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[Cancer Research 66, 3852-3858, April 1, 2006]
© 2006 American Association for Cancer Research

Immunology

Regeneration and Tolerance Factor: A Novel Mediator of Glioblastoma-Associated Immunosuppression

Patrick Roth1, Steffen Aulwurm1, Isabella Gekel1, Dagmar Beier1, Roxanne G. Sperry3, Michel Mittelbronn2, Richard Meyermann2, Kenneth D. Beaman3, Michael Weller1 and Jörg Wischhusen1

1 Laboratory of Molecular Neuro-Oncology, Department of General Neurology, Hertie Institute for Clinical Brain Research, and 2 Institute for Brain Research, University of Tübingen, Medical School, Tübingen, Germany and 3 Clinical Immunology Laboratory, Department of Microbiology/Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois

Requests for reprints: Patrick Roth, Department of General Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, Medical School, Hoppe-Seyler-Strasse 3, 72076 Tübingen, Germany. Phone: 49-7071-2981960; Fax: 49-7071-295742; E-mail: patrick.roth{at}uni-tuebingen.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Regeneration and tolerance factor (RTF) was originally identified in placenta where it is thought to be essential for fetal allograft survival. Here we report that RTF mRNA and protein are also expressed in human glioma cells in vitro and in vivo. Suppression of RTF expression by RNA interference promotes the lysis of glioma cells by natural killer (NK) and T cells in vitro. Moreover, RTF-depleted glioma cells are less tumorigenic than control cells in nude mice in vivo. Depletion of NK cells in these animals abolished this effect. RTF is thus a novel aberrantly expressed molecule which confers immune privilege to human malignant gliomas. (Cancer Res 2006; 66(7): 3852-8)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human glioblastoma is a highly lethal tumor that is paradigmatic for tumor-dependent immunosuppression (1, 2). Human glioma patients exhibit distinct deficits in cellular immunoreactivity. Despite the highly malignant cellular phenotype of glioma cells, these tumors rarely metastasize outside the brain but kill patients by locally destructive growth in the central nervous system. Intriguingly, several case reports of glioblastomas developing in recipients of peripheral organ transplants (3) illustrate that some glioma cells leave the brain and populate the periphery. Because such cells are incapable of forming clinically apparent metastases, they are possibly eliminated by the immune system in the periphery but not in the immunoprivileged site of their origin, the brain.

We have previously shown that glioblastoma cells express activatory natural killer (NK) and costimulatory T-cell ligands (4). However, inhibitory signals, notably transforming growth factor-ß (TGF-ß; refs. 5, 6) but also interleukin (IL)-10 (7), HLA-G (8), and B7-H1 (9), seem to dominate tumor-host interactions in vivo. More effective antitumor responses might be induced either by providing additional immune stimulation or by antagonizing tumor-derived immune inhibition.

Regeneration and tolerance factor (RTF; alternative name: TJ6) is a 70-kDa cell-surface protein first identified in peripheral cytotrophoblasts of early placentas (7-9 weeks; ref. 10). However, RTF transcripts have recently also been described in a broad spectrum of tissues with levels being lowest in brain and spinal cord (11). RTF is cleaved to yield a membrane-bound 50-kDa protein and a secreted, biologically active 20-kDa fragment (soluble RTF). Soluble RTF up-regulates the production of IL-10 and interferes with IL-2 signaling in peripheral blood mononuclear cells (PBMC) stimulated with anti-CD3 antibody (12), thus shifting the balance towards a Th2 T-cell response. RTF is physiologically expressed in activated PBMC. Because neutralizing RTF antibodies induce apoptosis in activated T cells (13), RTF may limit T-cell activation and thus prevent activation-induced cell death. A tolerogenic function of RTF is strongly supported by the reversal of fetal semi-allograft tolerance in mice treated with a monoclonal antibody (mAb) to RTF/TJ6. This treatment completely ablates pregnancy at early stages (14). An aberrant expression of membrane-bound RTF has been described in B-cell lymphomas (15) but not yet in solid tumors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and reagents. The human glioma cell lines, kindly provided by Dr. N. de Tribolet (Lausanne, Switzerland), have previously been characterized (16). The LN-229 cells used in this study have, unlike LN-229 cells grown in other laboratories, retained wild-type p53 function and were therefore renamed LNT-229 for clarification (T for Tübingen; ref. 17). Primary glioblastoma cells were established from freshly resected tumors, cultured in monolayers, and used between passages 4 and 9 (18). The cells were maintained in DMEM containing 10% FCS (Biochrom KG, Berlin, Germany) and penicillin (100 IU/mL)/streptomycin (100 µg/mL; Life Technologies, Inc., Karlsruhe, Germany). RPMI 8866 cells were cultured in RPMI 1640 containing 10% FCS, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, and antibiotics. The pSUPER plasmid was obtained from R. Agami (Amsterdam, the Netherlands). A puromycin cassette was inserted into the NaeI site. The RTF-specific oligonucleotide sequences GATCCCCCATCGTGGATGCTTATGGAttcaagagaTCCATAAGCATCCACGATGTTTTTGGAAA and TCGATTTCCAAAAACATCGTGGATGCTTATGGAtctcttgaaTCCATAAGCATCCACGATGGGG [nucleotides (nt) 1,148-1,166], GATCCCCGGCCATCTATCACATGCTGttcaagagaCAGCATGTGATAGATGGCCTTTTTGGAAA and TCGATTTCCAAAAAGGCCATCTATCACATGCTGtctcttgaaCAGCATGTGATAGATGGCCGGG (nt 923-941) were obtained from Metabion (Munich, Germany) and cloned into the BglII and SalI sites of pSUPER. The RTF-specific parts of the sequences are underlined. For the generation of stable siRTF transfectants, pSUPERpuro control or siRTF plasmids were introduced using FuGene6 transfection reagent (Roche, Mannheim, Germany). The cells were selected in medium containing 2 µg/mL puromycin (Sigma, Deisenhofen, Germany). For transient transfections, 2 x 105 LN-308 cells were seeded in a six-well plate. Twenty-four hours later, they were transfected with 10 nmol/L of either RTF siRNA, 5'-CAUCGUGGAUGCUUAUGGA(dTdT)-3' and 5'-UCCAUAAGCAUCCACGAUG(dTdT)-3', or irrelevant GL3 control siRNA, 5'-CUUACGCUGAGUACUUCGA(dTdT)-3' and 5'-UCGAAGUACUCAGCGUAAG(dTdT)-3', using TransIT-TKO Transfection Reagent (Mirus, Madison, WI). Cells were analyzed and used for functional assays 72 hours posttransfection. Anti-RTF antibody was prepared as described (19). Immunoglobulin G1 isotype–matched antibody was used as a control (BD PharMingen, Heidelberg, Germany). MHC class I–specific mAb clone W6/32 was purchased from Sigma. Rat anti-mouse Dx5 was from Caltag (Burlingame, CA); goat anti-mammalian ß-actin (I-19) was from Santa Cruz Biotechnology (Santa Cruz, CA); and rabbit anti-asialo GM1 was purchased from Wako Chemicals (Duesseldorf, Germany).

Real-time and semiquantitative reverse transcription-PCR. Total RNA was prepared using the RNeasy system (Qiagen, Hilden, Germany) and transcribed according to standard protocols. The conditions for all standard PCR were 5 min/95°C, 34 cycles 95°C/40 seconds, 1 min/57°C for RTF or 50.5°C for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 1 min/72°C with a final extension at 72°C for 10 minutes. The following primers were used: 5'-ACGACAGTCCATGCCATCAC-3' (nt 4,209-4,228 of human GAPDH), 5'-TCCACCACCCTGTTCCTGTA-3' (nt 4,761-4,742), 5'-TGAATCCCTTGAAGACCCTG-3' (nt 629-648 of human RTF cDNA), and 5'-GTGTAGAGATCCTGGATGCGG-3' (nt 829-809), yielding 553- and 201-bp fragments, respectively. For real-time PCR, cDNA amplification was monitored using SYBR Green chemistry on the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Weiterstadt, Germany). The conditions for these PCR reactions were 40 cycles, 95°C/15 seconds, 60°C/min, using the following specific primers: 18S up, 5'-CGGCTACCACATCCAAGGAA-3' (nt 450-469); 18S down, 5'-GCTGGAATTACCGCGGCT-3' (nt 636-619); RTF up, 5'-GCTGGAACTGATAGAGTACAC-3' (nt 371-391); RTF down, 5'-GTAATCCAACAAAGAATCGCTC-3' (nt 494-473). Data analysis was done using the {Delta}{Delta}CT method for relative quantification. Briefly, threshold cycles (CT) for 18S rRNA (reference) and RTF (sample) were determined in duplicates. We chose normal brain cDNA as calibrator tissue (100%) and determined the relative change (rI) in copy numbers according to the formula rI = 2–[(CTRTF normal brain – CT18S normal brain) – (CCTRTF glioma – CCT18S glioma)].

Immunoblot. Cellular soluble proteins (10 µg/lane) were separated on 8% acrylamide gels (Bio-Rad, Munich, Germany). After transfer to nitrocellulose (Bio-Rad), the blots were blocked in PBS containing 5% skim milk and 0.05% Tween 20 and incubated overnight at 4°C with antibodies to RTF or ß-actin. Visualization of protein bands was accomplished using horseradish peroxidase (HRP)–coupled secondary antibodies (Sigma) and enhanced chemiluminescence (ECL; Amersham, Braunschweig, Germany). Scanned blots were quantified using Corel PhotoPaint Software (Corel Corporation, Ottawa, Canada).

Flow cytometry. Glioma cells (106) were detached using Accutase (PAA, Wien, Austria), fixed with paraformaldehyde (4%) for 10 minutes, blocked with 2% FCS in PBS, and incubated for 30 minutes on ice using FITC-conjugated 2C1 anti-RTF mAb or matched isotype control antibody (5 µg/mL; Sigma). Fluorescence was detected in a Becton Dickinson FACSCalibur. Specific fluorescence indexes were calculated by dividing mean fluorescence obtained with specific antibody by mean fluorescence obtained with control antibody.

Immunohistochemistry. All tissue specimens were from the Brain Bank of the Institute of Brain Research of the University of Tübingen where they had been evaluated by at least two neuropathologists in routine diagnostics. Glioblastomas were embedded in paraffin and cut for histology. Sections of 5 µm were deparaffinized and rehydrated in a descending alcohol sequence, washed thrice in TBS, and incubated with 2C1 antibody (dilution 1:100). Avidin-biotin complex (AB complex, Dakopatts, Glostrup, Denmark) consisting of biotinylated secondary antibody and HRP-conjugated avidin was used at 1:400. For visualization, the stainings were developed for 1 to 2 minutes with diaminobenzidine. Nuclei were counterstained with hemalum. Expression levels were quantitated by two blinded raters, taking into account the percentage of positive cells of the respective stainings. On the chosen scale, 0 signifies the absence of staining, 1 corresponds to single positive cells in a focal pattern, 2 denotes positive cells in a diffuse pattern, 3 indicates up to 20% of positive cells, 4 was allotted when the percentage of positive cells was between 20% and 50%, and a score of 5 was given when >50% of the cells were positive.

Glioma cell proliferation. LNT-229 control or siRTF cells (5 x 103) were plated in 96-well flat-bottomed plates and cultured in full medium. Cultures were pulsed with [methyl-3H]thymidine (1 µCi; Amersham) on day 2 and collected 16 hours later using a cell harvester (Tomtec, Hamden, CT). Incorporated radioactivity was bound to a glass fiber filtermat (Wallac, Turku, Finland). The filtermat was wetted with Ultima Gold Scintillation Cocktail (Packard, Dreieich, Germany) and radioactivity was determined in a Wallac 1450 Microbeta Plus Liquid Scintillation Counter.

Purification of peripheral blood lymphocytes and isolation of NK cells. Peripheral blood lymphocytes (PBL) were obtained from healthy donors by density gradient centrifugation (Biocoll, Biochrom). Monocytic cells were depleted by adherence. PBLs were cultured on irradiated (30 Gy) RPMI 8866 feeder cells to obtain polyclonal NK-cell populations. Cytotoxicity was assessed in 4-hour 51Cr release assays with 104 51Cr-labeled targets per well and various effector/target (E/T) ratios in 100 µL of medium. Spontaneous 51Cr release was determined by incubating the target cells with medium alone. To obtain the maximum 51Cr release, NP40 (2%) was added. After coincubation for 4 hours, 50 µL of the supernatant were transferred to a Luma-Plate 96 (Packard), dried, and measured. The percentage of 51Cr release was calculated as follows: [(experimental release – spontaneous release) / (maximum release – spontaneous release)] x 100. The lytic activity of cytotoxic T cells was examined after 5 days of coculture with glioma cells. Irradiated glioma cells (5 x 105) were seeded into 6-cm dishes. PBLs (5 x 106) were added in 3 mL of RPMI 1640 containing 10% FCS. Primed alloreactive cytotoxic T cells were removed at day 5 and used at different E/T ratios in a 51Cr release assay as described above.

IL-2, IL-10, and IFN-{gamma} ELISA. Freshly isolated PBLs (5 x 106) were stimulated with LNT-229 control or siRTF cells (5 x 105) for 3 days in 6-cm dishes. Supernatants were harvested and IL-2 (eBioscience, San Diego, CA), IFN-{gamma} (PeproTech, London, United Kingdom) and IL-10 (Bender Med Systems, Vienna, Austria) concentrations in the supernatant were determined by ELISA.

Preparation of murine NK cells. Murine NK cells were prepared from splenocytes from CD1 nude mice. NK cells were positively selected using DX5 mAb-coupled magnetic beads with the corresponding column system (Miltenyi Biotech, Bergisch Gladbach, Germany). Polyclonal mouse NK cells were cultured with mouse IL-2 (5,000 units/mL; PreproTech) for at least 10 days before they were used in cytotoxicity assays.

Mice and animal experiments. Athymic CD1 nude mice were purchased from Charles River Laboratories (Sulzfeld, Germany). Mice, 6 to 12 weeks of age, were used in all experiments. The experiments were done according to NIH Guide for the Care and Use of Laboratory Animals. Groups of six mice were injected s.c. in the right flank with 106 transfected LNT-229 tumor cells in 0.1-mL PBS as indicated. Mice were examined regularly for tumor growth using a metric caliper and killed when tumors reached diameters >15 mm. NK-cell depletion was done by i.p. injection of 0.2 mL of a 1/20 diluted stock solution of rabbit AAGM1 antibody (20). Starting 2 days before tumor cell injection, the depletion antibody was given twice weekly. Flow cytometric staining of PBMC and splenocytes with anti-Dx5 (Ly49B) antibody in weekly intervals confirmed NK-cell depletion. Before all intracranial procedures, mice were anesthetized by an i.p. injection of 7% chloral hydrate. For intracranial implantation, the mice were placed in a stereotactic fixation device (Stoelting, Wood Dale, IL) and a burr hole was drilled in the skull 2 mm lateral to the bregma. The needle of a Hamilton syringe (Hamilton, Darmstadt, Germany) was introduced to a depth of 3 mm. LNT-229 glioma cells (105) in a volume of 2-µL PBS were injected into the right striatum. The mice were observed daily and sacrificed when developing neurologic symptoms.

Statistics. Where indicated, data were screened for outliers and analysis of significance was done using two-tailed Student's t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Statistical analysis of immunohistochemistry RTF scores was done using Wilcoxon rank test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human malignant glioma cells express RTF in vitro and in vivo. Reverse transcription-PCR (RT-PCR) revealed that RTF mRNA expression was elevated in all of 12 examined glioma cell lines and 3 primary glioma cell cultures when compared with normal human brain white matter (Fig. 1A ). When quantified by real-time PCR, the overexpression of RTF mRNA in glioma cell lines relative to normal brain cDNA ranged from 3- to 30-fold (Fig. 1B). Immunoblot analysis allowed the detection of 70 and 50 kDa in all 12 glioma cell lines and 3 primary glioma cell cultures investigated. D247MG and A172 expressed lower levels than the other cell lines. Nevertheless, similar to the mRNA data, RTF protein levels were lowest in normal human brain white matter (Fig. 1C). In all cell lines, the signal obtained for the cleaved 50-kDa RTF isoform was much stronger than the signal for full-length 70 kDa RTF, suggesting that most RTF molecules are cleaved. Because the 2C1 antibody does not detect the soluble 20-kDa fragment, the presence of soluble RTF in glioma cell supernatant can only be deduced from the strong signal for the residual presumably membrane-bound 50-kDa fragment. No 50- or 70-kDa RTF was detected in the supernatant of any cell line (data not shown). Flow cytometry revealed membrane-associated RTF in all glioma cell lines, too, as shown representatively for LNT-229 cells (Fig. 1D) using the lymphoma cell line RPMI 8866 as a positive control (15). Specific fluorescence index values for cell-surface RTF levels are shown below the histograms.


Figure 1
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  		Figure 1. Human malignant glioma cells express RTF in vitro. A and B, RTF expression was analyzed by standard RT-PCR using GAPDH as a reference (A) and by real-time RT-PCR using 18S RNA as a reference, with quantitative data expressed relative to normal brain cDNA (B). C, RTF levels in whole-cell or tissue lysates were assessed by immunoblot using ß-actin as a reference. D, RTF levels at the cell-surface of nonpermeabilized cells were measured by flow cytometry [RTF-specific 2C1 antibody (open profile, thick line); isotype control antibody (closed profile)]. Representative flow cytometry profiles are shown for RPMI 8866 cells as a positive control and for LNT-229 glioma cells. Specific fluorescence index values (SFI) for RPMI 8866 and 12 different glioma cell lines are shown below the histograms.

 
Immunohistochemistry of paraffin-embedded sections from human glioblastomas showed RTF expression in 24 of 25 glioblastoma samples. RTF-positive cells were identified as tumor cells by morphologic criteria. Both nuclear and cytoplasmic staining patterns were observed (Fig. 2A, top right and middle ). Further, RTF was also detected in endothelial cells and lymphocytes whereas, in normal brain, RTF expression was barely detectable in white matter (Fig. 2A, bottom). Only a faint purely cytoplasmic staining in some scattered cortical neurons was found (data not shown). No staining was seen with isotype control antibodies (Fig. 2A, top). Placenta was used as a positive control (Fig. 2A, bottom). The findings from 6 normal brain and 25 glioblastoma specimens are summarized in Fig. 2B. Wilcoxon rank test for RTF levels detected by immunohistochemistry revealed a highly significant up-regulation of RTF in tumor tissue (P = 0.0012).


Figure 2
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  		Figure 2. Human glioblastomas express RTF in vivo. A, paraffin-embedded tissue sections of normal human brain, WHO grade 4 gliomas, and mature human placenta were immunostained with mAb 2C1 or isotype control antibody. Whereas the immunoglobulin G control did not result in any labeling on tissue sections (shown here for a WHO grade 4 glioma; top left), both nuclear and cytoplasmic staining patterns were observed in gliomas (top right and middle). No such RTF immunoreactivity was detected in the white matter of normal human brain (bottom left) whereas mature human placenta, used as a positive control, showed intense labeling of the periphery of the microvilli (bottom right). Bar, 50 µm. B, RTF expression levels were quantified and the respective scores for 6 normal human brain sections and 25 WHO grade 4 glioma sections are shown. Scoring was based on the percentage of positive cells of the respective stainings. Scores for the two groups were compared by Wilcoxon rank test.

 
RNA interference defines an immunoprotective role for RTF in human malignant glioma cells. RNA interference using the target sequence CATCGTGGATGCTTATGGA (1,148-1,166) resulted in a reduction of RTF mRNA expression by 85% and a reduction in RTF protein of 95% of 70-kDa RTF and of 89% of 50-kDa RTF in LNT-229 cells determined by densitometry (Fig. 3A ). Targeting of the sequence GGCCATCTATCACATGCTG (923-941) yielded an 85% reduction in RTF mRNA and decreased RTF protein levels by 80%. MHC class I expression was unaltered in these transfectants as assessed by flow cytometry (data not shown). Both LNT-229 siRTF cell lines were significantly more susceptible to polyclonal human NK cells than control cells (Fig. 3B). Having established that different siRNA sequences directed against RTF yield similar results, which strongly argues against potential off-target effects, we chose the LNT-229 subline with the more profound RTF depletion (designated LNT-229 siRTF) for all ensuing experiments. To investigate the biological effects of shed soluble RTF (12), we preincubated polyclonal NK cells with supernatant obtained from LNT-229 control or siRTF cells and assessed these effectors subsequently in a 51Cr release assay using siRTF LNT-229 cells as targets. Whereas both supernatants impaired NK-cell activity to a certain extent, most likely due to TGF-ß and other immunosuppressive cytokines present in glioma cell supernatant, there was nevertheless a highly significant difference between the two effector cell populations: NK cells that had been preincubated with the supernatant of siRTF LNT-229 cells were lytically much more active than NK cells that had been treated with supernatant of control LNT-229 cells. LNT-229 siRTF cells were used as targets here to minimize effects of membrane-bound RTF and ongoing RTF cleavage during the effector phase of the assay (Fig. 3C).


Figure 3
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  		Figure 3. siRNA-mediated inhibition of RTF expression enhances the susceptibility of glioma cells towards NK cell–mediated lysis. A, LNT-229 control or siRTF cells were assessed for RTF mRNA expression by real-time PCR (left) and for RTF protein levels by immunoblot (right). Two different siRNA target sequences cloned into pSUPERpuro were used with siRTF1 targeting nt 1,148-1,166 and siRTF2 directed against nt 923-941 of human RTF. B, LNT-229 control ({blacksquare}) or siRTF ({bigtriangleup}, {lozenge}) cells were used as targets (104 per well) for polyclonal NK cell–mediated lysis at various E/T ratios in a 4-hour 51Cr release assay. siRNA sequences correspond to those in (A). C, NK cells were preincubated with serum-free SN collected during 48 hours from LNT-229 control or siRTF (nt 1,148-1,166) cells for 16 hours and subsequently used as effector cells in a 51Cr release assay using LNT-229 siRTF cells as targets. D, LN-308 glioma cells were transiently transfected with control or RTF siRNA oligonucleotides (irrelevant control: firefly luciferase GL3, RTF target sequence: nt 1,148-1,166). Down-regulation of RTF was assessed by real-time RT-PCR (left) and immunoblot (right) at 72 hours after transfection and simultaneously the immunogenicity of GL3 or RTF siRNA-treated cells was assessed as in B (bottom).

 
To ascertain that the enhanced immunogenicity of LNT-229 cells following down-regulation of RTF was not specific to this single glioma cell line, we transiently transfected LN-308 cells with siRTF oligonucleotides, which resulted in an ~75% reduction in RTF mRNA and protein levels. Accordingly, polyclonal NK cells killed LN-308 siRTF cells more efficiently than LN-308 cells transfected with GL3 control (Fig. 3D).

To investigate the effect of RTF on T-cell responses, T cells were cocultured for 5 days with LNT-229 control or siRTF cells and then used as effectors against fresh LNT-229 siRTF targets. LNT-229 siRTF cells were efficiently killed by T cells primed with LNT-229 siRTF cells whereas priming with LNT-229 control cells did not induce significant lytic activity (Fig. 4A ). When the T cells were primed with LNT-229 siRTF cells, LNT-229 control cells were still more resistant to T-cell lysis than LNT-229 siRTF targets (Fig. 4B), indicating that RTF inhibits T-cell–mediated lysis during the effector phase, too. The killing of LNT-229 control cells by T cells primed with these cells was <10% at all E/T ratios (data not shown). Consistent with the generation of active immune effector cells (Fig. 4A), PBLs cocultured with LNT-229 siRTF cells generated high levels of IL-2 whereas PBLs cocultured with LNT-229 control cells did not (P < 0.001). In contrast, there was no difference in IFN-{gamma} levels. Conversely, PBLs cocultured with LNT-229 siRTF generated decreased levels of IL-10 compared with control cocultures (P < 0.05; Table 1 ).


Figure 4
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  		Figure 4. Down-regulation of RTF expression enhances the immunogenicity of glioma cells both in the priming and effector phase of allogeneic recall lysis. A, PBLs were cocultured (primed) with LNT-229 control or siRTF cells for 5 days. Lytic activity of primed T cells against LNT-229 siRTF cells was determined in a 4-hour 51Cr release assay. B, LNT-229 siRTF cells were used to stimulate PBLs for 5 days. Primed T cells were then assessed for the lysis of LNT-229 siRTF or control cells as targets.

 

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  		Table 1. Cytokine release by cocultures of PBL with glioma cells (determined by ELISA)

 
RNA interference–mediated RTF depletion delays growth of glioma xenografts in nude mice. The relevance of RTF as an immune inhibitory molecule paralyzing NK-cell responses was subsequently assessed in vivo in a nude mouse model. Importantly, LNT-229 control and siRTF cells displayed equivalent proliferative capacity in vitro (Fig. 5A ). Further, the enhanced NK-cell sensitivity of RTF-depleted human glioma cells (Fig. 3B) was retained in a xenogenic encounter using LNT-229 transfectants as targets and polyclonal NK cells from athymic CD1 nude mice as effector cells (Fig. 5B). This strongly suggests that the yet unidentified murine receptor for RTF also recognizes human RTF. A modulation of the tumorigenicity conferred by RTF depletion was assessed in s.c. and intracerebral glioma xenograft models. The monitoring of s.c. tumor growth showed a significantly delayed tumor growth in siRTF tumors compared with control transfectants (P < 0.01 from day 15; P < 0.001 from day 39 until the end of the experiment). This effect was abolished when NK cells were depleted with AAGM1 (Fig. 5C). The depletion of NK cells was verified by flow cytometry. The equivalent growth of control and siRTF gliomas in NK cell–depleted mice confirmed the pivotal role of NK cells in delaying the growth of siRTF gliomas. When LNT-229 cells were implanted stereotactically into the brains of nude mice, animals carrying control cells developed neurologic symptoms and had to be sacrificed earlier than animals carrying tumors formed by siRTF cells. The median survival was prolonged from 33 days with control tumors to 36 days with siRTF tumors (P < 0.05 by two-sided, unpaired Student's t test, neglecting one outlier which fell outside of the confidence interval with P < 0.01). Survival at 37 days was 0% with control tumors but 33% with siRTF tumors (Fig. 5D).


Figure 5
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  		Figure 5. RNA interference targeting RTF promotes glioma cell lysis by murine NK cells and delays the growth of s.c. and intracerebral human glioma xenografts in NK cell–proficient nude mice. A, the respective growth rates of LNT-229 control or siRTF cells were assessed by [methyl-3H]thymidine incorporation for 16 hours. B, LNT-229 transfectants were used in a 4-hour 51Cr release assay as targets for NK cells derived from athymic CD1 nude mice. C, athymic CD1 nude mice were treated (dotted lines) or not (solid lines) with the NK cell–depleting antibody AAGM1 2 days before they were injected s.c. with LNT-229 control (filled symbols) or siRTF (open symbols) cells. AAGM1 was administered twice weekly thereafter. Tumor growth was monitored every 3 days. D, LNT-229 control or siRTF transfectants were inoculated intracerebrally in athymic CD1 nude mice. Survival data for six animals per group are presented as a Kaplan-Meier plot.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Tumor cells accumulate a set of genetic alterations that could promote their recognition as "altered self" by the immune system. However, tumor-derived factors which suppress NK and T-cell functions are a substantial barrier to effective antitumor immunity in vivo. We here show by PCR, immunoblot, and flow cytometry that RTF, a pregnancy-associated tolerogenic factor, is expressed in glioma cell lines and primary glioma cell cultures in vitro (Fig. 1). RTF cell-surface expression determined by flow cytometry did not exactly parallel the RTF levels found in whole-cell lysates. This might be due to the fact that RTF protein levels at the cell-surface may depend on cleavage and possibly subsequent degradation. Further, it is not known whether the 50-kDa fragment can be internalized and thus escape detection by flow cytometry.

RTF expression in vivo was barely detectable in the normal brain. In contrast, RTF was detected in 24 of 25 glioblastoma samples investigated, suggesting that RTF expression is a general characteristic of gliomas. Of note, in some samples, scattered cortical neurons also displayed very faint RTF immunoreactivity, raising the possibility that RTF expression can be induced in the normal brain under particular neurologic conditions (Fig. 2).

Suppression of RTF mRNA (Fig. 3A and D) and protein (Fig. 3B) expression by stable or transient RNA interference results in an increase of NK cell–mediated lysis of allogeneic glioma cells (Fig. 3B and D). This effect was observed with two different siRNA target sequences and two different cell lines. A similar, albeit weaker, effect of RTF on NK-cell activity was found when NK cells were preincubated with supernatant from siRTF and control cells (Fig. 3C). This strongly argues for an immunoregulatory activity of soluble RTF unrelated to the recently described autocrine effect of RTF on organellar acidification that was found to reside in the transmembrane domains (11). In fact, our data show that both soluble and membrane-bound RTF compromise NK-cell function. In vivo, the effects of membrane-bound and soluble RTF are likely to be additive. Also in vitro, the effect of membrane-bound RTF cannot be fully dissociated from that of soluble RTF secreted freshly during the course of a 4-hour lysis assay. However, the amount of soluble RTF newly released during the assays will probably be too low to account for all NK-cell inhibition observed in these assays. We further show that RTF protects glioma cells both during the priming and the effector phase of T-cell–mediated lysis (Fig. 4A and B).

The potency of RTF as a novel candidate molecule for glioblastoma-dependent immune suppression was confirmed in vivo using xenografts of LNT-229 siRTF and LNT-229 control cells in nude mice. Whereas down-regulation of RTF expression by siRNA did not affect glioma cell growth in vitro, s.c. injected LNT-229 control transfectants formed larger tumors than LNT-229 siRTF transfectants. This difference was no longer apparent in animals that had undergone NK-cell depletion, confirming that the differences in in vivo growth were, in fact, due to immunologic effects. In line with these findings, NK cell–proficient animals injected with LNT-229 control cells into the brain died earlier than animals which had received LNT-229 siRTF cells.

These data show that anti-RTF strategies may partly relieve glioma-induced NK-cell defects and enable effective antitumor responses in vivo. The effect of RTF on T cells, in particular on the generation of tumor-specific CTL, could not be evaluated in the animal model used. The survival advantage achieved by anti-RTF strategies may become even greater in a fully immune competent model, which awaits to be established.

Intriguingly, J6B7, the rat homologue of RTF, was one of three glioma antigens identified in a SEREX screening using the 9L rat glioma model (21). Because 9L cells show a lower expression of J6B7/RTF mRNA than any other rodent (C6, 9L, GL-261, SMA-497, and SMA-560) or human glioma cell line we have tested (data not shown), there is a considerable chance that human glioblastomas may also induce anti-RTF immunoreactivity in vivo. Given the scarcity of well-defined cancer-associated antigens in brain tumors (22, 23) and the low level expression of RTF in normal brain tissue, RTF may therefore also be of interest for immunotherapeutic approaches aiming at enhancing an antitumor T-cell response by vaccination. Whereas the wide distribution of RTF in human gliomas as well as the growth of 9L cells in syngeneic Fischer 344 rats shows a priori that the net effect of RTF-induced immune suppression and anti-RTF immunoreactivity is in favor of immune inhibition, RTF may represent an attractive target for novel immune modulatory approaches.


   Acknowledgments
 
Grant support: Wilhelm Sander Foundation (M. Weller) and the Hertie Foundation (P. Roth).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8/26/05. Revised 12/27/05. Accepted 1/26/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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