Infusing virus-specific T cells is effective treatment for rare Epstein-Barr virus (EBV)–associated posttransplant lymphomas, and more limited success has been reported using this approach to treat a far more common EBV-associated malignancy, nasopharyngeal carcinoma (NPC). However, current approaches using EBV-transformed lymphoblastoid cell lines to reactivate EBV-specific T cells for infusion take 2 to 3 months of in vitro culture and favor outgrowth of T cells targeting viral antigens expressed within EBV+ lymphomas, but not in NPC. Here, we explore T-cell receptor (TCR) gene transfer to rapidly and reliably generate T cells specific for the NPC-associated viral protein LMP2. We cloned a human leukocyte antigen (HLA) A*1101-restricted TCR, which would be widely applicable because 40% of NPC patients carry this HLA allele. Studying both the wild-type and modified forms, we have optimized expression of the TCR and demonstrated high-avidity antigen-specific function (proliferation, cytotoxicity, and cytokine release) in both CD8+ and CD4+ T cells. The engineered T cells also inhibited LMP2+ epithelial tumor growth in a mouse model. Furthermore, transduced T cells from patients with advanced NPC lysed LMP2-expressing NPC cell lines. Using this approach, within a few days large numbers of high-avidity LMP2-specific T cells can be generated reliably to treat NPC, thus providing an ideal clinical setting to test TCR gene transfer without the risk of autoimmunity through targeting self-antigens. Cancer Immunol Res; 3(10); 1138–47. ©2015 AACR.

Nasopharyngeal carcinoma (NPC) is unusually common throughout Southeast Asia especially in southern China where it is the third most common cancer in men with annual incidence rates of up to 28 cases per 100,000 men (1). Early-stage disease responds well to radiotherapy (±chemotherapy), but a study of 2,687 patients treated in Hong Kong reported that over half of these patients presented with advanced disease (stage III–IV) and have a 5-year disease-specific survival rate of only 72% (2). Survivors are also at risk of treatment-related toxicities, including secondary malignancies (3). Therefore, there is clear need to develop improved therapies for this cancer.

Epstein-Barr virus (EBV) is consistently detected in malignant cells of patients with undifferentiated NPC, and is strongly implicated in the pathogenesis of this and other human tumors (4). Despite its oncogenic potential, EBV is ubiquitous in the human population and it normally persists as an asymptomatic life-long infection under the control of virus-specific T cells (4). The presence of this virus within NPC, therefore, raises the possibility of a T-cell–based therapy for this disease.

Treatments based on infusing tumor-specific T cells have yielded impressive clinical responses in some cancers. Indeed some of the earliest data supporting this approach came from trials targeting EBV+ lymphomas. Infusing EBV-specific polyclonal T-cell lines is highly effective as a therapeutic and prophylactic treatment for rare EBV+ lymphomas that occur in transplant recipients (5). However, to extend this treatment to more common EBV+ tumors such as NPC, two issues must be addressed. First, polyclonal T-cell lines initially used to treat EBV+ lymphomas were reactivated in vitro using the autologous EBV-transformed lymphoblastoid cell line (LCL). Within an LCL (and most posttransplant EBV+ lymphomas), the virus expresses at least six nuclear antigens, EBNA-1, -2, -3A, -3B, -3C, -LP, and two latent membrane proteins, LMP1 and LMP2. Of these, members of the EBNA3 family are immunodominant antigens for CD8+ T cells. However, in NPC, EBV protein expression is restricted to EBNA1, LMP1 (variable), and LMP2. Nevertheless, attempts to treat NPC by infusing LCL-reactivated T-cell lines have yielded objective responses in a minority of patients (6–9). Low frequencies of LMP2-specific T cells were detectable within some infused cell preparations, and these may have mediated antitumor effects, but the procedure is clearly suboptimal because the majority of virus-specific T cells targeted EBV proteins not expressed in the tumor (7, 9). Second, generating T cells by LCL reactivation takes over 2 months of in vitro culture, including the time required to establish an LCL, and then the selective expansion of EBV-specific effector cells. This is labor intensive and does not always generate detectable T-cell responses specific for NPC-associated EBV antigens (7–9). More recently, selective reactivation of T cells targeting these antigens has been attempted using recombinant viral vectors or peptides (10–12), but again this requires several weeks of in vitro culture and/or often results in products with very low frequencies of tumor-specific T cells.

Therefore, we have explored the use of T-cell receptor (TCR) gene transfer, an approach that is rapid, reliable, and capable of generating large quantities of T cells (>108–1010 cells/patient) with the desired specificity, regardless of the patient's preexisting immune repertoire. TCRs are expressed on the surface of all T cells and determine antigenic specificity. Having identified a tumor antigen-specific T cell, by cloning the genes encoding its TCR into a retroviral vector it is then possible within a few days to engineer a patients' T cells expressing the same TCR and targeting the same tumor antigen. The efficacy of this approach to treat melanoma and synovial cell sarcoma has already been demonstrated in clinical trials (13, 14).

To ensure TCR gene transfer could be widely applicable to NPC patients, we focused on a T-cell response to an epitope derived from the NPC-associated EBV protein LMP2, presentation of which is restricted through human leukocyte antigen (HLA)-A*1101, an allele carried by >50% of the Chinese population. This epitope comprises the sequence SSCSSCPLSK (referred to subsequently as “SSC”). Here, we report the cloning of an SSC-specific TCR and studies to determine the expression and function of both the wild-type and modified forms of this receptor in transduced T cells. We thereby demonstrate that TCR transfer using this receptor offers a rapid and efficient means to generate T cells to target NPC.

Cells and cell lines

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by density gradient centrifugation on lymphoprep (Axis Shield). LCLs were generated using Caucasian (B95.8) or Chinese (CKL) prototype 1 EBV strains (15). Phoenix amphotropic packaging cells were kindly provided by Gary Nolan (Stanford University). The T2 cell line transduced with HLA A*1101 gene was kindly provided by M. Masucci (Karolinska Institute, Stockholm, Sweden). NPC cell lines HK1 (16) and c666.1 (17) were transduced with retrovirus (pQCXIH and pQCXIN, respectively; Clontech, CA) into which we had cloned the gene encoding HLA A*1101. These cell lines were then cultured under drug selection using 20 μg/mL hygromycin or 50 μg/mL G418 (Life technologies), respectively. Though originally described as an NPC cell line, and used here because it naturally expresses HLA A*1101, HONE-1 now appears to be a Hela-related somatic cell hybrid (18). The breast cancer cell line MDA-MB-231 (19) was transduced with three retroviruses (pQCXIH, pLXSN, and pMSCV) carrying genes encoding HLA A*1101, LMP2, and luciferase, respectively, and cultured under drug selection using 300 μg/mL hygromycin, 600 μg/mL G418, and 1 μg/mL puromycin. All of the above cells lines were cultured in RPMI-1640 (Sigma) containing 10% FBS (PAA), 2 mmol/L glutamine, 100 IU/mL penicillin, and 100 pg/mL streptomycin (standard medium). Fibroblasts were grown from a skin biopsy cultured in DMEM (Sigma) supplemented as described above. All T, B, and fibroblast cell lines were derived from healthy donors or NPC patients of known HLA type. All cancer cell lines were authenticated by short tandem repeat analysis and passaged for fewer than 6 months before experiments. The use of human materials for this study was approved by the National Research Ethics Service, UK, and the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee. Work was conducted according to the declaration of Helsinki protocols and all donors provided written informed consent.

Synthetic peptides and recombinant vaccinia viruses

Peptides were synthesized using Fluorenylmethoxycarbonyl chemistry by Alta Bioscience, Birmingham, UK. Recombinant vaccinia and modified vaccinia Ankara viruses–expressing LMP2 and corresponding control vectors have been described previously (20, 21).

TCR gene cloning

RNA from the T-cell clone was isolated using an RNeasy mini kit (Qiagen) and reverse transcribed. TCRα and -β genes were then amplified with the BD SMART RACE cDNA Amplification Kit (BD Biosciences) according to the manufacturer's instructions using the following primers: TCRα constant region: 5′-agcacaggctgtcttacaatcttgc-3′; TCRβ2 constant region: 5′-ggacacagattgggagcagg-3′. TCR genes were subcloned into the pCR2.1 (Life Technologies) vector and sequenced. The TCRα (TCRVA22) and TCRβ (TRBV4.01) chains were then cloned into the same retroviral pMP71 vector (kindly provided by C. Baum, Hannover, Germany; ref. 22) separated by a 2A peptide linker from porcine teschovirus (Supplementary Fig. S3). The sequence has been deposited in GenBank, accession code KU163590. Modified TCR genes were designed and produced by GeneArt.

Retroviral transduction of human T cells

Phoenix amphotropic packaging cells were transfected with pMP71 retroviral vector and pCL ampho (Imgenex) using FuGENE HD (Roche) according to the manufacturer's instructions and retroviral supernatant harvested 48 hours later. PBMCs were preactivated for 48 hours using anti-CD3 antibody (OKT3; 30 ng/mL) and IL2 (600 U/mL; Chiron) in standard medium containing 1% human AB serum (TCS Biosciences). These cells were then transduced with retroviral supernatant (or mock transduced with conditioned supernatant from nontransfected phoenix cells) using retronectin-coated (Takara) 6-well plates according to the manufacturer's instructions. Cells were then maintained in standard medium containing 1% human AB serum and IL2 (100 U/mL).

Flow cytometry

Cells were stained for 10 minutes at room temperature with an HLA-A*1101/SSC pentamer (5 μg/mL; ProImmune) according to the manufacturer's instructions. Cells were then washed and stained on ice for 30 minutes with Pro5 Fluorotag (APC or R-PE-labeled; ProImmune) and saturating concentrations of anti-CD3 (PE-conjugated), anti-CD4 (FITC-conjugated; Pharmingen), and anti-CD8 (tricolor- or ECD-conjugated; Caltag) antibodies. For intracellular cytokine staining T cells were stimulated for 2 hours with T2-A11 cells prepulsed with or without SSC peptide (5 μg/mL). Brefeldin A (10 μg/mL; Sigma) was then added and cells cultured for another 5 hours. Cells were then stained with pentamer and antibodies to surface markers (CD4-FITC, CD8-ECD; BD Pharmingen) as described above. After treatment with fixation and permeabilization buffers (E-bioscience) according to the manufacturer's instructions, cells were incubated for 30 minutes at 4°C with anti-cytokine antibodies (IL2-PE, IFNγ-PECy7, and TNFα-APC) or an isotype- and concentration-matched control antibody (BD Pharmingen), then washed twice in PBS. Cells were analyzed using an LSRII cytometer (Becton Dickinson) and FlowJo software (Tree Star).

CFSE labeling

T cells were washed twice with PBS and incubated with 2.5 μmol/L Carboxyfluorescein succinimidyl ester (CFSE) for 10 minutes at 37°C. The labeling reaction was quenched by addition of RPMI-1640 containing 10% FBS. Cells were washed, resuspended in standard growth medium at 2 × 106 cells/mL, cocultured for 5 days with T2-A*1101 cells prepulsed with SSC peptide (10 μg/mL), then analyzed by flow cytometry as described above.

IFNγ release assay

Stimulator cells (5 × 104/well) were cocultured in triplicate with T cells at responder:stimulator ratios as indicated. Cells were incubated at 37°C/5% CO2 in 100 μL per well of Iscove's modified dulbecco's medium (Life Technologies) supplemented with 10% FBS and IL2 (25 U/mL). After 18 hours, culture supernatants were tested for secreted IFNγ using an ELISA (Pierce Endogen) according to the manufacturer's instructions.

Cytotoxicity assays

Chromium release assays, using vaccinia-infected or peptide-pulsed targets, were set up at known effector:target ratios (2,500 targets/well) and harvested after 5 or 8 hours. These protocols have been described in detail previously (23).

In vivo tumor protection experiments

Six- to 8-week-old female NSG mice (Charles River Laboratories) were inoculated s.c. on the flank with MDA-MB-231 cells (expressing A*1101, LMP2, and luciferase; 5 × 106 cells/mouse) in Matrigel (BD Biosciences). One day later, mice received 107 TCR-transduced (or mock-transduced) T cells intravenously. Intraperitoneal injections of 104 U IL2 were given on days 2, 4, 7, 9, and 11. Tumor growth was measured in a blinded fashion with calipers and bioluminescence imaging (IVIS Spectrum; Caliper Life Sciences). All experiments were performed under UK Home Office authorization.

Expression and function of a wild-type HLA A*1101-restricted LMP2-specific TCR

EBV-specific T cells from a healthy Chinese donor were reactivated in vitro with the autologous LCL and cloned by limiting dilution as previously described (23). Clones were screened for reactivity to the A*1101-restricted LMP2 epitope SSC and clone 85 was selected. The avidity of this CD8+ clone for SSC peptide was determined using a cytotoxicity assay with A*1101+ targets pulsed with titrated concentrations of peptide. The clone displayed high avidity, with clear recognition of target cells pulsed with only 10−10 mol/L peptide (Fig. 1A). When tested for IFNγ production in response to A*1101-matched and -mismatched LCL targets, a clear A*1101-restricted response was observed (Fig. 1B). Importantly, this clone recognized not only A*1101+ LCLs carrying the standard EBV strain B95.8 (derived from a Caucasian population) but also those carrying EBV strains from the Chinese population, which is the most at risk of NPC.

Figure 1.

Characterizing an A*1101-restricted SSC-specific CD8+ cytotoxic T-cell clone. A, avidity for SSC peptide was determined by cytotoxicity assay (E:T = 3:1). B, response to LMP2 expressed in A*1101-matched or -mismatched LCLs carrying EBV strains from Caucasian or Chinese populations was measured by IFNγ production. Target cells alone produced <100 pg/mL IFNγ. The responder:stimulator ratio = 1:10. Results show mean + SD and are representative of three separate experiments.

Figure 1.

Characterizing an A*1101-restricted SSC-specific CD8+ cytotoxic T-cell clone. A, avidity for SSC peptide was determined by cytotoxicity assay (E:T = 3:1). B, response to LMP2 expressed in A*1101-matched or -mismatched LCLs carrying EBV strains from Caucasian or Chinese populations was measured by IFNγ production. Target cells alone produced <100 pg/mL IFNγ. The responder:stimulator ratio = 1:10. Results show mean + SD and are representative of three separate experiments.

Close modal

Genes encoding TCRα and -β chains from clone 85 were isolated and cloned into the same MP71 retroviral expression vector separated by a 2A peptide-linker from porcine teschovirus to ensure equimolar expression of these chains (Fig. 2A). Activated T cells from healthy donors and NPC patients were then transduced with the recombinant retrovirus and surface expression of SSC-specific TCR determined using an A*1101/SSC pentamer. Figure 2B shows results with T cells from a patient with advanced NPC. SSC-specific T cells are rare/undetectable in most NPC patients and healthy virus carriers (as indicated by mock-transduced cells), but 3 days after transduction with recombinant retrovirus, surface expression of SSC-specific TCR was clearly detectable in 13.6% of CD8+ T cells. Note that 12% of CD4+ T cells also expressed this TCR following transduction. These data are representative of those from 9 healthy donors and 5 NPC patients.

Figure 2.

Expression and function of wild-type SSC-specific TCR. A, design of the pMP71 retroviral expression vector. B, SSC-specific TCR expression on transduced PBMCs from a patient with advanced NPC (TCR-T) compared with that with mock-transduced cells (Mock-T). Values shown refer to the percentage of pentamer+ CD8+ or CD4+ cells. C, avidity for SSC peptide of TCR-transduced T cells (TCR-T) and T-cell clone 85 was measured by ELISA for IFNγ release. Mock-transduced T cells (mock-T) were included as a control. The responder:stimulator ratio = 1:4. Results show mean + SD and are representative of three repeat experiments. D, TCR-transduced (but not mock transduced) T cells lyse autologous fibroblasts expressing LMP2 from a recombinant vaccinia vector (closed symbols), but not fibroblasts infected with a control vaccinia vector (open symbols). Data representative of three separate experiments.

Figure 2.

Expression and function of wild-type SSC-specific TCR. A, design of the pMP71 retroviral expression vector. B, SSC-specific TCR expression on transduced PBMCs from a patient with advanced NPC (TCR-T) compared with that with mock-transduced cells (Mock-T). Values shown refer to the percentage of pentamer+ CD8+ or CD4+ cells. C, avidity for SSC peptide of TCR-transduced T cells (TCR-T) and T-cell clone 85 was measured by ELISA for IFNγ release. Mock-transduced T cells (mock-T) were included as a control. The responder:stimulator ratio = 1:4. Results show mean + SD and are representative of three repeat experiments. D, TCR-transduced (but not mock transduced) T cells lyse autologous fibroblasts expressing LMP2 from a recombinant vaccinia vector (closed symbols), but not fibroblasts infected with a control vaccinia vector (open symbols). Data representative of three separate experiments.

Close modal

Functional testing of this wild-type TCR began using transduced polyclonal T cells to explore their ability to produce IFNγ in response to T2:A*1101 cells pulsed with SSC peptide at titrating concentrations. TCR-transduced T cells clearly recognized peptide-pulsed targets with as low as 10−10 mol/L peptide, whereas mock-transduced T cells did not respond at any peptide concentration tested (Fig. 2C). Testing Clone 85, from which the TCR genes were derived, at the same input cell number as SSC-specific effectors within the transduced T cells yielded almost identical results (Fig. 2C). Transduced T cells also mediated specific cytotoxic function when tested against autologous fibroblasts expressing LMP2 protein from a recombinant vaccinia vector, compared with that against fibroblasts infected with the empty control vector (Fig. 2D).

Optimization of the TCR gene construct

Previous studies have suggested that function and/or expression of transduced TCRs can be improved by codon optimization to increase translation efficiency (24), and addition of a second disulphide bond in the TCR constant domains to aid preferential pairing of the introduced TCR chains (25). The latter also helps prevent exogenous TCR chains mispairing with endogenous TCR chains naturally expressed by T cells. Such mispairing not only reduces the number of SSC-specific TCRs expressed, but also risks generating novel, potentially autoreactive TCRs. Therefore, we generated two variants of our wild-type SSC-specific TCR, a codon-optimized version (coTCR) and a codon-optimized TCR in which amino acid residue 48 of the TCRα chain and residue 57 of the TCRβ chain were both changed to cysteine, thus introducing a second disulfide bond (coTCRcys; refs. 25). A series of experiments then compared expression and function of these two variants with wild-type SSC-specific TCR (WT TCR). The main difference observed was TCR surface expression. Pentamer staining of CD8+ T cells, transduced with increasing volumes of the three retroviral supernatants produced in parallel, showed similar expression of WT TCR and coTCR, but a clear increase was observed with the coTCRcys construct (Fig. 3A). Similar results were obtained with CD4+ T cells (data not shown). Not only was the coTCRcys receptor expressed on a greater proportion of T cells, but the levels of expression on individual cells were increased (Fig. 3B). These data are consistent with previous reports that introducing a second disulphide bond reduces mispairing with endogenous TCR chains (25). Staining transduced cells with an antibody to Vβ4.1 showed similar results to the same cells stained with the SSC pentamer (Supplementary Fig. S1), suggesting that there is little if any mispairing between this exogenous β-chain and the endogenous α-chains. However, an antibody was not available to stain the exogenous Vα22-chain; therefore, it is possible that increased expression of coTCRcys is a result of reduced mispairing between the exogenous α-chain and the endogenous β-chains. Although expression was improved with coTCRcys, when an equivalent number of transduced effectors were tested for each TCR construct, T-cell function was unaffected (Fig. 3C). Although codon optimization alone (coTCR) affected neither surface expression nor functional activity (Fig. 3), other studies have shown that despite such lack of in vitro effects, codon optimization can nevertheless improve both frequency of TCR-modified T cells detectable post-infusion and antitumor activity in vivo (26, 27). Therefore, the coTCRcys construct was selected for further testing. Analyzing the differentiation status of coTCRcys-transduced cells showed that they contained a mixture of mainly naïve, central–memory and effector–memory cells (Supplementary Fig. S2).

Figure 3.

Optimizing TCR gene construct. A, SSC-specific TCR expression 3 days after transduction with wild-type TCR (WT TCR), a codon-optimized version (coTCR) or a codon-optimized TCR incorporating an additional disulphide bond (coTCRcys). B, intensity of pentamer staining for the different TCR constructs. C, avidity for SSC peptide of T cells transduced with each of the TCR constructs was compared using an ELISA for IFNγ release. T-cell input numbers were adjusted to ensure equivalent numbers of transduced effectors were tested for each TCR construct. The responder:stimulator ratio = 1:3. Results show mean + SD. Mock-transduced T cells (Mock-T) were included as a control. All results shown are representative of at least three separate experiments.

Figure 3.

Optimizing TCR gene construct. A, SSC-specific TCR expression 3 days after transduction with wild-type TCR (WT TCR), a codon-optimized version (coTCR) or a codon-optimized TCR incorporating an additional disulphide bond (coTCRcys). B, intensity of pentamer staining for the different TCR constructs. C, avidity for SSC peptide of T cells transduced with each of the TCR constructs was compared using an ELISA for IFNγ release. T-cell input numbers were adjusted to ensure equivalent numbers of transduced effectors were tested for each TCR construct. The responder:stimulator ratio = 1:3. Results show mean + SD. Mock-transduced T cells (Mock-T) were included as a control. All results shown are representative of at least three separate experiments.

Close modal

Functional analysis of coTCRcys in CD8+ and CD4+ T cells

Having optimized expression of the SSC-specific TCR, to explore its therapeutic potential, we then determined the ability of coTCRcys-transduced T cells to recognize LMP2 protein expressed at physiologic levels in an LCL. For this, we used cloned populations of TCR-transduced cells to study the functional activity in CD8+ cells, which can have direct antitumor effects in vivo, and CD4+ cells, which can help generate and maintain effective CD8+ responses and can also be cytotoxic. To ensure SSC-specific CD8+ clones had been engineered and were not naturally occurring effectors, we used PCR to detect the retroviral construct (data not shown). Both engineered CD8+ and CD4+ cells responded by IFNγ production in an A*1101-restricted manner when tested against a panel of A*1101-matched and -mismatched LCLs (Fig. 4A). Thus, this TCR can function in a CD8-independent manner.

Figure 4.

Function of coTCRcys-expressing CD8+ and CD4+ T cells. A, response of transduced T-cell clones to LMP2 expressed in A*1101-matched or -mismatched LCLs was measured by IFNγ production. Target cells alone produced <100pg/mL IFNγ. The responder:stimulator ratio = 1:10. Results show mean + SD and are representative of seven clones for each subset. B, proliferation of coTCRcys-expressing T cells measured by CFSE staining after stimulation with T2-A*1101 cells alone (dotted line) or T2-A*1101 cells pulsed with SSC peptide (solid line). C, cytotoxic activity of coTCRcys-transduced CD8+ and CD4+ T-cell clones against HONE1 cells expressing LMP2 +/−pulsed with SSC peptide, or HONE1 cells alone. Results show mean + SD and are representative of four clones for each subset.

Figure 4.

Function of coTCRcys-expressing CD8+ and CD4+ T cells. A, response of transduced T-cell clones to LMP2 expressed in A*1101-matched or -mismatched LCLs was measured by IFNγ production. Target cells alone produced <100pg/mL IFNγ. The responder:stimulator ratio = 1:10. Results show mean + SD and are representative of seven clones for each subset. B, proliferation of coTCRcys-expressing T cells measured by CFSE staining after stimulation with T2-A*1101 cells alone (dotted line) or T2-A*1101 cells pulsed with SSC peptide (solid line). C, cytotoxic activity of coTCRcys-transduced CD8+ and CD4+ T-cell clones against HONE1 cells expressing LMP2 +/−pulsed with SSC peptide, or HONE1 cells alone. Results show mean + SD and are representative of four clones for each subset.

Close modal

Using CFSE-labeling, we explored the ability of coTCRcys-transduced T cells to proliferate following antigen encounter. Both engineered CD8+ and CD4+ T cells underwent several rounds of division following stimulation with SSC peptide-loaded T2-A*1101 cells (compared with T2-A*1101 alone; Fig. 4B). Furthermore, both engineered CD8+ and CD4+ T cells were cytotoxic, lysing A*1101-positive HONE1 cells expressing LMP2 from a recombinant vaccinia vector with or without addition of the SSC peptide (Fig. 4C).

An increased frequency of CD4 T cells with multifunctional capacity for cytokine production is associated with improved control of some infections (28). Using intracellular staining, we showed that coTCRcys-transduced CD4+ T cells can simultaneously produce multiple cytokines (IL2, IFNγ, and TNFα) following antigen-specific stimulation (Fig. 5).

Figure 5.

coTCRcys-expressing CD4+ T cells produce multiple cytokines following stimulation with T2-A*1101 cells prepulsed with SSC peptide. A, IL2 production by coTCRcys-T cells stimulated with T2-A*1101 + SSC (solid line) compared with coTCRcys-T cells stimulated with T2-A*1101 alone (dashed line), or mock-T cells stimulated with T2-A*1101 + SSC (gray area). B, the percentage of these IL2-producing coTCRcys-T cells that also produced TNFα and/or IFNγ. All data shown were gated on CD4+ T cells. Thresholds for positive cytokine staining were determined from coTCRcys-T cells stimulated with T2-A*1101 alone. Results are representative of five separate experiments.

Figure 5.

coTCRcys-expressing CD4+ T cells produce multiple cytokines following stimulation with T2-A*1101 cells prepulsed with SSC peptide. A, IL2 production by coTCRcys-T cells stimulated with T2-A*1101 + SSC (solid line) compared with coTCRcys-T cells stimulated with T2-A*1101 alone (dashed line), or mock-T cells stimulated with T2-A*1101 + SSC (gray area). B, the percentage of these IL2-producing coTCRcys-T cells that also produced TNFα and/or IFNγ. All data shown were gated on CD4+ T cells. Thresholds for positive cytokine staining were determined from coTCRcys-T cells stimulated with T2-A*1101 alone. Results are representative of five separate experiments.

Close modal

In vivo studies with an LMP2+ epithelial tumor model

Currently, there are no appropriate animal models of NPC to test the therapeutic potential of these T cells. Therefore, we engineered another human epithelial tumor (MDA-MB-231) to coexpress LMP2 and A*1101 as well as luciferase for bioluminescence imaging. Immunodeficient mice carrying this tumor were treated with coTCRcys-expressing T cells. Flow cytometric analysis showed the infused T cells contained a CD4:CD8 ratio of 3:2, with 50% CD4 and 60% CD8 T cells expressing the SSC-specific TCR. Tumor growth in these mice was significantly reduced compared with that in control mice that received mock-transduced T cells (Fig. 6).

Figure 6.

coTCRcys-transduced T cells control tumor growth in vivo. NSG mice were injected with A*1101+ LMP2+ MDA-MB-231 tumor cells then treated with T-cell infusions (6 mice/group). Tumor size, measured by calipers (A) or bioluminescence (B), showed significant inhibition of tumor growth by coTCRcys-transduced T cells compared with mock-T cells. Bioluminescence images were taken 17 days after T-cell infusion.

Figure 6.

coTCRcys-transduced T cells control tumor growth in vivo. NSG mice were injected with A*1101+ LMP2+ MDA-MB-231 tumor cells then treated with T-cell infusions (6 mice/group). Tumor size, measured by calipers (A) or bioluminescence (B), showed significant inhibition of tumor growth by coTCRcys-transduced T cells compared with mock-T cells. Bioluminescence images were taken 17 days after T-cell infusion.

Close modal

TCR transduction of T cells from patients with advanced NPC and recognition of NPC cell lines

Finally, we sought to determine whether coTCRcys-transduced T cells from patients with advanced NPC could respond to NPC cell lines expressing LMP2. All NPC tumors are EBV+, with the exception of c666.1, NPC lines established in vitro have lost the EBV genome, and even c666.1 does not express LMP2 protein. Therefore, having introduced the restricting HLA allele into c666.1 by retroviral transduction (c666.1/A*1101), we expressed LMP2 from a recombinant modified vaccinia (Ankara) vector with or without addition of the SSC peptide. Transduced T cells from two advanced NPC patients clearly responded by producing IFNγ in an antigen-specific manner to LMP2-expressing c666.1/A*1101 cells. Similar levels of response were seen with antigen-loaded A*1101-matched fibroblasts and HONE1 cells (Fig. 7A). These T cells were also tested for cytotoxic activity toward NPC cell lines, and here, we included a second NPC line HK1, which again had to be transduced to express A*1101 (HK1/A*1101). Transduced (but not mock-transduced) T cells lysed both HK1/A*1101 and c666.1/A*1101 cells in an LMP2-specific manner (Fig. 7B).

Figure 7.

Functional testing of coTCRcys-transduced T cells from patients with advanced NPC. A, IFNγ production following stimulation with A*1101+ targets infected with a recombinant modified vaccinia vector expressing LMP2 (MVA LMP2) or empty vector (MVA control). MVA LMP2–infected targets were also tested after pulsing with SSC peptide. Mock-transduced T cells from the same donors were used as controls. Target cells alone produced <10 pg/mL IFNγ. B, cytotoxic activity of coTCRcys- or mock-transduced T cells from a patient with advanced NPC when cocultured with NPC cell lines (HK1/A*1101 and c666.1/A*1101; effector:target = 6:1). Targets were infected with recombinant vaccinia vector–expressing LMP2 (vacc LMP2) or with empty vector (vacc control). Some vacc LMP2–infected targets were prepulsed with SSC peptides. All results shown represent mean + SD and are representative of three to five separate experiments.

Figure 7.

Functional testing of coTCRcys-transduced T cells from patients with advanced NPC. A, IFNγ production following stimulation with A*1101+ targets infected with a recombinant modified vaccinia vector expressing LMP2 (MVA LMP2) or empty vector (MVA control). MVA LMP2–infected targets were also tested after pulsing with SSC peptide. Mock-transduced T cells from the same donors were used as controls. Target cells alone produced <10 pg/mL IFNγ. B, cytotoxic activity of coTCRcys- or mock-transduced T cells from a patient with advanced NPC when cocultured with NPC cell lines (HK1/A*1101 and c666.1/A*1101; effector:target = 6:1). Targets were infected with recombinant vaccinia vector–expressing LMP2 (vacc LMP2) or with empty vector (vacc control). Some vacc LMP2–infected targets were prepulsed with SSC peptides. All results shown represent mean + SD and are representative of three to five separate experiments.

Close modal

That NPC is responsive to EBV-specific T-cell–based therapies is apparent from studies using adoptive T-cell therapy (6–9). However, current approaches to generate such cells for infusion are both time consuming and unreliable. Therefore, we explored the use of TCR gene transfer, a technology that can reliably generate large quantities of specific T cells in a few days, regardless of the patient's preexisting immune response. Having identified a T-cell clone with high avidity for the HLA A*1101-restricted LMP2 epitope SSC, we cloned the genes encoding the TCR and through retroviral-mediated gene transfer expressed them in T cells from healthy donors and advanced NPC patients. T cells from healthy donors engineered to express a modified form of the TCR responded in an antigen-specific manner by proliferating, generating cytokines (IFNγ, TNFα, and IL2), lysing target cells and inhibiting LMP2+ tumor growth in vivo. TCR-transduced T cells from advanced NPC patients could also recognize NPC cell lines expressing the LMP2 protein.

As described in the methods, retroviral transduction requires only 48 hours of culture to preactivate T cells, and scaling up the process by starting with large numbers (109−1010) of T cells available from leukapheresis of patients, it should be possible to engineer >108−109 T cells for infusion in a few days. Including a few days more for in vitro expansion, trials of TCR gene transfer have infused 109−1011 T cells per patient (13, 14). This greatly exceeds the dose used to successfully treat patients with NPC by adoptive therapy with LCL-reactivated T cells (7), in which patients received only 4 × 107 to 4 × 108 cells/m2, and LMP-specific and SSC-specific T cells comprised <1% and <0.05% of this product, respectively (29). T cells transduced with the coTCRcys receptor contained a mixture of naïve, central–memory, and effector–memory cells (Supplementary Fig. S2). The presence of less differentiated T cells suggests that they should persist and display greater antitumor responses in vivo (30).

We focused on an A*1101-restricted TCR because this HLA allele is very common in the populations most at risk for NPC. Indeed, approximately 40% of NPC patients are A*1101+ (31, 32), and are therefore available for treatment with an A*1101-restricted SSC-specific TCR. Encouragingly, several studies have also reported that A*1101 is associated with decreased risk of NPC (31, 32), supporting our hypothesis that SSC peptide is a good target for T-cell therapy. Furthermore, transiently boosting of T-cell responses to this epitope in A*1101+ NPC patients using SSC peptide-pulsed dendritic cells is safe and can induce partial clinical responses (33). The SSC epitope sequence, originally identified using standard laboratory strain B95.8, is largely conserved in EBV strains within the Southern Chinese population, including virus isolates from NPC tumors (23, 34). In Northern China an S–T mutation in residue 9 of the epitope has been detected in 50% of NPC patients (35). However, from our previous studies, we found no evidence that this mutation affects antigenicity of the epitope (23).

T-cell–based therapies targeting a single epitope could lead to selection of tumor cells carrying epitope-loss EBV variants. However, this could be avoided by using multiple TCRs targeting additional epitopes in NPC-associated EBV proteins. Indeed several epitopes have already been described, some of which are again restricted through HLA class I and II alleles present at relatively high frequency in the Chinese population (23, 36), thereby increasing the number of patients available for a TCR gene transfer-based therapy. Combining TCR gene transfer with vaccination (37) could also amplify and broaden the EBV-specific T-cell response in vivo.

If T-cell therapy is to be effective for NPC, antigen-presenting function in the tumor cells must be intact. Results from immunohistochemical analysis of NPC tissues have indicated that critical components of the HLA class I antigen-processing pathway may be downregulated in some NPC tumors (38). Furthermore, there is evidence for other potential immune evasion mechanisms in NPC, including the presence of regulatory T cells (39) and transforming growth factor beta (40). Nevertheless, results from in vitro studies on NPC cell lines (41), including data presented in this report, and the association of A*1101 with reduced risk for NPC (31, 32) suggest that the malignant cells can present antigen to T cells. More importantly, clinical responses following adoptive T-cell therapy (6–9) and vaccination (33) indicate that immune evasion mechanisms can be overcome at least in some patients. Indeed, effective delivery of large numbers of tumor-specific IFNγ-producing cytotoxic T cells may be sufficient to overwhelm immunosuppressive factors. Additional genetic modifications of infused T cells, such as expression of a dominant negative TGFβ receptor (42) may also help. If the patient's antigen-presenting function is compromised, successful treatment may yet be possible by targeting stromal cells if they cross-present tumor antigens. Cross-presentation appears dependent on HLA binding affinity of the target epitope (43), which suggests that SSC [predicted affinity (IC50) = 14 nmol/L based on the Immune Epitope Database Analysis Resource] should be readily cross-presented, thereby also reducing the risk of tumor relapse through escape variants.

TCR gene transfer has been tested in the clinic to treat advanced melanoma and synovial cell sarcoma (13, 14). Combining these studies, objective clinical responses were seen in 22 of 87 patients treated. However, significant autoimmune reactions occurred in some patients in whom TCRs targeted self-proteins expressed on normal cells (13). In this respect, NPC is an ideal setting to test the potential of TCR gene transfer because foreign (viral) rather than self-antigens can be targeted using naturally occurring high-affinity TCRs. EBV is present in some normal lymphocytes, but only 1 to 50 per million circulating B cells and most of these lack viral protein expression (44). Therefore, the risk of on-target toxicity with an EBV-specific TCR is minimal.

TCR gene transfer carries a potential risk of off-target toxicity due to mispairing of TCR chains generating novel autoreactive receptor specificities (45). Although such toxicity has not yet been reported in clinical trials, we have incorporated several approaches to reduce this risk with the coTCRcys receptor. Thus, genes encoding the TCR α- and β-chains were cloned into a single retroviral vector with a 2A peptide-linker to ensure equimolar expression in the same T cell. Furthermore, we incorporated a second disulphide bond between the α- and β-constant domains, which also improved TCR surface expression. To reduce this risk further, it is possible to knockdown expression of endogenous TCR chains using shRNA (46). Nevertheless, it may be prudent to incorporate a suicide gene (47) for selective deletion of infused cells should autoimmunity develop.

Several studies have highlighted the importance of CD4+ T cells in controlling tumor growth (48, 49), and the ability of our SSC-specific TCR to function in these cells is important for two reasons. First, a concurrent antigen-specific CD4+ T-cell response aids expansion and efficacy of cytotoxic CD8+ T cells (50). Indeed, when NPC patients were immunized with dendritic cells expressing SSC peptide, CD8+ T-cell responses to this epitope were boosted but only temporarily (33). The implication was that boosting EBV-specific CD4+ T cells was also required. When stimulated with SSC peptide, CD4+ T cells transduced with coTCRcys produced cytokines, including IL2, indicating that they could help sustain coTCRcys-transduced CD8+ T cells. Secondly, coTCRcys-transduced CD4+ T cells were cytotoxic, indicating that they might destroy NPC cells directly. Therefore, the ability of this TCR to function in both CD8 and CD4 T cells increases its potential for treating NPC.

No potential conflicts of interest were disclosed.

Conception and design: Y. Zheng, A.T.C. Chan, S.P. Lee

Development of methodology: Y. Zheng, G. Parsonage, L.R. Machado, A. Salman, S.P. Lee

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): G. Parsonage, X. Zhuang, L.R. Machado, P.F. Searle, E.P. Hui, S.P. Lee

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Zheng, G. Parsonage, X. Zhuang, L.R. Machado, S.P. Lee

Writing, review, and/or revision of the manuscript: G. Parsonage, X. Zhuang, L.R. Machado, E.P. Hui, A.T.C. Chan, S.P. Lee

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Zheng, C.H. James

Study supervision: G. Parsonage, S.P. Lee

The authors thank Beatrice Johnson for excellent technical assistance in this work.

This work was supported by a Cancer Research UK Senior Cancer Fellowship Award to S.P. Lee (grant number C489/A5798) and Hong Kong Cancer Fund.

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

1.
Curado
MP
,
Edwards
B
,
Shin
HR
,
Storm
H
,
Ferlay
J
,
Heanue
M
, et al
Cancer incidence in five continents
.
Volume IX
.
International Agency for Research on Cancer
; 
2007
.
2.
Lee
AW
,
Sze
WM
,
Au
JS
,
Leung
SF
,
Leung
TW
,
Chua
DT
, et al
Treatment results for nasopharyngeal carcinoma in the modern era: the Hong Kong experience
.
Int J Radiat Oncol Biol Phys
2005
;
61
:
1107
16
.
3.
Wang
CC
,
Chen
ML
,
Hsu
KH
,
Lee
SP
,
Chen
TC
,
Chang
YS
, et al
Second malignant tumors in patients with nasopharyngeal carcinoma and their association with Epstein-Barr virus
.
Int J Cancer
2000
;
87
:
228
31
.
4.
Rickinson
AB
,
Kieff
E
,
Knipe
DM
,
Howley
PM
. 
Epstein-Barr virus. Fields virology
.
Vol 5.
Philadelphia, PA
:
Lippincott Williams and Wilkins
; 
2007
. p
2655
700
.
5.
Heslop
HE
,
Slobod
KS
,
Pule
MA
,
Hale
GA
,
Rousseau
A
,
Smith
CA
, et al
Long-term outcome of EBV specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients
.
Blood
2010
;
115
:
925
35
.
6.
Louis
CU
,
Straathof
K
,
Bollard
CM
,
Ennamuri
S
,
Gerken
C
,
Lopez
TT
, et al
Adoptive transfer of EBV-specific T cells results in sustained clinical responses in patients with locoregional nasopharyngeal carcinoma
.
J Immunother
2010
;
33
:
983
90
.
7.
Straathof
KC
,
Bollard
CM
,
Popat
U
,
Huls
MH
,
Lopez
T
,
Morriss
MC
, et al
Treatment of nasopharyngeal carcinoma with Epstein-Barr virus–specific T lymphocytes
.
Blood
2005
;
105
:
1898
904
.
8.
Comoli
P
,
Pedrazzoli
P
,
Maccario
R
,
Basso
S
,
Carminati
O
,
Labirio
M
, et al
Cell therapy of stage IV nasopharyngeal carcinoma with autologous Epstein-Barr virus-targeted cytotoxic T lymphocytes
.
J Clin Oncol
2005
;
23
:
8942
49
.
9.
Louis
CU
,
Straathof
K
,
Bollard
CM
,
Gerken
C
,
Huls
MH
,
Gresik
MV
, et al
Enhancing the in vivo expansion of adoptively transferred EBV-specific CTL with lymphodepleting CD45 monoclonal antibodies in NPC patients
.
Blood
2009
;
113
:
2442
50
.
10.
Bollard
CM
,
Gottschalk
S
,
Torrano
V
,
Diouf
O
,
Ku
S
,
Hazrat
Y
, et al
Sustained complete responses in patients with lymphoma receiving autologous cytotoxic T lymphocytes targeting Epstein-Barr virus latent membrane proteins
.
J Clin Oncol
2014
;
32
:
798
808
.
11.
Smith
C
,
Tsang
J
,
Beagley
L
,
Chua
D
,
Lee
V
,
Li
V
, et al
Effective treatment of metastatic forms of Epstein-Barr virus-associated nasopharyngeal carcinoma with a novel adenovirus-based adoptive immunotherapy
.
Cancer Res
2012
;
72
:
1116
25
.
12.
Gerdemann
U
,
Keirnan
JM
,
Katari
UL
,
Yanagisawa
R
,
Christin
AS
,
Huye
LE
, et al
Rapidly generated multivirus-specific cytotoxic T lymphocytes for the prophylaxis and treatment of viral infections
.
Mol Ther
2012
;
20
:
1622
32
.
13.
Johnson
LA
,
Morgan
RA
,
Dudley
ME
,
Cassard
L
,
Yang
JC
,
Hughes
MS
, et al
Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen
.
Blood
2009
;
114
:
535
46
.
14.
Robbins
PF
,
Morgan
RA
,
Feldman
SA
,
Yang
JC
,
Sherry
RM
,
Dudley
ME
, et al
Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1
.
J Clin Oncol
2011
;
29
:
917
24
.
15.
Midgley
RS
,
Bell
AI
,
McGeoch
DJ
,
Rickinson
AB
. 
Latent gene sequencing reveals familial relationships among Chinese Epstein-Barr virus strains and evidence for positive selection of A11 epitope changes
.
J Virol
2003
;
77
:
11517
30
.
16.
Huang
DP
,
Ho
JH
,
Poon
YF
,
Chew
EC
,
Saw
D
,
Lui
M
, et al
Establishment of a cell line (NPC/HK1) from a differentiated squamous carcinoma of the nasopharynx
.
Int J Cancer
1980
;
26
:
127
32
.
17.
Cheung
ST
,
Huang
DP
,
Hui
ABY
,
Lo
KW
,
Ko
CW
,
Tsang
YS
, et al
Nasopharyngeal carcinoma cell line (c666–1) consistently harbouring Epstein-Barr virus
.
Int J Cancer
1999
;
83
:
121
26
.
18.
Strong
MJ
,
Baddoo
M
,
Nanbo
A
,
Xu
M
,
Puetter
A
,
Lin
Z
. 
Comprehensive high-throughput RNA sequencing analysis reveals contamination of multiple nasopharyngeal carcinoma cell lines with hela cell genomes
.
J Virol
2014
;
88
:
10696
704
.
19.
Cailleau
R
,
Young
R
,
Olive
M
,
Reeves
WJ
 Jr
. 
Breast tumor cell lines from pleural effusions
.
J Natl Cancer Inst
1974
;
53
:
661
74
.
20.
Murray
RJ
,
Kurilla
MG
,
Brooks
JM
,
Thomas
WA
,
Rowe
M
,
Kieff
E
, et al
Identification of target antigens for the human cytotoxic T-cell response to Epstein–Barr virus (EBV)—implications for the immune control of EBV-positive malignancies
.
J Exp Med
1992
;
176
:
157
68
.
21.
Taylor
GS
,
Haigh
TA
,
Gudgeon
NH
,
Phelps
RJ
,
Lee
SP
,
Steven
NM
, et al
Dual stimulation of Epstein-Barr virus (EBV)-specific CD4+- and CD8+-T-cell responses by a chimeric antigen construct: potential therapeutic vaccine for EBV-positive nasopharyngeal carcinoma
.
J Virol
2004
;
78
:
768
78
.
22.
Engels
B
,
Cam
H
,
Schuler
T
,
Indraccolo
S
,
Gladow
M
,
Baum
C
, et al
Retroviral vectors for high-level transgene expression in T lymphocytes
.
Hum Gene Ther
2003
;
14
:
1155
68
.
23.
Lee
SP
,
Tierney
RJ
,
Thomas
WA
,
Brooks
JM
,
Rickinson
AB
. 
Conserved CTL epitopes within EBV latent membrane protein 2—a potential target for CTL-based tumor therapy
.
J Immunol
1997
;
158
:
3325
34
.
24.
Scholten
KB
,
Kramer
D
,
Kueter
EW
,
Graf
M
,
Schoedl
T
,
Meijer
CJ
, et al
Codon modification of T cell receptors allows enhanced functional expression in transgenic human T cells
.
Clin Immunol
2006
;
119
:
135
45
.
25.
Kuball
J
,
Dossett
ML
,
Wolfl
M
,
Ho
WY
,
Voss
RH
,
Fowler
C
, et al
Facilitating matched pairing and expression of TCR chains introduced into human T cells
.
Blood
2007
;
109
:
2331
38
.
26.
de Witte
MA
,
Jorritsma
A
,
Kaiser
A
,
van den Boom
MD
,
Dokter
M
,
Bendle
GM
, et al
Requirements for effective antitumor responses of TCR transduced T cells
.
J Immunol
2008
;
181
:
5128
36
.
27.
Jorritsma
A
,
Gomez-Eerland
R
,
Dokter
M
,
van de Kasteele
W
,
Zoet
YM
,
Doxiadis
II
, et al
Selecting highly affine and well-expressed TCRs for gene therapy of melanoma
.
Blood
2007
;
110
:
3564
72
.
28.
Darrah
PA
,
Patel
DT
,
De Luca
PM
,
Lindsay
RW
,
Davey
DF
,
Flynn
BJ
, et al
Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major
.
Nat Med
2007
;
13
:
843
50
.
29.
Straathof
KC
,
Leen
AM
,
Buza
EL
,
Taylor
G
,
Huls
MH
,
Heslop
HE
, et al
Characterization of latent membrane protein 2 specificity in CTL lines from patients with EBV-positive nasopharyngeal carcinoma and lymphoma
.
J Immunol
2005
;
175
:
4137
47
.
30.
Gattinoni
L
,
Klebanoff
CA
,
Palmer
DC
,
Wrzesinski
C
,
Kerstann
K
,
Yu
Z
, et al
Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells
.
J Clin Invest
2005
;
115
:
1616
26
.
31.
Tang
M
,
Zeng
Y
,
Poisson
A
,
Marti
D
,
Guan
L
,
Zheng
Y
, et al
Haplotype-dependent HLA susceptibility to nasopharyngeal carcinoma in a Southern Chinese population
.
Genes Immun
2010
;
11
:
334
42
.
32.
Hu
SP
,
Day
NE
,
Li
DR
,
Luben
RN
,
Cai
KL
,
Ou-Yang
T
, et al
Further evidence for an HLA-related recessive mutation in nasopharyngeal carcinoma among the Chinese
.
Br J Cancer
2005
;
92
:
967
70
.
33.
Lin
CL
,
Lo
WF
,
Lee
TH
,
Ren
Y
,
Hwang
SL
,
Cheng
YF
, et al
Immunization with Epstein-Barr virus (EBV) peptide-pulsed dendritic cells induces functional CD8+ T-cell immunity and may lead to tumor regression in patients with EBV-positive nasopharyngeal carcinoma
.
Cancer Res
2002
;
62
:
6952
58
.
34.
Kwok
H
,
Wu
CW
,
Palser
AL
,
Kellam
P
,
Sham
PC
,
Kwong
DL
, et al
Genomic diversity of Epstein-Barr virus genomes isolated from primary nasopharyngeal carcinoma biopsy samples
.
J Virol
2014
;
88
:
10662
72
.
35.
Wang
X
,
Liu
X
,
Jia
Y
,
Chao
Y
,
Xing
X
,
Wang
Y
, et al
Widespread sequence variation in the Epstein-Barr virus latent membrane protein 2A gene among northern Chinese isolates
.
J Gen Virol
2010
;
91
:
2564
73
.
36.
Tsang
CW
,
Lin
X
,
Gudgeon
NH
,
Taylor
GS
,
Jia
H
,
Hui
EP
, et al
CD4+ T-cell responses to Epstein-Barr virus nuclear antigen EBNA1 in chinese populations are highly focused on novel C-terminal domain-derived epitopes
.
J Virol
2006
;
80
:
8263
66
.
37.
Hui
EP
,
Taylor
GS
,
Jia
H
,
Ma
BB
,
Chan
SL
,
Ho
R
, et al
Phase I trial of recombinant modified vaccinia Ankara encoding Epstein-Barr viral tumor antigens in nasopharyngeal carcinoma patients
.
Cancer Res
2013
;
73
:
1676
88
.
38.
Ogino
T
,
Moriai
S
,
Ishida
Y
,
Ishii
H
,
Katayama
A
,
Miyokawa
N
, et al
Association of immunoescape mechanisms with Epstein-Barr virus infection in nasopharyngeal carcinoma
.
Int J Cancer
2007
;
120
:
2401
10
.
39.
Lau
KM
,
Cheng
SH
,
Lo
KW
,
Lee
SA
,
Woo
JK
,
van Hasselt
CA
, et al
Increase in circulating Foxp3+CD4+CD25(high) regulatory T cells in nasopharyngeal carcinoma patients
.
Br J Cancer
2007
;
96
:
617
22
.
40.
Xu
J
,
Ahmad
A
,
Jones
JF
,
Dolcetti
R
,
Vaccher
E
,
Prasad
U
, et al
Elevated serum transforming growth factor beta1 levels in Epstein-Barr virus-associated diseases and their correlation with virus-specific immunoglobulin A (IgA) and IgM
.
J Virol
2000
;
74
:
2443
6
.
41.
Lee
SP
,
Chan
AT
,
Cheung
ST
,
Thomas
WA
,
Croomcarter
D
,
Dawson
CW
, et al
CTL control of EBV in nasopharyngeal carcinoma (NPC): EBV-specific CTL responses in the blood and tumors of NPC patients and the antigen-processing function of the tumor cells
.
J Immunol
2000
;
165
:
573
82
.
42.
Bollard
CM
,
Rossig
C
,
Calonge
MJ
,
Huls
MH
,
Wagner
HJ
,
Massague
J
, et al
Adapting a transforming growth factor beta-related tumor protection strategy to enhance antitumor immunity
.
Blood
2002
;
99
:
3179
87
.
43.
Engels
B
,
Engelhard
VH
,
Sidney
J
,
Sette
A
,
Binder
DC
,
Liu
RB
, et al
Relapse or eradication of cancer is predicted by peptide-major histocompatibility complex affinity
.
Cancer Cell
2013
;
23
:
516
26
.
44.
Babcock
GJ
,
Decker
LL
,
Freeman
RB
,
Thorley-Lawson
DA
. 
Epstein–Barr virus-infected resting memory B cells, not proliferating lymphoblasts, accumulate in the peripheral blood of immunosuppressed patients
.
J Exp Med
1999
;
190
:
567
76
.
45.
Bendle
GM
,
Linnemann
C
,
Hooijkaas
AI
,
Bies
L
,
deWitte
MA
,
Jorritsma
A
, et al
Lethal graft-versus-host disease in mouse models of T-cell receptor gene therapy
.
Nat Med
2010
;
16
:
565
70
.
46.
Bunse
M
,
Bendle
GM
,
Linnemann
C
,
Bies
L
,
Schulz
S
,
Schumacher
TN
, et al
RNAi-mediated TCR knockdown prevents autoimmunity in mice caused by mixed TCR dimers following TCR gene transfer
.
Mol Ther
2014
;
22
:
1983
91
.
47.
Di Stasi
A
,
Tey
SK
,
Dotti
G
,
Fujita
Y
,
Kennedy-Nasser
A
,
Martinez
C
, et al
Inducible apoptosis as a safety switch for adoptive cell therapy
.
N Engl J Med
2011
;
365
:
1673
83
.
48.
Frankel
TL
,
Burns
WR
,
Peng
PD
,
Yu
Z
,
Chinnasamy
D
,
Wargo
JA
, et al
Both CD4 and CD8 T cells mediate equally effective in vivo tumor treatment when engineered with a highly avid TCR targeting tyrosinase
.
J Immunol
2010
;
184
:
5988
98
.
49.
Quezada
SA
,
Simpson
TR
,
Peggs
KS
,
Merghoub
T
,
Vider
J
,
Fan
X
, et al
Tumor-reactive CD4(+) T cells develop cytotoxic activity and eradicate large established melanoma after transfer into lymphopenic hosts
.
J Exp Med
2010
;
207
:
637
50
.
50.
Bevan
MJ
. 
Helping the CD8(+) T-cell response
.
Nat Rev Immunol
2004
;
4
:
595
602
.