Glioblastomas Induce T-Lymphocyte Death by Two Distinct Pathways Involving Gangliosides and CD70

Research Article

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

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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 Â 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 the ganglioside mixtures into separate components each of tumor type and grade was made in accordance with the WHO histologic which is then introduced into an electrospray needle at 0.3 mL/min. ESI-MS- typing. Single-cell suspension of GBM cells were obtained following MS spectra were taken using 0.1 mTorr argon as the collision gas at 60 eV. digestion as previously described (14). They were then seeded in flasks Analysis of T-cell apoptosis. T-cell death was assessed by staining and subcultured for two to four passages before being verified to be GBM lymphocytes with trypan blue and counting the number of viable as well as cells (14). Three short-term cultures (CCF3, CCF4, and CCF52) were the number of trypan blue positive cells. obtained from three different patients. Quantitation of DNA fragmentation in T cells was determined by the Expression of CD70, Fas ligand, and tumor necrosis factor-A by terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) glioblastoma multiforme lines. GBM lines were stained with anti-human assay. Fixed cells (1% paraformaldehyde) were stained and analyzed for CD70, anti-human FasL (BD PharMingen) or immunoglobulin G isotype apoptosis using the APO-BRDU kit system (Phoenix Flow Systems, San control for 60 minutes at 4jC. The cells were then incubated with a PE- Diego, CA; ref. 19). Percentages of apoptotic T cells were obtained using conjugated goat anti-mouse immunoglobulin G antibody for 30 minutes at quadrant analysis software (LYSIS II, Becton Dickinson). 4jC. The cells were fixed in 1% paraformaldehyde. At least 10,000 events Coculturing T cells with tumor cell lines or tumor-derived ganglio- were acquired on a FACScan (BD Biosciences, San Jose, CA) with a 488-nm sides. T cells or Jurkats were resuspended at a concentration of 2 Â 106 argon ion laser. Flow cytometric analysis was done using CellQuest v3.3 cells/mL in complete RPMI 1640 before being added to the 100 mm dishes software (BD Biosciences). containing GBM cells at a 1.5:1 tumor/T-cell target ratio. Following a 72- The GBM lines supernatants were harvested and assessed for TNF-a hour incubation period, the nonadherent T cells were removed from the protein using an ELISA kit (Pierce Biotechnology, Rockford, IL). Total RNA GBM monolayers and assessed for cell death by trypan blue and for was extracted from GBM and reverse transcription-PCR was done using apoptosis by TUNEL. 5.0 Ag RNA and the Taqman Reverse Transcription Reagents (Applied Blocking experiments were done with the addition of anti-CD70 Biosystems, Foster City, CA) and cycled on a Perkin-Elmer Cetus DNA antibody, anti-TNF-a, or Fas fusion protein at the time that T cells were thermal cycler. cDNA (2.0 AL) was used in the PCR reaction using SYBR incubated with the GBM monolayer. Activated T cells suspended at a green PCR core reagents (Applied Biosystems) with primers for TNF-a concentration of 1 Â 106 cells/mL in 1 mL of complete RPMI 1640 in 24-well (forward primer 5V-CTTCTCCTTCCTGATCGTGGC-3Vand reverse primer 5V- plates were treated with 7 to 10 Ag of isolated gangliosides (based on total GATGATCTGACTGCCTGGG-3V; in addition, forward primer 5V-CTCTCTG- sialic acid content). Seventy-two hours later, the T cells were assessed for CCATCAAGAGCCC-3Vand reverse primer 5V-CCGATTGATCTCAGCGCTG- cell death by trypan blue staining and the TUNEL assay. 3V). The control gene was b-GUS (forward primer: 5V-TCATTGGAGGTG- Inhibition of glucosylceramide synthase in glioblastoma multiforme CAGCTGAC-3V, h-Gus reverse primer: 5V-ACTGGCTCTTGGTGACAGCC-3V). cell lines by PPPP. The glucosylceramide synthase inhibitor, 1-phenyl-2- The cycle threshold (cT) values were used to calculate the fold differences hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP), was added to the of cytokine expression relative to the expression of the housekeeping gene, 50% confluent GBM cell lines to give the desired effective concentration of b-GUS. 1.0 Amol/L (20). Following a 5-day exposure of the GBM cells to PPPP, the T-cell isolation. T cells are isolated from peripheral blood first with drug was washed away, and the flask was replenished with fresh medium a ficoll-paque gradient (Amersham, Uppsala, Sweden) followed by nega- containing T cells. Gangliosides levels were evaluated by HPLC as previously tive selection using StemSep antibody cocktail (StemCell Technologies, described (21). Vancouver, British Columbia). Stimulation of T cells with cross-linked anti-CD3 antibody (OKT3) plus anti-CD28 antibody was done as previously described (15). The T cells were Results expanded for 2 to 3 weeks in the presence of 200 units/mL IL-2 before use. Glioblastoma multiforme cell lines induce T-lymphocyte Ganglioside isolation and profiling by high-performance liquid death that is most pronounced in activated cells. Glioma cells chromatography. A total lipid extraction of GBM cells was done using chloroform/methanol, and gangliosides were isolated from the lipids by a are likely responsible for the increase in lymphocyte death, because diisopropyl ether/1-butanol partition and subsequent Sephadex G-25 gel we show here that GBM cell lines can induce peripheral blood T- filtration step (16). cell death (Fig. 1). Four long-term and three primary glioma lines The water-soluble gangliosides were further fractionated by high- were cocultured with resting T lymphocytes for 72 hours before performance liquid chromatography (HPLC) using a Beckmann-Coulter assessing lymphocyte death. Four out of these seven GBM cell lines www.aacrjournals.org 5429 Cancer Res 2005; 65: (12). June 15, 2005

Similar results to those observed with cell viability staining were obtained when the readout was DNA breaks, a marker of apoptosis (data not shown). Role of death receptors in glioblastoma multiforme– induced T-cell death. We examined the role that different TNF receptor (TNFR) family members play in GBM-induced T-cell death. This included TNFR family members that have a death domain in their cytoplasmic tails (Fas and TNFR1) and hence initiate apoptosis via the recruitment of adaptor proteins followed by activation of caspases (22). TNF-a production by the GBM lines does not contribute to their apoptogenic activity, because none of the glioma lines expressed any detectable TNF-a mRNA or the secreted protein (data not shown). Furthermore, the addition of neutralizing antibody to TNF-a did not block the ability of GBM cells to induce apoptosis in T lymphocytes (data not shown); although this antibody did block T-cell death initiated by some renal cell carcinoma lines.4 These findings suggest that TNF-a does not play a significant role in the ability of the GBM lines used here to induce T-cell killing. The role of FasL in GBM-induced apoptosis of T cells was also examined, because activated T cells express Fas/APO-1 (23) and some glioma lines express FasL (24). Staining with anti-FasL antibody, followed by flow cytometry analysis, a significant number of CCF52 (48%), U87 (27%), and D54 (95%) cells, which all induce T-cell death, expressed FasL (Fig. 2A). The nonapoptogenic GBM line, U138, and one of the apoptogenic GBM lines, CCF4, only stained weakly for FasL (10% and 5% positive cells, respectively; Fig. 2A). Addition of the inhibitory Fas fusion protein had no effect on the ability of CCF4 to induce T-cell death, because this line expressed very little FasL (Fig. 2B). However, the Fas fusion protein only had a modest ability to inhibit T-cell death initiated by the GBM lines (CCF52, 19% inhibition; U87, 19% and D54, 28%) in which a high percentage expressed FasL (Fig. 2B). We found similar results with Jurkats (data not shown). The effectiveness of Fas:Fc to block (60%) T-cell death induced by anti-Fas antibody and by soluble FasL but not GBM-induced cell death suggests that Figure 1. GBM lines can induce T-cell death. A, seven GBM cell lines were cocultured with resting peripheral blood T cells from normal donors. After 72 GBM induced cell death is mostly FasL independent (Fig. 2B). hours, the nonadherent T cells were removed from the tumor monolayer Role of CD70 ligand in glioblastoma multiforme–induced and tested for cell viability. Four of the cell lines (CCF4, CCF52, U87, and D54) T-cell death. CD27 is a TNFR family member that can mediate induced 17.6% to 33% death. Columns, mean of four or more separate experiments; bars, FSD. B, seven GBM cell lines are cocultured with activated apoptosis when activated by its cell surface ligand, CD70. Unlike T cells for 72 hours before assessing lymphocyte viability. Five of the cell lines TNFR1 and Fas receptor, CD27 lacks the cytoplasmic death domain (CCF4, CCF52, U87, U251, and D54) induced 38.6% to 54.5% death of activated (25). Instead, CD70 transduces its apoptotic signal through a T cells. Columns, mean of five or more separate experiments; bars, FSD. cytoplasmic protein, Siva, which initiates apoptosis by mediating mitochondrial damage and caspase activation (26). Here we show (U87, D54, CCF4, and CCF52) induced T-cell death, whereas, U251, that three of the apoptogenic GBM lines had a high percentage of U138, and CCF3 were ineffective (<10% cell death; Fig. 1A). On CD70-positive cells, CCF52 (40%), U87 (47%), and D54 (46%; average CCF4 (n = 4) caused 33% T-cell death, CCF52 (n = 4) 24%, Fig. 2C), whereas another apoptogenic line, CCF4, had a low U87 (n = 6) 22.4%, and D54 (n = 6) 17.6% (Fig. 1A). percentage (10%) of cells expressing CD70. The two nonapopto- It was determined that activated T cells are even more genic tumor lines (CCF3 and U138) had minimal CD70 expression sensitive than resting T cells to GBM-mediated T-cell killing. (f1%). CD70 expressed by the tumor lines did contribute to T-cell The heightened sensitivity of activated T cells to apoptosis death, because T cells cocultured with the CD70-positive GBM cell would be counter productive to the development of an lines, CCF52, U87, and D54, were partially protected from apoptosis effective antitumor immune response. Indeed, a high percent- when anti-CD70 antibody was added to the cultures (40%, 58%, 60% age of tumor infiltrating T cells express activation markers and respectively; Fig. 2D). Mouse immunoglobulin G was used as a are most sensitive to apoptosis (1). As illustrated in Fig. 1B, negative control and had no inhibitory effect. T-cell death induced the four cell lines that induced cell death in resting T by CCF4, however, was minimally affected by anti-CD70 antibody cells showed even greater ability to kill activated T cells: CCF4 (n = 5) 54.5%, CCF52 (n = 5) 38.6%, U87 (n = 6) 53%, and D54 (n = 6) 40.8%. Interestingly, the U251 cell line that did not induce cell death in resting T cells (10% cell death) had significant activity against activated T cells (35.5%, n = 3). 4 Hilston et al., submitted for publication.

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Figure 2. CD70 but not FasL expression on GBM lines is important for inducing T-cell death. A, all four apoptogenic GBM lines and one nonapoptogenic line were assessed for surface expression of FasL by flow analysis following staining with anti-FasL antibody. CCF52, U87, and D54, which all induce T-cell death, stained positive for FasL, 48%, 27%, 95%, respectively, whereas CCF4 and the nonapoptogenic line U138 only showed low level of FasL staining. B, five GBM cell lines were cocultured with activated T cells for 72 hours in the presence of Fas fusion protein (blocking agent), fusion control, or medium alone. To show the efficiency of Fas-Fc, two positive controls, anti-Fas activating antibody, and supernatants from ecotropic retroviral packaging cells (GP + E 86) carrying human FasL-expressing retroviral particles (13) were used. Fas fusion protein only blocked cell death of activated T cells by 19% in CCF52 and U87 and 28% in D54. It had no effect with CCF4 or U138. Representative data. C, GBM lines were stained with anti-CD70 antibody and the percentage of CD70-positive cells was assessed by flow cytometry. Three of the four cell lines that induce T-cell death had high percentage of CD70 (CCF52 40%, U87 47%, and D54 46%). Only 10% of the apoptogenic line, CCF4 expressed CD70. D, the same GBM cell lines were cocultured for 72 hours with activated T cells in the presence of CD70 blocking antibody, immunoglobulin G control, or medium alone. Thereafter, cell viability was determined by trypan blue staining. CD70 antibody showed partial protection against T-cell death with three of the apoptogenic lines (40% block with CCF52, 58% with U87, and 60% with U87) Only 15% protection was observed with CCF4 cells when incubated with anti-CD70 antibody. Similar results were observed in a second experiment.

(15%; Fig. 2D) as predicted, because only a low percentage (10%) of cell lines pretreated with PPPP (CCF4, CCF52, U87, and D54) were CCF 4 cells express CD70. significantly less effective at inducing T-cell apoptosis as compared Gangliosides expressed by glioblastoma multiforme lines with untreated GBM lines (Fig. 3B). These show that PPPP has an contribute to tumor-induced T-cell death. Additional experi- inhibitory effect on glycosphingolipids synthesis in GBM cell lines, ments determined whether gangliosides expressed by the GBM which consequently reduces their ability to kill T cells. lines are involved in T-cell killing. To address this possibility, we The importance of GBM-derived gangliosides in T-cell killing is tested whether reducing the expression of glycosphingolipids by further highlighted by the demonstration that gangliosides isolated GBM lines would impair their ability to induce T-cell apoptosis. from the tumor lines and their supernatants were sufficient to TheGBMlinesweretreatedwithPPPP,aninhibitorof induce cell death of activated T cells following 72 hours of glucosylceramide synthase, for 5 days before adding T cells for an treatment. Gangliosides isolated from the apoptogenic GBM lines additional 72 hours (21). PPPP pretreatment of U87 cells caused a and supernatants, but not the nonapoptogenic line, caused T-cell 50% to 60% reduction in expression of GM3, GM2 GD3, and GD1a death ranging anywhere from 52% to 80% (with the apoptogenic gangliosides as determined by HPLC analysis (Fig. 3A). A similar lines’ gangliosides; Fig. 4A) and 44% to 48% (with the supernatants’ decrease in gangliosides expression by the other tumor lines gangliosides; Fig. 4B). These gangliosides also induced DNA breaks cultured with PPPP was noted (data not shown). Pretreatment of as determined by the TUNEL assay, to an extent that varied GBM cell lines with PPPP did not alter the growth or the viability of depending on the cell type (CCF4, 29%; CCF52, 81%; U87, 35%; and the tumor cells. Five days after culturing 300,000 cells, the number D54, 31%; Fig. 4A). The concentration of gangliosides isolated from of viable cells recovered from medium and PPPP-treated cells, the GBM lines was determined by measuring total lipid bound respectively, was CCF4 1.93 Â 106 and 1.69 Â 106, CCF52 3.2 Â 106 sialic acid (18), and they ranged from 7 to 10 Ag/mL. Based on the and 3.5 Â 106, U87 2.59 Â 106 and 2.49 Â 106, U138 2.46 Â 106 and above findings, we propose that gangliosides are a second class of 2.19 Â 106, and D54 2.75 Â 106 and 3.058 Â 106. However, tumor molecules involved in the GBM-induced T-cell killing. www.aacrjournals.org 5431 Cancer Res 2005; 65: (12). June 15, 2005

HPLC analysis revealed that the gangliosides isolated from the U87 cell lines were subjected to HPLC separation, and individual different GBM lines consist of a mixture of gangliosides that vary in fractions eluting off the column were tested for their induction of composition (Fig. 3A and Fig. 4C). Based on the elution times of T-cell killing using activated T cells. Similar results were observed standard bovine brain–derived gangliosides off the HPLC column, with gangliosides fractionated from both CCF52 and U87 showing it was determined that the gangliosides isolated from the two major peaks of apoptotic activity. The initial peak coeluted off apoptogenic GBM lines (CCF4, CCF52, U87, and D54) were the HPLC column with a retention time similar to the bovine brain enriched in GM2 and GD1a compared with those isolated from standard GM2, whereas the other peak coeluted with a retention the nonapoptogenic tumor lines (CCF3 and U138). Furthermore, time near GD1a and GD3 (Fig. 5A). Mass spectroscopy employing CCF4 showed increased levels of GM1 (data not shown) when parental scan showed that the masses for GM2 (Fig. 5B) and GD1a/ compared with other isolated GBM gangliosides. On the other GD3 (data not shown) are present in the first and second peaks, hand, GD3 levels were relatively high in all of the GBM lines tested, respectively. ESI-MS-MS analysis was used to determine if there is including the nonapoptogenic lines. The apoptogenic lines, unlike more than one ganglioside in the HPLC fraction that contained the nonapoptogenic lines, also contained additional gangliosides GM2 by noting if there were several parent ion peaks that yield the that did not coelute with the purified standard brain-derived characteristic daughter ion mass of N-acetylneuraminic acid gangliosides (Fig. 3A and Fig. 4C). HPLC with online LC-ESI-MS-MS (NeuAc, 291.09 MW). Indeed, we did detect the masses of GM2 analyses employing parental scan confirmed that gangliosides (GM2 masses 1,382 and 1,410). The parental scan also detected expressed by CCF52 included GM2, ganglioside coeluting with ganglioside masses distinct from GM2 but that elute with GM2: GM2, GD1a, and GD3 (Table 1). We also analyzed the gangliosides 1,466, 1,465, 1,371, 1,354, and 1,352 (Fig. 5B). shed by CCF52 with mass spectroscopy and found that they were Based on the result that fractions with the most T-cell killing mostly comprised of several molecular weights (MW) of GM2 and contained multiple gangliosides including GM2, GD1a, and GD3, several gangliosides coeluting with GM2. However, the shed we wanted to determine if combination of gangliosides would gangliosides had no detectable GD1a and GD3 which was observed improve the induction of T-cell apoptosis. Therefore, we tested in the gangliosides isolated from the CCF52 cells (Table 1). purified bovine gangliosides individually and in combination to To address which gangliosides expressed by the GBM are capable assess T-cell killing effect. Activated T cells were treated with of inducing T-cell apoptosis, gangliosides isolated from CCF52 and purified bovine gangliosides GM1, GM2, GM3, GD1a, and GD3. All

Figure 3. A reduction in glycosphingolipid synthesis by the GBM lines partially blocked their ability to induce T-cell death. A, the U87 cell line was incubated with the glucosylceramide synthase inhibitor PPPP (1 Amol/L) for 5 days. Thereafter, HPLC analysis was performed on gangliosides extracted from the same number of U87 cells (4 Â 107) pretreated or not with PPPP. The retention time of gangliosides isolated from U87 was compared with that of purified bovine brain derived ganglioside run in the same experiment. PPPP treatment caused a 2-fold reduction in ganglioside expression by U87, including GM3, GM2, GD3, and GD1a when compared with untreated U87. B, activated T-cell were cocultured with GBM cell lines treated or not with PPPP (for 5 days) and assessed for tumor-induced cell death by trypan blue staining. T-cell death was reduced by 40% when lymphocytes were cocultured with CCF4, CCF52, and D54 cells pretreated with PPPP and 50% when cocultured with PPPP treated U87. Columns, mean of three different experiments; bars, FSD.

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Figure 4. Gangliosides isolated from GBM lines induced T-cell death, with the apoptogenic GBM lines expressing elevated levels of select gangliosides compared with the nonapoptogenic GBM lines. Activated T cells were incubated with (A) isolated gangliosides from five GBM cell lines or with (B) isolated gangliosides from GBM supernatant. After 72 hours, T cells were harvested and assessed for apoptosis by staining with trypan blue and by TUNEL. Gangliosides isolated from all the apoptogenic GBM lines or their supernatant but not the nonapoptogenic line caused T-cell death anywhere from 44% to 80% by trypan blue staining and 29% to 81% by TUNEL. The final concentration of isolated ganglioside’s lipid-bound sialic acid in this experiment ranged from 7 to 10 Ag/mL. C, gangliosides isolated from an apoptogenic line (CCF52) and a nonapoptogenic tumor line (CCF3) were then subjected to HPLC fractionation. The retention times of gangliosides derived from the GBM were compared with the retention times of purified bovine brain gangliosides. HPLC analysis revealed that the gangliosides isolated from the apoptogenic GBM line CCF52 showed increased levels of GM2 and GD1a when compared with those isolated from the nonapoptogenic tumor line CCF3. Similar increases in GM2 and GD1a expression were observed with gangliosides isolated from other apoptogenic GBM lines, (CCF4, U87, and D54) but not from the nonapoptogenic line U138. It was noted that GD3 was expressed to similar levels by all of the GBM lines. www.aacrjournals.org 5433 Cancer Res 2005; 65: (12). June 15, 2005

Table 1. Gangliosides expressed by tumor versus shed some functions following removal of the primary tumor, although they lose it again upon recurrence (37). These findings suggest that Gangliosides expressed Gangliosides shed soluble products secreted by gliomas contribute to the lymphocyte c by CCF52 (mass)* by CCF52 (mass)*, dysfunction observed within the tumor and peripheral blood. The immunosuppression initiated by the high-grade gliomas Shared GM2: 1,382 and 1,398 GM2: 1,382 and 1,398 may be a barrier to the development of an effective immune Coeluting with GM2: Coeluting with GM2: response to GBM immunotherapy. Our findings reveal that many 1,354, 1,371, 1,354, 1,371, but not all GBM lines can induce apoptosis in peripheral blood T and 1,468 and 1,468 cells from healthy individuals and that activated T lymphocytes are Unique GD1a: 790, 930, and 918 significantly more susceptible to tumor-induced apoptosis than GM2: 1,412 resting cells. Most relevant is the observation that the GBM lines GD3: 1,441, 763, and 770 we tested kill T cells by two different mechanisms. One involves a receptor-mediated pathway of apoptosis that is initiated by the *Isolated gangliosides were subjected to mass spectroscopy. ligand CD70 expressed on some of the GBM lines. The other cGangliosides were isolated from supernantant-derived CCF52 col- pathway is receptor independent and is mediated by select lected on day 7. Supernatant fluid from CCF52 collected on the third, gangliosides expressed on the GBM lines. Interestingly, three of fifth, seventh, but not first day induced T-cell death. the GBM lines induced T-cell death by both mechanisms, whereas another line induced death through a receptor-independent ganglioside-mediated pathway. but GM3 were capable to induce T-cell killing at high Several different molecules have been proposed to mediate concentration (150 Ag/mL; Fig. 5C) but none at low concentration immune escape by human tumors including GBMs. FasL has been (40 Ag/mL; Fig. 5D). However, when GM1, GM2, GD1a, and GD3 reported to be expressed by various histologically distinct tumors were used in combination at low concentration (10 Ag/mL each and thereby can induce apoptosis of FasR-positive T cells (38–40). for a total of 40 Ag/mL) a synergistic effect in T-cell killing was However, this mechanism remains controversial due to conflicting observed (Fig. 5D). data regarding FasL expression on some tumor types (41). For some GBM lines (CCF52, U87, and D54), the ability to induce Furthermore, it has been suggested that T-cell apoptosis may not T-cell death involves both CD70 and gangliosides expressed by the be initiated by FasL-positive tumors in coculture experiments but tumors. Whether blocking CD70 function and ganglioside expres- may result from FasL-expressing T cells interacting with FasR- sion together would provide better protection from GBM-induced expressing T cells (41). Other groups have described FasL apoptosis than blocking either one alone was examined. The GBM expression on high-grade glioma specimens (5, 24), GBM cell lines lines were pretreated with PPPP for 5 days before adding anti-CD70 (42), and GBM tissue endothelial cells (43). Indeed, we observed antibody and activated T cells to the tumor monolayer. As varying levels of FasL on three of the apoptogenic GBM lines previously shown, pretreatment of GBM lines with either PPPP (CCF52, U87, and D54). However, Fas fusion protein only blocked a or the addition of CD70 antibody partially blocked T-cell death. For modest amount of T-cell death, suggesting that for these GBM cell the three lines that expressed CD70, blocking both CD70 function lines, FasL expression is not a major mechanism of tumor escape. and ganglioside synthesis provided greater protection than block- Likewise, TNF-a does not seem involved in the GBM-induced ing either one alone. This combined effect seems additive. As apoptosis of T cells, although under certain conditions, it can expected, there was little protective value observed by combining mediate apoptosis via the TNFRI (44). In fact, none of our GBM cell PPPP and anti-CD70 antibody in coculture experiments with the lines expressed either TNF-a mRNA or protein, which is not CD70-negative GBM line (CCF4; Fig. 6). surprising because gliomas have been found to secrete TNF-a only upon stimulation (45). Furthermore, the addition of anti-TNF-a neutralizing antibody did not block T-cell death induced by GBM Discussion lines. One role of T cells is to recognize and respond to neoplastic cells One mechanism by which some GBM cell lines can kill T cells is anywhere in the body including the central nervous system (27–29). through their expression of CD70, a surface protein that is the Immune effector cells do enter the brain in response to molecules ligand for CD27, a member of the TNFR family (46). CD70 is expressed by the endothelial cells and astrocytomas (30). Even when normally expressed on activated B and T lymphocytes, whereas the immune system is stimulated in the periphery, immune effector the CD27 receptor is constitutively expressed on both resting and mechanisms are activated in the brain as well (31). Furthermore, activated lymphocytes (47). The interaction of CD70 with CD27 between 30% to 60% of gliomas are infiltrated with inflammatory can provide a costimulatory signal important for T-cell activation cells (28, 32) with a predominance of CD8+ T cells (33). Whereas (48). However, CD70/CD27 interaction can also lead to T-cell lymphocyte infiltration does occur in gliomas (27–29), a significant apoptosis, probably due to persistent stimulation (49, 50). We now number of these immune cells undergo apoptosis (5, 34). Thus, show that CD70 expressed on some GBM lines can also induce tumor progression occurs despite lymphocyte infiltration, likely due death of T lymphocytes as shown by the observation that anti- to tumor-induced immune dysfunction (27). CD70 antibody when added to cocultures partially blocked T-cell Patients with gliomas are characterized by systemic immune killing. Our results are similar to a recent report (50) which dysfunction, as shown by impaired cell-mediated immunity, showed immune escape of high-grade gliomas via the interactions lymphopenia (2, 3, 6), and inability to mount delayed-type of CD70 ligand expressed on glioma cells and CD27 expressed on hypersensitivity reaction (3). The degree of immunosuppression T cells to induce lymphocyte apoptosis. The one difference observed in glioma patients may correlate with tumor size and between our findings and those of Wischhusen et al. (50) was with grade (35, 36). Interestingly, peripheral T cells are observed to regain the U138 cell line that was CD70 positive and apoptogenic in their

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Figure 5. HPLC fractionation of CCF52 and U87 gangliosides revealed two distinct peaks of apoptogenic activity. A, gangliosides isolated from CCF52 and U87 cell lines were subjected to HPLC separation and individual fractions were tested for their induction of apoptosis using activated T cells (following a 72-hour incubation period). Both CCF52 and U87 fractionation showed two major peaks of T-cell killing activity. The initial peak had a retention time similar to the bovine brain standard GM2, while the other peak had a retention time near GD1a and GD3. All fractions and the crude gangliosides have the same amount of ganglioside (7 Ag/mL). B, GM2 and other gangliosides coeluting with GM2 are present in the first apoptogenic peak following HPLC fractionation of the GBM ganglioside CCF52. HPLC with online LC-ESI-MS-MS analyses, employing parent scan, was used to define the gangliosides masses present in the first peak that had apoptogenic activity against T cells (fraction 6). The parent scan (inset) showed that this HPLC fraction 6 contains several parent ion peaks including two that are characteristic of GM2 (GM2 masses 1,382 and 1,410). Additionally, this fraction contained five predominant masses that correspond to gangliosides distinct from GM2 but that coelute with GM2 and have the masses of 1,466, 1,465, 1,371, 1,354, and 1,352. C, GM1, GM2, GD1a, and GD3 purified bovine gangliosides but not GM3 induce 60% to 70% T-cell killing at 150 Ag/mL. D, at lower concentration (40 Ag/mL), individual gangliosides are incapable of killing T cells; whereas in combination (10 Ag/mL each to make a total of 40 Ag/mL), they can induce 30% T-cell death. www.aacrjournals.org 5435 Cancer Res 2005; 65: (12). June 15, 2005

Figure 6. For GBM lines CCF52, U87, and D54, both CD70 and gangliosides cooperate to induce T-cell death. Activated T cells were cocultured with GBM cell lines with or without anti-CD70 blocking antibody (10 Ag/mL), glucosylceramide synthase inhibitor PPPP (1 Amol/L), or a combination of anti-CD70 and PPPP. GBM lines were pretreated with PPPP for 5 days before adding anti-CD70 blocking antibody and activated T cells to the GBM monolayer. Pretreatment of GBM lines with either PPPP or the addition of CD70 antibody partially blocked T-cell death. In the three lines that expressed high CD70 (CCF52, U87, and D54), combined treatment of anti-CD70 and PPPP provided greater protection than blocking either one alone.

study but CD70 negative and nonapoptogenic in our study. This gangliosides coeluted with GM2. Analysis of isolated gangliosides of difference may be because they irradiated the U138 lines before CCF52 supernatant by mass spectrometry also showed high coculture with T cells, which is known to up-regulate CD70 on the concentration of GM2 and several other gangliosides coeluting GBM cells and would make the tumor line more apoptogenic for with GM2. Using commercial bovine gangliosides, we determined lymphocytes (50). that GM1, GM2, GD1a, and GD3 induced T-cell death at Although the signaling events stimulated by CD70-positive GBM high concentration (150 Ag/mL) but not at low concentration cells have not been well defined, it likely involves the activation of (40 Ag/mL). However, when combined, they achieved a synergistic the proapoptotic protein Siva known to be associated with CD27 effect and an appreciable killing of T cells at 40 Ag/mL. This finding (25). Interestingly, our study and the one by Wischhusen et al. (50) supports our HPLC and mass spectroscopy results, suggesting that both showed that treatment of glioma cells with anti-CD70 multiple gangliosides contribute to T-cell death induced by GBM antibody provided only partial protection (f50% reduction), lines. It was also shown that, in the HPLC fractionation data, the leaving open the possibility that other tumor-derived products two peaks with apoptogenic activity were not as potent as the may be involved in GBM-induced T-cell death. unfractionated gangliosides isolated from CCF52 suggesting The data presented here indicate that gangliosides expressed by maximal T cell killing is the result of multiple gangliosides. It may the apoptogenic GBM lines can also mediate T-cell death be that the inability of gangliosides isolated from CCF3 (10 Ag/mL) independently of CD70. We initially showed that the glucosylcer- to induce apoptosis is due to the fact that CCF3 expressed less amide synthase inhibitor, PPPP, blocked ganglioside expression on GM2 (plus GM2 like) and GD1a than the apoptogenic CCF52 line GBM lines by 50% and comparably reduced their ability to induce T- although both had similar amounts of GD3. The fact that the cell death (50%), suggesting that gangliosides play a significant role isolated GBM gangliosides (containing multiple gangliosides) were in T-cell killing. Because PPPP additionally blocks the synthesis of able to kill at much lower concentrations (7-10 Ag/mL) than the other glycosphingolipids, it was not possible to conclude from these individual purified bovine brain–derived gangliosides further experiments that gangliosides were the major lipid involved in T-cell suggest that there is cooperation among individual gangliosides death. Direct involvement of gangliosides in T-cell killing was to induce potent apoptogenic activity. supported by the observation that gangliosides isolated from Gangliosides are overproduced in a number of different tumor apoptogenic tumor lines and their supernatants were able to induce types including gliomas (51–53). Gangliosides are known to be shed apoptosis in T cells at relatively low concentrations. As expected, from tumors and can be detected in the peripheral blood of cancer gangliosides isolated from the two nonapoptogenic tumor lines and patients. Elevated GD2 levels in neuroblastoma patients seemed to their supernatants did not induce significant T-cell killing, and their correlate with tumor progression and a lower survival rate (54). HPLC analysis showed reduced expression of several ganglioside Although gangliosides play important roles in cell differentiation, when compared with the apoptogenic GBM lines (Fig. 3A and Fig. 4C). cell adhesion (55), and modulation of receptor function (56) and This reduction in ganglioside expression by the nonapoptogenic lines participate in tumor progression (57), they also inhibit host included those with retention times of GM2 and GD1a. immunity (12, 21). Administration of tumor-derived GM1b Analysis of individual HPLC fractions from two apoptogenic inhibited the in vivo development of antitumor immune responses GBM lines (CCF52 and U87) identified two distinct peaks with and promoted malignant growth (12). Gangliosides isolated from significant T-cell death activity. One peak had a retention time different tumors are known to inhibit immune responses (21, 58), similar to bovine brain–derived ganglioside GM2, whereas the which may be related to their capacity to suppress T-cell other peak had a retention time similar to GD1a and GD3. Mass proliferation and Th-1 cytokine production in vitro (15, 59). spectrometry analysis of the first peak did reveal that the mass for Gangliosides have also been shown to induce apoptosis in various GM2 was present but it also showed that several other unidentified cell types including lymphocytes (15).

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GBM tumors are known to either overexpress or synthesize associated CD70 and gangliosides expressed and shed by GBM, modified forms of GD3, GD2, GM1, GM2, 3Viso-LM1, and 3V6V- both mediate T-cell apoptosis, providing the glioblastomas isoLD1 (8, 10, 53, 60). Others have reported that gangliosides mechanisms by which they both evade host immunity and hence (mostly GD3 and GM3) are shed into the serum of GBM patients can continue their progressive growth. (8, 61). This shedding of gangliosides by GBMs may partly explain lymphopenia, apoptosis, and depressed T-cell function in the Acknowledgments periphery of these patients (3, 62, 63). Although the responsible Received 12/8/2004; revised 3/15/2005; accepted 4/7/2005. agents were not defined in these studies, it was proposed that Grant support: NIH grants RO1-CA56937, CA90995, and CA87644 and the perhaps unknown or known molecules such as transforming American Brain Tumor Association award (A. Chahlavi). h The costs of publication of this article were defrayed in part by the payment of page growth factor- 1,2, prostaglandin E2, IL-10, or even other unknown charges. This article must therefore be hereby marked advertisement in accordance molecules were involved (64–66). Our findings suggest that tumor- with 18 U.S.C. Section 1734 solely to indicate this fact.

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