Research Article Glioblastomas Induce T-Lymphocyte Death by Two Distinct Pathways Involving Gangliosides and CD70 Ali Chahlavi,1 Patricia Rayman,2 Amy L. Richmond,2 Kaushik Biswas,2 Renliang Zhang,3 Michael Vogelbaum,1 Charles Tannenbaum,2 Gene Barnett,1 and James H. Finke2 1Department of Neurosurgery, Brain Tumor Institute, 2Department of Immunology, Lerner Research Institute, and 3Department of Cell Biology, Cleveland Clinic Foundation, Cleveland, Ohio Abstract T lymphocytes, can occur through mechanisms that increase the sensitivity of these cells to apoptosis. Here we report that glioblastoma multiforme (GBM) mediates immunosuppression by promoting T-cell death via tumor- Studies on the mechanisms by which tumor cells induce T-cell associated CD70 and gangliosides that act through receptor- apoptosis have shown a role for tumor-associated Fas ligand (FasL) dependent and receptor-independent pathways, respectively. and other tumor necrosis factor (TNF)–related ligands that activate GBM lines cocultured with T cells induced lymphocyte death. the receptor-dependent apoptotic pathway (4, 5). However, similar The GBM lines were characterized for their expression of immune dysfunction can also be mediated by tumor cell culture CD70, Fas ligand (FasL), and tumor necrosis factor-A (TNF-A), supernatants, which when added to normal activated human T and the possible participation of those molecules in T-cell cells, also induce T-cell death (6). Likewise, tumor cyst fluid and killing was assessed by doing GBM/T cell cocultures in the cerebrospinal fluid from patients with gliomas suppressed normal presence of anti-CD70 antibodies, Fas fusion proteins, or anti- lymphocyte activity (7). This is reminiscent of the finding that isolated, peripheral blood T cells from glioma patients are TNF-A antibodies. CD70 but not TNF-A or FasL is responsible for initiating T-cell death via the receptor-dependent pathway. apoptotic (6), suggesting that gliomas secrete soluble products Of the four GBM cell lines that induced T-cell death, three capable of distant immunosuppression. Given that GBM over- highly expressed CD70. Two nonapoptogenic GBM lines (CCF3 express various gangliosides (8–10) with known immunosuppres- and U138), on the other hand, had only minimally detectable sive and apoptotic activities (11, 12), it is possible that these CD70 expression. Blocking experiments with the anti-CD70 gangliosides are also involved in the observed apoptosis of T cells. antibody confirmed that elevated CD70 levels were involved in We defined the mechanisms of GBM-induced T-cell death in the apoptogenicity of the three GBM lines expressing that coculture experiments using multiple cell lines. GBM-induced molecule. Gangliosides were found to participate in the apoptosis of T cells is most pronounced in activated T cells induction of T-cell apoptosis, because the glucosylceramide suggesting that the activation status of lymphocytes is an synthase inhibitor (PPPP) significantly reduced the abilities of important determinant of sensitivity to tumor-mediated apoptosis. all four apoptogenic lines to kill the lymphocytes. High- T-cell death induced by some GBM lines is partially receptor performance liquid chromatography (HPLC) and mass spec- dependent, involving CD70 expression by the tumor cells. CD70, the ligand for CD27, seems the predominant receptor-dependent troscopy revealed that GM2, GM2-like gangliosides, and GD1a were synthesized in abundance by all four apoptogenic GBM pathway involved in T-cell death by GBM lines. The addition of lines but not by the two GBMs lacking activity. Furthermore, anti-CD70 antibody to the T-cell tumor cultures partially blocked a gangliosides isolated from GBM lines as well as HPLC T-cell death, whereas neither the Fas fusion protein nor anti-TNF- fractions containing GM2 and GD1a were directly apoptogenic antibody had any significant protective effect. Furthermore, death for T cells. Our results indicate that CD70 and gangliosides are induced by both receptor-dependent GBM lines (CCF52, U87, D54, both products synthesized by GBMs that may be key mediators and U251) and the receptor-independent line (CCF4) involved the of T-cell apoptosis and likely contribute to the T-cell participation of tumor associated gangliosides. Blocking glyco- dysfunction observed within the tumor microenvironment. sphingolipid production with the glucosylceramide synthase inhibitor PPPP partially protected T cells from GBM-mediated (Cancer Res 2005; 65(12): 5428-38) apoptosis. Additionally, gangliosides isolated from the GBM lines independently induced apoptosis of T lymphocytes. Our results Introduction also indicate that some GBM tumor lines may induce T-cell Depressed T-cell function, resulting in ineffective antitumor apoptosis via a cooperative interaction between CD70 and immune response, is common among cancer patients (1). The gangliosides, because greater protection was observed when PPPP tumor microenvironment has a deleterious effect on tumor- and anti-CD70 antibody were given in combination. infiltrating T cells, and there is evidence that the immunosuppres- sive effects extend into the periphery as well (1). Even glioblastoma Materials and Methods multiforme (GBM) that develops and remains usually within the brain can induce local and systemic host immunosuppression (2, 3). Reagents. The inhibitor of glucosylceramide synthase, 1-phenyl-2- hexadeanoylamino-3-pyrrolidino-1-propanol (PPPP), was purchased from This impairment of the immune system, specifically when it affects Matreya, Inc. (Pleasant Gap, PA). Monoclonal anti-CD3 (OKT3, Ortho Biotech, Raritan, NJ) and anti-CD28 antibody (Becton Dickinson Immuno- cytometry Systems, San Jose, CA) were used for the stimulation of T Requests for reprints: James H. Finke, Department of Immunology, NB30, Lerner lymphocytes. Human recombinant interleukin-2 (200 units/mL; IL-2, Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195. Phone: 216-444- 5186; Fax: 216-444-9329; E-mail: [email protected]. [Proleukin], CHIRON Corp., Emeryville, CA) was used to maintain the I2005 American Association for Cancer Research. viability of activated T cells. Mouse monoclonal anti-human CD70 and Cancer Res 2005; 65: (12). June 15, 2005 5428 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. Glioblastoma Multiforme Induced T-Cell Apoptosis BU69 (Ancell, Bayport, MN) was used at a dilution of 10 Ag/mL for blocking HPLC system. A normal phase Varian Microsorb-NH2 column (25 cm, 100 A˚) CD70 ligand. The isotype control for the CD70 antibody was purified mouse was used as the stationary phase. Ganglioside separation was carried out immunoglobulin G (BD PharMingen, San Jose, CA). Human Fas:Fc chimeric with a gradient of the following solvent mixtures, used as the mobile phase: fusion protein (BD PharMingen) was used at 7.5 Ag/mL (optimal Solvent A, Acetonitrile/5 mmol/L phosphate buffer, pH 5.6 (83:17); Solvent concentration). Mouse P-Selectin-immunoglobulin G fusion protein (BD B, Acetonitrile/20 mmol/L phosphate buffer, pH 5.6 (50:50). The gradient PharMingen) was used as the control at 7.5 Ag/mL. Surface expression of elution program was the same as described earlier (17). The elution profile FasL was detected using anti-FasL antibody from BD PharMingen. Positive was monitored by flow-through detection of UV absorbance at 197 nm. The controls used for the efficiency of Fas:Fc to block apoptosis included anti- retention times of individual gangliosides were compared with the retention Fas activating antibody (Upstate, Charlottesville, VA) and supernatants from times of purified bovine brain–derived gangliosides (Matreya). ecotropic retroviral packaging cells (GP+E 86) carrying human FasL- Measurement of total lipid-bound sialic acid. The concentration of expressing retroviral particles (13). isolated ganglioside expressed as lipid bound sialic acid was determined as Established cell lines and tissue culture conditions. The well described previously (18). Ganglioside (50 AL) was extracted with chloro- characterized, long-term GBM cell lines: U87, U138, U251, and D54 form/methanol (2:1). Proteins are precipitated with phosphotungstic acid. were obtained courtesy of Dr. Vogelbaum (Cleveland Clinic Foundation, Precipitated ganglioside was dissolved in boiling resorcinol-HCL reagent. The Cleveland, OH). These lines were maintained in DMEM (Bio Whittaker, concentration of LSA is determined by the absorption at 580 nm. Walkersville, MD) supplemented with 10% fetal bovine serum Mass spectrometric analysis of gangliosides. Mass spectrometric (HyClone, Logan, UT), 2 mmol/L L-glutamine, 50 Ag/mL gentamicin. analysis of the isolated gangliosides were done using a Waters Alliance 2690 The Jurkat leukemic T-cell line (American Type Culture Collection, HPLC connected to a Micromass Quattro-Ultima triple quadruple electro- Rockville, MD) was maintained in complete RPMI 1640 (Bio Whittaker) spray tandem mass spectrometer (LC-ESI-MS-MS). A Supelco Supelcosil LC- supplemented as described above. All the cell lines were found free of NH2 column (7.5 cm  2.1 mm, 3 Am) was used as the stationary phase for Mycoplasma (ELISA, Roche Technologies). the HPLC. Samples were dissolved in 83% acetonitrile and injected into the Primary cell cultures. GBM specimens were obtained from our Brain HPLC with a Waters-Alliance-2690 separation module autosampler. The Tumor Registry after being reviewed by a pathologist. Classification of column resolves