Antigen Receptor Stimulation Research Articles

Recombination activation gene-2-deficient blastocyst complementation analysis reveals an essential role for nuclear factor I-A transcription factor in T-cell activation

Nuclear factor I (NFI)-A is a member of the NFI family of transcription factors implicated in regulation of granulocyte differentiation. However, its role in the lymphoid lineage is not known. NFI-A deficiency results in perinatal lethality, thus precluding analysis of the role of NFI-A in lymphocyte development and function. Using recombination activation gene-2-deficient (RAG-2(-/-)) blastocysts and embryonic stem cells with homozygous NFI-A gene deletion, we show an essential role for NFI-A in T-cell activation. NFI-A(-/-)→RAG-2(-/-) chimeric mice had normal distributions of CD4(-)CD8(-) double negative, CD4(+)CD8(+) double positive, CD4(+)CD8(-) and CD4(-)CD8(+)-single positive cells in the thymus and CD4(+)CD8(-) and CD4(-)CD8(+) cells in spleen and lymph nodes. However, NFI-A(-/-)→RAG-2(-)(/)(-) mice had severely reduced thymus size and hypocellularity. The decrease in thymocytes and peripheral T cells in NFI-A(-/-)→RAG-2(-/-) chimeric mice is attributed to proliferative defects associated with decreased blast transformation, CD69 expression and DNA synthesis in response to T antigen receptor stimulation. Interestingly, NFI-A-null T cells showed increased levels of c-myc transcription that is inhibited in response to antigen receptor-mediated activation. These studies demonstrate for the first time a requirement for the NFI-A transcription factor in antigen receptor-induced T-cell activation events.

Dynamic cortical actin remodeling by ERM proteins controls BCR microcluster organization and integrity

Signaling microclusters are a common feature of lymphocyte activation. However, the mechanisms controlling the size and organization of these discrete structures are poorly understood. The Ezrin-Radixin-Moesin (ERM) proteins, which link plasma membrane proteins with the actin cytoskeleton and regulate the steady-state diffusion dynamics of the B cell receptor (BCR), are transiently dephosphorylated upon antigen receptor stimulation. In this study, we show that the ERM proteins ezrin and moesin influence the organization and integrity of BCR microclusters. BCR-driven inactivation of ERM proteins is accompanied by a temporary increase in BCR diffusion, followed by BCR immobilization. Disruption of ERM protein function using dominant-negative or constitutively active ezrin constructs or knockdown of ezrin and moesin expression quantitatively and qualitatively alters BCR microcluster formation, antigen aggregation, and downstream BCR signal transduction. Chemical inhibition of actin polymerization also altered the structure and integrity of BCR microclusters. Together, these findings highlight a crucial role for the cortical actin cytoskeleton during B cell spreading and microcluster formation and function.

The role of Stat4 in IL-23-dependent IL-17 production (114.2)

Abstract Signal transducers and activators of transcription (STAT) proteins are induced upon cytokine and growth factor stimulation and play a pivotal role in immune and inflammatory responses. Stat4 is a critical mediator of inflammatory immunity and is required for all known IL-12 biological responses, including the induction of IFN-γ and development of Th1 cells. IL-23, an IL-12-related cytokine, also induces Stat4 activation, which may be important for optimal IL-17 secretion from CD4+ T cells. We further characterized the requirement for Stat4 in IL-23-mediated cytokine production using cells from Stat4-deficient mice. Stat4-deficient CD4+ T cells display reduced cytokine-induced IL-17 production in comparison to control cells, and this phenotype is most prominent in IL-23-primed memory CD4+ T cells. Furthermore, innate IL-17-producing cells, such as γδ T cells, natural killer T (NKT) cells and natural killer (NK) cells display distinct dependence on Stat4 activation for IL-17 production following antigen receptor or cytokine stimulation. Taken together, these data demonstrate a restricted requirement for Stat4 in the responses of innate and adaptive IL-17-secreting cells and define candidates for further defining the role of Stat4 in inflammatory immunity.

Participation of the Cell Polarity Protein PALS1 to T-Cell Receptor-Mediated NF-κB Activation

BackgroundBeside their established function in shaping cell architecture, some cell polarity proteins were proposed to participate to lymphocyte migration, homing, scanning, as well as activation following antigen receptor stimulation. Although PALS1 is a central component of the cell polarity network, its expression and function in lymphocytes remains unknown. Here we investigated whether PALS1 is present in T cells and whether it contributes to T Cell-Receptor (TCR)-mediated activation.Methodology/Principal FindingsBy combining RT-PCR and immunoblot assays, we found that PALS1 is constitutively expressed in human T lymphocytes as well as in Jurkat T cells. siRNA-based knockdown of PALS1 hampered TCR-induced activation and optimal proliferation of lymphocyte. We further provide evidence that PALS1 depletion selectively hindered TCR-driven activation of the transcription factor NF-κB.ConclusionsThe cell polarity protein PALS1 is expressed in T lymphocytes and participates to the optimal activation of NF-κB following TCR stimulation.

NF-κB signaling pathways regulated by CARMA family of scaffold proteins

The NF-κB family of transcription factors plays a crucial role in cell activation, survival and proliferation. Its aberrant activity results in cancer, immunodeficiency or autoimmune disorders. Over the past two decades, tremendous progress has been made in our understanding of the signals that regulate NF-κB activation, especially how scaffold proteins link different receptors to the NF-κB-activating complex, the IκB kinase complex. The growing number of these scaffolds underscores the complexity of the signaling networks in different cell types. In this review, we discuss the role of scaffold molecules in signaling cascades induced by stimulation of antigen receptors, G-protein-coupled receptors and C-type Lectin receptors, resulting in NF-κB activation. Especially, we focus on the family of Caspase recruitment domain (CARD)-containing proteins known as CARMA and their function in activation of NF-κB, as well as the link of these scaffolds to the development of various neoplastic diseases through regulation of NF-κB.

Characterisation of B Cell Receptor-Induced NF-KB Activation In Chronic Lymphocytic Leukaemia Cells

AbstractAbstract 375B cell receptor (BCR) signaling promotes survival of the malignant clone in chronic lymphocytic leukaemia (CLL) through its ability to stimulate NFkB pathway signaling. In lymphoid cells, antigen receptor stimulation of this pathway is achieved by engaging the Carma-1 – Bcl10 – MALT1 (CBM) complex for eventual activation of I-kB kinases (IKKs). In B cells, protein kinase C beta (PKCbeta) is an important mediator of CBM complex activation. However, in CLL cells we found that PKCs do not appear to have a role in BCR-mediated NFkB pathway signaling, despite high expression levels of PKCbeta, because the presence of specific inhibitors of this kinase (LY379196 and bisindolylmaleimide-I) has no effect on the induction of IKK phosphorylation during BCR crosslinking. Examination of CBM complex expression suggests an explanation for this phenomenon; the expression levels of Carma-1 and MALT-1 are largely similar in CLL and normal B cells, but the expression of Bcl10 is much reduced in CLL cells. These findings, taken together with the established role of Bcl10 in the pathway of BCR-induced NFkB activation, suggest that CLL cells may employ a different mechanism to activate this pathway during BCR stimulation. Tyrosine kinases are known to play a role in BCR-induced IKK activation in CLL cells because compounds like dasatinib and PP2 inhibit NFkB pathway activation by BCR. One possible tyrosine kinase is c-Abl because we have shown this protein to be overexpressed in CLL cells, where it plays a role in activation of the NFkB pathway. To investigate the role of c-Abl in BCR-induced IKK activation, we used the inhibitor imatinib and found that the presence of this compound partially inhibited IKK phosphorylation in BCR-stimulated CLL cells. However, imatinib can also inhibit Lck, a T cell-specific src-family tyrosine kinase that is expressed by CLL cells. To differentiate between Lck- and c-Abl-mediated BCR signals we used the specific inhibitor 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[3,2d] pyrimidin-7-yl-cyclopentane (Lck-i). We found that the presence of this compound in CLL cell cultures undergoing BCR stimulation almost completely inhibited the induction of IKK activation. Investigation of Lck-i specificity revealed this compound did not inhibit either c-Abl or Lyn at the concentration used to inhibit Lck in CLL cell cultures. Further investigation of the effects of Lck-i showed that this compound was also effective in inhibiting BCR-induced activation of the Akt and ERK signaling pathways. Taken together, these data suggest a major role for Lck in BCR-mediated signaling in CLL cells, and question the existing paradigm on the importance of Lyn. Disclosures:No relevant conflicts of interest to declare.

SPPL3 regulation of Ca2+ homeostasis modulates NFAT activation during antigen-receptor stimulation in lymphocytes (50.11)

Abstract During T cell receptor signaling, activation of several transcription factors initiates new gene expression, leading to the generation of specific T cell responses. One important event in this pathway involves store-operated calcium entry (SOCE) through Ca2+-release-activated-Ca2+ (CRAC) channels, which activates the key transcription factor NFAT. Induction of this Ca2+-dependent signaling necessitates tight regulation of intracellular Ca2+ levels, and while the identification of STIM1 and CRACM1 has significantly helped elucidate how SOCE is achieved, other players in this pathway and alternative unknown pathways also controlling Ca2+ levels are not well understood. Here, we have used an expression cloning screen to identify signal peptide peptidase like 3 (SPPL3) as a novel activator of NFAT. Interestingly, SPPL3 is a polytopic membrane protein which localizes to the ER or Golgi compartment. Using Ca2+-sensitive dyes, we determined that SPPL3 overexpression elevated basal intracellular Ca2+ levels, dampened the thapsigargin-stimulated release of ER Ca2+ stores, and enhanced Ca2+ influx. SPPL3-mediated NFAT activation was blocked by the SOCE inhibitor SKF 96365 and a dominant negative CRACM1. By influencing intracellular Ca2+ levels, SPPL3 appears to be a novel regulator of calcium homeostasis that can positively modulate antigen receptor-mediated activation of NFAT.

CD5-dependent CK2 activation pathway regulates T cell activation and threshold for anergy (143.27)

Abstract We have reported that a major biological activity of CD5 is to regulate the survival of activated T cells, a property mediated by the CD5-dependent CK2 activation pathway. In order to address the physiological role of the CD5-dependent CK2 activation pathway in T cell activation, the generation of Th effector cells, and autoimmunity, we generated by gene targeting a mouse (CD5ΔCK2BD) that expressed CD5 lacking the ability to bind and activate CK2. CD4 and CD8 T cells from CD5ΔCK2BD mice exhibited enhanced AICD and hyporeactivity to antigen receptor stimulation compared to T cells from CD5WT and CD5-/- mice. The ability to mobilize calcium and upregulate CD69 were greatly enhanced in CD5-/- T cells but not in CD5WT T cells or CD5ΔCK2BD T cells, indicating that the CD5-dependent CK2 activation pathway did not regulate TCR-proximal signaling pathways. CD5-/- mice developed attenuated experimental autoimmune encephalomyelitis (EAE) when immunized with 150 μg MOG, yet the disease in CD5ΔCK2BD mice was comparable to that seen in CD5WT mice. In contrast, immunization with tolerogenic doses of MOG resulted in attenuated EAE in CD5WT mice but not in CD5-/- and CD5ΔCK2BD mice. The results indicated that loss of the CD5-dependent CK2 pathway made T cells refractory to induction of anergy, a prediction that was recapitulated in in vitro recall assays. Our results reveal a novel role for CD5 in setting thresholds for anergy in T cells. Supported by grants from the NIH and NMSS.

Vav1 couples the T cell receptor to cAMP response element activation via a PKC-dependent pathway

The transcription factor cAMP-responsive element binding protein (CREB) is a regulator of the expression of several genes important for lymphocyte activation and proliferation. However, the proximal signaling events leading to activation of CREB in T cells upon antigen receptor stimulation remain unknown. Here we identify a role for Vav1 in the activation of the cAMP response element (CRE), the binding site for CREB. T cell receptor (TCR)/CD28 — induced costimulation of Jurkat T cells expressing Vav1 but not a GEF-deficient mutant showed increased CRE activation (7.2 ± 2.4 fold over control), whereas Vav1 downregulation by siRNA reduced activation of CRE by 2.6 ± 1.3 fold. Inhibition of PKC and MEK but not p38 could reduce Vav1-mediated CRE activation, suggesting that Vav1 transmits TCR and CD28 signals to activation of CRE via PKC and ERK signaling pathways. As a consequence, downregulation of Vav1 impaired the expression of several CRE-containing genes like cyclin D1, INFγ and IL-2, whereas overexpression of Vav1 enhanced CRE-dependent gene expression. Furthermore, cAMP-induced CRE-dependent transcription and gene expression was also modulated by Vav1, but did not require activation of PKC and the GEF function of Vav1. Our data provide insights into the signal transduction events regulating CRE-mediated gene expression in T cells, which affects T cell development, proliferation and activation. We identify Vav1 as an essential component of TCR-induced CRE activation and gene expression, which underlines the central role for Vav1 as key player for TCR signal transduction and gene expression.

Roles of autophagy in lymphocytes: reflections and directions.

Recent studies have revealed that autophagy, a fundamental intracellular process, plays many different roles in lymphocyte development and function. Autophagy regulates naive T-lymphocyte homeostasis, specifically by regulating mitochondrial quality and turnover, and is necessary for the proliferation of mature T cells. Autophagy also acts as a cellular death pathway in lymphocytes, both upon prolonged cytokine withdrawal and during acute antigen-receptor stimulation if improperly regulated. Furthermore, during HIV infection, hyperinduction of autophagy leads to massive T-cell death in uninfected CD4(+) T cells, and is rescued by inhibiting autophagic initiation. Constitutively high levels of autophagy in thymic epithelial cells are necessary for optimal processing and presentation of endogenous antigens, and required for proper positive and negative selection of developing thymocytes. Autophagy also promotes the survival of B lymphocytes, as well as the development of early B-cell progenitors. In B cells, autophagy is an alternative death pathway, as antigen-receptor stimulation in the absence of costimulation induces a potent autophagic death. Thus, autophagy plays a complex role in lymphocytes and is regulated during their lifespan to ensure a healthy immune system.

Grb2, a simple adapter with complex roles in lymphocyte development, function, and signaling

Lymphocyte development, activation, and tolerance depend on antigen receptor signaling transduced via multiple intracellular signalosomes. These signalosomes are assembled by different adapters. Given that signaling molecules can be either positive or negative regulators for a biochemical target, the complex of a target with different regulator may dictate the final signaling outcome. Grb2 is a simple adapter known to be involved in a variety of growth factor receptor signaling. However, its role in antigen receptor signaling as well as lymphocyte development and function has emerged only recently. Despite its simple molecular structure, recent experiments show that Grb2 may play a complex role in T and B-cell antigen receptor signaling. In this article, we review recent findings about the physiological role of Grb2 in T and B-cell development and activation and summarize the current mechanistic understanding of how Grb2 exerts its function following T and B-cell antigen receptor stimulation.

T cell factor 1 initiates the T helper type 2 fate by inducing the transcription factor GATA-3 and repressing interferon-γ

The differentiation of activated CD4(+) T cells into the T helper type 1 (T(H)1) or T(H)2 fate is regulated by cytokines and the transcription factors T-bet and GATA-3. Whereas interleukin 12 (IL-12) produced by antigen-presenting cells initiates the T(H)1 fate, signals that initiate the T(H)2 fate are not completely characterized. Here we show that early GATA-3 expression, required for T(H)2 differentiation, was induced by T cell factor 1 (TCF-1) and its cofactor beta-catenin, mainly from the proximal Gata3 promoter upstream of exon 1b. This activity was induced after T cell antigen receptor (TCR) stimulation and was independent of IL-4 receptor signaling through the transcription factor STAT6. Furthermore, TCF-1 blocked T(H)1 fate by negatively regulating interferon-gamma (IFN-gamma) expression independently of beta-catenin. Thus, TCF-1 initiates T(H)2 differentiation of activated CD4(+) T cells by promoting GATA-3 expression and suppressing IFN-gamma expression.

Cytoskeletal protein 4.1R negatively regulates T-cell activation by inhibiting the phosphorylation of LAT

AbstractProtein 4.1R (4.1R) was first identified in red cells where it plays an important role in maintaining mechanical stability of red cell membrane. 4.1R has also been shown to be expressed in T cells, but its function has been unclear. In the present study, we use 4.1R-deficient mice to explore the role of 4.1R in T cells. We show that 4.1R is recruited to the immunologic synapse after T cell–antigen receptor (TCR) stimulation. We show further that CD4+ T cells of 4.1R−/− mice are hyperactivated and that they displayed hyperproliferation and increased production of interleukin-2 (IL-2) and interferon γ (IFNγ). The hyperactivation results from enhanced phosphorylation of LAT and its downstream signaling molecule ERK. The 4.1R exerts its effect by binding directly to LAT, and thereby inhibiting its phosphorylation by ZAP-70. Moreover, mice deficient in 4.1R display an elevated humoral response to immunization with T cell–dependent antigen. Thus, we have defined a hitherto unrecognized role for 4.1R in negatively regulating T-cell activation by modulating intracellular signal transduction.

BCR-induced cell death of B cells from CD22 deficient mice is mediated by a novel ssRNA-directed endonuclease (136.33)

Abstract B cells from CD22 deficient mice backcrossed onto a C57BL/6 genetic background (B6 CD22-/-) undergo B cell antigen receptor (BCR)-induced apoptosis. By contrast, B cells from parental inbred CD22-/- mice on a mixed B6x129 genetic background (B6/129 CD22-/-) survive and proliferate normally following BCR ligation. Through an extensive reverse genetic screen, a single locus was identified as being responsible for the proliferation defect of B6 CD22-/- B cells. In B6 CD22-/- mice this locus was homozygous for B6 germline DNA (locusB6), and in B6/129 CD22-/- mice this region was homozygous for 129 germline DNA (locus129). In locusB6 CD22-/- B cells, a novel single-stranded RNA (ssRNA)-directed endonuclease was specifically overexpressed compared to locus129 CD22-/- B cells, suggesting a direct role in BCR-induced cell death. Targeted disruption of the endonuclease gene in locusB6 CD22-/- mice restored BCR-mediated B cell survival and proliferation to normal levels. These results identify a novel ssRNA-directed endonuclease as a potent inhibitor of B cell survival when expressed following antigen receptor stimulation. This research was supported by NIH grant R01CA096547.

Critical role of CIN85 in Cbl-mediated regulation of B cell function after antigen receptor stimulation (34.11)

Abstract Upon B-cell receptor (BCR) ligation by autoantigens, B cells normally undergo deletion (apoptosis), inactivation (anergy), or receptor editing. These processes are finely tuned by positive and negative signals downstream of B cell receptor (BCR). Aberrant regulation of BCR signals may thus lead to activation and expansion of autoreactive B cells. B cell adaptors function as molecular conduits to effectively link upstream signals to specific downstream branches. Cbl is an E3 ubiquitin ligase that is highly expressed in self-tolerant lymphocytes. Notably, B cell-specific ablation of Cbl in mice manifests SLE-like autoimmune disease, suggesting a pivotal role of Cbl in BCR-mediated tolerance induction. A novel Cbl-interacting adaptor termed CIN85 (c-Cbl interacting protein of 85 kDa) was recently identified, however little is known about a role of CIN85 in B cell function. In this study we demonstrate that upon BCR stimulation, CIN85 strongly associated with Cbl as well as BLNK. This association required the SH3 domain of CIN85. The experiments using CIN85-overexpressing and CIN85 knockdown B cells showed that CIN85 is critical for Cbl phosphorylation, which in turn regulates BCR-induced calcium flux and activation of Syk and PLCγ2. CIN85 knockdown in human primary B cells resulted in their enhanced survival and proliferation by upregulating expression of bcl-2 family and cyclin genes following BCR stimulation. Together, these findings suggest a critical role of CIN85 in Cbl-mediated regulation of B cell function after BCR stimulation.

PTPN22 deficiency cooperates with the CD45 E613R wedge allele to break tolerance on a non-autoimmune background (49.18)

Abstract Pep and CD45 are tyrosine phosphatases whose targets include the Src-family kinases, critical mediators of antigen receptor signaling. A polymorphism in PTPN22, the gene that encodes the human Pep ortholog Lyp, confers susceptibility to multiple human autoimmune diseases. However, the significance of the R620W risk allele is not clear. We report that misexpression of wild type or R620W Lyp/Pep in Jurkat cells, in the context of their binding partner Csk, unmasks the risk allele as a hypomorph. Although Pep deficient mice on the B6 background have hyper-responsive memory T cells, they fail to develop autoimmune disease. Mice containing a point mutation in CD45 (E613R) develop a B cell-driven lupus-like disease on the mixed 129/B6, but not on the B6 background. To model in mice how human susceptibility loci such as PTPN22 cooperate to provoke autoimmune disease, we studied the ability of Pep deficiency to act as a genetic modifier of the CD45 E613R mutation on the non-autoimmune B6 background. Double mutant mice develop a lupus-like disease as well as a lymphoproliferative syndrome. Following antigen receptor stimulation, peripheral B cells in the double mutant mice phenocopy hyper-responsive CD45 E613R B cells, whereas peripheral T cells respond like Pep-/- T cells. These studies suggest that Pep-/- T cells in the context of a susceptible microenvironment can drive hyper-responsive CD45 E613R B cells to break tolerance.

Orchestration of B cell function by the ERM protein ezrin (34.2)

Abstract Appropriate B cell responses hinge on finely balanced signal transduction pathways as well as regulated membrane remodeling. Membrane remodeling requires its uncoupling from the underlying cortical cytoskeleton but its regulation and impact on B cell function is poorly understood. We have identified ezrin, a member of the ezrin-radixin-moesin (ERM) family, as a regulator of membrane-cytoskeletal uncoupling in response to both B cell antigen receptor (BCR) and chemokine stimulation. Both stimuli induce dephosphorylation of ezrin on amino acid T567 in the actin-binding domain of ezrin, resulting in conformational inactivation of ezrin. Expression of a mutant of ezrin (T567D) that constitutively links the membrane to the actin cytoskeleton interferes with BCR-induced lipid raft coalescence, and SDF-1-dependent B cell migration. Our data support a critical role for T567 dephosphorylation in the early membrane remodeling events associated with antigen and chemokine signaling. Mice with a conditional deletion of ezrin in the B cell lineage exhibit defects in peripheral B cell homeostasis. Ezrin-deficient B cells from these mice show impaired in vitro migration and in vivo homing. Our data suggest that ezrin has the potential to shape activation and migration associated with B cell homeostasis and immune response. Supported by grants from NIH (DK068292), Cancer Research Institute, Department of Defense.

C-Abl-deficient mice exhibit reduced numbers of peritoneal B-1 cells and defects in BCR-induced B cell activation

A role for c-Abl in B cell development and signaling has been suggested by previous work showing that c-Abl-deficient mice have defects in bone marrow B cell development and that c-Abl-deficient B cells are hypoproliferative in response to antigen receptor stimulation. Here we show that in addition to defects in early B cell development, c-Abl-deficient mice have defects in peripheral B cell development, including reduced percentages of peritoneal B-1 cells as well as transitional and marginal zone B cells in the spleen. It has been shown that c-Abl kinase activity increases upon B cell receptor (BCR) stimulation and that one of the targets of tyrosine phosphorylation by c-Abl is CD19. However, the consequences of c-Abl activity on B cell activation and CD19 signaling remain unknown. Here, we show that c-Abl-deficient splenic B cells exhibit reduced calcium flux in response to CD19 cross-linking, consistent with a role for c-Abl in CD19-dependent signaling. Additionally, we show that c-Abl-deficient B cells are defective in their ability to be activated in response to antigen receptor engagement, suggesting a functional role for c-Abl in BCR-dependent activation signaling pathways.

Casein kinase 1α governs antigen-receptor-induced NF-κB activation and human lymphoma cell survival

The transcription factor NF-kappaB is required for lymphocyte activation and proliferation as well as the survival of certain lymphoma types. Antigen receptor stimulation assembles an NF-kappaB activating platform containing the scaffold protein CARMA1 (also called CARD11), the adaptor BCL10 and the paracaspase MALT1 (the CBM complex), linked to the inhibitor of NF-kappaB kinase complex, but signal transduction is not fully understood. We conducted parallel screens involving a mass spectrometry analysis of CARMA1 binding partners and an RNA interference screen for growth inhibition of the CBM-dependent 'activated B-cell-like' (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Here we report that both screens identified casein kinase 1alpha (CK1alpha) as a bifunctional regulator of NF-kappaB. CK1alpha dynamically associates with the CBM complex on T-cell-receptor (TCR) engagement to participate in cytokine production and lymphocyte proliferation. However, CK1alpha kinase activity has a contrasting role by subsequently promoting the phosphorylation and inactivation of CARMA1. CK1alpha has thus a dual 'gating' function which first promotes and then terminates receptor-induced NF-kappaB. ABC DLBCL cells required CK1alpha for constitutive NF-kappaB activity, indicating that CK1alpha functions as a conditionally essential malignancy gene-a member of a new class of potential cancer therapeutic targets.

Induction of autophagy by B cell antigen receptor stimulation and its inhibition by costimulation

Autophagy is a major pathway for degradation of cytoplasmic components, and is induced by some apoptotic stimuli mostly in cancer cells under the condition in which apoptosis is blocked. Ligation of the B cell antigen receptor (BCR) induces apoptosis and plays a crucial role in self-tolerance. However, whether BCR ligation induces autophagy is not clear. Here, we demonstrate that autophagosomes are extensively formed in normal mouse B cells as well as the WEHI-231 B cell line upon induction of BCR ligation-induced apoptosis regardless of whether apoptosis is blocked by overexpression of Bcl-2. In contrast, autophagosomes were not formed during apoptosis of spleen B cells cultured with medium alone or in BCR-ligated BAL17 cells which do not undergo apoptosis. Moreover, autophagy is not induced when apoptotic BCR signaling is abrogated by CD40 signaling. These results indicate that autophagy is induced specifically by apoptotic BCR signaling even in unmanipulated normal B cells.