Reduced susceptibility to Fas-mediated apoptosis in B-1 cells

Elimination of activated T and B cells by Fas-dependent apoptosis may contribute to the maintenance of peripheral tolerance. CD40 ligation was recently shown to up-regulate Fas expression and enhance susceptibility to Fas-mediated apoptosis in mouse splenic B cells. In the present study, we have investigated the regulation of Fas expression and Fas-triggered apoptotis in mouse peritoneal B-1 cells. B-1 cells expressed a similar level of CD40 as that on B-2 cells, and proliferated in response to a soluble CD40 ligand (CD40L)-CD8α chimeric protein, suggesting that CD40 on B-1 cells is functional. In contrast to B-2 cells, B-1 cells expressed Fas at only low levels in response to CD40L-CD8α alone or CD40L-CD8α+interleukin-4, and were resistant to Fas-mediated apoptosis following these treatments. While Fas expression could be induced in B-1 cells to a comparable level as that in B-2 cells by cross-linking CD40L-CD8α with an anti-CD8α antibody, the sensitivity to Fas-mediated apoptosis in B-1 cells was significantly reduced compared with B2 cells. These results suggest that peritoneal B-1 cells from normal mice have a lower susceptibility to Fas-mediated apoptosis and may distinguish B-1 from B-2 cells. Similarly, B-1 cells from the peritoneal cavity and spleen of autoimmune-prone NZB mice exhibited reduced susceptibility to Fas-mediated apoptosis relative to their B-2 counterparts. NZB splenic B-1 cells, however, were more susceptible to Fas-mediated apoptosis than NZB peritoneal B-1 cells. The results presented here raise the possibility that the reduced susceptibility to Fas-triggered apoptosis in B-1 cells might be an accelerating factor for the autoantibody production in NZB mice.     

Studies of the mechanism of cytolysis by tumour-infiltrating lymphocytes.

In order to determine the mechanism of tumour destruction by tumour-infiltrating lymphocytes (TIL), we examined the ability of both CD4+ and CD8+ effector TIL, and TIL clones, to manifest granzyme-mediated and Fas-mediated destruction of tumour targets. In many in vitro studies TIL have been shown to manifest anti-tumour reactivity, yet many tumours escape immunological destruction. To investigate the role of Fas expression and the concomitant sensitivity to the inducibility of apoptotic death, we derived TIL from four melanomas and one glioma. The glioma, and all but one of the melanomas, expressed Fas, but Fas-mediated apoptosis could only be detected if the targets were treated with cyclohexamide. The melanomas and the glioma all expressed detectable cytoplasmic Bcl-2 protein, known to exert anti-apoptotic activity. Lysis of tumours by CD8-enriched cultures and CD8+ clones was Ca2+-dependent and could not be modified by an anti-Fas MoAb. In CD4-enriched cultures or CD4+ clones with cytotoxic potential against tumour cells, cytotoxicity was also Ca2+-dependent. As Ca2+-dependent cytotoxicity is usually the result of secretion of perforin/granzyme-B, we investigated the presence of perforin in cytotoxic CD4+ clones and demonstrated the presence of granular deposits of this enzyme in some of the CD4+ clones. Although an anti-Fas MoAb did not block the lysis of melanoma targets by CD4+ clones, the examination of Fas-dependent targets demonstrated that these clones also had the potential to kill by the Fas/Fas ligand system. These data suggest that the predominant mechanism in tumour killing by TIL appears to be perforin-granzyme-dependent, and that the solid tumour cell lines we studied are less susceptible to Fas-mediated apoptosis. As non-apoptotic pathways may enhance tumour immunogenicity, exploitation of the perforin-granzyme-dependent cytotoxic T lymphocyte (CTL) pathways may be important for achieving successful anti-tumour responses.     

抗人P185erbB2 scFv-Fc-IL-2增强LAK样和ADCC杀伤作用及机制的实验研究

目的 将抗人P185erbB2 scFv-Fc-IL-2融合蛋白(HFI)分别作用于表达高、中、低3个水平erbB2受体的SKOV3、MCF-7、SGC-7901三株肿瘤细胞和健康人外周血单个核细胞(PBMC),探讨HFI调变肿瘤细胞表面分子,激活免疫效应细胞的机制;模拟体内肿瘤组织,将HFI-PBMC-肿瘤细胞混合培养,探讨HFI对分别表达高、中、低3个水平erbB2肿瘤细胞的淋巴因子激活的杀伤细胞(LAK)样和抗体依赖性细胞介导的细胞毒(ADCC)作用,为临床应用提供实验依据.方法 MTF法检测细胞增殖、杀伤活性;流式细胞术检测细胞表面分子的表达变化;应用非核素细胞毒试剂盒观察HFI介导ADCC杀伤作用.结果 HFI处理后SKOV3细胞表面杀伤相关分子细胞间黏附分子-1(ICAM-1)、Fas表达水平分别由24.85%、0.53%增高到85.36%、59.19%;SKOV3、MCF-7、SGC-7901三株肿瘤细胞erbB2表达水平分别由98.48%、42.60%、36.66%下降到94.01%、30.95%、12.36%.HFI刺激后人PBMC的增殖活性显著增强,呈时间依赖效应,CD3+ CD8+ T细胞和CD3- CD16+ CD56+NK细胞分别由24.37%、6.90%提高到38.80%、13.45%,CD25、淋巴细胞功能抗原-1(LFA-1)、FasL表达水平分别由3.99%、86.52%、5.02%提高到12.96%、99.06%、16.19%.HFI活化的人PBMC对不同erbB2表达水平肿瘤细胞的LAK样杀伤活性,在各效靶比均比对照组明显提高.HFI能够介导和增强人PBMC对表达erbB2肿瘤细胞的ADCC杀伤作用.结论 HFI上调SKOV3细胞表面杀伤相关分子ICAM-1、Fas表达,下调肿瘤细胞表面erbB2表达.HFI对人PBMC具有明显的激活增殖作用,活化的人PBMC对表达不同水平erbB2肿瘤细胞的LAK样杀伤作用均显著增强.HFI能够介导和增强人PBMC对肿瘤细胞的ADCC杀伤作用,且杀伤活性的高低与肿瘤细胞表面erbB2表达水平的高低呈平行关系.     

Interactions of dendritic cells with cancer cells and modulation of surface molecules affect functional properties of CD8+ T cells

To understand the interaction of dendritic cells (DCs) with cancer cells, we investigated molecular changes in DCs following co-culture with cancer cells. DCs co-cultured with Jurkat cancer cells showed remarkable down-regulation of MHC class I molecules, while DCs co-cultured with MCF-7 cancer cells showed minimal changes. Interestingly, down-regulation of MHC class I on DCs was not observed upon treatment with Jurkat cell lysate or culture supernatant, suggesting the importance of direct cell-cell interactions. The expressions of CD40, CD80, CD83, MHC class II, and IL-12p40 on DCs co-cultured with Jurkat cells were only slightly affected. In contrast, DCs co-cultured with MCF-7 cells showed increased expressions of CD80, CD83, CD86, and IL-12p40. Furthermore, DCs co-cultured with Jurkat cells showed a down-regulation of low molecular weight polypeptides (LMP) 7, and of transporter associated with antigen processing (TAP) 1 and 2 at the mRNA expression level. LMP7, TAP2 and β2-microglobulin (β2M) were also down-regulated at the protein level. We further demonstrated how altered expression of MHC class I on DCs caused by co-culture with cancer cells affected autologous CD8(+) T cells, using the model MHC class I-presented HSV antigen. We found that DCs that had been HSV-treated and co-cultured with Jurkat cells showed a reduced potency to activate CD8(+) T cells. In contrast, HSV-treated DCs that had been co-cultured with MCF-7 cells induced activation of CD8(+) T cells, including high expression of CD25, CD69, granzyme B and cytokines, TNF-α and IFN-γ.     

Resistance to Fas-mediated apoptosis of human T-cell lines expressing human T-lymphotropic virus type-2 (HTLV-2) Tax protein

The susceptibility to Fas-mediated apoptosis was evaluated in seven T-cell lines (two infected with HTLV-2, one with HTLV-1, and four HTLV-free) as well as in Jurkat cells transfected with a Tax-2 expressing vector. Fas-mediated apoptosis was significantly reduced in the HTLV-1- and HTLV-2-infected lines in comparison with the HTLV-free lines regardless of the surface expression of Fas antigen (which was no different in the infected and uninfected cells). Fas-mediated apoptosis was also significantly inhibited in Jurkat cells transfected with the Tax-2 expressing vector without any modification in Fas expression. There was significantly more antiapoptotic Bcl-x(L) mRNA and protein in the transfected than in the untransfected Jurkat T cells. In conclusion, our results suggest that HTLV-2 is capable of inhibiting Fas-mediated apoptosis by means of a mechanism involving the tax-2 gene and probably the expression of bcl-x(L) messenger and protein.     

Antioestrogens Enhance Tumour Necrosis Factor Receptor 2 (TNF-R2) Expression and TNF-R2-Mediated Proliferation in Activated T Cells

In the present study we demonstrate that the non-steroidal antioestrogens toremifene and tamoxifen inhibit mitogen-induced proliferation and up-regulation of tumour necrosis factor (TNF) receptor family molecules on peripheral blood T cells. In activated T cells, however, toremifene and tamoxifen increase the surface expression of tumour necrosis factor receptor 2 (TNF-R2). This up-regulation is functionally important as TNF-R2-mediated proliferation is significantly enhanced in antioestrogen-treated activated T cells. The regulation of TNF-R2 expression in activated T cells seems to involve the c-Jun amino terminal kinase (JNK) pathway, as activation of JNK with anisomycin down-regulates TNF-R2. In activated T cells toremifene clearly inhibits phorbol 12-myristate 13-acetate (PMA)-induced JNK activity, suggesting that the JNK pathway may also be involved in the up-regulation of TNF-R2 expression by antioestrogens. Taken together, the enhancement of TNF-R2 expression and TNF-R2-mediated proliferation in activated T cells represents a novel feature for the effects of antioestrogens. The inhibitory effects of toremifene on the JNK pathway demonstrates that antioestrogens can influence not only cell growth, but also a variety of other cellular responses by inhibiting protein kinase C (PKC).     

Expression and function of Fas antigen on activated murine B cells

We have studied the expression and function of Fas antigen on murine B lymphocytes. While Fas was present on only a few B cells in the bone marrow, spleen, lymph node or peripheral blood, its expression could be strongly up-regulated by stimulation with soluble CD40 ligand (CD40L). Treatment with anti-IgM and interleukin-4 (IL-4) alone did not induce significant Fas expression but enhanced CD40L-mediated up-regulation of Fas expression. The T cell-derived signal via CD40 is therefore a potent inducer of Fas expression by B lymphocytes. The sensitivity to Fas-mediated apoptosis was found to depend on the duration of B cell activation. B cells activated for 1 day were resistant to Fas-mediated cell death, whereas B cells activated for 3 days were relatively sensitive. Interestingly, different sensitivity to Fas-mediated death signal was observed in 2-day activated B cells. It was found that B cells stimulated with CD40 L alone were more sensitive to Fas-mediated apoptosis than were cells stimulated with CD40L plus anti-IgM or IL-4, and in particular, the combination of the two. The greater sensitivity exhibited by B cells stimulated with CD40L alone seems to be related to limited activation of these cells in the absence of additional stimulation. Co-stimulation of B cells in the presence of CD40L and anti-Fas antibody resulted initially in activation of B lymphocytes, as reflected by the expression of activation markers and cell growth, but this was followed by growth inhibition and cell death. The data demonstrate that the B cell response can be regulated positively and negatively by signaling through CD40 and Fas antigens, respectively.     

The HCV core protein acts as a positive regulator of fas-mediated apoptosis in a human lymphoblastoid T cell line

Hepatitis C virus (HCV) is a major human pathogen causing mild to severe liver disease worldwide and is remarkably efficient at establishing persistent infections. Previously, we have shown that the core protein has an immunomodulatory function including the suppression of T lymphocyte responses to viral infection. To investigate the underlying mechanism for the role of core protein in immune modulation, we examined the effect of core on the sensitivity of the human T cell line, Jurkat, to Fas-mediated apoptosis. The transient and stable expression of core protein in Jurkat cells increased the sensitivity of cells to Fas-mediated apoptosis when compared to control cells expressing vector DNA alone. In addition, we demonstrated that the core protein binds to the cytoplasmic domain of Fas which may enhance the downstream signaling event of Fas-mediated apoptosis. The expression of core protein did not alter the cell surface expression of Fas, indicating that the increased sensitivity of core-expressing cells to Fas ligand was not due to upregulation of Fas. Furthermore, we observed the augmentation of caspase-3 activity in core-expressing cells. These results suggest that the core protein may promote the apoptosis of immune cells during HCV infection via the Fas signaling pathway, thus facilitating HCV persistence.     

Butyric Acid-Induced T-Cell Apoptosis Is Mediated by Caspase-8 and -9 Activation in a Fas-Independent Manner

Our previous study demonstrated that butyric acid, an extracellular metabolite of periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat cells. In this study, we examined whether CD95 ligand-receptor interaction is involved in butyric acid-induced T-cell apoptosis. Flow cytometry analysis indicated that expression of Fas in Jurkat and T cells from peripheral blood mononuclear cells was not affected by butyric acid treatment. Furthermore, the expression of Fas and FasL protein in Western blotting was not affected by butyric acid treatment. Coincubation with blocking anti-Fas antibodies prevented Fas-induced apoptosis but not butyric acid-induced apoptosis. Anti-FasL antibodies also did not prevent butyric acid-induced apoptosis at any dose examined. Although cytotoxic anti-Fas antibody affected butyric acid-induced apoptosis, a synergistic effect was not seen. Time-dependent activation of caspase-8 and -9 was recognized in butyric acid- as well as Fas-mediated apoptosis. IETD-CHO and LEHD-CHO, specific inhibitors of caspase-8 and -9, respectively, completely blocked Fas-mediated apoptosis and partially prevented butyric acid-induced apoptosis. These results suggest that the Fas-FasL interaction is not involved in butyric acid-induced apoptosis and that caspase-8 and -9-dependent apoptosis plays an important role in butyric acid-induced apoptosis, as well as Fas-induced apoptosis.     

Butyric acid-induced T-cell apoptosis is mediated by caspase-8 and -9 activation in a Fas-independent manner

Our previous study demonstrated that butyric acid, an extracellular metabolite of periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat cells. In this study, we examined whether CD95 ligand-receptor interaction is involved in butyric acid-induced T-cell apoptosis. Flow cytometry analysis indicated that expression of Fas in Jurkat and T cells from peripheral blood mononuclear cells was not affected by butyric acid treatment. Furthermore, the expression of Fas and FasL protein in Western blotting was not affected by butyric acid treatment. Coincubation with blocking anti-Fas antibodies prevented Fas-induced apoptosis but not butyric acid-induced apoptosis. Anti-FasL antibodies also did not prevent butyric acid-induced apoptosis at any dose examined. Although cytotoxic anti-Fas antibody affected butyric acid-induced apoptosis, a synergistic effect was not seen. Time-dependent activation of caspase-8 and -9 was recognized in butyric acid- as well as Fas-mediated apoptosis. IETD-CHO and LEHD-CHO, specific inhibitors of caspase-8 and -9, respectively, completely blocked Fas-mediated apoptosis and partially prevented butyric acid-induced apoptosis. These results suggest that the Fas-FasL interaction is not involved in butyric acid-induced apoptosis and that caspase-8 and -9-dependent apoptosis plays an important role in butyric acid-induced apoptosis, as well as Fas-induced apoptosis.     

CD44 stimulation down-regulates Fas expression and Fas-mediated apoptosis of lung cancer cells

Cytotoxic T lymphocytes (CTL) play a major role in the rejection of tumor cells, but tumor rejection does not always occur in vivo, indicating that defects in anti-tumor immune responses may be common. We here document a novel function for CD44--using lung cancer cells, we showed that stimulation of CD44 reduced Fas expression and Fas-mediated apoptosis: (i) lung cancer cells expressed high levels of CD44; (ii) engagement of CD44 on the cells by a specific antibody or fragmented hyaluronan reduced Fas expression; (iii) CD44 cross-linking reduced Fas-mediated apoptosis; (iv) stimulation of CD44 on lung cancer cells decreased IFN-gamma production by autologous CTL; and (v) CD44 stimulation prevented killing of lung cancer cells by autologous CTL. Based on these findings, we postulate a new concept--that interaction of CD44 on lung cancer cells with fragments of extracellular hyaluronan present in the surrounding extracellular matrix reduces Fas expression as well as Fas-mediated apoptosis of cancer cells. This leads to reduced susceptibility of the cells to CTL-mediated cytotoxicity through the Fas-Fas ligand pathway.     

Fas receptor‐mediated apoptosis: a clinical application?

Fas is a membrane protein belonging to the death receptor family. Cross-linking of Fas by its ligand, FasL, or agonistic anti-Fas antibodies, induces apoptosis of cells expressing Fas on the membrane by triggering a cascade of caspases. Since many different tumours express Fas on their membrane, targeting Fas-mediated apoptosis by anti-Fas antibodies may be a promising anticancer therapy. Unfortunately, not all Fas-expressing cells are sensitive to Fas-mediated apoptosis. This has resulted in the discovery of many different inhibition mechanisms of Fas-mediated apoptosis. In addition, mutations in the Fas or p53 gene can also influence the sensitivity for Fas-mediated apoptosis. However, the role of wild-type p53 in Fas expression is still controversial. Because several different cytotoxic drugs are able to induce Fas membrane expression, combination therapy of anticancer drugs with anti-Fas antibodies or FasL is conceivable as an anticancer strategy. The efficiency of the induction of Fas-mediated apoptosis by anti-Fas antibodies, FasL-expressing cells or recombinant FasL (rFasL) in tumours has been demonstrated in vivo in solid tumours implanted in mice. Unfortunately, systemic treatment with anti-Fas antibodies or rFasL causes severe damage to the liver, so most preclinical studies are now focusing on circumvention of this problem by local administration of FasL, or on the use of inducible FasL-expressing vectors as gene therapy.     

Overexpression of CD82 on human T cells enhances LFA-1 / ICAM-1-mediated cell-cell adhesion: functional association between CD82 and LFA-1 in T cell activation

We previously demonstrated that CD82, expressed on both T cells and antigen-presenting cells (APC), plays an important role as a co-stimulatory molecule especially in the early phase of T cell activation. We also showed that the CD82 expression level is up-regulated on activated T cells and memory T cells. This up-regulation enhances both T cell-T cell and T cell-APC interactions. In this study, we further investigated the mechanism of CD82-mediated cell-cell adhesion. The enhanced adhesion between CD82-overexpressing Jurkat cells was completely blocked by anti-ICAM-1 / LFA-1 monoclonal antibodies. Increased interaction of LFA-1 with ICAM-1 was further confirmed by enhanced adhesion of CD82-overexpressing Jurkat cells to immobilized ICAM-1-Ig. CD82 co-immunoprecipitated with LFA-1 from Jurkat cells and CD82 and LFA-1 colocalized at an adhesion foci. These results suggest that the T cell stimulation via anti-CD3 cross-linking or phorbol myristate acetate treatment up-regulates CD82 expression, leading to the colocalization of CD82 and LFA-1, and results in enhanced interaction between LFA-1 and ICAM-1. In addition, a blocking experiment using monoclonal antibodies suggested that CD82 and LFA-1 molecules on APC are also important for the optimal activation of T cells. This is the first report that describes the enhancement of cell-cell interaction through LFA-1 and ICAM-1 by the overexpression of another cell surface molecule, CD82.     

CD4+ T cell-mediated cytotoxicity toward thyrocytes: the importance of Fas/Fas ligand interaction inducing apoptosis of thyrocytes and the inhibitory effect of thyroid-stimulating hormone

The accumulation of activated CD4+ T cells and antigen (Ag)-dependent cellular interactions between thyrocytes and CD4+ T cells have been determined in thyroid gland from patients with Graves' disease. The Fas/Fas ligand (FasL) interaction between antigen-presenting cells and T cells regulates the apoptosis of the former cells triggered by the latter cells. The inhibition of Fas-mediated apoptosis in thyrocytes could be a underlying mechanism of hyperplasia of thyrocytes in patients with Graves' disease. We investigated the potential role of Fas/FasL interaction between thyrocytes and CD4+ T cells in the induction of Fas-mediated apoptosis of the former cells induced by the latter cells. The presence of only a few specific T cells responsive to a putative autoantigen has hampered the investigation of specific T cell activation toward antigen-presenting cells (APCs). Therefore, we used a superantigen, staphylococcal enterotoxin B (SEB), to examine specific T cell activation toward thyrocytes in vitro since it stimulates a large proportion of T cells with particular Vbeta elements. Spontaneous apoptosis of thyrocytes in culture was not found even in the presence of various kinds of cytokines. In contrast, a clear induction of Fas-mediated apoptosis by anti-Fas IgM was determined in interferon-gamma (IFN-gamma)-stimulated thyrocytes. In addition, a significant cytotoxicity of purified CD4+ T cells toward IFN-gamma-stimulated thyrocytes in the presence of SEB was induced, and the addition of anti-HLA-DR and -DQ monoclonal antibodies (mAbs) or blockade of the Fas/FasL interaction reduced this cytotoxicity. FasL expression of CD4+ T cells cocultured with IFN-gamma-stimulated thyrocytes in the presence of SEB was clearly induced. Furthermore, the addition of mAbs against CD54 and CD58 inhibited both cytotoxicity and FasL expression of CD4+ T cells. The cytotoxicity of CD4+ T cells toward IFN-gamma-stimulated, SEB-pulsed thyrocytes was markedly inhibited when we used thyrocytes cultured with IFN-gamma in the presence of thyroid-stimulating hormone (TSH) as target cells. Our results suggest that 1) CD4+ T cells were activated by thyrocytes expressing MHC class II molecules in an SEB-dependent manner and then expressed FasL. 2) These activated FasL+ CD4+ T cells killed thyrocytes by interacting with Fas on thyrocytes and FasL on activated CD4+ T cells. The presence of costimulating molecules such as CD54 and CD58 on thyrocytes was also necessary to generate activated FasL+ CD4+ T cells. 3) Since the actions of thyroid stimulating antibody (TSAb) toward thyrocytes are similar to those of TSH, one goitrogenic activity of TSAb may, in part, be due to the inhibitory effect on Fas-mediated apoptosis of thyrocytes triggered by activated CD4+ T cells.     

Activation of cAMP signaling enhances Fas-mediated apoptosis and activation-induced cell death through potentiation of caspase 8 activation

Defects in Fas receptor signaling lead to compromised maintenance of lymphocyte homeostasis and peripheral immune tolerance, leading in turn to autoimmune disorders. Therefore, agents that can enhance Fas-mediated apoptosis may be therapeutically useful in management of such disorders. In this study, we focused on the effect of cAMP on Fas-mediated apoptosis in human T cells. We show that elevation of intracellular cAMP levels by forskolin, an activator of adenylyl cyclase, 3-isobutyl-1-methylxanthine, an inhibitor of cyclic nucleotide phosphodiesterases, or prostaglandin E(2) potentiates Fas-induced apoptosis in Jurkat cells. Accordingly, cAMP was found to enhance the cleavage of caspase 8 at death-inducing signaling complex and lead to augmentation of the processing of Fas effector proteins. We also demonstrate that cAMP enahnaces Fas-induced apoptosis in normal human T cells and activation-induced cell death in Jurkat cells. These findings provide a rationale for investigating the feasibility of using cAMP-elevating agents to potentiate apoptosis in T cells with aberrant Fas signaling.