparts.As anticipated, the action of the BH3 mimetic compounds such as venetoclax/ABT-199 to inhibit BCL2 or small molecule inhibitors to selectively target relatives of BCL2, such as BCLxL and MCL1, rely exclusively on BAX and BAK. In the their absence, the treated cells remained viable. While the action of some drugs such as dexamethasone is already known to be mediated in part by BAX/BAK-driven apoptosis, we were very surprised that lenalidomide, an immunomodulatory drug (IMiD), can also kill some myeloma cell lines through BAX/BAK mediated apoptosis. A more potent relative of lenalidomide, pomalidamide, does likewise. While the cytotoxic action of these IMiDs, most notably the induction of cell cycle arrest upon activation of the E3 ligase cereblon (CRBN) is well known, our data is the first to definitively demonstrate that the IMiDs can actively induce BAX/BAK-mediated apoptosis.Next we explored the mechanisms by which the IMiDs can trigger apoptosis. Most stress signals and many cytotoxic drugs activate the BH3-only proteins such as BIM, BAD, BID or PUMA to initiate apoptosis. We checked whether killing by lenalidomide might also depend on specific BH3-only proteins and found that the genetic deletion of BIM conferred almost complete resistance to treatment with lenalidomide in vitro. We are currently investigating the precise mechanism by which the IMiDs can activate BIM to drive apoptosis. While lenalidomide can trigger BAX/BAK-mediated apoptosis in some myeloma cell lines, many others were spared.We expect that by deciphering the mechanisms by which anti-myeloma agents can induce BAX/BAK-mediated apoptosis of myeloma cell lines, we can probably improve the utility of drugs such as the IMiDs and understand why they might fail in some patients. DisclosuresGong:The Walter and Eliza Hall Institute of Medical Research: Employment. Segal:The Walter and Eliza Hall Institute of Medical Research: Employment. Riffkin:The Walter and Eliza Hall Institute of Medical Research: Employment. van Delft:The Walter and Eliza Hall Institute of Medical Research: Employment. Roberts:Walter and Eliza Hall Institute of Medical Research: Employment. Huang:The Walter and Eliza Hall Institute of Medical Research: Employment.

Abstract 3552: Regulation of immune homeostasis by direct activator BH3-only proteins

Abstract The BH3-only proteins BIM, BID, and PUMA have emerged as both inhibitors of anti-apoptotic BCL-2 family proteins and direct activators of the pro-apoptotic executioner proteins BAX and BAK, and have been implicated in regulating immune cell homeostasis. For example, BIM is a master regulator of B and T cells and its deletion in mice causes splenomegaly and lymphocytosis. Although deletion of Bid or Puma do not grossly impact murine hematopoietic organs, loss of each can protect lymphocytes from certain forms of cell death. Combined deletion of Puma and Bim causes increased lymphocytosis and spontaneous development of B-cell lymphoma, while loss of BID and BIM leads to delayed T cell contraction following antigen-mediated proliferation. To investigate the role of direct activator BH3-only proteins in regulating the adaptive immune response over time, we performed peripheral lymphocyte analyses on single and combinatorial knock-outs of Bim, Puma, and Bid. Select genotypes, such as Bim−/−, Bim−/−Puma−/−, and Bim−/−Puma−/−Bid−/− mice, show lymphocytosis early in life but, unexpectedly, counts were observed to gradually normalize by 40 weeks of age. Given the dominant role of BIM in immune cell homeostasis, we investigated whether this phenotype was dictated by global deletion of Bim or intrinsic to T or B cell-specific Bim deletion. In generating and analyzing Lck-cre and CD19-cre Bim floxed animals, we found that T cell-specific Bim deletion recapitulated early lymphocyte expansion followed by contraction, whereas the lymphocytosis of mice with B cell-specific Bim deletion showed no age-related changes. Further characterization of aged Lck-cre Bim floxed mice revealed spleen weights similar to wild type mice, and thymocytes with increased sensitivity to a range of apoptotic stimuli; these mice also have an increased percentage of regulatory T cells compared to similarly aged wild-type mice, as has been reported for globally-deleted Bim−/− animals. To determine the mechanism underlying the observed lymphocyte contraction, we performed qRT-PCR analysis for a panel of BCL-2 family proteins. We find that aged Lck-cre Bim floxed mice manifest upregulation of a series of BH3-only transcripts, including Puma and Bid, reflective of an intriguing compensatory mechanism to remedy, over time, the lymphocytosis that derives from Bim deletion in T cells. These studies provide new insight into the complexity and dynamism underlying BH3-only regulation of immune cell homeostasis during health, disease, and aging. Citation Format: Lindsey M. Ludwig, Lauren E. Roach, Jill K. Fisher, Loren D. Walensky, James L. LaBelle. Regulation of immune homeostasis by direct activator BH3-only proteins. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3552.

Bim expression in early DP thymocytes limits development of TCRαβ+DN thymocytes and CD8αα intestinal intraepithelial lymphocytes (HEM2P.243)

Abstract Thymocyte negative selection is thought to cull T cells with strong self-reactivity and maintain T cells with non-self-specificity. While initial work implicated Bim as a critical mediator of thymocyte negative selection, its role in the process has been controversial. Thus, we re-examined thymocyte development in mice with global and T cell specific loss of Bim. We found that global Bim-/- mice had a striking accumulation of DN4 thymocytes expressing high levels of surface TCR, which failed to generate DP cells in OP9-DL1 culture. Interestingly, CD4Cre-driven deletion of Bim promoted DN4 cells, but later, dLckCre-driven deletion of Bim did not. Combined, these data show that the aberrant DN4 cells in Bim-/- mice were once DP cells, but had lost co-receptor expression. Investigating the fate of these cells, we found that splenic DN T and gut CD8αα T cells were increased in Bim-/- mice. While IL-15 is critical for both DN/CD8αα T cells, the additional loss of Bim partially restored DN/CD8αα T cells in IL-15 deficient mice, suggesting that IL-15 signals antagonize Bim to promote DN T and CD8αα T cells. Finally, TCR or IL-15 signals were sufficient to promote expression of both CD8αα and the gut homing receptor CCR9 on splenic DN T cells. In conclusion, these data further delineate the role of Bim on thymocyte fate and DN/CD8αα T cell development; and show that dLckCre-Bimf/f mice can isolate the effects of Bim on peripheral T cell survival without affecting thymic selection.

Impact of conditional deletion of the pro-apoptotic BCL-2 family member BIM in mice.

The pro-apoptotic BH3-only BCL-2 family member BIM is a critical determinant of hematopoietic cell development and homeostasis. It has been argued that the striking hematopoietic abnormalities of BIM-deficient mice (accumulation of lymphocytes and granulocytes) may be the result of the loss of the protein throughout the whole animal rather than a consequence intrinsic to the loss of BIM in hematopoietic cells. To address this issue and allow the deletion of BIM in specific cell types in future studies, we have developed a mouse strain with a conditional Bim allele as well as a new Cre transgenic strain, Vav-CreER, in which the tamoxifen-inducible CreER recombinase (fusion protein) is predominantly expressed in the hematopoietic system. We show that acute loss of BIM in the adult mouse rapidly results in the hematopoietic phenotypes previously observed in mice lacking BIM in all tissues. This includes changes in thymocyte subpopulations, increased white blood cell counts and resistance of lymphocytes to BIM-dependent apoptotic stimuli, such as cytokine deprivation. We have validated this novel conditional Bim knockout mouse model using established and newly developed CreER strains (Rosa26-CreER and Vav-CreER) and will make these exciting new tools for studies on cell death and cancer available.

Deletion of Bim compromises the quality of CD8 T cell memory after acute infection (P6060)

Abstract One important feature of T-cell memory formation is the process of cell death during the contraction phase of the immune response. CD8 T-cell contraction after acute infections is mediated by the intrinsic apoptosis pathway, regulated by the interaction of anti-apoptotic BCL2 with pro-apoptotic Bim. In a murine model of influenza A virus infection, we found that Bim-deficient animals experienced a total ablation of the CD8+ T cell contraction phase leading to the significant accumulation of antigen-specific cells. While these cells displayed typical memory phenotypes, in vivo adoptive transfer and in vitro functional studies show that Bim-deficient memory CD8 T cells are non-responsive to stimulation by antigenic peptides and fail to expand properly on secondary challenge, indicating an intrinsic defect of this spent effector population. These functional deficiencies begin to emerge even at the peak of the primary response. Taken together, these results demonstrate that efficient memory generation entails the generation of a distinct, productive memory lineage that is capable of evading Bim-mediated apoptosis.