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RAPID COMMUNICATION
By
From the Laboratory of Molecular Cytology, the Department of Internal Medicine, University of Innsbruck; the Department of Oncology, Wilhelminen Spital, Vienna; and the Division of Molecular Pathophysiology, the Department of General and Experimental Pathology, University of Innsbruck, Innsbruck, Austria.
The Fas (Apo-1/CD95) ligand (FasL) plays a central role in the elimination of target cells by effector T lymphocytes and in the suppression of cellular immune responses against nonmalignant and malignant cells. We show the expression of FasL on the surface of neoplastic plasma cells. We provide evidence that the FasL is functionally active because five of five neoplastic plasma cell lines tested killed CEM-C7H2 T-acute lymphoblastic leukemia (T-ALL) cells. The effect was mediated via the Fas (Apo-1/CD95) receptor molecule because blocking of Fas on the target cells or the FasL on the tumor cells by receptor- and ligand-specific monoclonal antibodies (MoAbs), respectively, protected T cells from being killed by myeloma cells. In addition, overexpression of the cowpox virus protein CrmA, a molecule with inhibitory potential on caspase-1 and caspase-8, specifically involved in Fas-induced signaling, protected T cells from being destroyed by the neoplastic cells or the agonistic anti-Fas MoAb. The potential of the malignant plasma cells to extinguish target T cells was independent of their own sensitivity to the agonistic anti-Fas MoAb, and FasL-positive (FasL+) CEM-C7H2 T cells were incapable of killing myeloma cells. Our results suggest that tumor cell-induced suppression of the immune system may be exerted via the FasL active on malignant plasma cells. Furthermore, loss of Fas expression or insensitivity to the agonistic anti-Fas MoAb do not seem to be prerequisites for myeloma cells to defeat T cells via Fas/FasL interaction.
THE FAS LIGAND (FasL) recognizes and cross-links the Fas (Apo-1/CD95) receptor, a member of the tumor necrosis factor/nerve growth factor (TNF/NGF) receptor supergene family, which is expressed in a wide variety of normal and neoplastic tissues.1-3 The extracellular portion of the FasL protein has been shown to share significant homology with other members of the TNF-family like TNF- The recognized biologic roles of the FasL lie in its participation in the process of the acquisition of self tolerance by clonal deletion of thymocytes,8 as well as in T-cell-mediated target cell killing as a part of the host-defense against virally infected or transformed cells.9 Until recently, T cells were thought to actively control the direction of Fas-induced target cell killing and to be the major source of active FasL molecules10,11 The present study proves the expression of functional FasL on neoplastic plasma cells and their ability to kill target T cells regardless of the myeloma cells expression level of the Fas receptor and their own sensitivity to Fas-mediated signaling. This not only points to a possible mechanism by which myeloma cells escape the natural or therapeutically activated immune system, but may also open opportunities for novel therapeutic strategies. Thus, downregulation of the FasL by means of antisense or other immunomodulatory strategies could prove helpful in the treatment of this disease by disarming myeloma cells and, thereby, disabeling them from deleting activated T-cell clones.
Cell Lines and Culture Conditions
Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Northern Analysis Ten micrograms of total RNA was analyzed by electrophoresis using 1% agarose-formaldehyde gels, followed by capillary transfer overnight to a positively charged nitrocellulose membrane (Boehringer Mannheim). After transfer, the membrane was dried, UV cross-linked, and blocked for 2 hours in 25 mL (pre)-hybridization solution (5 × sodium/sodium citrate [SSC], 50% deionized formamide, 0.1% sodium-lauryl-sarcosine, 0.02% sodium dodecyl sulfate [SDS] 10 × Denhardt's reagent) at 55°C. The membrane was hybridized with 2 × 106 cpm/mL 32[P]-labeled FasL DNA probe overnight at the same temperature. For posthybridization washes, 2 × SSC/0.1% SDS (2 × 10 minutes, room temperature) as well as 0.1 × SSC/0.1% SDS (1 × 5 minutes room temperature, 1 × 5 minutes, 55°C) were used. The membrane was stripped and reprobed at 50°C using a human GAPDH probe. The FasL-specific probe was prepared as follows: CEM-C7H2 cDNA was amplified using FasL-specific primers as detailed in section "RT-PCR." After gel electrophoresis, the FasL PCR product was excised out of the gel, purified using a Bio-101 Gen-clean kit (San Francisco, CA) and labeled with 20 µCi 32[P]-CTP using a random primer labeling kit (Boehringer Mannheim) to a specific activity of ~5 × 106 cpm/µg.
Immunoblotting Immunoblotting of cellular proteins was performed as previously described.26 Cells (2 × 106) were resuspended in 200 µL lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 2 mmol/L EDTA, 1 mmol/L EGTA pH 7.5, supplemented with 25 µg/mL aprotinin, 25 µg/mL leupeptin, and 1% Triton X-100 [Sigma, St Louis, MO]). The samples were cleared by centrifugation (16,000g, 30 minutes, 4°C) and corrected for protein concentrations. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (14%) was performed under reducing conditions on Tris/glycine-buffered gels (Novex, San Diego, CA). Proteins were transferred onto a polyvinyl-difluoridon (PVDF) membrane (Millipore, Bedford, MA) by tank-blotting (220 mA, 80 minutes, 4°C). The anti-FasL MoAb (Transduction Laboratories, Lexington, KY) was diluted 1:2,000 in Tris-buffered saline (TBS) containing 0.05% Tween-20 and 5% nonfat dry milk for incubation. Rabbit-antimouse peroxidase-conjugated antibodies (Dako, Copenhagen, Denmark) served as secondary antibodies (1:1,000). Incubations were performed for 1 hour each at room temperature. Diaminobenzidine diluted in TBS served as substrate solution.Immunofluorescence FasL staining. Cells (0.2 × 106) were washed twice in phosphate-buffered saline (PBS), resuspended, and fixed in 1 mL paraformaldehyde (4%) and incubated for 15 minutes at 4°C. After an additional wash with PBS the pellet was resuspended in chilled methanol (100%) and incubated for 60 minutes on ice. Cells were washed twice in PBS, incubated with 1 µg anti-FasL MoAb NOK-1 (Pharmingen, San Diego, CA) for 30 minutes at 4°C. Again, cells were washed twice with PBS and incubated with 5 µL of fluorescein isothiocyanate (FITC)-labeled goat-antimouse antibody F0479 (DAKO, Copenhagen, Denmark) for 30 minutes at 4°C. The pellets were resuspended in 200 µL PBS/1% bovine serum albumin (BSA) and analyzed immediately by FACScan (5,000 cells per sample). Negative controls were carried out simultaneously using a mouse-antihuman IgG1 MoAb (no. X0931; DAKO) instead of the FasL MoAb.Determination of FasL Expression on Native Neoplastic Plasma Cells Bone marrow (BM) samples of six patients with multiple myeloma and one patient with breast cancer were analyzed. Samples were collected during routinely scheduled examinations after informed consent had been obtained. The patients characteristics are given in Table 2 (Results section). BM samples were pressed through a 0.2 × 20-mm syringe and diluted in RPMI-1640 medium 1:1 (vol/vol). The solution was applied onto a FICOLL density gradient (Lymphoprep; NYCOMED, Oslo, Norway) [2:1 (vol/vol)] and centrifuged with 500g for 30 minutes at room temperature. The mononuclear cell fraction was carefully aspirated, washed twice with PBS, and BM cells (0.5 × 106) were forwarded to FasL or isotype staining, respectively (see "FasL Staining"). After FasL staining, the cells were washed three times in PBS and thereafter subjected to CD38 immunostaining (30 minutes on ice) using 20 µL of a phycoerythrin (PE)-conjugated mouse-antihuman CD38 MoAb (Pharmingen). The plasma cell population was gated according to their unique position in the correlation of forward light scattering, orthogonal light scattering, and immunofluorescent-labeled CD38.29 Plasma cells stained with the FasL MoAb were compared with samples stained with the relevant FasL isotype control to determine FasL expression on the plasma cells.
Assay for Fas/FasL-Induced Cell Death Target cell death of CEM-C7H2 T-ALL cells resulting from their cocultivation with "effector" myeloma cells was quantified by measuring target cell DNA fragmentation using the JAM-Test.30,31 For this purpose, T-ALL cells were incubated with 10 µCi/mL 3[H]-thymidine (Amersham, Buckinghamshire, UK) for 5 hours, washed three times with PBS, resuspended in regular culture medium, and 100 µL of the suspension (2 × 104/mL) was cocultivated in 96-well plates with or without 100 µL of the suspension of the relevant myeloma cell lines (2 × 105/mL). The myeloma cells were incubated with 2.5 µg/mL anti-FasL MoAb (NOK-2) or a relevant negative control IgG2a MoAb (no. X0943; DAKO), respectively, 30 minutes before cocultivation with the labeled CEM-C7H2 cells. Alternatively, the labeled T cells were coincubated with 0.25 µg/mL of the Fas receptor blocking ZB4 MoAb (Kamiya, Thousand Oaks, CA) or a relevant negative control IgM MoAb (no. X0942; DAKO), respectively, 30 minutes before cocultivation with the myeloma cells. Treatment of labeled T cells with 0.25 µg/mL of the death-inducing anti-Fas MoAb (clone CH11; Kamiya) served as a positive control for Fas-mediated apoptosis. To determine the effect of CEM-C7H2 T cells on myeloma cells, myeloma cells were endogenously labeled with 3[H]-thymidine and exposed to unlabeled CEM-C7H2 cells using adequately adapted effector cell/target cell (E/T) ratios. Cocultivation of cells was performed for 72 hours at 37°C. Cells were obtained automatically, transferred onto filter papers, and washed six times. Incorporated radioactivity from undegraded chromosomal DNA was measured with a -scintillation counter. The reduction in incorporated radioactivity was used to calculate the percentage of specific target cell killing [(cpm Untreated Cells - cpm Cocultured Cells)/cpm Untreated Cells × 100].
Assay for Anti-Fas MoAb-Induced Apoptosis Cells were taken out of continuous culture, washed, and seeded at a density of 0.2 × 106/mL in 10% RPMI 1640 medium. For induction of apoptosis, 0.25 µg/mL of the IgM anti-Fas MoAb no. 1504, clone CH11 (Kamiya), was used. After 8 hours cells were procured, washed, and analyzed for morphologic changes characteristic for apoptosis32 as well as for the diminution of DNA-content by the propidium iodide (PI) assay. Cells were procured, washed by centrifugation in PBS, and incubated with 300 µL/well PI solution (50 µg/mL PI, 0.1% sodium citrate, and 0.1% Triton X-100) for permeabilization and DNA-staining. Analysis of cell size and fluorescence intensity was performed in the forward/side scatter program of a FACScan (Becton Dickinson, Vienna, Austria) as recently described.27 After exclusion of necrotic debris, apoptotic cells in PI assays were defined by a decrease in DNA content as compared to G1 phase cells of myeloma cell lines and normal T lymphocytes.
Determination of FasL protein expression in malignant plasma cell lines. (a) Cellular protein was extracted from the indicated cell lines. Protein extract from endothelial cells was provided by the manufacturer of the FasL MoAb and served as a positive control. Equal amounts of protein were separated on 14% Tris-glycine gels by PAGE and transferred onto a nylon membrane. Immunostaining showed a specific signal in all myeloma extracts at the predicted molecular weight of the FasL protein (ie, 37 kD) matching the signal obtained with control and T-cell extract. (b) LP-1 plasma cell leukemia cells were stained with the FasL-specific MoAb and characterized for staining by means of flow cytometry. A shift in fluorescence intensity in samples stained with the NOK-1 FasL antibody (red line) was observed when compared with samples treated with the isotype-matched control MoAbs only (black line). Fig 2.
Transfection of CEM-C7H2 T-ALL Cells Logarithmically growing CEM-C7H2 cells were washed in PBS, pelleted at 300g, and resuspended at a density of 1 × 107 cells/400 mL PBS. Cells were mixed with 20 µg of plasmid pHD1.2, a CrmA expression vector driven by the chicken-actin promoter, kindly provided by V. Gagliardini,33 incubated for 10 minutes on ice, and permeabilized with the eletroporator (Biorad Lab, Vienna, Austria) set at 960 mF and 300 V. After electroporation, cells were again placed on ice for 10 minutes diluted in 20 mL growth medium, and seeded on 96-well flat-bottom plates. Selection of stably transfected cells was initiated 48 hours after electroporation using 1 mg/mL G418 (bioactivity 70%). CrmA expression was monitored by RNA dot-blot analysis28 using the CrmA insert as a probe. The CrmA expressing subclone 2E8 was used for analysis. IC3 is a CEM-C7H2 subclone stably transfected with the "empty" control vector.
The FasL mRNA and Protein Are Expressed in Malignant Plasma Cells Since it has been shown in murine B lymphocytes that treatment with PMA induces the expression of FasL mRNA which was undetectable before such induction,25 we analyzed expression levels of FasL mRNA in myeloma cells in response to treatment with PMA/ionomycin. CEM-C7H2 T-ALL cells treated for 4 hours with PMA/ionomycin served as a positive control for FasL mRNA expression. When MC/CAR plasmacytoma cells were treated with PMA/ionomycin for up to 8 hours and the extracted total RNA was subjected to RT-PCR analysis, an FasL-specific PCR product could be detected (Fig 1a). The PCR product appeared exactly at the product size predicted for the chosen FasL primer set used and at the same product size as the T-cell-derived PCR product. The FasL mRNA was already detected in unstimulated MC/CAR plasmacytoma cells and slightly increased in response to PMA/ionomycin when a ratio between the relative expression levels of FasL and-actin mRNA was determined by image analysis. Similar results were observed in IM-9 cells and the RPMI-8226 myeloma cell line, too. However, basal FasL expression was not detected in the epithelial carcinoma cell lines HELA and KB (data not shown). We subsequently analyzed FasL mRNA expression by Northern analysis. In all neoplastic plasma cell lines tested, the RNA analysis showed a solitary signal (Fig 1b) at the size expected for the mRNA transcript specific for FasL (~2 kb, Fig 1b), as determined by comparison with the GAPDH signal.
FasL Expressed on Malignant Plasma Cells Is Functionally Active and Able to Induce Cell Death in CEM-C7H2 T Cells
Transfection of a CrmA Expression Plasmid Protects CEM-C7H2 T Cells From Myeloma Cell-Induced Cytotoxicity
Our experiments indicate the constitutive expression of FasL in seven of seven neoplastic plasma cell lines as shown at the RNA level by means of RT-PCR and Northern blot analysis (Fig 1a and b). These FasL-specific transcripts are translated into detectable amounts of protein as shown by immunoblotting experiments as well as by single cell analysis using flow cytometry (Figs 2a and b, Table 1). Furthermore, FasL protein could also be detected in the plasma cell fraction of BM samples from four of six patients with multiple myeloma (Fig 3, Table 2). The FasL expressed on the malignant plasma cell lines was functionally active, because five of five cell lines tested in the JAM assay30,31 were able to kill CEM-C7H2 T-ALL target cells (Figs 4 and 5). This cytotoxic effect of the neoplastic cells could specifically be inhibited by an MoAb neutralizing the FasL on myeloma cells6 as well as by the MoAb ZB4, which prevents the binding of the ligand to its receptor (Figs 4a and b). To confirm the role of the Fas/FasL system in the achievement of the cytotoxic effect on the level of intracellular signal transduction, CEM-C7H2 target cells were replaced by the subclone 2E8 transfected with a vector overexpressing the cowpox virus CrmA protein known to inhibit particulary ICE/caspase-135 and FLICE/caspase-8.36 Both caspases have been implicated in the effector phase of apoptosis triggered by antibodies to the Fas/Apo-1 membrane protein.34,37 Furthermore, the central role of caspase1 in the mediation of Fas/Apo-1-induced apoptosis was supported by experiments using ICE-deficient mice.38 Our results obtained in cocultivation experiments with CrmA-expressing CEM-C7H2 cells confirmed the data obtained in the antibody inhibition experiments, because CrmA-expressing cells were essentialy resistant to cell death caused by cocultivated myeloma cells or by treatment with anti-Fas MoAb (Fig 5). The degree of cytotoxicity blocked by transfection of CEM-C7H2 T-ALL cells with the vector allowing overexpression of the CrmA protein was superior to the degree of inhibition obtained with MoAbs against the FasL or its receptor. This phenomenon might be explained by submaximal inhibitory capacity of the antibodies used in this investigation, since in all cocultivation experiments protection of target cells from undergoing anti-Fas MoAb-induced apoptosis was achieved more efficiently by CrmA-transfection than by blocking of the Fas/FasL system with the inhibitory MoAbs (Figs 4 and 5). As an alternative explanation, other molecules like cytotoxic cytokines might in part be involved in the tumor cell-induced target cell killing. Signaling along the TNF- Submitted March 10, 1997;
accepted April 15, 1997.
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| Copyright © 1997 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Abstract
Introduction
Abstract
a supposition that is in good agreement with the functions mentioned above as well as with the rather broad expression profile of the Fas/Apo-1 receptor. However, this picture was incomplete because, in addition to T cells, cells of the murine testis and retina also were shown to express FasL mRNA and protein.
). Treatment of T cells with 0.25 µg/mL of the agonistic anti-Fas MoAb served as an internal control of the assay. Blockage of the Fas receptor with the antagonistic ZB4 anti-Fas MoAb (0.25 µg/mL, a) or neutralization of FasL molecules using the NOK-2 FasL MoAb (2.5 µg/mL, b) was performed to prove the dependence of the observed cytotoxicity on active Fas signaling (
). Mean values ± SEM (n = 8) are depicted.
