Characterization of peptide aptamers targeting Bfl-1 anti-apoptotic protein.

Bfl-1, an anti-apoptotic protein of the Bcl-2 family, has been identified as a potential therapeutic target for B-cell malignancies. We describe herein the first characterization of peptide aptamers selected against Bfl-1. We show that most of the Bfl-1 peptide aptamers do not interact with Bcl-2, Bcl-xL, or Mcl-1 in yeast and that some of them restore the pro-apoptotic activity of Bax in yeast in which Bax and Bfl-1 proteins are coexpressed. When expressed in mammalian cells, peptide aptamers interact with Bfl-1 and sensitize B-cell lines to apoptosis induced by chemotherapeutic agents. We further demonstrate that a nonconstrained peptide derived from one aptamer variable region reverses Bfl-1 anti-apoptotic activity in HeLa cells through disruption of Bax-Bfl-1 interaction. This peptide also promotes cell death in lymphoma B-cell lines expressing a high level of Bfl-1 and sensitizes these cells to drug-induced apoptosis. Taken together, these results further validate Bfl-1 as a therapeutic target for malignant B-cells and suggest that peptide aptamers may be a useful tool for guiding the identification of small compounds that target the anti-apoptotic Bfl-1 protein.

Overexpression of the antiapoptotic gene Bfl-1 in B cells from patients with familial systemic lupus erythematosus.

Genetic determinants taking part in the development of systemic lupus erythematosus (SLE) are complex and not fully characterized. Dysregulated expression of genes involved in the control of apoptosis has been previously suggested. We report here a consanguineous family with SLE manifestations in three siblings associated in one of them with severe lymphoproliferative features. Laboratory studies showed no defect in CD95-mediated cell death. Screening expression of Bcl-2 family genes that regulate mitochondrial apoptosis pathway showed an overexpression of the antiapoptotic Bfl-1 gene. Real time RT-PCR analysis indicated that overexpression of Bfl-1 was restricted to B-cells, with normal expression in T-cells. Those results suggest that overexpression of Bfl-1 could result in impaired B-lymphocyte homeostasis and inappropriate immune response leading to autoimmune manifestations.

Downregulation of Bfl-1 protein expression sensitizes malignant B cells to apoptosis.

Elevated expression of the antiapoptotic protein Bfl-1 (A1) was previously reported in several cancer cell lines. Recently, molecular profiling of large B-cell lymphoma identified Bfl-1 as a gene signature in 'OxPhos' diffuse large B-cell lymphoma subtype and in primary mediastinal large B-cell lymphoma, suggesting that in addition to Bcl-2, Bcl-xL and Mcl-1, Bfl-1 may be a relevant target in the design of new strategies for cancer therapy. Using short hairpin RNA strategy, we show here that Bfl-1 silencing in one lymphoblastoid B-cell line and in two diffuse large B-cell lymphoma cell lines potently induces their apoptosis and sensitizes those cell lines to anti-CD20 (Rituximab)-mediated cell death as well as to apoptosis induced by chemotherapeutic molecules such as doxorubicin, vincristine, cisplatin and fludarabine. These results demonstrate for the first time that Bfl-1 is an essential protein for survival of malignant B cells and suggest Bfl-1 may represent a potential target for future drug development against B cell lymphoma.

Link between immune cell infiltration and mitochondria-induced cardiomyocyte death during acute cardiac graft rejection.

Acute cardiac graft rejection (ACGR) is associated with cardiomyocyte apoptosis. We investigated the respective role of the Fas/FasL and mitochondrial permeability transition pore (mPTP) pathways in cardiomyocyte apoptosis accompanying ACGR. Heterotopic cardiac transplantations were performed in 7-9-week old C57BL6 or C3H mice. Wild type or Fas-deficient (lpr) mice underwent syngeneic (GS) or allogeneic (GA) transplantation, and received either saline or NIM811, a specific inhibitor of the mPTP. At day 5, we assessed ACGR by histology, cardiomyocyte apoptosis by caspase-3 activity and cytochrome c release, Ca(2+)-induced mPTP opening by a potentiometric approach, and expression of Fas, FasL, TNFalpha, perforin, granzyme using RT-PCR. Myocardial infiltration of CD8(+) T lymphocytes was performed by immunohistochemistry. Allogenic transplantation increased infiltration of inflammatory cells, upregulated FasL, perforin, granzyme, and TNFalpha, favored Ca(2+)-induced mPTP opening and increased caspase-3 activity and cytochrome c release in WT grafts. NIM811, but not Fas-deficiency, significantly reduced all these effects. NIM811 also limited infiltration of CD8(+) into WT and lpr transplants. These data suggest that the mPTP pathway plays a major role in cardiomyocyte apoptosis associated with ACGR. Inhibition of mPTP opening may attenuate cardiomyocyte apoptosis either directly or indirectly via a limitation of CD8(+) T-cell activation.

Overexpression of the antiapoptotic protein A1 promotes the survival of double positive thymocytes awaiting positive selection.

As it has been shown for Mcl-1, Bcl-xl and Bcl-2, proteins of the Bcl-2 family play a crucial role during T-cell development in the thymus. We here show that the expression of the antiapoptotic gene A1 is specifically enhanced at the DN3/DN4 transition and in DP thymocytes that have been positively selected suggesting that A1 expression might be considered as a transcriptional signature of thymocytes that have received pre-TCR or TCR survival signal. Furthermore, we observed that A1-a overexpression in recombination activation gene 1-deficient mice transgenic for the major histocompatibillity complex class I-restricted F5 TCR enhances cell survival of DP thymocytes and permits accumulation of DP cells awaiting positive selection. However, A1-a overexpression has no effect on negative selection. Therefore, our results suggest that A1 plays a specialized role in allowing survival of DP thymocytes and that its role can be distinguished from that of Mcl-1, Bcl-xl and Bcl-2.

A1/Bfl-1 expression is restricted to TCR engagement in T lymphocytes.

We analyzed regulation of the prosurvival Bcl-2 homologue A1, following T-cell receptor (TCR) or cytokine receptor engagement. Activation of CD4(+) or CD8(+) T cells by antigenic peptides induced an early but transient IL-2-independent expression of A1 and Bcl-xl mRNA and proteins, whereas expression of Bcl-2 was delayed and required IL-2. Cytokines such as IL-2, IL-4, IL-7 or IL-15 prevented apoptosis of activated T cells that effect being associated with the maintenance of Bcl-2, but not of A1 expression. However, restimulation of activated or posteffector T cells with antigenic peptide strongly upregulated A1 mRNA and maintained A1 protein expression. IL-4, IL-7 or IL-15 also prevented cell death of naive T cells. In those cells, cytokines upregulated Bcl-2, but not A1 expression. Therefore, in naive, activated and posteffector T cells, expression of A1 is dependent on TCR but not on cytokine receptor engagement, indicating that A1 is differently regulated from Bcl-xl and Bcl-2.

Anthracyclines trigger apoptosis of both G0-G1 and cycling peripheral blood lymphocytes and induce massive deletion of mature T and B cells.

The anthracyclines daunorubicin and doxorubicin were shown to induce apoptosis of hematopoietic cell lines. Here we report that they induce apoptosis of both nonactivated and phytohemagglutinin-activated human peripheral blood lymphocytes. Apoptosis demonstrated by surface expression of phosphatidylserine and typical nuclear alterations reached a maximum after 48 h of incubation with these agents. In contrast to topoisomerase inhibitors (etoposide and camptothecin) and antimetabolites (methotrexate and 5-fluorouracil) that induced apoptosis of activated cells only, daunorubicin and doxorubicin triggered apoptosis of cells in the G0-G1 phases of the cell cycle. In agreement with in vitro data, a single i.p. injection of daunorubicin or doxorubicin in BALB/c mice induced T- and B-cell depletion in spleen, lymph nodes, and to a lesser extent in the thymus. Soluble Fas-Fc, CD95 antagonistic antibodies, as well as the p55 tumor necrosis factor receptor-immunoglobulin fusion protein, did not inhibit drug-induced apoptosis. The level of reactive oxygen species was significantly increased in the presence of daunorubicin or doxorubicin only in nonactivated lymphocytes. However, antioxidants such as N-acetyl-L-cysteine or glutathione did not prevent apoptosis. Activation of caspase-3 after daunorubicin or doxorubicin treatment of either nonactivated or activated lymphocytes was demonstrated by the cleavage of poly(ADP-ribose) polymerase, which was, as apoptosis, inhibited by the peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Finally, daunorubicin and doxorubicin induced a rapid production of ceramides. These data indicate that anthracyclines may induce major peripheral T-cell deletion, a property not shared by many cytotoxic agents.

The topoisomerase inhibitors camptothecin and etoposide induce a CD95-independent apoptosis of activated peripheral lymphocytes.

The effect of etoposide and camptothecin, two topoisomerase inhibitors directed against topoisomerases II and I, respectively, was evaluated on human peripheral blood lymphocytes. Etoposide and camptothecin induced apoptosis of mitogen-activated but not resting CD4+ and CD8+ T lymphocytes. Cell sensitivity to these agents required G1 to S-phase transition of the cell cycle. Conversely, daunorubicin, an intercalating agent and topoisomerase II inhibitor, induced apoptosis of both resting and activated lymphocytes. Although etoposide and camptothecin induced CD95-ligand mRNA expression, drug-induced apoptosis of activated human lymphocytes was not inhibited by CD95 antagonists. Drug-induced cell death was also not inhibited by p55 TNFR-Ig fusion protein. Activation of the caspases cascade was suggested by the partial inhibitory effect of the tripeptide zVAD-fmk and documented by activation of caspase 3. Finally etoposide and camptothecin induced a rapid production of ceramide in activated but not resting peripheral blood lymphocytes, suggesting that ceramide might initiate the signaling apoptotic cascade in sensitive cells.

A noncomitogenic CD2R monoclonal antibody induces apoptosis of activated T cells by a CD95/CD95-L-dependent pathway.

Clonal expansion of activated T and B cells is controlled by homeostatic mechanisms resulting in apoptosis of a large proportion of activated cells, mostly through interaction between CD95 (Fas or Apo-1) receptor and its ligand CD95-L. CD2, which is considered as a CD3/TCR alternative pathway of T cell activation, may trigger activation-induced cell death, but the role of CD95/CD95-L interaction in CD2-mediated apoptosis remains controversial. We show here that the CD2R mAb YTH 655.5, which does not induce comitogenic signals when associated with another CD2 mAb, triggers CD95-L expression by preactivated but not resting T cells, resulting in CD95/CD95-L-mediated apoptosis. The critical role of CD95/CD95-L interaction was supported by complete inhibition in the presence of the antagonist CD95 mAb ZB4 and by blocking CD95-L synthesis and surface expression by cycloheximide, cyclosporin A, EGTA, or cytochalasin B. YTH 655.5 was shown to stimulate p56lck phosphorylation and enzymatic activity. However, p56lck activation is not sufficient to trigger apoptosis, because other CD2R and CD4 mAbs that activate p56lck do not induce apoptosis. In conclusion, CD2 can mediate nonmitogenic signals, resulting in CD95-L expression and apoptosis of CD95+ cells.

Induction of Fas (Apo-1, CD95)-mediated apoptosis of activated lymphocytes by polyclonal antithymocyte globulins.

Polyclonal horse antilymphocyte and rabbit antithymocyte globulins (ATGs) are currently used in severe aplastic anemia and for the treatment of organ allograft acute rejection and graft-versus-host disease. ATG treatment induces a major depletion of peripheral blood lymphocytes, which contributes to its overall immunosuppressive effects. Several mechanisms that may account for lymphocyte lysis were investigated in vitro. At high concentrations (.1 to 1 mg/mL) ATGs activate the human classic complement pathway and induce lysis of both resting and phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells. At low, submitogenic, concentration ATGs induce antibody-dependent cell cytotoxicity of PHA-activated cells, but not resting cells. They also trigger surface Fas (Apo-1, CD95) expression in naive T cells and Fas-ligand gene and protein expression in both naive and primed T cells, resulting in Fas/Fas-L interaction-mediated cell death. ATG-induced apoptosis and Fas-L expression were not observed with an ATG preparation lacking CD2 and CD3 antibodies. Susceptibility to ATG-induced apoptosis was restricted to activated cells, dependent on IL-2, and prevented by Cyclosporin A, FK506, and rapamycin. The data suggest that low doses of ATGs could be clinically evaluated in treatments aiming at the selective deletion of in vivo activated T cells in order to avoid massive lymphocyte depletion and subsequent immunodeficiency.

Caspase-dependent ceramide production in Fas- and HLA class I-mediated peripheral T cell apoptosis.

We recently demonstrated that the engagement of HLA class I alpha1 domain induced Fas-independent apoptosis in human T and B lymphocytes. We analyzed the signaling pathway involved in HLA class I-mediated apoptosis in comparison with Fas (APO-1, CD95)-dependent apoptosis. The mouse mAb90 or the rat YTH862 monoclonal antibodies which bind the human HLA class I alpha1 domain induced the production of ceramide which was blocked by addition of the phosphatidylcholine-dependent phospholipase C inhibitor, D609. Furthermore, HLA class I-mediated apoptosis involved at least two different caspases, an interleukin-1 converting enzyme-like protease and another protease inhibited by the CPP32-like protease inhibitor Ac-DEVD-CHO. Despite similarity between Fas and HLA class I signaling pathways, we failed to demonstrate any physical association between these two molecules. We also report that the pan-caspase inhibitory peptide zVAD-fmk, but not Ac-DEVD-CHO and Ac-YVAD-CHO, inhibited decrease of mitochondrial transmembrane potential and generation of ceramide induced by anti-HLA class I and anti-Fas monoclonal antibodies, whereas all three peptides efficiently inhibited apoptosis. Altogether these results suggest that signaling through Fas and HLA class I involve caspase(s), targeted by zVAD-fmk, which act upstream of ceramide generation and mitochondrial events, whereas interleukin-1 converting enzyme-like and CPP32-like proteases act downstream of the mitochondria.

Fas-independent apoptosis of activated T cells induced by antibodies to the HLA class I alpha1 domain.

In addition to their major function in antigen presentation and natural killer cell activity regulation, HLA class I molecules may modulate T-cell activation and proliferation. Monoclonal antibodies (MoAbs) that recognize distinct epitopes of HLA class I molecules were reported to interfere with T-cell proliferation. We show here that two MoAbs (mouse MoAb90 and rat YTH862) that bind to an epitope of the alpha1 domain of HLA class I heavy chain induce apoptotic cell death of activated, but not resting, peripheral T lymphocytes. Other reference anti-HLA class I antibodies specific for distinct epitopes of the alpha1 (B9.12.1), alpha2 (W6/32), or alpha3 (TP25.99) domains of the heavy chain decreased T-cell proliferation but had little or no apoptotic effect. Apoptosis shown by DNA fragmentation, phosphatidylserine externalization, and decrease of mitochondrial transmembrane potential was observed whatever the type of T-cell activator. Apoptosis did not result from Fas/Fas-L interaction and distinct though partly overlapping populations of activated T cells were susceptible to Fas- and HLA class I-mediated apoptosis, respectively. Induction of apoptosis did not require HLA class I cross-linking inasmuch as it could be observed with monovalent Fab' fragments. The data indicate that MoAb90 and YTH862 directed against the alpha1 domain of HLA class I trigger apoptosis of activated T lymphocytes by a pathway which does not involve Fas-ligand.

T cell sensitivity to HLA class I-mediated apoptosis is dependent on interleukin-2 and interleukin-4.

Antibody interaction with a specific epitope of the HLA class I alpha1 domain triggers apoptosis of activated but not resting T and B cells by a pathway which involves neither Fas ligand nor tumor necrosis factor-alpha. We have investigated at which stage of activation and proliferation T cells become sensitive to HLA class I-mediated apoptosis, using two monoclonal antibodies (mAb) which recognize the same monomorphic epitope of the HLA class I alpha1 domain (mAb9O, mouse IgG1, and YTH862, rat IgG2b) and can induce apoptosis of phytohemagglutinin (PHA)-activated peripheral blood lymphocytes. Sensitivity to apoptosis develops after the expression of G1 markers (CD69 expression) but it is accelerated by addition of recombinant interleukin-2 (rIL-2). Blocking the IL-2 pathway by cyclosporin A, FK506, rapamycin, anti-IL-2 or CD25 antibodies, prevented the development of sensitivity to apoptosis. Addition of IL-2 and, to a lesser extent, IL-4, reversed the inhibitory effect of cyclosporin A. Conversely, rIL-7 and recombinant interferon-gamma restored proliferation of peripheral blood lymphocytes stimulated by PHA in the presence of cyclosporin A but did not restore sensitivity to class I-mediated apoptosis. Finally cells stimulated in the presence of the DNA polymerase inhibitor aphidicolin did not enter into S phase of the cell cycle but secreted IL-2 and underwent apoptosis when exposed to mAb90 or YTH862. Together, the data indicate that sensitivity of peripheral T cells to HLA class I-mediated apoptosis depends on both activation signals and IL-2 or IL-4, but does not require cell proliferation. These data suggest that YTH862 and mAb90 might be used for achieving clonal deletion of antigen-activated peripheral T cells in vivo, provided that the IL-2 pathway is not blocked by other immunosuppressive agents.

Direct interaction between protein kinase C theta (PKC theta) and 14-3-3 tau in T cells: 14-3-3 overexpression results in inhibition of PKC theta translocation and function.

Recent studies have documented direct interactions between 14-3-3 proteins and several oncogene and proto-oncogene products involved in signal transduction pathways. Studies on the effects of 14-3-3 proteins on protein kinase C (PKC) activity in vitro have reported conflicting results, and previous attempts to demonstrate a direct association between PKC and 14-3-3 were unsuccessful. Here, we examined potential physical and functional interactions between PKC theta, a Ca(2+)-independent PKC enzyme which is expressed selectively in T lymphocytes, and the 14-3-3 tau isoform in vitro and in intact T cells. PKC theta and 14-3-3 tau coimmunoprecipitated from Jurkat T cells, and recombinant 14-3-3 tau interacted directly with purified PKC theta in vitro. Transient overexpression of 14-3-3 tau suppressed stimulation of the interleukin 2 (IL-2) promoter mediated by cotransfected wild-type or constitutively active PKC theta, as well as by endogenous PKC in ionomycin- and/or phorbol ester-stimulated cells. This did not represent a general inhibition of activation events, since PKC-independent (but Ca(2+)-dependent) activation of an IL-4 promoter element was not inhibited by 14-3-3 tau under similar conditions. Overexpression of wild-type 14-3-3 tau also inhibited phorbol ester-induced PKC theta translocation from the cytosol to the membrane in Jurkat cells, while a membrane-targeted form of 14-3-3 tau caused increased localization of PKC theta in the particulate fraction in unstimulated cells. Membrane-targeted 14-3-3 tau was more effective than wild-type 14-3-3 tau in suppressing PKC theta-dependent IL-2 promoter activity, suggesting that 14-3-3 tau inhibits the function of PKC theta not only by preventing its translocation to the membrane but also by associating with it. The interaction between 14-3-3 and PKC theta may represent an important general mechanism for regulating PKC-dependent signals and, more specifically, PKC theta-mediated functions during T-cell activation.

Activation-modulated association of 14-3-3 proteins with Cbl in T cells.

14-3-3 proteins have recently been implicated in the regulation of intracellular signaling pathways via their interaction with several oncogene and protooncogene products. We found recently that 14-3-3 associates with several tyrosine-phosphorylated proteins and phosphatidylinositol 3-kinase (PI3-K) in T cells. We report here the identification of the 120-kDa 14-3-3tau-binding phosphoprotein present in activated T cell lysates as Cbl, a protooncogene product of unknown function which was found recently to be a major protein-tyrosine kinase (PTK) substrate, and to interact with several signaling molecules including PI3-K, in T lymphocytes. The association between 14-3-3tau and Cbl was detected both in vitro and in intact T cells and, in contrast to Raf-1, was markedly increased following T cell activation. The use of truncated 14-3-3tau fusion proteins demonstrated that the 15 C-terminal residues are required for the association between 14-3-3 and three of its target proteins, namely, Cbl, Raf-1, and PI3-K. The findings that 14-3-3tau binds both PI3-K and Cbl, together with recent reports of an association between Cbl and PI3-K, suggest that 14-3-3 dimers play a critical role in signal transduction processes by promoting and coordinating protein-protein interactions of signaling proteins.

Mechanisms of immunosuppression induced by antithymocyte globulins and OKT3.

OKT3 monoclonal antibody and polyclonal antithymocyte or antilymphocyte globulins are among the most potent immunosuppressive agents which have been used in organ transplantation for many years. Both induce a rapid and profound lymphocytopenia classically attributed to several mechanisms, such as complement-dependent cytolysis, cell-mediated antibody-dependent cytolysis, as well as opsonization and subsequent phagocytosis by macrophages. However, the relative contribution of these three Fc-dependent mechanisms in vivo is difficult to ascertain and may be less important than previously thought. In addition OKT3 induces T-cell receptor modulation in vivo and modulated T cells no longer interact with antigen-presenting cells. Modulation of the T-cell receptor complex has not been documented for antithymocyte and antilymphocyte globulins as yet. The monoclonal antibody OKT3, which is directed against the epsilon chain of the CD3 molecule on the T-cell surface, but also antithymocyte and antilymphocyte globulins, which contain antibodies directed against CD3 and other functional molecules on the surface of T and B cells, generate various transduction signals to the target cells which can affect their functions in different ways. Recent in vitro studies suggest that these antibodies interfere with activation signals. Indeed, antithymocyte and antilymphocyte globulins, at low concentrations, inhibit T-cell activation induced by alloantigens, whereas they induce polyclonal T-cell activation at higher concentrations. Mitogenic antibodies can trigger an activation-induced cell death phenomenon as documented with anti-CD3 antibodies. Anti-CD3 antibodies can also induce a state of specific unresponsiveness (clonal anergy) which may contribute to their long-lasting immunosuppressive effect. One may hypothesize from in vitro data that in spite of their nonspecific immunosuppressive effects which may result in severe iatrogenic immunodeficiency, antilymphocyte antibodies may also act on stimulated alloreactive T-cell clones and therefore contribute to donor-specific graft adaptation.