Galectin-1 induced activation of the mitochondrial apoptotic pathway: evidence for a connection between death-receptor and mitochondrial pathways in human Jurkat T lymphocytes

Galectin-1 (gal-1) triggers T cell death by several distinct intracellular pathways including the activation of the death-receptor pathway. The aim of this study was to investigate whether gal-1 induced activation of the death-receptor pathway in Jurkat T lymphocytes mediates apoptosis via the mitochondrial pathway linked by truncated Bid (tBid). We demonstrate that gal-1 induced proteolytic cleavage of the death agonist Bid, a member of the Bcl-2/Bcl-xL family and a substrate of activated caspase-8, was inhibited by caspase-8 inhibitor II (Z-IETD-FMK). Downstream of Bid, gal-1 stimulated mitochondrial cytochrome c release as well as the activation and proteolytic processing of initiator procaspase-9 were effectively decreased by caspase-8 inhibitor II. Blocking of gal-1 induced cleavage of effector procaspase-3 by caspase-8 inhibitor II as well as by caspase-9 inhibitors I (Z-LEHD-FMK) and III (Ac-LEHD-CMK) indicates that receptor and mitochondrial pathways converged in procaspase-3 activation and contribute to proteolytic processing of effector procaspase-6 and -7. Western blot analyses and immunofluorescence staining revealed that exposure of Jurkat T cells to gal-1 resulted in the cleavage of the DNA-repair enzyme poly (ADP-ribose) polymerase, cytoskeletal α-fodrin, and nuclear lamin A as substrates of activated caspases. Our data demonstrate that Bid provides a connection between the death receptor and the mitochondrial pathway of gal-1 induced apoptosis in human Jurkat T lymphocytes.     

The role of apoptosis in the development and function of T lymphocytes

Apoptosis plays an essential role in T cell biology. Thymocytes expressing nonfunctional or autoreactive TCRs are eliminated by apoptosis during development. Apoptosis also leads to the deletion of expanded effector T cells during immune responses. The dysregulation of apoptosis in the immune system results in autoimmunity, tumorogenesis and immunodeficiency. Two major pathways lead to apoptosis： the intrinsic cell death pathway controlled by Bcl-2 family members and the extrinsic cell death pathway controlled by death receptor signaling. These two pathways work together to regulate T lymphocyte development and function.     

Caspase-independent cell death by Fas ligation in human thymus-derived T cell line, HPB-ALL cells

In HPB-ALL cells, a human thymus-derived T-cell line, Fas (CD95)-mediated cell death was inhibited by about only 50% as a result of treatment with an amount of benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)-CH(2)F (zVAD-fmk) sufficient to block the caspase activity. Fas-mediated caspase-independent cell death was not observed in other lymphoblast cell lines or mouse activated splenocytes, but this type of cell death was observed in mouse and rat thymocytes, the same as for HPB-ALL cells. This suggests that Fas-mediated caspase-independent cell death is a common feature in thymocytes. The signaling pathway of caspase-independent cell death has not yet been fully elucidated. In HPB-ALL cells, DNA fragmentation, one of the features of apoptotic cells, did not occur in the caspase-independent cell death after Fas ligation. On the other hand, this type of cell death and the surface exposure of phosphatidylserine were recovered by pretreatment with geldanamycin, which brought about a decrease in receptor interacting protein (RIP) kinase expression. These results suggested that HPB-ALL cells have a caspase-independent RIP kinasedependent pathway for Fas ligation.     

Mechanism of apoptosis induced by zinc deficiency in peripheral blood T lymphocytes

Alterations in intracellular Zn²⁺ concentrations are believed to play a crucial role in modulating apoptosis. The observation that Zn²⁺ deficiency can induce cell death both in vivo and in vitro has been attributed to the fact that exchange of Zn²⁺ for Ca²⁺ and Mg²⁺ within the nuclei may directly activate endogenous endonucleases therefore inducing DNA fragmentation independent of cytoplasmic factors. Here we show that the membrane-permeable zinc chelator, N,N,N-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces translocation of cytochrome c from the mitochondrial intramembranous space into the cytosol in human peripheral blood T lymphocytes (PBL) with subsequent activation of caspases-3, -8, and -9. Pretreatment of T lymphocytes with caspase inhibitors Z-VAD.fmk or DEVD.fmk prevented DNA fragmentation in response to TPEN indicating that apoptosis triggered by zinc deficiency is entirely dependent on activation of caspase family members. The release of cytochrome c and activation of downstream caspases precedes changes in the mitochondrial transmembrane potential ( m). Therefore, cytoplasmic and mitochondrial events are critical to this process.     

Self-nonself discrimination by T lymphocytes

Tolerance of T lymphocytes to self-antigens is mainly achieved at the level of the primary lymphoid organ, the thymus, and probably to a lesser extent in the secondary lymphoid tissues. Whether self-reactive lymphocytes ignore their target autoantigen, or are tolerized by the various mechanisms discussed, depends on the circumstances.     

Galectin-1 induced activation of the apoptotic death-receptor pathway in human Jurkat T lymphocytes

Galectin-1 (gal-1), a member of the family of β-galactoside binding proteins, participates in several biological processes such as immunomodulation, cell adhesion, regulation of cell growth and apoptosis. The aim of this study was to investigate whether gal-1 interferes with the Fas (Apo-1/CD95)-associated apoptosis cascade in the T-cell lines Jurkat and MOLT-4. Gal-1 and an Apo-1 monoclonal antibody (mAb) induced DNA-fragmentation in Jurkat T-cells whereas MOLT-4 cells were resistant. Gal-1 stimulated DNA-fragmentation could be efficiently inhibited by caspase-8 inhibitor II (Z-IETD-FMK) and a neutralizing Fas mAb. Fas could be identified as a target for gal-1 recognition as demonstrated by immunofluorescence staining, binding of the receptor glycoprotein to immobilized gal-1 and analyses by immunoblotting as well as by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gal-1 stimulates the activation and proteolytic processing of procaspase-8 and downstream procaspase-3 in Jurkat-T cells. Inhibition of gal-1 induced procaspase-8 activation by a neutralizing Fas mAb strongly suggests that gal-1 recognition of Fas is associated with caspase-8 activation. Our data provide the first experimental evidence for targeting of gal-1 to glycotopes on Fas and the subsequent activation of the apoptotic death-receptor pathway.     

Annexin V expression in apoptotic peripheral blood lymphocytes: An electron microscopic evaluation

Loss of plasma membrane asymmetry, resulting in the exposure of phosphatidylserine (PS), is considered to be an early event in apoptosis. It is generally accepted to precede nuclear condensation, independent of the apoptosis inductive agent.In the present study we focus on 2 apoptotic parameters: PS exposure in comparison with morphological alterations. Peripheral blood lymphocytes were irradiated in vitro (5 Gy Co--rays) or incubated with staurosporine (1 M, 6 hours). PS exposure was measured flow cytometrically using FITC-labelled annexin V, combined with PI. Morphological alterations were evaluated by electron microscopy (EM). Results are based on 3 independent experiments.For the irradiated lymphocytes the amount of viable cells (annexin V-/PI-) as scored by flow cytometry was comparable or slightly lower than the number of viable cells as scored by EM (75% compared to 79%). However, for the staurosporine treated lymphocytes only about 24% of the cells were designated as viable by EM, whereas by flow cytometry about 65% of the cells were annexin V-/PI-. Examination by EM showed about 40% cells with a morphology distinct from that of a normal viable cell, but without the clear-cut characteristics of apoptotic cells. Time studies revealed that these cells went into apoptosis after prolonged incubation times up to 18 hours.Application of biotinilated annexin V for EM detection with gold-conjugated anti-biotin, showed that only clear-cut apoptotic, apoptotic necrotic and oncotic cells showed the gold-label at their membranes. Cells that could be detected under the EM as non-viable but without the clear-cut characteristics of apoptotic cells, were not labelled. Data indicate that, dependent on the apoptosis inductive mechanism, morphological alterations can occur before PS exposure.     

Immortalization of human T lymphocytes by oncogenes

Immortalized human T cell lines were established by cotransfecting c-Ha-ras and c-myc oncogenes to lymph node lymphocytes. The cell lines kept growing for 3 months after establishment without a decrease in growth rate. The cells did not require interleukin-2(IL-2) for their growth, but addition of IL-2 stimulated the growth of these cells. Flow cytometric analysis revealed that these cells were T cells expressing CD4 or CD8 antigens. A CD4 positive (CD4⁺) cell line produced IL-6, indicating that the cell line belongs to helper T cells. The CD8 positive (CD8⁺) cell line possessed cytotoxicity to tumor cells, indicating that the cell line were killer T cells. Both cell lines were able to proliferate in serum-free medium indefinitely.     

Syphilitic infection impairs immunity by inducing both apoptosis and pyroptosis of CD4+ and CD8+ T lymphocytes

Syphilis is an important health problem worldwide; however, few studies have probed the impact of syphilitic infection on T cell turnover. The mechanisms behind the frequency of T cell subset changes and the associations between these subsets during syphilitic infection remain unclear. Herein, we used a cell-staining method and flow cytometry to explore changes in T cell subpopulations and potential contribution of apoptosis and pyroptosis that triggered therein. We investigated caspase-1-mediated pyroptosis and caspase-3-mediated apoptosis of CD4⁺ and CD8⁺ T cells, the major effector lymphocytes with pivotal roles in the pathogenesis of infectious diseases. We found that the levels of caspase-1 and caspase-3 increased in both the circulation and intracellularly in CD4⁺ and CD8⁺ T cells. Caspase-1 showed a continual increase from early latent stage infection through to phase 2 disease, whereas caspase-3 increased through to phase 1 disease but declined during phase 2. In addition, serum levels and intracellular expression of caspase-1 and caspase-3 were positively correlated. Overall, this study increases our understanding of how syphilitic infection influences CD4⁺ and CD8⁺ T-cell turnover, which may help with designing novel and effective strategies to control syphilis infection and prevent its transmission.     

Effects of N-glycan processing inhibitors on signaling events and induction of apoptosis in galectin-1-stimulated Jurkat T lymphocytes

To elucidate the role of N-linked glycans in triggering T-cell functions, the effects of the N-glycan processing inhibitors 1-deoxymannojirimycin (1-DMM) and swainsonine (SW) were investigated on signaling events and induction of apoptosis in galectin-1 (gal-1)-stimulated Jurkat T lymphocytes. The treatment of Jurkat E6.1 cells with 1-DMM and SW strongly reduced the cell binding of gal-1-biotin, conjugate binding to cell lysate glycoproteins, and to cluster of differentiation (CD) 3 immunoprecipitates on blots as well as the binding of CD2 and CD3 to immobilized gal-1. The mannosidase inhibitors efficiently decreased gal-1-induced calcium mobilization. Both phases originated from a transient Ca(2+) release of internal stores, and the sustained influx across the plasma membrane was found to be involved. Both inhibitors suppressed in transiently transfected Jurkat T lymphocytes the gal-1-induced expression of the luciferase (luc) reporter gene constructs pNFAT-TA-Luc and pAP1(phorbol-12-myristate-13-acetate [PMA])-TA-Luc. The data provide evidence that gal-1 triggers through binding to N-linked glycans a Ca(2+)-sensitive apoptotic pathway.     

紫杉醇诱导凋亡的信号传导通路及与凋亡相关的基因和蛋白

紫杉醇对临床多种恶性肿瘤都有疗效,并具广阔的市场应用前景,其抗癌机制是致使肿瘤细胞发生细胞周期阻滞而凋亡.作者结合课题组多年来的研究,对紫杉醇诱导凋亡过程中的信号通路、相关基因及蛋白进行介绍,包括微生物发酵生产紫杉醇的基因、细胞周期蛋白、端粒酶等.     

牙龈卟啉单胞菌诱导T淋巴细胞活化、凋亡的体外研究

目的检测牙龈卟啉单胞菌对人外周血中T淋巴细胞活化及凋亡的作用,并检测Fas/FasL在牙龈卟啉单胞菌(Porphyrom onas gingivalis,Pg)诱导的T淋巴细胞凋亡中的表达。方法选取10例全身及牙周组织健康受试者,分离外周血中T淋巴细胞,在有/无Pg情况下培养0~96 h,用荧光探针(Annexin V-FITC、PI、CD69)及特殊的单克隆抗体(Fas、FasL)进行标记,并进行流式细胞仪检测。结果 CD69+淋巴细胞+Pg组Annexin V+/PI-细胞百分数在各个时间点上都明显高于T淋巴细胞+Pg组(P0.01)。Fas和FasL的表达量明显上调。用抗Fas单克隆抗体阻滞Fas-FasL相互作用导致T细胞凋亡的明显减少,百分比为(20.56±2.43)%,未加抗体的为(50.41±2.68)%。但残余的细胞凋亡活动与阴性对照相比仍高。结论 Pg能够诱导人外周血中T淋巴细胞活化,并且能够通过活化促进其凋亡,Pg诱导T淋巴细胞凋亡主要通过Fas-FasL途径,并具有时间依赖性。     

Cytolytic T lymphocytes: an overview of their characteristics

Cloned T cells have been useful for assessing the lytic potential of distinct T cell subsets and for determining the relative contribution of different effector mechanism involved in the lytic process. Alloreactive CD8⁺ murine T cell clones and cloned murine CD4⁺ T_H1 and T_H2 T cells reactive with nominal antigen (ovalbumin) lysed nucleated target cells bearing antigen or coated with anti-CD3 monoclonal antibody in a short term⁵¹Cr-release assay. These clones were also evaluated for their ability to lyse efficiently sheep erythrocyte (SRBC) target cells coated with anti-CD3 mAb by a mechanism (presumably involving membrane damage) that does not involve nuclear degradation. Three patterns of lysis were observed: CD8⁺ and some CD4⁺ T_H2 effector cells lysed efficiently nucleated target cells and anucleated SRBC coated with anti-CD3 mAb. However, CD4⁺ T_H1 (and a few T_H2) T cells which lysed nucleated target cells bearing antigen or coated with anti-CD3 mAb didnotlyse efficiently the SRBC coated with anti-CD3 mAb. One CD4 bearing T_H2 cell failed to lyse efficiently either nucleated target cells or anucleated SRBC coated with anti-CD3 mAb. These results indicate that both T_H1 and T_H2 clones have lytic capabilities. Furthermore, they suggest that some but not all T_H2 murine T cell clones have lytic characteristics similar to those of conventional CD8⁺ CTL. However, it is not certain how these patterns of lysis of target cellsin vitro relates to the capacity of CTL to lyse such target cellsin vivo.     

Newly synthesized AIFM1 determines the hypersensitivity of T lymphocytes to STING activation-induced cell apoptosis

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Recycled addition of CD4+ T cell-rich population for induction of human autologous cytotoxic T lymphocytes: A practically efficient method

When CD4⁺ T cell-rich population appears in theinitial trial in induction cultures of humanautologous cytotoxic T lymphocytes (CTL), the cultureresults frequently in no or weak killing activity andtherefore usually be discarded as an `unsuccessful'CTL induction culture. However, addition of theinitial CD4⁺ T cell-rich population enabledefficient induction of the autologous CTL in theensuing trials. The CTL thus generated exhibitedstronger killing activities against autologous braintumor cells and ovarian tumor cells than previouslyobserved. This simple recycling of the primed butinert CD4⁺ T cell-rich population for CTLinduction will promote clinical practice of adoptiveimmunotherapy of human tumors with autologous CTL.     

Bispecific-monoclonal-antibody-directed lysis of ovarian carcinoma cells by activated human T lymphocytes

Summary Two different bispecific hybrid antibodies were established by fusing a hybridoma producing monoclonal antibody (mAb) against the pancarcinoma antigen KS1/4 with either of the two hybridomas OKT3 and 9.3, secreting antibodies reactive with the T cell determinants CD3 and CD28, respectively. The KS1/4 antibody reacts with a 40-kDa cell-surface glycoprotein antigen that is expressed on the surface of a variety of adenocarcinoma cells, including ovarian carcinoma. The ability of the bispecific antibodies 9.3KS1/4 and OKT3KS1/4 to direct peripheral blood mononuclear cells (PBMC) specifically against OVCAR-3 ovarian carcinoma target cells was measured in a 4-h⁵¹Cr-release assay. The bispecific antibodies were four to six times more potent in killing the OVCAR-3 target cells when compared to their parental antibodies either alone or in combination. A dose-dependent response was observed in the 10–10000 ng/ml range. The specificity of the targeting was demonstrated by the complete inhibition of cytotoxic activity following pre-incubation of tumor target cells with the parental mAb and by the lack of killing of KS1/4-negative target cell lines. An evaluation of the efficacy of PBMC from ovarian cancer patients as effector cells revealed that their specific cytotoxicity against OVCAR-3 cells was enhanced severalfold by bispecific antibodies as compared to parental antibodies. Furthermore, stimulation of PBMC with immobilized CD3 and interleukin-2 for 4 days resulted in an enhanced directed killing of human ovarian carcinoma cells by human T effector cells and the bispecific antibodies.