SEARCH:   Advanced

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Bollard, C. M. Articles by Rooney, C. M. Search for Related Content PubMed PubMed Citation Articles by Bollard, C. M. Articles by Rooney, C. M. Related Collections Immunotherapy Gene Therapy Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 May 2002, Vol. 99, No. 9, pp. 3179-3187 GENE THERAPY Adapting a transforming growth factor -related tumor protection strategy to enhance antitumor immunity Catherine M. Bollard, Claudia Rössig, M. Julia Calonge, M. Helen Huls, Hans-Joachim Wagner, Joan Massague, Malcolm K. Brenner, Helen E. Heslop, and Cliona M. Rooney From the Center for Cell and Gene Therapy, Departments of Pediatrics, Molecular Virology and Microbiology, and Medicine, Baylor College of Medicine, Houston, TX; and Memorial Sloan Kettering Cancer Center, New York, NY.     Abstract Top Abstract Introduction Materials and methods Results Discussion References Transforming growth factor  (TGF-), a pleiotropic cytokine that regulates cell growth and differentiation, is secreted by many human tumors and markedly inhibits tumor-specific cellular immunity. Tumors can avoid the differentiating and apoptotic effects of TGF- by expressing a nonfunctional TGF- receptor. We have determined whether this immune evasion strategy can be manipulated to shield tumor-specific cytotoxic T lymphocytes (CTLs) from the inhibitory effects of tumor-derived TGF-. As our model we used Epstein-Barr virus (EBV)-specific CTLs that are infused as treatment for EBV-positive Hodgkin disease but that are vulnerable to the TGF- produced by this tumor. CTLs were transduced with a retrovirus vector expressing the dominant-negative TGF- type II receptor HATGF-RII-cyt. HATGF-RII-cyt- but not green fluorescence protein (eGFP)-transduced CTLs was resistant to the antiproliferative and anticytotoxic effects of exogenous TGF-. Additionally, receptor-transduced cells continued to secrete cytokines in response to antigenic stimulation. TGF- receptor ligation results in phosphorylation of Smad2, and this pathway was disrupted in HATGF-RII-cyt-transduced CTLs, confirming blockade of the signal transduction pathway. Long-term expression of TGF-RII-cyt did not affect CTL function, phenotype, or growth characteristics. Tumor-specific CTLs expressing HATGF-RII-cyt should have a selective functional and survival advantage over unmodified CTLs in the presence of TGF--secreting tumors and may be of value in treatment of these diseases. (Blood. 2002;99:3179-3187) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References Immunotherapy strategies to boost cellular immune responses to tumors are increasingly applied as more tumor antigens are identified.1,2 The most successful use of adoptively transferred antigen-specific cytotoxic T cells has occurred in severely immunocompromised individuals whose tumors require few immune evasion strategies.3 By contrast, tumor immunotherapy in immunocompetent hosts has been of more limited benefit. In the presence of a normal immune system, tumors frequently develop immune evasion strategies that may influence every stage of the generation of a tumor-specific cellular immune response, from the activation of professional antigen-presenting cells (APCs) to T-cell recruitment, activation, and effector function.4 Tumor secretion of transforming growth factor  (TGF-) is among the most widely used evasion strategies. TGF- is a ubiquitous cytokine with pleiotropic effects on cell growth, differentiation, and matrix production. As a stimulator of mesenchymal, fibroblast, smooth muscle, and osteoblast cell growth,5,6 TGF- induces the synthesis of extracellular matrix proteins and promotes angiogenesis.7 As a growth inhibitor, it plays a role in T-cell homeostasis by limiting immune responses to antigens and by inducing tolerance.8,9 Hence, secretion of this cytokine by malignant cells such as neuroblastoma and Hodgkin Reed-Sternberg tumor cells may diminish the effectiveness of antitumor T-cell immune responses.10-12 The binding of TGF- to either TGF- receptor I (TGF-RI) or TGF- receptor II (TGF-RII), results in the formation of a tetramer complex, involving the dimers of the type I and II receptors, which is required for signaling.13-17 With this interaction between the 2 receptors and the ligand, phosphorylation occurs, rendering TGF-RI active and able to phosphorylate Smad 2 and 3, resulting in their translocation to the nucleus.18,19 Once in the nucleus, the Smads interact with transcription factors such as those involved in the regulation of cell growth and differentiation.20,21 TGF- may also have adverse effects on tumor cells themselves by promoting terminal differentiation and apoptosis. Tumors may avoid this activity by mutation of their TGF- receptors (TGF-RI and TGF-RII).13,22,23 Each of these receptors possesses an extracellular region, a single transmembrane domain, and a cytoplasmic signaling domain containing a serine/threonine kinase domain. Mutations in the TGF-RII gene that correlate with loss of sensitivity to TGF- have been identified in many tumors in which inhibition of differentiation contributes to unregulated growth.24,25 Mutant forms of the TGF-RII have also been created. HATGF-RII-cyt was created with a stop codon and a BamHI site introduced after the 10th cytoplasmic codon (nt597).16 This mutant receptor lacks the entire kinase domain and most of the juxtamembrane region. HATGF-RII-cyt has been shown to act in a dominant-negative fashion when transfected into the mink lung epithelial cell line, diminishing the cells' antiproliferative and transcriptional responses to TGF-.16 We have determined whether the strategy used by tumor cells to protect themselves against the effects of TGF- can be manipulated to shield tumor-specific cytotoxic T lymphocytes (CTLs) from the inhibitory effects of tumor-secreted TGF-. As our model, we have taken Epstein-Barr virus (EBV) antigen-positive Hodgkin disease, a tumor that expresses virus-specific antigens and should be susceptible to specific CTLs.26 Indeed, tracking of genetically marked EBV-specific CTL infusions shows homing to Hodgkin tumor sites and CTL survival in the patient's circulation for up to 9 months after infusion.27 In addition, CTL infusions enhanced EBV-specific immune responses and reduced virus load, but only limited tumor responses of brief duration were observed. This failure may be associated with the demonstrated ability of Hodgkin tumor cells to secrete TGF- and consequent inactivation of CTLs entering the tumor environment.12 We now show that forced expression of a dominant-negative TGF-RII in ex vivo-expanded EBV tumor-specific CTLs from patients with relapsed Hodgkin disease renders them resistant to the inhibitory effects of TGF-, while enabling them to retain their dependence on other growth regulatory signals.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Cell lines The ecotropic packaging cell line Phoenix28 was provided by Gary P. Nolan (Stanford, CA). PG-13 (obtained from American Type Culture Collection [ATCC], Manassas, VA) is an amphotropic retrovirus packaging cell line that produces virus pseudotyped with the gibbon-ape leukemia virus (GALV). HSB-2 (ATCC) is a T-cell lymphoma that is sensitive to lymphokine-activated killer cells and was used as a target in cytotoxicity assays. Lymphoblastoid cell lines (LCLs) were generated as described below. pMEP5/HATGF-RII-cyt plasmid A human type II TGF- receptor complimentary DNA (cDNA) was truncated at nt597, thereby deleting most of its cytoplasmic tail and all of its cytoplasmic kinase domain, leaving only 7 amino acids remaining in the intracellular domain.16 A sequence encoding the influenza virus hemagglutinin peptide epitope HA1 was spliced into the human TGF-RII cDNA.14,16,29 The HA sequence was inserted after the signal sequence in the human TGF-RII so that the HA epitope is retained near the amino terminus of the mature receptor. The presence of the HA tag does not affect ligand binding and allows the mutant construct to be distinguished from the wild type TGF-RII receptor with an anti-HA antibody.15,16 The function and the biochemistry of pMEP5/HATGF-RII-cyt have been extensively characterized.16 HATGF-RII-cyt was placed into the BamHI and NcoI sites of the retroviral vector SFG30 (provided by R.C. Mulligan, Cambridge, MA). Production of recombinant retrovirus Cells of the ecotropic packaging cell line Phoenix-eco were transiently transfected with vector DNA by using FuGENE 6 transfection reagent (Roche, Indianapolis, IN) in Dulbecco modified Eagle medium (DMEM; Biowhittaker, Walkersville, MD) supplemented with 10% fetal calf serum (FCS; Hyclone, Logan, UT). Twenty-four hours after transfection, Iscoves modified Dulbecco medium (IMDM; Biowhittaker) supplemented with 20% FCS (20% IMDM) was added, and cells were incubated at 32°C for 24 hours. Fresh retrovirus supernatants were then collected, filtered through a 0.45-µm filter, and used to infect the packaging cell line PG13 in the presence of polybrene (8 µg/mL) for 48 hours at 32°C. The infected cells were incubated overnight at 37°C in fresh 10% DMEM and then subjected to a second round of infection under the same conditions by using freshly generated Phoenix-eco cell supernatants. Viral supernatants were harvested from the resulting bulk producer lines by adding 20% IMDM to the PG-13 cells and incubated at 32°C for 24 hours. The supernatant was harvested, filtered by using a 0.45-µm filter, and used directly to transduce the CTLs. Generation of EBV-transformed B cell lines Peripheral blood-derived mononuclear cells (PBMCs) (5 × 106) were incubated with 100 µL concentrated supernatant from the EBV producer cell line B95-8 in a total of 200 µL complete medium (RPMI 1640 medium [GIBCO-BRL, Gaithersburg, MD] containing 10% FCS [Hyclone], and 2 mM L-glutamine [Biowhittaker]) for 30 minutes. The cells were plated at 106 cells per well in a flat-bottomed 96-well plate (Costar; Corning, Corning, NY) containing complete medium and 1 µg/mL cyclosporin A (Sandoz Pharmaceuticals, Washington, DC). Cells were fed weekly until LCLs were established.31 Generation and transduction of EBV-specific CTL cultures EBV-specific CTLs were prepared by stimulating PBMCs with the autologous EBV-transformed LCL.32,33 PBMCs (2 × 106) were cocultured with 5 × 104 gamma-irradiated (40 Gy) autologous LCLs per well in a 24-well plate. Starting on day 10, the responder cells were restimulated weekly with irradiated (40 Gy) LCLs at a responder-to-stimulator ratio of 4:1. Two weekly doses of recombinant human interleukin 2 (rhIL-2; 50 IU/mL) were added from day 14. Twenty-four hours after LCL stimulation, CTLs ready for transduction were transferred to a 24-well plate (Costar), precoated with OKT3 (1 µg/mL; Ortho Pharmaceuticals, Raritan, NJ) and anti-CD28 antibody (1 µg/mL; Pharmingen, San Diego, CA) at 1 × 106 cells per well and incubated for 48 hours for optimal activation before transduction.34 Transductions were carried out in 24-well nontissue culture-treated plates (Becton Dickinson, Franklin Lakes, NJ), coated with recombinant fibronectin fragment (FN CH-296; Retronectin; Takara Shuzo, Otsu, Japan) at a concentration of 4 µg/cm2. The prestimulated CTL lines were resuspended at 1 × 106 cells/mL in complete medium supplemented with 45% EHAA (Clicks; GIBCO-BRL) and rhIL-2 (100 IU/mL), then incubated with equal volumes of freshly generated viral supernatant for 36 hours at 37°C and 5% CO2. Two weeks after transduction, 3 CTL lines from healthy donors were positively selected for cell surface expression of the HA-tag by using flow cytometry. Flow cytometry For immunophenotyping, cells were stained with fluorescein-conjugated monoclonal antibodies (Becton Dickinson, San Jose, CA) directed against CD3, CD4, CD8, CD16, CD56, and CD25 surface proteins. For each sample, 10 000 cells were analyzed by FACSCalibur with the Cell Quest Software (Becton Dickinson). Surface expression of the HA-epitope was analyzed after incubation of CTLs (1 × 106) with the HA antibody (Sigma, St Louis, MO) at a concentration of 200 ng/5 × 105 in the presence of normal donkey serum (Jackson Immuno Research Laboratories, West Grove, PA) for 30 minutes at room temperature. This analysis was followed by incubation with fluorescein isothiocyanate (FITC)-labeled donkey antirabbit antibody (Jackson Immuno). The perforin assay was performed by fixing the CTLs in 4% paraformaldehyde (Sigma) for 20 minutes. The cells were then washed in permeabilizing buffer (1 × phosphate-buffered saline [PBS; GIBCO-BRL] + 0.1% saponin [Sigma] + 1% FCS [Hyclone]). CTLs were incubated in 3 mL permeabilizing buffer with 5% human AB-serum (C-6 Diagnostics, Germantown, WI) for 10 minutes at room temperature. CTLs were spun down and resuspended in 100 µL permeabilizing buffer. To each sample, 20 µL of either phycoerythrin (PE)-labeled antiperforin antibody (Pharmingen) or PE-labeled IgG1 isotype control (Pharmingen) was added and incubated for 30 minutes at room temperature. CTLs were washed again with permeabilizing buffer and resuspended in 1 × PBS + 1% FCS and analyzed immediately. Analysis of transcriptional activation by Western blot Cell pellets were resuspended in Tris sodium EDTA (TNE) buffer (100 mM Tris, 150 mM NaCl, 0.5% NP-40, 10 mM EDTA, 1 mM dithiothreitol) with phosphatase inhibitors (20 mM -glycerol phosphate and 20 mM NaVO3) and protease inhibitors. After 5 seconds of sonication, the lysates were centrifuged at 14 000 rpm for 5 minutes. Protein concentration of the supernatants was determined by protein assay (BIO-RAD No. 500-0006, Hercules, CA). Protein (50 µg) was loaded on a 9% sodium dodecyl sulfate-polyacrylamide gel. Western blot was performed with either antiphospho-Smad 2 antibody (Upstate Biotechnology No. 06-829, Lake Placid, NY) at a final concentration of 1 µg/mL or purified anti-Smad2/3 rabbit polyclonal antisera.35 Measurement of cytokine production by enzyme-linked immunosorbent assay To assess the effect of the truncated TGF-RII on cytokine release in the presence of TGF-, duplicate samples of transduced and nontransduced effector cells (5 × 104/well) were cocultured with irradiated, EBV-transformed LCLs at stimulator-to-effector ratios of 1:4 in rhIL-2 (50 U/mL) ± 5 ng/mL TGF-1 (R&D Systems, Minneapolis, MN) in 96-well round-bottom plates (Costar). After 24 hours, the supernatants were harvested and analyzed for human granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interferon  (IFN-) by using 96-well plates precoated with either antihuman GM-CSF monoclonal antibody (R&D Systems) or antihuman IFN- monoclonal antibody (Pharmingen) by enzyme-linked immunosorbent assay according to the manufacturer's instructions. Cytotoxicity assays To compare the cytotoxic specificity of transduced and nontransduced CTLs in the presence of TGF-1, standard 51Cr release assays were performed. At 72 to 96 hours before performing the cytotoxicity assay, 5 ng/mL TGF-1 (R&D Systems) was added to 8 × 106 transduced and nontransduced CTLs. Doubling dilutions of CTLs were coincubated in triplicate for 4 hours with 5000 51Cr-labeled target cells (Amersham Pharmacia Biotech, Piscataway, NJ) in a total volume of 200 µL in a V-bottom 96-well plate (Costar) as previously described.31 The targets tested were autologous LCLs, HLA class I and II mismatched LCLs, and HSB-2. Target cells incubated in RPMI 1620 alone or in 5% Triton X-100 (Sigma) were used to determine spontaneous and maximum 51Cr release, respectively. At the end of a 4-hour incubation period at 37°C and 5% CO2, supernatants were harvested, and 51Cr release was measured on a gamma counter (Tri-CARB 4640; Packard BioScience, Downers Grove, IL). The mean percentage of specific lysis of triplicate wells was calculated as follows: [(test counts  spontaneous counts)/(maximum counts  spontaneous counts)] × 100%. Proliferation assays Transduced CTLs were coincubated in triplicate at 5 × 104 cells/well with irradiated autologous EBV-LCLs at a 4:1 stimulator-to-responder ratio ± titrated concentrations of TGF-1 up to 20 ng/mL. After a 72-hour coincubation period, wells were pulsed with 0.037 MBq (1 µCi)/well of [3H]thymidine (Amersham Pharmacia Biotech) for 18 hours, and the samples were harvested onto glass fiber filter paper for -scintillation counting (TriCarb 2500 TR; Packard BioScience). Quantification of the transduction rate by real-time polymerase chain reaction DNA was extracted from cytotoxic T cells by using the DNeasy Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. For quantification of the transduction rate of CTL, real-time polymerase chain reaction (RT PCR) assays specific for the HA sequence were developed by using 5' nuclease PCR technology and the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA).36 RT PCR amplification was performed with 2× TaqMan Universal Master Mix (PE Applied Biosystems) adjusted to 50 µL with 300 nM of each primer, 200 nM probe, template, and nuclease-free water. The forward primer (5'-GTGGACGCGTATCGCCAG-3') binds 12 base pair (bp) upstream of the HA sequence, the reverse primer (5'-TGTCAGTGACTATCATGTCGTTATTAACC-3') 15 bp downstream of the HA sequences, whereas the probe (5'-VIC-CCACCGTATGATGTTCCTGATTATGCTAGCC-TAMRA-3') spans the entire HA sequence. DNA solution (250 ng) was analyzed in triplicate for each sample. As positive controls, samples were analyzed for the -actin gene in parallel by using the TaqMan Beta-actin Detection Reagents (PE Applied Biosystems). PCR consisted of 2 minutes at 50°C (inactivation of possible carry-over contamination by uracil N'-glycosylase [UNG]), 10 minutes at 95°C (UNG inactivation and activation of DNA polymerase), and 40 2-step cycles of 15 seconds at 95°C and 60 seconds at 60°C. For quantification, serial 1:4-fold dilutions of the plasmid pMEP5/HATGF-RII-cyt16 were used as the standard. A correlation coefficient of more than 0.99 was found over at least 5 orders of magnitude after amplification of the HA sequence. Statistical analysis The Student t test was used to test for significance in each set of values, assuming equal variance. Mean values ± SE are given unless otherwise stated.     Results Top Abstract Introduction Materials and methods Results Discussion References Truncated TGF- receptor mutant was expressed by CTLs after transduction with HATGF-RII-cyt Four EBV-specific CTL lines from healthy individuals and 4 from patients with EBV-positive Hodgkin disease were transduced with HATGF-RII-cyt after 21 to 105 days of culture (mean, 52 days). We used RT PCR analysis to compare the transgene copy number per cell in bulk HATGF-RII-cyt-transduced CTLs with that in transduced CTLs that had been sorted by flow cytometry for HA epitope expression. Assuming that 100% of the cells sorted for the HA tag contained at least one copy of SFG:HATGF-RII-cyt DNA, the transduction efficiency in unsorted CTLs ranged from 6.5% to 55% (mean = 27%) (Table 1), which is not significantly different from the transduction efficiency with SFG-eGFP (19%-51.5%; mean, 31%) (Figure 1A,B). Mutant TGF-RII surface expression was also detected by flow cytometry (Figure 1C-E). By using the anti-HA antibody, the percentage of expression of HATGF-RII-cyt on CD8+ cells ranged from 3.5% to 49.3% (mean, 17%), which is consistent with RT PCR results. Similar transduction efficiencies were also seen for CD4+ cells (Figure 1F). CTLs sorted for the HA tag showed 53.15% to 62.6% HA expression on CD8+ cells 6 weeks after sorting (Figure 1G,H).                                View this table: [in this window] [in a new window]   Table 1. Transduction rate of HATGF-RII-cyt ranges from 6.5% to 55% as determined by quantitative real-time polymerase chain reaction View larger version (45K): [in this window] [in a new window]   Figure 1. Transduced CTLs surface express HA tag of mutant TGF-RII receptor. Cells of an EBV-specific CTL line 14 days after retroviral transduction with SFG:eGFP were stained with either PE-labeled anti-IgG1 (A) or anti-CD3 antibodies (B). CTLs transduced with SFG:HATGF-RII-cyt (truncated TGF-RII containing HA tag) gene were stained with FITC-labeled donkey antirabbit antibody alone (isotype control) (C). Nontransduced (D) or HATGF-RII-cyt-transduced CTLs (E,F) were then stained with anti-HA monoclonal antibody followed by incubation with FITC-labeled donkey antirabbit antibody and PE-labeled CD8 or CD4 antibody. Six weeks after sorting CTLs for the HA tag, HA expression was measured on CD8+ cells (G [isotype control] and H). Surface immunofluorescence was analyzed by flow cytometry. HATGF-RII-cyt-transduced CTLs are resistant to the antiproliferative effects of exogenous TGF-1 To investigate whether expression of the truncated TGF-RII by CTLs could overcome the antiproliferative effects of TGF- on CTLs, we compared thymidine uptake by HATGF-RII-cyt-transduced CTLs with eGFP-transduced and -nontransduced CTLs after addition of TGF-1 for 72 hours (Figure 2). TGF-1 had a dramatic antiproliferative effect on established eGFP-transduced and -nontransduced EBV-CTLs generated from both healthy donors and patients, inhibiting uptake by a mean of 59.5% (range, 44%-75%). By contrast, the mean inhibition of thymidine uptake by HATGF-RII-cyt-transduced CTLs was 16% (range, 0%-30%). This resistance to the antiproliferative effects of TGF- in HATGF-RII-cyt-transduced CTLs was statistically significant when compared with the mock or nontransduced CTLs (P = .03). Importantly, when EBV-specific CTLs were maintained under normal growth conditions with the addition of TGF-, they failed to proliferate, and most died within 12 days. HATGF-RII-cyt-transduced CTLs, however, continued to proliferate and grow normally, showing that the transduced cells were resistant to the antiproliferative effects of the TGF-1 (Figure 3A,B). View larger version (17K): [in this window] [in a new window]   Figure 2. Ability of TGF- to inhibit T-cell proliferation is significantly greater in nontransduced CTLs when compared with HATGF-RII-cyt-transduced CTLs. Nontransduced CTLs, eGFP-transduced CTLs, and HATGF-RII-cyt-transduced CTLs were stimulated with irradiated autologous LCLs and IL-2 ± 5 ng/mL TGF-1. Proliferative responses were measured after 72 hours of incubation by measurement of 3[H] thymidine uptake. The mean percentage of inhibition by TGF- was measured in nontransduced CTLs (black), eGFP-transduced CTLs (white), and HATGF-RII-cyt-transduced CTLs (gray). The graph represents a pooled analysis of the mean inhibition of TGF- on 3[H] thymidine uptake in the 8 CTL lines tested. View larger version (11K): [in this window] [in a new window]   Figure 3. In the presence of TGF-, HATGF-RII-cyt-transduced CTLs continue to proliferate and grow normally in long-term cultures. HATGF-RII-cyt-transduced, nontransduced, and eGFP-transduced CTLs were harvested 2 weeks after transduction. From each group, 1 × 106 cells were stimulated weekly with LCLs and fed twice weekly with IL-2 ± 5 ng/mL TGF-. The figures show results from one representative experiment. (A) This panel represents CTL cell numbers (× 106) recorded from weekly cell counts in the 3 CTL groups grown without TGF- () and with TGF- (). (B) This panel shows 3[H] thymidine uptake in nontransduced CTLs (black) and HATGF-RII-cyt-transduced CTLs (gray) before addition of TGF- then on days 4 and 12. Phosphorylation of Smad2 is inhibited in TGF-RII-cyt-transduced CTLs with addition of TGF- To confirm that downstream signaling by TGF- is abrogated in CTLs transduced with TGF-RII-cyt, TGF- was added to nontransduced, eGFP-transduced, and TGF-RII-cyt-transduced CTLs at a concentration of 5 ng/mL. After 60 minutes, the cells were harvested, and whole cell lysates were prepared. All the CTL extracts were subjected to Western immunoblotting by using anti-Smad2/3 antibody and antiphospho-Smad2 antibody (Figure 4). Western blot analysis demonstrated the presence of Smad-2 (S2) in all the CTL groups in the presence and absence of TGF-. However, phosphorylated Smad-2 (P-S2) was only detected in nontransduced and eGFP-transduced CTLs treated with TGF-. In contrast, there was no expression of P-S2 in TGF-RII-cyt-transduced CTLs with the addition of TGF-, confirming that signal transduction was blocked by the presence of the dominant-negative TGF-RII. View larger version (55K): [in this window] [in a new window]   Figure 4. Western immunoblotting shows absence of phosphorylated Smad2 in TGF-RII-cyt-transduced CTLs with addition of TGF-. Nontransduced, eGFP-transduced, and TGF-RII-cyt-transduced CTLs were incubated for 1 hour with 5 ng/mL TGF-, as indicated. The presence or absence of Smad2 (S2) and phosphorylated Smad2 (P-S2) was detected by Western immunoblotting by using anti-Smad2 and antiphospho-Smad2 antibodies, respectively. TGF-RII-cyt-transduced CTLs continue to produce cytokines in response to antigenic stimulus in the presence of TGF-1 TGF- inhibited IFN- and GM-CSF release from nontransduced EBV-specific CTLs after they were stimulated with irradiated LCL (effector-to-target ratio of 4:1) and 50 U/mL rhIL-2 for 24 hours. The level of inhibition was 60.5% (range, 47%-71%) for IFN- and 71% (range, 63%-83%) for GM-CSF. By contrast, the mean inhibition of cytokine release by HATGF-RII-cyt-transduced CTLs was 43% (range, 17%-56%) for GM-CSF and 22.5% (range, 6%-39%) for IFN-. The effect of TGF- on IFN- and GM-CSF release in HATGF-RII-cyt-transduced CTLs compared with the cytokine release in nontransduced and eGFP-transduced CTLs was statistically significant (P = .05 and P = .01, respectively). This protection was even greater with HA-sorted CTLs in which there was just 4.5% (range, 0%-9%) GM-CSF inhibition (Figure 5A) and 2.2% (range, 0%-9%) inhibition of IFN- release (P = .002) (Figure 5B). View larger version (15K): [in this window] [in a new window]   Figure 5. HATGF-RII-cyt-transduced CTLs sorted for the HA epitope demonstrate a significant resistance to the inhibitory effects of TGF- on secretion of GM-GSF and IFN- when compared with nontransduced CTLs. EBV-specific CTLs were transduced with the mutant TGF-RII receptor and then positively selected for the HA tag by flow cytometry by using an anti-HA antibody. TGF-RII-cyt-transduced/sorted CTLs and mock-transduced CTLs were stimulated with irradiated autologous LCLs and IL-2 ± 5 ng/mL TGF-1. Supernatant removed after 24 hours was analyzed for GM-CSF (A) and IFN- (B). The graphs represent a pooled analysis of the mean percentage of inhibition of TGF- on IFN- and GM-CSF release in 8 nontransduced (black) and 3 eGFP-transduced CTL (white) lines versus the 3 HATGF-RII-cyt-transduced CTL (gray) lines sorted for the HA-epitope. CTLs transduced with retrovirus TGF-RII-cyt maintain their cytolytic activity and specificity in the presence of TGF- The cytotoxic activity of HATGF-RII-cyt-transduced, mock-transduced, and nontransduced CTLs were compared in standard 4-hour 51Cr release assays in the presence of TGF-1. CTL lines were tested up to 26 days after transduction in the presence of TGF-1 (Table 2). At an effector-to-target ratio of 20:1, the percentage of autologous LCLs lysed by nontransduced CTLs was inhibited by 51% to 100% (mean, 74%) compared with a range of 27% to 57% (mean, 37.7%) after more than a 72-hour incubation with 5 ng/mL TGF-1 (P = .02). By comparison, at the same effector-to-target ratio, the percentage of autologous LCLs lysed by transduced (unsorted) CTLs ranged from 40% to 81% (mean, 61.3%) in the absence of TGF-1 and 41% to 100% (mean, 61.3%) in the presence of TGF-1 (P = .7). No CTL lines had significant (> 20%) reactivity with allogeneic LCL or HSB-2 targets (Figure 6A,B).                                View this table: [in this window] [in a new window]   Table 2. Cytolytic characteristics of HATGF-RII-cyt-transduced versus nontransduced cytotoxic T-lymphocyte lines with and without exposure to exogenous transforming growth factor   View larger version (25K): [in this window] [in a new window]   Figure 6. TGF- decreases CTL-specific lysis against autologous EBV-LCL targets in mock-transduced or nontransduced CTLs but not in HATGF-RII-cyt-transduced CTLs. Percentage of specific 51Cr release was determined 4 hours after coincubation with autologous LCLs, allogeneic LCLs, and HSB-2 targets. TGF- was added to CTL cultures 96 hours before cytotoxicity assay. The graphs show the percentage of specific lysis at effector-to-target ratios of 20:1, 10:1, and 5:1 in representative CTL lines generated from a healthy donor (A) and from a patient with Hodgkin disease (B).  indicates CTLs cultured without TGF- and autologous LCL target; , CTLs cultured with TGF- and autologous LCL target; , CTL versus HSB-2 target; , CTLs versus allogeneic (HLA class I mismatch) LCL target. Effects of exogenous TGF- on intracellular perforin levels in TGF-RII-cyt-expressing and -nonexpressing CTLs To identify a possible mechanism by which the cytolytic activity of CTLs is reduced by TGF-, the intracellular perforin levels of the CTLs were measured by flow cytometry after CTLs were stained by PE-labeled antiperforin antibody (Figure 7). The intracellular perforin levels in untransduced and eGFP-transduced CTLs were significantly reduced by 50% to 96% (mean, 73%, P = .002) in the presence of TGF- (compare 7A with 7D and 7B with 7E). By comparison, CTLs transduced with HATGF-RII-cyt had no significant reduction in perforin (range, 4%-11%, mean 6.8%, P = .7) (7C compared with 7F). View larger version (32K): [in this window] [in a new window]   Figure 7. TGF- significantly reduces intracellular perforin levels in nontransduced or eGFP-transduced CTLs, whereas perforin release is unaffected by TGF- in HATGF-RII-cyt-transduced CTLs. Ninety-six hours after the addition of TGF- (5 ng/mL), nontransduced (A,D), eGFP-transduced (B,E), and HATGF-RII-cyt-transduced CTLs (C,F) were stained for intracellular perforin by using PE-labeled antiperforin antibody and were detected by flow cytometry. (A-F) These panels show representative histograms for CTLs from one donor cultured without TGF- (A-C) versus with TGF- (D-F). (G) This panel shows mean perforin levels from 6 CTL lines with and without TGF-. Expression of HATGF-RII-cyt does not affect the phenotype or cytotoxic specificity of transduced CTLs Although the dominant-negative TGF- receptor clearly protects CTLs from the growth inhibitory effects of TGF-, it is important to show that the transduced CTLs can continue to function normally and remain under normal growth control. The CTLs were phenotyped before and after transduction with HATGF-RII-cyt. The CTLs were then maintained in culture for up to 35 days after transduction and were phenotyped weekly from day 7 after transduction. Most of the CTL lines generated had a characteristic immunophenotype with more than 90% CD3+ T cells, of which about 90% were also CD8+, whereas up to 10% of the cells had a T-cell helper phenotype (CD3+CD4+). These lines were compared with nontransduced lines for phenotypic differences. Transduction of CTLs did not result in any change in immunophenotype when compared with nontransduced cells (Figure 8). Nor was there any interference with cytolytic function. 51Cr release assays were performed between 14 and 26 days after transduction. There was no significant difference in the cytolytic specificity or activity of the transduced lines when compared with otherwise identical nontransduced lines from the same donor over several time points. As outlined in Table 2 after a range of 14 to 26 days after transduction with TGF-RII-cyt, the CTL lines maintained their cytotoxic activity (Table 2) and specificity (Figure 6A). View larger version (27K): [in this window] [in a new window]   Figure 8. CTL phenotype is unchanged after transduction. To determine CTL phenotypes, the nontransduced (black) and TGF-RII-cyt-transduced (gray) EBV-specific CTL cultures were stained with antibodies against T-cell surface antigens CD3, CD4, CD8, CD56, T-cell receptor , and T-cell receptor , and surface immunofluorescence was analyzed by flow cytometry. TGF-RII-cyt-transduced CTLs proliferate normally in long-term culture but die rapidly in the absence of exogenous growth factors and antigenic stimulation Unresponsiveness to TGF- might lead to a loss of dependence on other growth regulatory signals and, hence, to uncontrolled T lymphoproliferation. To exclude this possibility, the growth of the mature CTLs in the absence of growth stimuli was assessed. Transduced and nontransduced CTLs were cultured in the absence of antigenic stimulation (LCLs) and the growth factor rhIL-2 for 3 weeks. Both transduced and nontransduced CTLs failed to proliferate and became nonviable after 3 weeks in the absence of IL-2 and LCL stimulation (Figure 9). View larger version (27K): [in this window] [in a new window]   Figure 9. HATGF-RII-cyt-transduced CTLs will fail to proliferate in the absence of IL-2 or LCL stimulation. The proliferation of long-term HATGF-RII-cyt-transduced CTL (gray) and nontransduced CTL lines (black) were determined by 3[H]thymidine uptake. The 3[H]thymidine uptake of CTL lines left in culture for 4 weeks after transduction and stimulated weekly with LCL and IL-2 were compared with the same CTL lines grown in the absence of LCL/IL-2 stimulation for 3 weeks.     Discussion Top Abstract Introduction Materials and methods Results Discussion References Secretion of TGF- is a strategy commonly used by tumors to thwart cellular immune responses. It prevents the maturation of professional APCs and inhibits T-cell proliferation, cytokine release, and cytolytic activity.37 This effect may severely affect adoptively transferred tumor-specific CTLs used in cellular immunotherapy. Tumor cells may themselves be sensitive to TGF--induced differentiation and apoptosis but can avoid this fate if they also possess mutant receptors for the cytokine.24,38,39 We now show that such mutants can be adapted for the protection of tumor-specific CTLs. A dominant-negative mutant TGF- type II receptor, in which the cytoplasmic signaling domain is deleted, protects virus-specific CTLs from the inhibitory effects of TGF- signaling.16 Expression of the mutant receptor did not result in alterations in phenotype, cytotoxic specificity, or requirement for growth-regulatory signals. This tumor-derived defense may have clinical value when adoptively transferred T cells are used for the treatment of TGF--secreting tumors, including Hodgkin lymphoma. We chose EBV-positive Hodgkin lymphoma to investigate this approach because the tumor cells express well-defined (viral) tumor antigens to which CTLs can readily be generated. Hodgkin tumors also secrete TGF-, which may contribute to the limited clinical effectiveness of CTLs in this disease, compared with other EBV-positive malignancies.26 The experiments were performed by using polyclonal CTLs, as these cells have been successfully used in the clinical setting.3 Polyclonal CTLs are less likely to be successfully evaded by escape mutants.40 In addition, the presence of CD4+ T cells has been shown to be important for long-term CTL persistence in vivo as well as the maintenance of CD8+ T-lymphocyte-mediated antiviral or antitumor immunity.40,41 The polyclonal EBV-specific CTL lines generated from healthy individuals and patients with Hodgkin disease were readily transduced with a GALV-pseudotyped retrovirus expressing a dominant-negative TGF-RII, and transduced CTLs were resistant to the antiproliferative effects of TGF-. Not only was there significantly less inhibition of proliferation after 72 hours of culture with TGF-, but also transduced CTLs continued to grow in the presence of the cytokine, whereas untransduced CTLs were nonviable after 12 days of culture. Additionally, CTLs could be transduced from day 21 to day 105 of culture, resulting in reproducible effects that were maintained for up to 120 days, thereby testifying to the robust nature of this modification. Inhibition of the TGF- signal transduction pathway in the TGF-RII-cyt-transduced CTL was demonstrated by an absence of detectable phosphorylated Smad2 on Western blot in the presence of TGF-. In nontransduced and transduced CTLs, TGF- binds to TGF-RII on the cell surface. However, in mock-transduced CTLs, the ligand-bound TGF-RII then interacts with and phosphorylates TGF-RI. Phosphorylation activates the intrinsic kinase activity of TGF-RI, allowing the receptor to phosphorylate and thereby activate Smad proteins as confirmed by the detection of phosphorylated Smad2 on Western analysis in the mock-transduced CTLs. Once activated by phosphorylation, the Smad complexes migrate to the nucleus, where they recruit other transcription factors and stimulate the expression of genes, including mediators of cell growth.42 In contrast, when TGF- is added to cells expressing the truncated dominant-negative TGF-RII, the lack of the intracellular domain prevents phosphorylation of TGF-RI16 and the CTL gene-modified abrogation of all downstream signaling events (including Smad phosphorylation). The complete lack of detection of phosphorylated Smad2 in the HATGF-RII-cyt-transduced CTL suggests either that transduction efficiency was higher than suggested by HA detection and/or the presence of transacting protection. TGF- also prevented the secretion of IFN- and GM-CSF from EBV-CTLs in response to stimulation with autologous LCLs. In contrast, cytokine release from transduced CTLs was minimally inhibited, and protection was almost complete when the CTLs were selected for expression of the transgene by using the HA tag. Although these CTLs, which were sorted by using an anti-HA antibody, did show a near complete resistance to these TGF- effects, the marked resistance demonstrated with the unsorted-transduced CTLs is important when we consider using such gene-modified CTLs clinically, because the approval of such a clinical protocol may be less likely with the use of antibody-selected gene-modified CTL populations. Further, selection for TGF--resistant tumor-specific CTLs is likely to occur in vivo and, therefore, should be unnecessary in vitro. Finally, although the cytolytic activity of EBV-specific CTLs was greatly reduced after culture in the presence of TGF-, the cytotoxic activity of the unsorted HATGF-RII-cyt-transduced CTLs was unaffected. TGF- has been shown to reduce the cytolytic activity of alloreactive cytotoxic CD8+ and CD4+ T cells,43 and, in the presence of IL-10, TGF- anergizes alloreactive CD4+ T cells, which remain tolerant in vivo.44 In addition, TGF- has been shown to inhibit IL-12 and IL-2-induced cell proliferation and IFN- production by T cells.45-47 This finding is partly due to a down-regulation of the IL-12 receptor 2 chain expression by TGF-, resulting in the inhibition of antigen-specific activation and cytokine secretion.47 The antiproliferative effects of TGF- on cell growth appear to be secondary to the effect of TGF- on tyrosine phosphorylation, which results in altered control of expression and activation of cell cycle regulatory molecules.44 The anticytotoxic activities of TGF- are mediated by 2 mechanisms. First, TGF- inhibits early signal transduction events such as Janus kinase 2, Tyk2, and STAT4 phosphorylation after the interaction of IL-12 with its receptor on activated T cells.48 This suppression of IL-12 signaling likely explains the reduction of IFN- release seen in our nontransduced and eGFP-transduced established EBV-CTL lines by TGF-.46,49 Second, the regulatory effect of TGF- on human alloreactive CTL cytotoxic activity is associated with down-regulation of perforin and granzyme B gene expression.43,50 We also found that inhibition of EBV-specific CTL killing coincides with a significant reduction in intracellular perforin expression. In addition, we found a reduction in GM-CSF release with the addition of TGF- to CTL cultures. Although this finding has not been previously described, it may be the result of TGF- interference of the GM-CSF signaling pathway, including an inhibitory effect on Janus kinase 2 and STAT5.51-53 All of these effects can be prevented by expression of the HATGF-RII-cyt receptor. Although the approach we describe may counteract one of the most important tumor immune evasion strategies, many others remain intact. For example, Hodgkin tumor cells down-regulate the immunodominant viral latency proteins, EBNAs 3A, 3B, and 3 that are expressed in EBV-LPD of immunosuppressed individuals and express at least one other cytokine (IL-10) that shares with TGF- the ability to inhibit professional APC and, indirectly, CTL activation.23,48,54 Hodgkin cells also secrete the chemokine TARC, which specifically recruits IL-4-secreting Th2 cells, and a Th2 growth factor, IL-13.55-58 Both favor the creation of a noncytotoxic Th2 environment. Although this is a daunting list of evasion mechanisms, their very profligacy argues that multiple defenses are required to protect the tumor cells from attack. Abrogation of even one or two may, therefore, produce a significant change in the effectiveness of cellular immunotherapy. Adoptive immunotherapy with ex vivo-expanded tumor-specific CTLs may evade the inhibition of professional APC function, whereas transferred resistance to TGF- may ensure that these infused CTLs can continue to proliferate and function even in a tumor environment rich in this cytokine. One potential concern facing the clinical use of TGF--resistant CTLs is that lack of response to this inhibitory cytokine may undesirably impair homeostasis of the tumor-specific lymphocytes. Indeed, mice made transgenic for a human dominant-negative TGF-RII (DNRII) expressed exclusively in T cells, develop CD8+ lymphoproliferation.59 However, the T cells involved in the lymphoproliferation were naive and IL-2 independent, and they exhibited patterns of recirculation and homeostasis that were distinct from the mature "memory" T cells studied here.60,61 Other mechanisms, such as Fas and tumor necrosis factor pathways as well of lack of growth stimulation by antigen and growth factors are also available to maintain mature T-cell homeostasis.23,62,63 Certainly, we found no evidence that even long-term expression of the mutant TGF-RII had any deleterious effects on the transduced CTL lines. In particular, their phenotype and cytotoxic specificity were unmodified, and, although they continued to grow and secrete cytokines in response to stimulation and culture in IL-2, withdrawal of these stimulants led to cell death that followed identical temporal kinetics to nontransduced cells. These results were reproducible in all 4 healthy donor CTLs and in all 4 lines derived from patients with Hodgkin disease. Another possible concern is that CTLs transduced with a mutant TGF-RII may be recognized and eliminated by the host immune response. This situation is unlikely, because the truncation used to create the mutant receptor creates no new epitopes. Hence, CTLs expressing this construct should have a selective growth and functional advantage in vivo in patients with TGF--secreting tumors but should not be capable of autonomous growth. We conclude that the expression of a transdominant-negative TGF- receptor II (HATGF-RII-cyt) in tumor-specific CTLs may allow at least one human tumor evasion strategy to be overcome.     Acknowledgments We thank the Texas Childrens' Hospital, The Methodist Hospital, and Baylor College of Medicine for their contribution.     Footnotes Submitted August 24, 2001; accepted December 4, 2001. Supported by the Department of Pediatrics, Baylor College of Medicine, Houston, TX, research grant CA61384 from the National Institutes of Health and by Royal Australasian College of Physicians Odlin Fellowship (C.M.B.) and a Distinguished Clinical Scientist Award from the Doris Duke Foundation (H.E.H.). The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Cliona M. Rooney, Center for Cell and Gene Therapy, Baylor College of Medicine, 6621 Fannin St, Houston, TX 77030; e-mail:cmrooney{at}txccc.org.     References Top Abstract Introduction Materials and methods Results Discussion References 1. Greenberg PD, Riddell SR. Deficient cellular immunityfinding and fixing the defects. Science. 1999;285:546-551[Abstract/Free Full Text]. 2. Zhai Y, Yang JC, Kawakami Y, et al. Antigen-specific tumor vaccines. Development and characterization of recombinant adenoviruses encoding MART1 or gp100 for cancer therapy. J Immunol. 1996;156:700-710[Abstract]. 3. Rooney CM, Smith CA, Ng CY, et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood. 1998;92:1549-1555[Abstract/Free Full Text]. 4. Chouaib S, Asselin-Paturel C, Mami-Chouaib F, Caignard A, Blay JY. The host-tumor immune conflict: from immunosuppression to resistance and destruction. Immunol Today. 1997;18:493-497[CrossRef][Medline] [Order article via Infotrieve]. 5. Massague J. The transforming growth factor-beta family. Annu Rev Cell Biol. 1990;6:597-641[CrossRef]. 6. Border WA, Noble NA. Transforming growth factor beta in tissue fibrosis. N Engl J Med. 1994;331:1286-1292[Free Full Text]. 7. Roberts AB, Sporn MB, Assoian RK, et al. Transforming growth factor type beta: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci U S A. 1986;83:4167-4171[Abstract/Free Full Text]. 8. Palladino MA, Morris RE, Starnes HF, Levinson AD. The transforming growth factor-betas. A new family of immunoregulatory molecules. Ann N Y Acad Sci. 1990;593:181-187[Medline] [Order article via Infotrieve]. 9. Fargeas C, Wu CY, Nakajima T, Cox D, Nutman T, Delespesse G. Differential effect of transforming growth factor beta on the synthesis of Th1- and Th2-like lymphokines by human T lymphocytes. Eur J Immunol. 1992;22:2173-2176[Medline] [Order article via Infotrieve]. 10. Hsieh CL, Chen DS, Hwang LH. Tumor-induced immunosuppression: a barrier to immunotherapy of large tumors by cytokine-secreting tumor vaccine. Hum Gene Ther. 2000;11:681-692[CrossRef][Medline] [Order article via Infotrieve]. 11. Scarpa S, Coppa A, Ragano-Caracciolo M, et al. Transforming growth factor beta regulates differentiation and proliferation of human neuroblastoma. Exp Cell Res. 1996;229:147-154[CrossRef][Medline] [Order article via Infotrieve]. 12. Poppema S, Potters M, Visser L, van Den Berg AM. Immune escape mechanisms in Hodgkin's disease. Ann Oncol. 1998;9(suppl 5):S21-S24. 13. Attisano L, Carcamo J, Ventura F, Weis FM, Massague J, Wrana JL. Identification of human activin and TGF beta type I receptors that form heteromeric kinase complexes with type II receptors. Cell. 1993;75:671-680[CrossRef][Medline] [Order article via Infotrieve]. 14. Wrana JL, Attisano L, Carcamo J, et al. TGF beta signals through a heteromeric protein kinase receptor complex. Cell. 1992;71:1003-1014[CrossRef][Medline] [Order article via Infotrieve]. 15. Wrana JL, Attisano L, Wieser R, Ventura F, Massague J. Mechanism of activation of the TGF-beta receptor. Nature. 1994;370:341-347[CrossRef][Medline] [Order article via Infotrieve]. 16. Wieser R, Attisano L, Wrana JL, Massague J. Signaling activity of transforming growth factor beta type II receptors lacking specific domains in the cytoplasmic region. Mol Cell Biol. 1993;13:7239-7247[Abstract/Free Full Text]. 17. Carcamo J, Zentella A, Massague J. Disruption of transforming growth factor beta signaling by a mutation that prevents transphosphorylation within the receptor complex. Mol Cell Biol. 1995;15:1573-1581[Abstract]. 18. Jayaraman L, Massague J. Distinct oligomeric states of SMAD proteins in the transforming growth factor-beta pathway. J Biol Chem. 2000;275:40710-40717[Abstract/Free Full Text]. 19. Massague J. TGF-beta signal transduction. Annu Rev Biochem. 1998;67:753-791[CrossRef][Medline] [Order article via Infotrieve]. 20. Depoortere F, Pirson I, Bartek J, Dumont JE, Roger PP. Transforming growth factor beta(1) selectively inhibits the cyclic AMP-dependent proliferation of primary thyroid epithelial cells by preventing the association of cyclin D3-cdk4 with nuclear p27(kip1). Mol Biol Cell. 2000;11:1061-1076[Abstract/Free Full Text]. 21. Sandhu C, Garbe J, Bhattacharya N, et al. Transforming growth factor beta stabilizes p15INK4B protein, increases p15INK4B-cdk4 complexes, and inhibits cyclin D1-cdk4 association in human mammary epithelial cells. Mol Cell Biol. 1997;17:2458-2467[Abstract]. 22. Ebner R, Chen RH, Shum L, et al. Cloning of a type I TGF-beta receptor and its effect on TGF-beta binding to the type II receptor. Science. 1993;260:1344-1348[Abstract/Free Full Text]. 23. Ranges GE, Figari IS, Espevik T, Palladino MA Jr. Inhibition of cytotoxic T cell development by transforming growth factor beta and reversal by recombinant tumor necrosis factor alpha. J Exp Med. 1987;166:991-998[Abstract/Free Full Text]. 24. Park K, Kim SJ, Bang YJ, et al. Genetic changes in the transforming growth factor beta (TGF-beta) type II receptor gene in human gastric cancer cells: correlation with sensitivity to growth inhibition by TGF-beta. Proc Natl Acad Sci U S A. 1994;91:8772-8776[Abstract/Free Full Text]. 25. Knaus PI, Lindemann D, DeCoteau JF, et al. A dominant inhibitory mutant of the type II transforming growth factor beta receptor in the malignant progression of a cutaneous T-cell lymphoma. Mol Cell Biol. 1996;16:3480-3489[Abstract]. 26. Roskrow MA, Suzuki N, Gan Y, et al. Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes for the treatment of patients with EBV-positive relapsed Hodgkin's disease. Blood. 1998;91:2925-2934[Abstract/Free Full Text]. 27. Bollard C, Gahn B, Aguilar L, et al. Cytotoxic T lymphocyte therapy for EBV+ Hodgkin disease [abstract]. Blood. 2000;96:576a. 28. Kinsella TM, Nolan GP. Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum Gene Ther. 1996;7:1405-1413[Medline] [Order article via Infotrieve]. 29. Wilson IA, Niman HL, Houghten RA, Cherenson AR, Connolly ML, Lerner RA. The structure of an antigenic determinant in a protein. Cell. 1984;37:767-778[CrossRef][Medline] [Order article via Infotrieve]. 30. Riviere I, Brose K, Mulligan RC. Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells. Proc Natl Acad Sci U S A. 1995;92:6733-6737[Abstract/Free Full Text]. 31. Smith LJ, Braylan RC, Edmundson KB, Nutkis JE, Wakeland EK. In vitro transformation of human B-cell follicular lymphoma cells by Epstein-Barr virus. Cancer Res. 1987;47:2062-2066[Abstract/Free Full Text]. 32. Heslop HE, Brenner MK, Rooney C, et al. Administration of neomycin-resistance-gene-marked EBV-specific cytotoxic T lymphocytes to recipients of mismatched-related or phenotypically similar unrelated donor marrow grafts. Hum Gene Ther. 1994;5:381-397[Medline] [Order article via Infotrieve]. 33. Rooney CM, Smith CA, Ng CY, et al. Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation. Lancet. 1995;345:9-13[CrossRef][Medline] [Order article via Infotrieve]. 34. Bonini C, Ferrari G, Verzeletti S, et al. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science. 1997;276:1719-1724[Abstract/Free Full Text]. 35. Kretzschmar M, Doody J, Timokhina I, Massague J. A mechanism of repression of TGFbeta/Smad signaling by oncogenic Ras. Genes Dev. 1999;13:804-816[Abstract/Free Full Text]. 36. Lie YS, Petropoulos CJ. Advances in quantitative PCR technology: 5' nuclease assays. Curr Opin Biotechnol. 1998;9:43-48[CrossRef][Medline] [Order article via Infotrieve]. 37. de Visser KE, Kast WM. Effects of TGF-beta on the immune system: implications for cancer immunotherapy. Leukemia. 1999;13:1188-1199[CrossRef][Medline] [Order article via Infotrieve]. 38. Inagaki M, Moustakas A, Lin HY, Lodish HF, Carr BI. Growth inhibition by transforming growth factor beta (TGF-beta) type I is restored in TGF-beta-resistant hepatoma cells after expression of TGF-beta receptor type II cDNA. Proc Natl Acad Sci U S A. 1993;90:5359-5363[Abstract/Free Full Text]. 39. Markowitz S, Wang J, Myeroff L, et al. Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. Science. 1995;268:1336-1338[Abstract/Free Full Text]. 40. Gottschalk S, Ng CY, Perez M, et al. An Epstein-Barr virus deletion mutant associated with fatal lymphoproliferative disease unresponsive to therapy with virus-specific CTLs. Blood. 2001;97:835-843[Abstract/Free Full Text]. 41. Matloubian M, Concepcion RJ, Ahmed R. CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection. J Virol. 1994;68:8056-8063[Abstract/Free Full Text]. 42. Zhou S, Kinzler KW, Vogelstein B. Going mad with Smads. N Engl J Med. 1999;341:1144-1146[Free Full Text]. 43. Asselin-Paturel C, Pardoux C, Gay F, Chouaib S. Failure of TGF beta1 and IL-12 to regulate human FasL and mTNF alloreactive cytotoxic T-cell pathways. Tissue Antigens. 1998;51:242-249[Medline] [Order article via Infotrieve]. 44. Boussiotis VA, Chen ZM, Zeller JC, et al. Altered T-cell receptor + CD28-mediated signaling and blocked cell cycle progression in interleukin 10 and transforming growth factor-beta-treated alloreactive T cells that do not induce graft-versus-host disease. Blood. 2001;97:565-571[Abstract/Free Full Text]. 45. Sudarshan C, Galon J, Zhou Y, O'Shea JJ. TGF-beta does not inhibit IL-12- and IL-2-induced activation of Janus kinases and STATs. J Immunol. 1999;162:2974-2981[Abstract/Free Full Text]. 46. Bonig H, Banning U, Hannen M, et al. Transforming growth factor-beta1 suppresses interleukin-15-mediated interferon-gamma production in human T lymphocytes. Scand J Immunol. 1999;50:612-618[CrossRef][Medline] [Order article via Infotrieve]. 47. Ludviksson BR, Seegers D, Resnick AS, Strober W. The effect of TGF-beta1 on immune responses of naive versus memory CD4+ Th1/Th2 T cells. Eur J Immunol. 2000;30:2101-2111[CrossRef][Medline] [Order article via Infotrieve]. 48. Pardoux C, Ma X, Gobert S, et al. Downregulation of interleukin-12 (IL-12) responsiveness in human T cells by transforming growth factor-beta: relationship with IL-12 signaling. Blood. 1999;93:1448-1455[Abstract/Free Full Text]. 49. Van Weyenbergh J, P Silva MP, Bafica A, Cardoso S, Wietzerbin J, Barral-Netto M. IFN-beta and TGF-beta differentially regulate IL-12 activity in human peripheral blood mononuclear cells. Immunol Lett. 2001;75:117-122[CrossRef][Medline] [Order article via Infotrieve]. 50. Smyth MJ, Strobl SL, Young HA, Ortaldo JR, Ochoa AC. Regulation of lymphokine-activated killer activity and pore-forming protein gene expression in human peripheral blood CD8+ T lymphocytes. Inhibition by transforming growth factor-beta. J Immunol. 1991;146:3289-3297[Abstract]. 51. Kelso A. Cytokines: principles and prospects. Immunol Cell Biol. 1998;76:300-317[CrossRef][Medline] [Order article via Infotrieve]. 52. Bright JJ, Kerr LD, Sriram S. TGF-beta inhibits IL-2-induced tyrosine phosphorylation and activation of Jak-1 and Stat 5 in T lymphocytes. J Immunol. 1997;159:175-183[Abstract]. 53. Pazdrak K, Justement L, Alam R. Mechanism of inhibition of eosinophil activation by transforming growth factor-beta. Inhibition of Lyn, MAP, Jak2 kinases and STAT1 nuclear factor. J Immunol. 1995;155:4454-4458[Abstract]. 54. Rickinson AB, Moss DJ. Human cytotoxic T lymphocyte responses to Epstein-Barr virus infection. Annu Rev Immunol. 1997;15:405-431[CrossRef][Medline] [Order article via Infotrieve]. 55. Graus F, Gultekin SH, Ferrer I, Reiriz J, Alberch J, Dalmau J. Localization of the neuronal antigen recognized by anti-Tr antibodies from patients with paraneoplastic cerebellar degeneration and Hodgkin's disease in the rat nervous system. Acta Neuropathol (Berl). 1998;96:1-7[CrossRef][Medline] [Order article via Infotrieve]. 56. Seo N, Tokura Y. Downregulation of innate and acquired antitumor immunity by bystander gammadelta and alphabeta T lymphocytes with Th2 or Tr1 cytokine profiles. J Interferon Cytokine Res. 1999;19:555-561[CrossRef][Medline] [Order article via Infotrieve]. 57. Sallusto F, Lenig D, Mackay CR, Lanzavecchia A. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J Exp Med. 1998;187:875-883[Abstract/Free Full Text]. 58. Skinnider BF, Elia AJ, Gascoyne RD, et al. Interleukin 13 and interleukin 13 receptor are frequently expressed by Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma. Blood. 2001;97:250-255[Abstract/Free Full Text]. 59. Lucas PJ, Kim SJ, Melby SJ, Gress RE. Disruption of T cell homeostasis in mice expressing a T cell-specific dominant negative transforming growth factor beta II receptor. J Exp Med. 2000;191:1187-1196[Abstract/Free Full Text]. 60. McHeyzer-Williams MG, Davis MM. Antigen-specific development of primary and memory T cells in vivo. Science. 1995;268:106-111[Abstract/Free Full Text]. 61. Picker LJ, Treer JR, Ferguson-Darnell B, Collins PA, Buck D, Terstappen LW. Control of lymphocyte recirculation in man. I. Differential regulation of the peripheral lymph node homing receptor L-selection on T cells during the virgin to memory cell transition. J Immunol. 1993;150:1105-1121[Abstract]. 62. Genestier L, Kasibhatla S, Brunner T, Green DR. Transforming growth factor beta1 inhibits Fas ligand expression and subsequent activation-induced cell death in T cells via downregulation of c-Myc. J Exp Med. 1999;189:231-239[Abstract/Free Full Text]. 63. Suda T, Zlotnik A. In vitro induction of CD8 expression on thymic pre-T cells. I. Transforming growth factor-beta and tumor necrosis factor-alpha induce CD8 expression on CD8 thymic subsets including the CD25+CD3CD4CD8 pre-T cell subset. J Immunol. 1992;148:1737-1745[Abstract]. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: G. Dotti Blocking PD-1 in cancer immunotherapy Blood, August 20, 2009; 114(8): 1457 - 1458. [Abstract] [Full Text] [PDF] S. Kim, G. Buchlis, Z. G. Fridlender, J. Sun, V. Kapoor, G. Cheng, A. Haas, H. K. Cheung, X. Zhang, M. Corbley, et al. Systemic Blockade of Transforming Growth Factor-{beta} Signaling Augments the Efficacy of Immunogene Therapy Cancer Res., December 15, 2008; 68(24): 10247 - 10256. [Abstract] [Full Text] [PDF] M. Zhang, B. E. Berndt, J.-J. Chen, and J. Y. Kao Expression of a Soluble TGF-{beta} Receptor by Tumor Cells Enhances Dendritic Cell/Tumor Fusion Vaccine Efficacy J. Immunol., September 1, 2008; 181(5): 3690 - 3697. [Abstract] [Full Text] [PDF] A. Wallace, V. Kapoor, J. Sun, P. Mrass, W. Weninger, D. F. Heitjan, C. June, L. R. Kaiser, L. E. Ling, and S. M. Albelda Transforming Growth Factor-{beta} Receptor Blockade Augments the Effectiveness of Adoptive T-Cell Therapy of Established Solid Cancers Clin. Cancer Res., June 15, 2008; 14(12): 3966 - 3974. [Abstract] [Full Text] [PDF] G Kapatai and P Murray Contribution of the Epstein Barr virus to the molecular pathogenesis of Hodgkin lymphoma J. Clin. Pathol., December 1, 2007; 60(12): 1342 - 1349. [Abstract] [Full Text] [PDF] J. M. Chemnitz, D. Eggle, J. Driesen, S. Classen, J. L. Riley, S. Debey-Pascher, M. Beyer, A. Popov, T. Zander, and J. L. Schultze RNA fingerprints provide direct evidence for the inhibitory role of TGF{beta} and PD-1 on CD4+ T cells in Hodgkin lymphoma Blood, November 1, 2007; 110(9): 3226 - 3233. [Abstract] [Full Text] [PDF] J. A. Westwood, M. J. Smyth, M. W. L. Teng, M. Moeller, J. A. Trapani, A. M. Scott, F. E. Smyth, G. A. Cartwright, B. E. Power, D. Honemann, et al. Adoptive transfer of T cells modified with a humanized chimeric receptor gene inhibits growth of Lewis-Y-expressing tumors in mice PNAS, December 27, 2005; 102(52): 19051 - 19056. [Abstract] [Full Text] [PDF] G. Dotti, B. Savoldo, M. Pule, K. C. Straathof, E. Biagi, E. Yvon, S. Vigouroux, M. K. Brenner, and C. M. Rooney Human cytotoxic T lymphocytes with reduced sensitivity to Fas-induced apoptosis Blood, June 15, 2005; 105(12): 4677 - 4684. [Abstract] [Full Text] [PDF] K. C. Straathof, M. A. Pule, P. Yotnda, G. Dotti, E. F. Vanin, M. K. Brenner, H. E. Heslop, D. M. Spencer, and C. M. Rooney An inducible caspase 9 safety switch for T-cell therapy Blood, June 1, 2005; 105(11): 4247 - 4254. [Abstract] [Full Text] [PDF] M. Ahmadzadeh and S. A. Rosenberg TGF-{beta}1 Attenuates the Acquisition and Expression of Effector Function by Tumor Antigen-Specific Human Memory CD8 T Cells J. Immunol., May 1, 2005; 174(9): 5215 - 5223. [Abstract] [Full Text] [PDF] J. E. Dierksheide, R. A. Baiocchi, A. K. Ferketich, S. Roychowdhury, R. P. Pelletier, C. F. Eisenbeis, M. A. Caligiuri, and A. M. VanBuskirk IFN-{gamma} gene polymorphisms associate with development of EBV+ lymphoproliferative disease in hu PBL-SCID mice Blood, February 15, 2005; 105(4): 1558 - 1565. [Abstract] [Full Text] [PDF] S. Poppema Immunobiology and Pathophysiology of Hodgkin Lymphomas Hematology, January 1, 2005; 2005(1): 231 - 238. [Abstract] [Full Text] [PDF] Y.-J. Kim, T. M. Stringfield, Y. Chen, and H. E. Broxmeyer Modulation of cord blood CD8+ T-cell effector differentiation by TGF-{beta}1 and 4-1BB costimulation Blood, January 1, 2005; 105(1): 274 - 281. [Abstract] [Full Text] [PDF] C. M. Bollard, L. Aguilar, K. C. Straathof, B. Gahn, M. H. Huls, A. Rousseau, J. Sixbey, M. V. Gresik, G. Carrum, M. Hudson, et al. Cytotoxic T Lymphocyte Therapy for Epstein-Barr Virus+ Hodgkin's Disease J. Exp. Med., December 20, 2004; 200(12): 1623 - 1633. [Abstract] [Full Text] [PDF] S. Poppema Regulatory T cells in Hodgkin lymphoma Blood, March 1, 2004; 103(5): 1565 - 1566. [Full Text] [PDF] K. C.M. Straathof, C. M. Bollard, C. M. Rooney, and H. E. Heslop Immunotherapy for Epstein-Barr Virus-Associated Cancers in Children Oncologist, February 1, 2003; 8(1): 83 - 98. [Abstract] [Full Text] [PDF] H. E. Heslop, F. K. Stevenson, and J. J. Molldrem Immunotherapy of Hematologic Malignancy Hematology, January 1, 2003; 2003(1): 331 - 349. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Bollard, C. M. Articles by Rooney, C. M. Search for Related Content PubMed PubMed Citation Articles by Bollard, C. M. Articles by Rooney, C. M. Related Collections Immunotherapy Gene Therapy Social Bookmarking                   What's this?

	Copyright © 2002 by American Society of Hematology         Online ISSN: 1528-0020