Pilar Lasierra

Differential Secretion of Fas Ligand- or APO2 Ligand/TNF-Related Apoptosis-Inducing Ligand-Carrying Microvesicles During Activation-Induced Death of Human T Cells

Preformed Fas ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL) are stored in the cytoplasm of the human Jurkat T cell line and of normal human T cell blasts. The rapid release of these molecules in their bioactive form is involved in activation-induced cell death. In this study, we show by confocal microscopy that FasL and APO2L/TRAIL are mainly localized in lysosomal-like compartments in these cells. We show also by immunoelectron microscopy that FasL and APO2L/TRAIL are stored inside cytoplasmic compartments ∼500 nm in diameter, with characteristics of multivesicular bodies. Most of these compartments share FasL and APO2L/TRAIL, although exclusive APO2L/TRAIL labeling can be also observed in separate compartments. Upon PHA activation, the mobilization of these compartments toward the plasma membrane is evident, resulting in the secretion of the internal microvesicles loaded with FasL and APO2L/TRAIL. In the case of activation with anti-CD59 mAb, the secretion of microvesicles labeled preferentially with APO2L/TRAIL predominates. These data provide the basis of a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation and could modify the interpretation of the role of FasL and APO2L/TRAIL as effector mechanisms in physiological and pathological situations.

Doxorubicin-induced apoptosis in human T-cell leukemia is mediated by caspase-3 activation in a Fas-independent way

It has recently been proposed that doxorubicin (DOX) can induce apoptosis in human T‐leukemia cells via the Fas/FasL system in an autocrine/paracrine way. We show here that treatment of Jurkat cells with either anti‐Fas antibodies, anthracyclin drugs or actinomycin D induces the activation of CPP32 (caspase‐3) and apoptosis. However, DOX treatment did not induce the expression of membrane FasL or the release of soluble FasL and co‐incubation with blocking anti‐Fas antibodies prevented Fas‐induced but not DOX‐induced apoptosis. All the morphological and biochemical signs of apoptosis induced by anti‐Fas or DOX can be prevented by Z‐VAD‐fmk, a general caspase inhibitor. DEVD‐cho, a specific inhibitor of CPP32‐like caspases which completely blocks Fas‐mediated apoptosis, prevented drug‐induced nuclear apoptosis but not cell death. We conclude that: (i) DOX‐induced apoptosis in human T‐leukemia/lymphoma is Fas‐independent and (ii) caspase‐3 is responsible of DOX‐induced nuclear apoptosis but other Z‐VAD‐sensitive caspases are implicated in cell death.

Resistance to apoptosis correlates with a highly proliferative phenotype and loss of Fas and CPP32 (caspase-3) expression in human leukemia cells

Apoptosis induced by effector cells of the immune system or by cytotoxic drugs is a main mechanism mediating the prevention or elimination of tumoral cells. For instance, the human T‐cell leukemia Jurkat is sensitive to Fas‐induced apoptosis and to activation‐induced cell death (AICD), and the promonocytic leukemia U937 is sensitive to Fas‐ and TNF‐induced apoptosis. In this work, we have analyzed the mechanisms of resistance to physiological or pharmacological apoptosis in human leukemia by generating highly proliferative (hp) sub‐lines derived from Jurkat and U937 cells. These hp sub‐lines were resistant to Fas‐ and TNF‐induced apoptosis, as well as to AICD. This was due to the complete loss of Fas and TNFR surface expression and, in the case of Jurkat‐derived sub‐lines, also of CD3, CD2 and CD59 molecules. The sub‐lines also completely lacked the expression of the apoptotic protease CPP32, present in parental cells. Moreover, these sub‐lines were no longer sensitive to doxorubicin‐induced apoptosis, which was efficiently blocked by the general caspase inhibitor Z‐VAD‐fmk in the parental cell lines. These data suggest a molecular mechanism for the development of resistance of leukemic cells to physiological and pharmacological apoptosis inducers, giving rise to highly proliferative tumoral phenotypes. These results also indicate that Fas and CPP32 could be useful prognostic markers for the progression and/or therapy outcome of human leukemias. Int. J. Cancer 75:473–481, 1998.

Tendon xanthomas in familial hypercholesterolemia are associated with a differential inflammatory response of macrophages to oxidized LDL

Tendon xanthomas (TX) are pathognomonic lipid deposits commonly found in familial hypercholesterolemia (FH) patients. The aim of this study was to determine whether macrophages from FH patients with TX (TX+) have higher predisposition to foam cells formation after oxidized LDL (oxLDL) overload than those from FH patients without TX (TX−), and if their differential gene expression profile could explain these different phenotypes. Total RNA pools from macrophages from FH patients TX+ and TX− were analyzed using Affymetrix oligonucleotide arrays to evaluate the gene expression profile in presence and absence of oxLDL. Also, the intracellular lipid content was measured by fluorescence flow cytometry. Results of these studies suggest that macrophages from FH subjects TX+ compared to those TX− have a differential response to oxLDL, since they show higher intracellular cholesterol ester accumulation and a differential gene expression profile. The gene array data were validated by relative quantitative real‐time RT‐PCR and quantitative ELISA in culture media and plasma samples. FH subjects TX+ showed increased plasma tryptase, TNF‐α, IL‐8 and IL‐6 concentrations. We propose that TX formation are associated with higher intracellular lipid content, and higher inflammatory response of macrophages in response to oxLDL.

CD59 cross-linking induces secretion of APO2 ligand in overactivated human T cells.

Jurkat cells and the derived TCR / CD3‐defective subline, J.RT3.T3.5 undergo activationinduced cell death (AICD) when stimulated with phytohemagglutinin (PHA). Since J.RT3.T3.5 cells do not express antigen receptor, we searched for the molecules that could be ligated by PHA and induce AICD in this cell line. We show here that the glycosylphosphatidylinositollinked CD59 molecule is expressed at the surface of Jurkat and J.RT3.T3.5 cells, and when cross‐linked by specific antibodies can induce cell death. The toxicity of supernatants from PHA‐stimulated Jurkat or J.RT3.T3.5 cells was prevented by a combination of the blocking anti‐Fas mAb SM1 / 23 and anti‐APO2L / TRAIL mAb 5C2. However, toxicity of supernatants from anti‐CD59 stimulated cells was specifically prevented by the anti‐APO2L blocking antibody. Anti‐CD59 cross‐linking induced AICD also in normal human T cell blasts, which secreted toxic molecules into the supernatant. The toxicity of these supernatants on Jurkat cells was fully prevented by the anti‐APO2L blocking antibody, showing that CD59 crosslinking induces the preferential release of APO2L also in normal T cells. The possible physiological and / or pathological consequences of this observation are discussed.

Human CD8+ T cell blasts are more sensitive than CD4+ T cell blasts to regulation by APO2L/TRAIL

The mechanisms responsible for the down‐modulation of the activation of separated CD4+ or CD8+ human T cell blasts were studied using cells obtained from healthy donors. In the presence of IL‐2, human CD8+ T cell blasts were more sensitive than CD4+ T cell blasts to regulation by APO2 ligand/TNF‐related apoptosis‐inducing ligand (APO2L/TRAIL), while both T cell subsets were equally sensitive to Fas/CD95 regulation. This regulation was defined as inhibition of IL‐2‐dependent T cell growth in the absence of cell death induction, characterized by cell cycle arrest in G2/M. The physiological validity of these observations was corroborated by the demonstration of intracellular FasL and APO2L/TRAIL expression in CD4+ and CD8+ T cell blasts, which were secreted in their bioactive form into the supernatant upon PHA, CD3 or CD59 reactivation. Additionally, the inhibition of IL‐2‐dependent CD4+ or CD8+ T cell blast growth upon CD3 or CD59 ligation was dependent, at least partially, on FasL and/or APO2L/TRAIL. These data precisely define the role of APO2L/TRAIL in the regulation of human T cell activation.

Results of a pilot trial of immunotherapy with dendritic cells pulsed with autologous tumor lysates in patients with advanced cancer.

Aims and background The purpose of the study was to test the immunological and clinical effects of infusions of dendritic cells pulsed with autologous tumor lysate in patients with advanced cancer. Patients and methods Peripheral blood mononuclear cells from 15 patients with metastatic cancer (melanoma in 10, lung cancer in 2, renal cell carcinoma in 1, sarcoma in 1, breast cancer in 1) were harvested by leukapheresis after mobilization with GM-CSF (5 μg/kg/day s.c. for 4 days). Mononuclear cells were separated and cultured in GM-CSF (1000 U/ml) and interleukin-4 (1000 U/ml) for 7 days. Phenotype was assessed by 2-color flow cytometry and immunocytochemistry. On day 6, dendritic cells were pulsed with 1 g of fresh autologous tumor lysate for 24 h and infused intravenously. Interleukin-2 (6 million IU), interferon a (4 million IU) and GM-CSF (400 μg) were injected s.c. daily for 10 days beginning on the day of dendritic cell infusion. Treatment was repeated every 21 days for 3 courses. Results The morphology, immunocytochemistry and phenotype of cultured cells was consistent with dendritic cells: intense positivity for HLA-DR and CD86, with negativity for markers of other lineages, including CD3, CD4, CD8 and CD14. More than 5 × 107 dendritic cells were injected in all patients. Nine patients developed >5 mm delayed type cutaneous hypersensitivity reactions to tumor lysate ± GM-CSF after the first immunization (larger than GM-CSF in all cases). Median delayed type cutaneous hypersensitivity to lysate + GM-CSF was 3 cm after the third immunization. One melanoma patient with skin, liver, lung and bone metastases had a partial response lasting 8 months (followed by progression in the brain). Seven patients had stable disease for >3 months, and 7 had progression. Conclusions Infusion of tumor lysate-pulsed dendritic cells induces a strong cell-mediated antitumor immune reaction in patients with advanced cancer and has some clinical activity.