doi:10.1182/blood.V99.9.31792002 99: 3179-3187   

 Massague, Malcolm K. Brenner, Helen E. Heslop and Cliona M. RooneyCatherine M. Bollard, Claudia Rössig, M. Julia Calonge, M. Helen Huls, Hans-Joachim Wagner, Joan to enhance antitumor immunity

related tumor protection strategy−βAdapting a transforming growth factor

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GENE THERAPY

Adapting a transforming growth factor�–related tumor protection strategy toenhance antitumor immunityCatherine M. Bollard, Claudia Rossig, M. Julia Calonge, M. Helen Huls, Hans-Joachim Wagner, Joan Massague, Malcolm K. Brenner,Helen E. Heslop, and Cliona M. Rooney

Transforming growth factor � (TGF-�), apleiotropic cytokine that regulates cellgrowth and differentiation, is secreted bymany human tumors and markedly inhib-its tumor-specific cellular immunity. Tu-mors can avoid the differentiating andapoptotic effects of TGF-� by expressinga nonfunctional TGF-� receptor. We havedetermined whether this immune evasionstrategy can be manipulated to shieldtumor-specific cytotoxic T lymphocytes(CTLs) from the inhibitory effects of tu-mor-derived TGF-�. As our model weused Epstein-Barr virus (EBV)–specificCTLs that are infused as treatment for

EBV-positive Hodgkin disease but thatare vulnerable to the TGF-� produced bythis tumor. CTLs were transduced with aretrovirus vector expressing the domi-nant-negative TGF-� type II receptorHATGF-�RII-�cyt. HATGF-�RII-�cyt– butnot green fluorescence protein (eGFP)–transduced CTLs was resistant to theantiproliferative and anticytotoxic effectsof exogenous TGF-�. Additionally, recep-tor-transduced cells continued to secretecytokines in response to antigenic stimu-lation. TGF-� receptor ligation results inphosphorylation of Smad2, and this path-way was disrupted in HATGF-�RII-�cyt–

transduced CTLs, confirming blockade ofthe signal transduction pathway. Long-termexpression of TGF-�RII-�cyt did not af-fect CTL function, phenotype, or growthcharacteristics. Tumor-specific CTLs ex-pressing HATGF-�RII-�cyt should have aselective functional and survival advan-tage over unmodified CTLs in the pres-ence of TGF-�–secreting tumors and maybe of value in treatment of these dis-eases. (Blood. 2002;99:3179-3187)

© 2002 by The American Society of Hematology

Introduction

Immunotherapy strategies to boost cellular immune responses totumors are increasingly applied as more tumor antigens areidentified.1,2 The most successful use of adoptively transferredantigen-specific cytotoxic T cells has occurred in severely immuno-compromised individuals whose tumors require few immuneevasion strategies.3 By contrast, tumor immunotherapy in immuno-competent hosts has been of more limited benefit. In the presenceof a normal immune system, tumors frequently develop immuneevasion strategies that may influence every stage of the generationof a tumor-specific cellular immune response, from the activationof professional antigen-presenting cells (APCs) to T-cell recruit-ment, activation, and effector function.4

Tumor secretion of transforming growth factor� (TGF-�) isamong the most widely used evasion strategies. TGF-� is aubiquitous cytokine with pleiotropic effects on cell growth, differ-entiation, 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 promotesangiogenesis.7 As a growth inhibitor, it plays a role in T-cellhomeostasis by limiting immune responses to antigens and byinducing tolerance.8,9 Hence, secretion of this cytokine by malig-nant cells such as neuroblastoma and Hodgkin Reed-Sternbergtumor cells may diminish the effectiveness of antitumor T-cellimmune responses.10-12

The binding of TGF-�to either TGF-�receptor I (TGF-�RI) orTGF-� receptor II (TGF-�RII), results in the formation of atetramer complex, involving the dimers of the type I and IIreceptors, which is required for signaling.13-17With this interactionbetween 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 thenucleus, the Smads interact with transcription factors such as thoseinvolved in the regulation of cell growth and differentiation.20,21

TGF-�may also have adverse effects on tumor cells themselvesby promoting terminal differentiation and apoptosis. Tumors mayavoid this activity by mutation of their TGF-� receptors (TGF-�RIand TGF-�RII).13,22,23Each of these receptors possesses an extracel-lular region, a single transmembrane domain, and a cytoplasmicsignaling domain containing a serine/threonine kinase domain.Mutations in the TGF-�RII gene that correlate with loss ofsensitivity to TGF-�have been identified in many tumors in whichinhibition 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 aBamHI siteintroduced after the 10th cytoplasmic codon (nt597).16 This mutantreceptor lacks the entire kinase domain and most of the juxtamem-brane region. HATGF-�RII-�cyt has been shown to act in adominant-negative fashion when transfected into the mink lung

From the Center for Cell and Gene Therapy, Departments of Pediatrics,Molecular Virology and Microbiology, and Medicine, Baylor College ofMedicine, Houston, TX; and Memorial Sloan Kettering Cancer Center, NewYork, NY.

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 Healthand 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.).

Reprints: Cliona M. Rooney, Center for Cell and Gene Therapy, Baylor Collegeof Medicine, 6621 Fannin St, Houston, TX 77030; e-mail:cmrooney@txccc.org.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.

© 2002 by The American Society of Hematology

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epithelial cell line, diminishing the cells’ antiproliferative andtranscriptional responses to TGF-�.16

We have determined whether the strategy used by tumor cells toprotect themselves against the effects of TGF-� can be manipulatedto shield tumor-specific cytotoxic T lymphocytes (CTLs) from theinhibitory effects of tumor-secreted TGF-�. As our model, we havetaken Epstein-Barr virus (EBV) antigen-positive Hodgkin disease,a tumor that expresses virus-specific antigens and should besusceptible to specific CTLs.26 Indeed, tracking of geneticallymarked EBV-specific CTL infusions shows homing to Hodgkintumor sites and CTL survival in the patient’s circulation for up to 9months after infusion.27 In addition, CTL infusions enhancedEBV-specific immune responses and reduced virus load, but onlylimited tumor responses of brief duration were observed. Thisfailure may be associated with the demonstrated ability of Hodgkintumor cells to secrete TGF-� and consequent inactivation of CTLsentering the tumor environment.12 We now show that forcedexpression of a dominant-negative TGF-�RII in ex vivo–expandedEBV tumor-specific CTLs from patients with relapsed Hodgkindisease renders them resistant to the inhibitory effects of TGF-�,while enabling them to retain their dependence on other growthregulatory signals.

Materials and methods

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 linethat produces virus pseudotyped with the gibbon-ape leukemia virus(GALV). HSB-2 (ATCC) is a T-cell lymphoma that is sensitive tolymphokine-activated killer cells and was used as a target in cytotoxicityassays. Lymphoblastoid cell lines (LCLs) were generated as described below.

pMEP5/HATGF-�RII-�cyt plasmid

A human type II TGF-� receptor complimentary DNA (cDNA) wastruncated at nt597, thereby deleting most of its cytoplasmic tail and all of itscytoplasmic kinase domain, leaving only 7 amino acids remaining in theintracellular domain.16 A sequence encoding the influenza virus hemagglu-tinin peptide epitope HA1 was spliced into the human TGF-�RIIcDNA.14,16,29 The HA sequence was inserted after the signal sequence in thehuman TGF-�RII so that the HA epitope is retained near the aminoterminus of the mature receptor. The presence of the HA tag does not affectligand binding and allows the mutant construct to be distinguished from thewild type TGF-�RII receptor with an anti-HA antibody.15,16 The functionand the biochemistry of pMEP5/HATGF-�RII-�cyt have been extensivelycharacterized.16 HATGF-�RII-�cyt was placed into the BamHI andNcoI 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 transientlytransfected 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, Iscovesmodified Dulbecco medium (IMDM; Biowhittaker) supplemented with20% FCS (20% IMDM) was added, and cells were incubated at 32°C for 24hours. Fresh retrovirus supernatants were then collected, filtered through a0.45-�m filter, and used to infect the packaging cell line PG13 in thepresence of polybrene (8 �g/mL) for 48 hours at 32°C. The infected cellswere incubated overnight at 37°C in fresh 10% DMEM and then subjectedto a second round of infection under the same conditions by using freshly

generated Phoenix-eco cell supernatants. Viral supernatants were harvestedfrom the resulting bulk producer lines by adding 20% IMDM to the PG-13cells and incubated at 32°C for 24 hours. The supernatant was harvested,filtered by using a 0.45-�m filter, and used directly to transduce the CTLs.

Generation of EBV-transformed B cell lines

Peripheral blood–derived mononuclear cells (PBMCs) (5 � 106) wereincubated with 100 �L concentrated supernatant from the EBV producercell line B95-8 in a total of 200 �L complete medium (RPMI 1640 medium[GIBCO-BRL, Gaithersburg, MD] containing 10% FCS [Hyclone], and 2mM L-glutamine [Biowhittaker]) for 30 minutes. The cells were plated at106 cells per well in a flat-bottomed 96-well plate (Costar; Corning,Corning, NY) containing complete medium and 1 �g/mL cyclosporin A(Sandoz Pharmaceuticals, Washington, DC). Cells were fed weekly untilLCLs were established.31

Generation and transduction of EBV-specific CTL cultures

EBV-specific CTLs were prepared by stimulating PBMCs with the autolo-gous EBV-transformed LCL.32,33 PBMCs (2 � 106) were cocultured with5 � 104 gamma-irradiated (40 Gy) autologous LCLs per well in a 24-wellplate. Starting on day 10, the responder cells were restimulated weekly withirradiated (40 Gy) LCLs at a responder-to-stimulator ratio of 4:1. Twoweekly doses of recombinant human interleukin 2 (rhIL-2; 50 IU/mL) wereadded from day 14. Twenty-four hours after LCL stimulation, CTLs readyfor transduction were transferred to a 24-well plate (Costar), precoated withOKT3 (1 �g/mL; Ortho Pharmaceuticals, Raritan, NJ) and anti-CD28antibody (1 �g/mL; Pharmingen, San Diego, CA) at 1 � 106 cells per welland 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 fibronec-tin fragment (FN CH-296; Retronectin; Takara Shuzo, Otsu, Japan) at aconcentration of 4 �g/cm2. The prestimulated CTL lines were resuspendedat 1 � 106 cells/mL in complete medium supplemented with 45% EHAA(Clicks; GIBCO-BRL) and rhIL-2 (100 IU/mL), then incubated with equalvolumes of freshly generated viral supernatant for 36 hours at 37°C and 5%CO2. Two weeks after transduction, 3 CTL lines from healthy donors werepositively selected for cell surface expression of the HA-tag by usingflow cytometry.

Flow cytometry

For immunophenotyping, cells were stained with fluorescein-conjugatedmonoclonal antibodies (Becton Dickinson, San Jose, CA) directed againstCD3, CD4, CD8, CD16, CD56, and CD25 surface proteins. For eachsample, 10 000 cells were analyzed by FACSCalibur with the Cell QuestSoftware (Becton Dickinson). Surface expression of the HA-epitope wasanalyzed after incubation of CTLs (1 � 106) with the HA� antibody(Sigma, St Louis, MO) at a concentration of 200 ng/5 � 105 in the presenceof normal donkey serum (Jackson Immuno Research Laboratories, WestGrove, PA) for 30 minutes at room temperature. This analysis was followedby incubation with fluorescein isothiocyanate (FITC)-labeled donkeyantirabbit antibody (Jackson Immuno). The perforin assay was performedby fixing the CTLs in 4% paraformaldehyde (Sigma) for 20 minutes. Thecells were then washed in permeabilizing buffer (1 � phosphate-bufferedsaline [PBS; GIBCO-BRL] � 0.1% saponin [Sigma] � 1% FCS [Hy-clone]). CTLs were incubated in 3 mL permeabilizing buffer with 5%human AB-serum (C-6 Diagnostics, Germantown, WI) for 10 minutes atroom temperature. CTLs were spun down and resuspended in 100 �Lpermeabilizing buffer. To each sample, 20 �L of either phycoerythrin(PE)-labeled antiperforin antibody (Pharmingen) or PE-labeled IgG1isotype control (Pharmingen) was added and incubated for 30 minutes atroom temperature. CTLs were washed again with permeabilizing buffer andresuspended 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 mMTris, 150 mM NaCl, 0.5% NP-40, 10 mM EDTA, 1 mM dithiothreitol) with

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phosphatase inhibitors (20 mM �-glycerol phosphate and 20 mM NaVO3)and protease inhibitors. After 5 seconds of sonication, the lysates werecentrifuged at 14 000 rpm for 5 minutes. Protein concentration of thesupernatants was determined by protein assay (BIO-RAD No. 500-0006,Hercules, CA). Protein (50 �g) was loaded on a 9% sodium dodecylsulfate–polyacrylamide gel. Western blot was performed with eitherantiphospho-Smad 2 antibody (Upstate Biotechnology No. 06-829, LakePlacid, NY) at a final concentration of 1 �g/mL or purified anti-Smad2/3rabbit polyclonal antisera.35

Measurement of cytokine production by enzyme-linkedimmunosorbent assay

To assess the effect of the truncated TGF-�RII on cytokine release in thepresence of TGF-�, duplicate samples of transduced and nontransducedeffector cells (5 � 104/well) were cocultured with irradiated, EBV-transformed LCLs at stimulator-to-effector ratios of 1:4 in rhIL-2 (50U/mL) � 5 ng/mL TGF-�1 (R&D Systems, Minneapolis, MN) in 96-wellround-bottom plates (Costar). After 24 hours, the supernatants wereharvested and analyzed for human granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interferon � (IFN-�) by using 96-wellplates 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’sinstructions.

Cytotoxicity assays

To compare the cytotoxic specificity of transduced and nontransducedCTLs in the presence of TGF-�1, standard 51Cr release assays wereperformed. At 72 to 96 hours before performing the cytotoxicity assay, 5ng/mL TGF-�1 (R&D Systems) was added to 8 � 106 transduced andnontransduced CTLs. Doubling dilutions of CTLs were coincubated intriplicate for 4 hours with 5000 51Cr-labeled target cells (AmershamPharmacia Biotech, Piscataway, NJ) in a total volume of 200 �L in aV-bottom 96-well plate (Costar) as previously described.31 The targetstested were autologous LCLs, HLA class I and II mismatched LCLs, andHSB-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 gammacounter (Tri-CARB 4640; Packard BioScience, Downers Grove, IL). Themean percentage of specific lysis of triplicate wells was calculated asfollows: [(test counts � spontaneous counts)/(maximum counts � sponta-neous counts)] � 100%.

Proliferation assays

Transduced CTLs were coincubated in triplicate at 5 � 104 cells/well withirradiated autologous EBV-LCLs at a 4:1 stimulator-to-responder ratio �titrated concentrations of TGF-�1 up to 20 ng/mL. After a 72-hourcoincubation period, wells were pulsed with 0.037 MBq (1 �Ci)/well of[3H]thymidine (Amersham Pharmacia Biotech) for 18 hours, and thesamples were harvested onto glass fiber filter paper for �-scintillationcounting (TriCarb 2500 TR; Packard BioScience).

Quantification of the transduction rate by real-time polymerasechain reaction

DNA was extracted from cytotoxic T cells by using the DNeasy Tissue Kit(Qiagen, Valencia, CA) according to the manufacturer’s instructions. Forquantification of the transduction rate of CTL, real-time polymerase chainreaction (RT PCR) assays specific for the HA sequence were developed byusing 5 nuclease PCR technology and the ABI PRISM 7700 SequenceDetection System (PE Applied Biosystems, Foster City, CA).36 RT PCRamplification was performed with 2� TaqMan Universal Master Mix (PEApplied Biosystems) adjusted to 50 �L with 300 nM of each primer, 200nM probe, template, and nuclease-free water. The forward primer (5-GTGGACGCGTATCGCCAG-3) binds 12 base pair (bp) upstream of theHA sequence, the reverse primer (5-TGTCAGTGACTATCATGTCGTTAT-

TAACC-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 intriplicate for each sample. As positive controls, samples were analyzed forthe �-actin gene in parallel by using the TaqMan Beta-actin DetectionReagents (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 DNApolymerase), and 40 2-step cycles of 15 seconds at 95°C and 60 seconds at60°C. For quantification, serial 1:4-fold dilutions of the plasmid pMEP5/HATGF-�RII-�cyt16 were used as the standard. A correlation coefficient ofmore than 0.99 was found over at least 5 orders of magnitude afteramplification 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 unlessotherwise stated.

Results

Truncated TGF-� receptor mutant was expressed by CTLs aftertransduction with HATGF-�RII-�cyt

Four EBV-specific CTL lines from healthy individuals and 4 frompatients with EBV-positive Hodgkin disease were transduced withHATGF-�RII-�cyt after 21 to 105 days of culture (mean, 52 days).We used RT PCR analysis to compare the transgene copy numberper cell in bulk HATGF-�RII-�cyt–transduced CTLs with that intransduced CTLs that had been sorted by flow cytometry for HAepitope expression. Assuming that 100% of the cells sorted for theHA tag contained at least one copy of SFG:HATGF-�RII-�cytDNA, the transduction efficiency in unsorted CTLs ranged from6.5% to 55% (mean 27%) (Table 1), which is not significantlydifferent from the transduction efficiency with SFG-eGFP (19%-51.5%; mean, 31%) (Figure 1A,B). Mutant TGF-�RII surfaceexpression was also detected by flow cytometry (Figure 1C-E). Byusing the anti-HA antibody, the percentage of expression ofHATGF-�RII-�cyt on CD8� cells ranged from 3.5% to 49.3%(mean, 17%), which is consistent with RT PCR results. Similartransduction efficiencies were also seen for CD4� cells (Figure 1F).CTLs sorted for the HA tag showed 53.15% to 62.6% HAexpression on CD8� cells 6 weeks after sorting (Figure 1G,H).

Table 1. Transduction rate of HATGF-�RII-�cyt ranges from 6.5% to 55% asdetermined by quantitative real-time polymerase chain reaction

No. of transduced copiesper 100 000 cells % transduced*

CTL-sorted (positive selection of

transduced cells by FACS) 310 000 100

Donor 1 (unsorted) 67 550 19

Donor 2 (unsorted) 28 055 10

Patient 1 (unsorted) 141 000 45

Patient 3 (unsorted) 170 000 55

Patient 4 (unsorted) 22 140 6.5

To assess the transduction efficiency, DNA was extracted from transducedcytotoxic T lymphocytes (CTLs) and transduced CTLs sorted for the hemagglutininpeptide epitope tag. These sorted cells were therefore assumed to be 100%transduced. The number of copies per 100 000 cells was calculated by using astandard curve generated by serial dilutions of plasmid DNA from 256 to 131 072copies. Sorted CTLs were compared with 5 unsorted transduced CTL lines, and thetransduction rates were calculated.

*Compared with 100% population of sorted CTLs.

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HATGF-�RII-�cyt–transduced CTLs are resistant to theantiproliferative effects of exogenous TGF-�1

To investigate whether expression of the truncated TGF-�RII byCTLs could overcome the antiproliferative effects of TGF-� onCTLs, we compared thymidine uptake by HATGF-�RII-�cyt–transduced CTLs with eGFP-transduced and -nontransduced CTLsafter addition of TGF-�1 for 72 hours (Figure 2). TGF-�1 had adramatic antiproliferative effect on established eGFP-transducedand -nontransduced EBV-CTLs generated from both healthy do-nors and patients, inhibiting uptake by a mean of 59.5% (range,44%-75%). By contrast, the mean inhibition of thymidine uptakeby HATGF-�RII-�cyt–transduced CTLs was 16% (range, 0%-30%). This resistance to the antiproliferative effects of TGF-� inHATGF-�RII-�cyt–transduced CTLs was statistically significantwhen compared with the mock or nontransduced CTLs (P .03).Importantly, when EBV-specific CTLs were maintained undernormal growth conditions with the addition of TGF-�, they failedto proliferate, and most died within 12 days. HATGF-�RII-�cyt–transduced CTLs, however, continued to proliferate and grownormally, showing that the transduced cells were resistant to theantiproliferative effects of the TGF-�1 (Figure 3A,B).

Phosphorylation of Smad2 is inhibited inTGF-�RII-�cyt–transduced CTLs with addition of TGF-�

To confirm that downstream signaling by TGF-� is abrogated inCTLs transduced with TGF-�RII-�cyt, TGF-� was added tonontransduced, eGFP-transduced, and TGF-�RII-�cyt–transducedCTLs at a concentration of 5 ng/mL. After 60 minutes, the cellswere harvested, and whole cell lysates were prepared. All the CTL

extracts were subjected to Western immunoblotting by usinganti-Smad2/3 antibody and antiphospho-Smad2 antibody (Figure4). 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 innontransduced and eGFP-transduced CTLs treated with TGF-�. Incontrast, there was no expression of P-S2 in TGF-�RII-�cyt–transduced CTLs with the addition of TGF-�, confirming thatsignal transduction was blocked by the presence of thedominant-negative TGF-�RII.

TGF-�RII-�cyt–transduced CTLs continue to producecytokines in response to antigenic stimulusin the presence of TGF-�1

TGF-� inhibited IFN-� and GM-CSF release from nontransducedEBV-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-� and71% (range, 63%-83%) for GM-CSF. By contrast, the meaninhibition of cytokine release by HATGF-�RII-�cyt–transducedCTLs was 43% (range, 17%-56%) for GM-CSF and 22.5% (range,6%-39%) for IFN-�. The effect of TGF-� on IFN-� and GM-CSFrelease in HATGF-�RII-�cyt–transduced CTLs compared with thecytokine release in nontransduced and eGFP-transduced CTLs wasstatistically significant (P .05 and P .01, respectively). Thisprotection was even greater with HA-sorted CTLs in which therewas just 4.5% (range, 0%-9%) GM-CSF inhibition (Figure 5A) and2.2% (range, 0%-9%) inhibition of IFN-� release (P .002)(Figure 5B).

CTLs transduced with retrovirus TGF-�RII-�cyt maintain theircytolytic activity and specificity in the presence of TGF-�

The cytotoxic activity of HATGF-�RII-�cyt–transduced, mock-transduced, and nontransduced CTLs were compared in standard4-hour 51Cr release assays in the presence of TGF-�1. CTL lineswere tested up to 26 days after transduction in the presence ofTGF-�1 (Table 2). At an effector-to-target ratio of 20:1, thepercentage of autologous LCLs lysed by nontransduced CTLs wasinhibited by 51% to 100% (mean, 74%) compared with a range of

Figure 1. Transduced CTLs surface express HA tag of mutant TGF-�RIIreceptor. Cells of an EBV-specific CTL line 14 days after retroviral transduction withSFG: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 HAtag) gene were stained with FITC-labeled donkey antirabbit antibody alone (isotypecontrol) (C). Nontransduced (D) or HATGF-�RII-�cyt–transduced CTLs (E,F) werethen stained with anti-HA monoclonal antibody followed by incubation with FITC-labeled donkey antirabbit antibody and PE-labeled CD8 or CD4 antibody. Six weeksafter 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.

Figure 2. Ability of TGF-� to inhibit T-cell proliferation is significantly greater innontransduced CTLs when compared with HATGF-�RII-�cyt–transduced CTLs.Nontransduced CTLs, eGFP-transduced CTLs, and HATGF-�RII-�cyt–transducedCTLs were stimulated with irradiated autologous LCLs and IL-2 � 5 ng/mL TGF-�1.Proliferative responses were measured after 72 hours of incubation by measurementof 3[H] thymidine uptake. The mean percentage of inhibition by TGF-� was measuredin nontransduced CTLs (black), eGFP-transduced CTLs (white), and HATGF-�RII-�cyt–transduced CTLs (gray). The graph represents a pooled analysis of the meaninhibition of TGF-� on 3[H] thymidine uptake in the 8 CTL lines tested.

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27% to 57% (mean, 37.7%) after more than a 72-hour incubationwith 5 ng/mL TGF-�1 (P .02). By comparison, at the sameeffector-to-target ratio, the percentage of autologous LCLs lysed bytransduced (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).

Effects of exogenous TGF-� on intracellular perforin levels inTGF-�RII-�cyt–expressing and –nonexpressing CTLs

To identify a possible mechanism by which the cytolytic activity ofCTLs is reduced by TGF-�, the intracellular perforin levels of theCTLs were measured by flow cytometry after CTLs were stainedby PE-labeled antiperforin antibody (Figure 7). The intracellularperforin levels in untransduced and eGFP-transduced CTLs weresignificantly reduced by 50% to 96% (mean, 73%, P .002) in thepresence of TGF-� (compare 7A with 7D and 7B with 7E). Bycomparison, CTLs transduced with HATGF-�RII-�cyt had nosignificant reduction in perforin (range, 4%-11%, mean 6.8%,P .7) (7C compared with 7F).

Expression of HATGF-�RII-�cyt does not affect the phenotypeor cytotoxic specificity of transduced CTLs

Although the dominant-negative TGF-� receptor clearly protectsCTLs from the growth inhibitory effects of TGF-�, it is importantto show that the transduced CTLs can continue to function

normally and remain under normal growth control. The CTLs werephenotyped before and after transduction with HATGF-�RII-�cyt.The CTLs were then maintained in culture for up to 35 days aftertransduction and were phenotyped weekly from day 7 aftertransduction. Most of the CTL lines generated had a characteristicimmunophenotype with more than 90% CD3� T cells, of whichabout 90% were also CD8�, whereas up to 10% of the cells had aT-cell helper phenotype (CD3�CD4�). These lines were comparedwith nontransduced lines for phenotypic differences. Transductionof CTLs did not result in any change in immunophenotype whencompared with nontransduced cells (Figure 8). Nor was there anyinterference with cytolytic function. 51Cr release assays were

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 andfed 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 fromweekly cell counts in the 3 CTL groups grown without TGF-� (■) and with TGF-� (Œ). (B) This panel shows 3[H] thymidine uptake in nontransduced CTLs (black) andHATGF-�RII-�cyt–transduced CTLs (gray) before addition of TGF-� then on days 4 and 12.

Figure 4. Western immunoblotting shows absence of phosphorylated Smad2 inTGF-�RII-�cyt–transduced CTLs with addition of TGF-�. Nontransduced, eGFP-transduced, and TGF-�RII-�cyt–transduced CTLs were incubated for 1 hour with5 ng/mL TGF-�, as indicated. The presence or absence of Smad2 (S2) andphosphorylated Smad2 (P-S2) was detected by Western immunoblotting by usinganti-Smad2 and antiphospho-Smad2 antibodies, respectively.

Figure 5. HATGF-�RII-�cyt–transduced CTLs sorted for the HA epitope demon-strate a significant resistance to the inhibitory effects of TGF-� on secretion ofGM-GSF and IFN-� when compared with nontransduced CTLs. EBV-specificCTLs were transduced with the mutant TGF-�RII receptor and then positivelyselected for the HAtag by flow cytometry by using an anti-HAantibody. TGF-�RII-�cyt–transduced/sorted CTLs and mock-transduced CTLs were stimulated with irradiatedautologous LCLs and IL-2 � 5 ng/mL TGF-�1. Supernatant removed after 24 hourswas analyzed for GM-CSF (A) and IFN-� (B). The graphs represent a pooled analysisof the mean percentage of inhibition of TGF-� on IFN-� and GM-CSF release in 8nontransduced (black) and 3 eGFP-transduced CTL (white) lines versus the 3HATGF-�RII-�cyt–transduced CTL (gray) lines sorted for the HA-epitope.

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performed between 14 and 26 days after transduction. There was nosignificant difference in the cytolytic specificity or activity of thetransduced lines when compared with otherwise identical nontrans-duced lines from the same donor over several time points. Asoutlined in Table 2 after a range of 14 to 26 days after transductionwith TGF-�RII-�cyt, the CTL lines maintained their cytotoxicactivity (Table 2) and specificity (Figure 6A).

TGF-�RII-�cyt–transduced CTLs proliferate normally inlong-term culture but die rapidly in the absence of exogenousgrowth factors and antigenic stimulation

Unresponsiveness to TGF-� might lead to a loss of dependence onother growth regulatory signals and, hence, to uncontrolled Tlymphoproliferation. To exclude this possibility, the growth of themature CTLs in the absence of growth stimuli was assessed.Transduced and nontransduced CTLs were cultured in the absenceof antigenic stimulation (LCLs) and the growth factor rhIL-2 for 3weeks. Both transduced and nontransduced CTLs failed to prolifer-ate and became nonviable after 3 weeks in the absence of IL-2 andLCL stimulation (Figure 9).

Table 2. Cytolytic characteristics of HATGF-�RII-�cyt–transduced versusnontransduced cytotoxic T-lymphocyte lines with and without exposure toexogenous transforming growth factor �

CTL line

% Specific lysis (ratio 10:1)

Day of culture*(d after

transduction)

NontransducedTGF-�RII-�cyt

transduced

Auto LCLAuto LCL� TGF-� Auto LCL

Auto LCL� TGF-�

Patient 1 69 27 63 66 70 (21)

Patient 2 43 18 40 41 96 (14)

Patient 3 41 24 30 35 88 (26)

Patient 4 26 3 27 23 120 (21)

Donor 1 37 19 44 54 42 (26)

Donor 2 95 69 44 53 56 (21)

Donor 3 64 54 48 42 52 (14)

Donor 4 61 36 22 23 52 (14)

Percentage of specific 51Cr release was determined after 4-hour coincubationwith autologous lymphoblastoid cell line (LCL) in cytotoxic T-lymphocyte (CTL) lineswith and without transforming growth factor � (TGF-�) added to CTL cultures 96hours before the cytotoxicity assay. The table shows the percentage of specific lysisat an effector-to-target ratio of 10:1 in the CTL lines generated from 4 healthy donorsand 4 patients with Hodgkin disease. The far right column shows the day of CTLculture/and day after transduction when the cytotoxicity assay was performed.

*Cytotoxicity of transduced and nontransduced CTLs was tested on the sameday of culture.

Figure 6. TGF-� decreases CTL-specific lysis against autologous EBV-LCLtargets in mock-transduced or nontransduced CTLs but not in HATGF-�RII-�cyt–transduced CTLs. Percentage of specific 51Cr release was determined 4hours after coincubation with autologous LCLs, allogeneic LCLs, and HSB-2 targets.TGF-� was added to CTL cultures 96 hours before cytotoxicity assay. The graphsshow the percentage of specific lysis at effector-to-target ratios of 20:1, 10:1, and 5:1in representative CTL lines generated from a healthy donor (A) and from a patientwith Hodgkin disease (B). Œ indicates CTLs cultured without TGF-� and autologousLCL target; F, CTLs cultured with TGF-� and autologous LCL target; u, CTL versusHSB-2 target; �, CTLs versus allogeneic (HLA class I mismatch) LCL target.

Figure 7. TGF-� significantly reduces intracellular perforin levels in nontrans-duced or eGFP-transduced CTLs, whereas perforin release is unaffected byTGF-� in HATGF-�RII-�cyt–transduced CTLs. Ninety-six hours after the additionof 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) Thesepanels show representative histograms for CTLs from one donor cultured withoutTGF-� (A-C) versus with TGF-� (D-F). (G) This panel shows mean perforin levelsfrom 6 CTL lines with and without TGF-�.

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Discussion

Secretion of TGF-� is a strategy commonly used by tumors tothwart cellular immune responses. It prevents the maturation ofprofessional APCs and inhibits T-cell proliferation, cytokine re-lease, and cytolytic activity.37 This effect may severely affectadoptively transferred tumor-specific CTLs used in cellular immu-notherapy. Tumor cells may themselves be sensitive to TGF-�–induced differentiation and apoptosis but can avoid this fate if theyalso possess mutant receptors for the cytokine.24,38,39 We now showthat such mutants can be adapted for the protection of tumor-specific CTLs. A dominant-negative mutant TGF-� type II recep-tor, in which the cytoplasmic signaling domain is deleted, protectsvirus-specific CTLs from the inhibitory effects of TGF-� signal-ing.16 Expression of the mutant receptor did not result in alterationsin phenotype, cytotoxic specificity, or requirement for growth-regulatory signals. This tumor-derived defense may have clinicalvalue when adoptively transferred T cells are used for the treatmentof TGF-�–secreting tumors, including Hodgkin lymphoma.

We chose EBV-positive Hodgkin lymphoma to investigate thisapproach because the tumor cells express well-defined (viral)tumor antigens to which CTLs can readily be generated. Hodgkintumors also secrete TGF-�, which may contribute to the limitedclinical effectiveness of CTLs in this disease, compared with otherEBV-positive malignancies.26 The experiments were performed byusing polyclonal CTLs, as these cells have been successfully usedin the clinical setting.3 Polyclonal CTLs are less likely to besuccessfully evaded by escape mutants.40 In addition, the presenceof CD4� T cells has been shown to be important for long-term CTLpersistence in vivo as well as the maintenance of CD8� T-lymphocyte–mediated antiviral or antitumor immunity.40,41 Thepolyclonal EBV-specific CTL lines generated from healthy individu-als and patients with Hodgkin disease were readily transduced witha GALV-pseudotyped retrovirus expressing a dominant-negativeTGF-�RII, and transduced CTLs were resistant to the antiprolifera-tive effects of TGF-�. Not only was there significantly lessinhibition of proliferation after 72 hours of culture with TGF-�, butalso transduced CTLs continued to grow in the presence of thecytokine, whereas untransduced CTLs were nonviable after 12days of culture. Additionally, CTLs could be transduced from day21 to day 105 of culture, resulting in reproducible effects that weremaintained for up to 120 days, thereby testifying to the robustnature of this modification.

Inhibition of the TGF-� signal transduction pathway in theTGF-�RII-�cyt–transduced CTL was demonstrated by an absenceof detectable phosphorylated Smad2 on Western blot in thepresence of TGF-�. In nontransduced and transduced CTLs,

TGF-� binds to TGF-�RII on the cell surface. However, inmock-transduced CTLs, the ligand-bound TGF-�RII then interactswith and phosphorylates TGF-�RI. Phosphorylation activates theintrinsic kinase activity of TGF-�RI, allowing the receptor tophosphorylate and thereby activate Smad proteins as confirmed bythe detection of phosphorylated Smad2 on Western analysis in themock-transduced CTLs. Once activated by phosphorylation, theSmad complexes migrate to the nucleus, where they recruit othertranscription factors and stimulate the expression of genes, includ-ing mediators of cell growth.42 In contrast, when TGF-� is added tocells expressing the truncated dominant-negative TGF-�RII, thelack of the intracellular domain prevents phosphorylation ofTGF-�RI16 and the CTL gene-modified abrogation of all down-stream signaling events (including Smad phosphorylation). Thecomplete lack of detection of phosphorylated Smad2 in theHATGF-�RII-�cyt–transduced CTL suggests either that transduc-tion efficiency was higher than suggested by HA detection and/orthe presence of transacting protection.

TGF-� also prevented the secretion of IFN-� and GM-CSFfrom EBV-CTLs in response to stimulation with autologous LCLs.In contrast, cytokine release from transduced CTLs was minimallyinhibited, and protection was almost complete when the CTLs wereselected for expression of the transgene by using the HA tag.Although these CTLs, which were sorted by using an anti-HAantibody, 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 suchgene-modified CTLs clinically, because the approval of such aclinical protocol may be less likely with the use of antibody-selected gene-modified CTL populations. Further, selection forTGF-�–resistant tumor-specific CTLs is likely to occur in vivoand, therefore, should be unnecessary in vitro. Finally, although thecytolytic activity of EBV-specific CTLs was greatly reduced afterculture in the presence of TGF-�, the cytotoxic activity of theunsorted HATGF-�RII-�cyt–transduced CTLs was unaffected.

TGF-� has been shown to reduce the cytolytic activity ofalloreactive cytotoxic CD8� and CD4� T cells,43 and, in thepresence of IL-10, TGF-� anergizes alloreactive CD4� T cells,which remain tolerant in vivo.44 In addition, TGF-� has beenshown to inhibit IL-12 and IL-2–induced cell proliferation andIFN-� production by T cells.45-47 This finding is partly due to adown-regulation of the IL-12 receptor �2 chain expression byTGF-�, resulting in the inhibition of antigen-specific activation andcytokine secretion.47 The antiproliferative effects of TGF-� on cellgrowth appear to be secondary to the effect of TGF-� on tyrosinephosphorylation, which results in altered control of expression and

Figure 9. HATGF-�RII-�cyt–transduced CTLs will fail to proliferate in theabsence of IL-2 or LCL stimulation. The proliferation of long-term HATGF-�RII-�cyt–transduced CTL (gray) and nontransduced CTL lines (black) were determined by3[H]thymidine uptake. The 3[H]thymidine uptake of CTL lines left in culture for 4 weeksafter transduction and stimulated weekly with LCL and IL-2 were compared with thesame CTL lines grown in the absence of LCL/IL-2 stimulation for 3 weeks.

Figure 8. CTL phenotype is unchanged after transduction. To determine CTLphenotypes, the nontransduced (black) and TGF-�RII-�cyt–transduced (gray) EBV-specific CTL cultures were stained with antibodies against T-cell surface antigensCD3, CD4, CD8, CD56, T-cell receptor ��, and T-cell receptor � , and surfaceimmunofluorescence was analyzed by flow cytometry.

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activation of cell cycle regulatory molecules.44 The anticytotoxicactivities 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-12with its receptor on activated T cells.48 This suppression of IL-12signaling likely explains the reduction of IFN-� release seen in ournontransduced and eGFP-transduced established EBV-CTL linesby TGF-�.46,49 Second, the regulatory effect of TGF-� on humanalloreactive CTL cytotoxic activity is associated with down-regulation of perforin and granzyme B gene expression.43,50 Wealso found that inhibition of EBV-specific CTL killing coincideswith a significant reduction in intracellular perforin expression. Inaddition, we found a reduction in GM-CSF release with theaddition of TGF-� to CTL cultures. Although this finding has notbeen previously described, it may be the result of TGF-� interfer-ence of the GM-CSF signaling pathway, including an inhibitoryeffect on Janus kinase 2 and STAT5.51-53 All of these effects can beprevented by expression of the HATGF-�RII-�cyt receptor.

Although the approach we describe may counteract one of themost important tumor immune evasion strategies, many othersremain intact. For example, Hodgkin tumor cells down-regulate theimmunodominant viral latency proteins, EBNAs 3A, 3B, and 3 thatare expressed in EBV-LPD of immunosuppressed individuals andexpress at least one other cytokine (IL-10) that shares with TGF-�the ability to inhibit professional APC and, indirectly, CTLactivation.23,48,54 Hodgkin cells also secrete the chemokine TARC,which specifically recruits IL-4–secreting Th2 cells, and a Th2growth factor, IL-13.55-58 Both favor the creation of a noncytotoxicTh2 environment. Although this is a daunting list of evasionmechanisms, their very profligacy argues that multiple defenses arerequired to protect the tumor cells from attack. Abrogation of evenone or two may, therefore, produce a significant change in theeffectiveness of cellular immunotherapy. Adoptive immunotherapywith ex vivo–expanded tumor-specific CTLs may evade theinhibition of professional APC function, whereas transferredresistance to TGF-� may ensure that these infused CTLs cancontinue to proliferate and function even in a tumor environmentrich in this cytokine.

One potential concern facing the clinical use of TGF-�–resistant CTLs is that lack of response to this inhibitory cytokinemay undesirably impair homeostasis of the tumor-specific lympho-cytes. 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 in-volved in the lymphoproliferation were naive and IL-2 indepen-dent, and they exhibited patterns of recirculation and homeostasisthat were distinct from the mature “memory” T cells studiedhere.60,61 Other mechanisms, such as Fas and tumor necrosis factorpathways as well of lack of growth stimulation by antigen andgrowth factors are also available to maintain mature T-cell homeosta-sis.23,62,63 Certainly, we found no evidence that even long-termexpression of the mutant TGF-�RII had any deleterious effects onthe transduced CTL lines. In particular, their phenotype andcytotoxic specificity were unmodified, and, although they contin-ued to grow and secrete cytokines in response to stimulation andculture in IL-2, withdrawal of these stimulants led to cell death thatfollowed identical temporal kinetics to nontransduced cells. Theseresults were reproducible in all 4 healthy donor CTLs and in all 4lines derived from patients with Hodgkin disease. Another possibleconcern is that CTLs transduced with a mutant TGF-�RII may berecognized and eliminated by the host immune response. Thissituation is unlikely, because the truncation used to create themutant receptor creates no new epitopes. Hence, CTLs expressingthis construct should have a selective growth and functionaladvantage in vivo in patients with TGF-�–secreting tumors butshould not be capable of autonomous growth.

We conclude that the expression of a transdominant-negativeTGF-� receptor II (HATGF-�RII-�cyt) in tumor-specific CTLsmay allow at least one human tumor evasion strategy tobe overcome.

Acknowledgments

We thank the Texas Childrens’ Hospital, The Methodist Hospital,and Baylor College of Medicine for their contribution.

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STRATEGY TO OVERCOME TUMOR PROTECTION BY TGF-� 3187BLOOD, 1 MAY 2002 � VOLUME 99, NUMBER 9

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