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The Journal of Immunology, 2000, 165: 573-582.
Copyright © 2000 by The American Association of Immunologists

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 Cells1

Steven P. Lee2,*, Anthony T. C. Chan{dagger}, Siu-Tim Cheung3,{ddagger}, Wendy A. Thomas*, Debbie CroomCarter*, Chris W. Dawson*, Ching-Hwa Tsai§, Sing-Fai Leung{dagger}, Philip J. Johnson{dagger} and Dolly P. Huang{ddagger}

* CRC Institute for Cancer Studies, University of Birmingham, Edgbaston, Birmingham, United Kingdom; Departments of {dagger} Clinical Oncology and {ddagger} Pathology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, People’s Republic of China; § Graduate Institute of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan, Republic of China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Undifferentiated nasopharyngeal carcinoma (NPC) is latently infected with EBV and expresses a restricted number of viral proteins. Studies in healthy virus carriers have demonstrated that at least some of these proteins can act as targets for HLA class I-restricted CTLs. Therefore we have explored the possibility of a CTL-based therapy for NPC by characterizing EBV-specific CTL responses in 10 newly diagnosed NPC cases and 21 healthy virus carriers from Southeast Asia. Using the autologous EBV-transformed lymphoblastoid cell line, virus-specific CTL were reactivated in vitro from PBMC, cloned, and screened for cytotoxicity against target cells expressing individual EBV proteins from recombinant vaccinia vectors. EBV-specific CTLs were identified in 6 of 10 patients and 14 of 21 controls and mainly targeted the EBV nuclear Ag 3 (EBNA3) family of viral latent proteins. However, in 3 of 10 patients and 11 of 21 controls, CTLs specific for the NPC-associated protein LMP2 were also detected, albeit at low frequency. EBV-specific CTLs were detected in tumor biopsy material obtained from 3 of 6 of the patients, indicating that functional CTL are present at the tumor site, but none was specific for tumor-associated viral proteins. To assess the Ag-presenting function in NPC we studied two NPC-derived cell lines (C15 and c666.1) and demonstrated that both were capable of processing and presenting endogenously synthesized protein to HLA class I-restricted CTL clones. Overall, our data provide a sound theoretical basis for therapeutic strategies that aim to boost or elicit LMP2-specific CTL responses in NPC patients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
There is considerable evidence that HLA class I-restricted CTLs play a major role in controlling EBV infection in humans. First, EBV-specific CTLs are present at high frequency in the blood during primary infection and throughout subsequent life-long persistent infection with this virus (1, 2, 3, 4). Second, if CTL-mediated control is reduced (e.g., in transplant patients receiving immunosuppressive treatment or in HIV-infected individuals) the cell growth-transforming ability of EBV is apparent, and life-threatening EBV-driven lymphoproliferations may occur (5); however, these often regress following relaxation of immunosuppressive treatment and recovery of the cellular immune response (6). Given these observations, there is considerable interest in the possibility of targeting this virus-specific immune response to treat the growing list of human tumors that carry EBV in the malignant cells. Indeed, clinical studies have already demonstrated that infusions of autologous EBV-specific CTLs expanded in vitro can safely and effectively control EBV-positive posttransplant lymphoproliferative disease in bone marrow transplant recipients (7).

One of the most important EBV-positive tumors in world health terms is nasopharyngeal carcinoma (NPC),4 which occurs at unusually high frequency in the Chinese population in Southeast Asia (incidence, 20–50/100,000 men/year) (8). Currently the favored form of treatment for NPC is radical external radiotherapy, which cures 80–90% of cases presenting with early stage disease. However, only 10–40% of cases presenting with advanced disease survive >5 years (9, 10), and once metastases have developed, 85% of patients will be dead within 1 year (10, 11). There is therefore a need to develop additional forms of treatment for NPC, and here we investigate the possibility of a CTL-based therapy.

EBV-specific CTLs can be reactivated in vitro by culturing PBMCs with the autologous EBV-transformed lymphoblastoid cell line (LCL). Within an LCL, the majority of cells are latently infected with EBV and express at least eight virus latent proteins: namely, six nuclear Ags (EBNA1, -2, -3A, -3B, -3C, and -LP) and two membrane proteins (latent membrane protein LMP1 and LMP2) (12). In addition, polyadenylated RNA transcripts derived from the BamHI-A region of the EBV genome have been detected, including the putative open reading frame BARFO (13). Studies of EBV-specific CTL reactivated in vitro with the autologous LCL have demonstrated a hierarchy of immunodominance among these virus latent proteins. Thus, across a wide background of HLA alleles, the majority of donors mount a CTL response that targets one or more of the EBNA3 family of proteins (EBNA3A, -3B, and -3C), with subdominant responses to LMP2 and only occasional responses to EBNA2, EBNA-LP, and LMP1 (1, 2). Endogenously expressed EBNA1 is protected from processing via the conventional HLA class I processing pathway because it contains a glycine-alanine repeat (GAr) domain that prevents degradation by the proteasome complex (14). These results have important implications for a possible CTL-based therapy for NPC. Thus, in contrast to posttransplant lymphoproliferative disease, where the full complement of EBV latent genes is expressed, including the immunodominant EBNA3 family of proteins (15), in NPC the only viral proteins present are EBNA1, LMP1 (in some cases), and, from the evidence of transcriptional analysis, LMP2 (16, 17). As mentioned above, EBNA1 is protected from processing and presentation to HLA class I-restricted T cells and so is unlikely to be an effective target for a CTL-based therapy. Therefore, attention has focussed on responses to the LMP proteins, which are thought to comprise only a minor component of the total EBV-specific response in most virus carriers. Polyadenylated RNA transcripts from the BARFO gene of EBV are expressed at high levels in NPC (18). No direct evidence exists for BARFO protein expression in this tumor, but CTL responses to cell lines transfected with the BARFO gene have been detected in some healthy virus carriers (19), suggesting that this putative viral protein may also be a target Ag in NPC.

For a CTL-based therapy for NPC to be effective, NPC patients must possess CTL precursors with the appropriate antigenic specificity, which can then be expanded in vivo through immunization strategies or expanded in vitro and then returned to the patient. Previous studies of immune responses in NPC patients using mitogens and skin test reagents have demonstrated a generalized immunosuppression in these individuals (20). Furthermore, in vitro studies using the regression of EBV transformation assay, which measures T cell-mediated regression and death of EBV-infected B cells, have shown that virus-specific T cell immunity is significantly reduced in NPC patients compared with that in healthy Chinese virus carriers (21). Nevertheless, because EBV-specific T cell responses are detectable in patients, albeit at lower levels than in healthy controls, the important question is whether they include responses to those viral proteins expressed in the tumor. Equally, it is important to determine whether such effectors can access and function at the tumor site. NPC is characterized by a large lymphocytic infiltrate comprised mainly of T cells, including both CD4+ and CD8+ cells (22), although the antigenic specificity of these lymphocytes is unknown. A further requirement for an effective CTL-based therapy is that the tumor must be capable of processing and presenting the target Ag to a CTL through the TAP-dependent HLA class I processing pathway. Malignant cells carrying a defect in this pathway could escape immune surveillance, and a number of examples of this have already been described; these include a second EBV-positive malignancy, Burkitt’s lymphoma, where the Ag-presenting function is blocked due to reduced TAP expression (23).

Here we examine the possibility of a CTL-based therapy for NPC by addressing these areas. First, we have characterized the EBV-specific CTL responses in peripheral blood samples from healthy Chinese virus carriers and newly diagnosed NPC patients and also attempted to reactivate EBV-specific CTL responses from NPC tumor biopsies. Second, using two NPC-derived cell lines we have assessed the Ag-presenting function in this tumor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Healthy virus carriers and NPC patients

Peripheral blood samples were taken from 21 healthy virus donors (C1–C21) and 10 NPC patients (NPC1–NPC10) all of Chinese origin. Donors C1, C2, C4, C6, C18, and C19 were residents of Taiwan; the remainder were born in Hong Kong (C3, C5, C7–15, C17, and C20) or mainland China (C16 and C21) and emigrated to the U.K. in adulthood. These control donors (excluding C1 and C2 for whom data were not available) had a mean age of 36.2 ± 9.5 years (range, 24–58). The 10 patients included in this study were all untreated and had undifferentiated NPC by accepted histopathologic criteria. All patients attended the Prince of Wales Hospital, Hong Kong, with the exception of NPC1 and NPC6, who attended the National Taiwan University Hospital, Taiwan. NPC patients had a mean age of 52 ± 16.8 years (range, 26–79). In three cases (NPC4, -5, and -9) patients presented with advanced disease (Ho stage IV or V (24)). Blood was fractionated on Ficoll density gradients, and PBMCs were collected and stored frozen before analysis in the U.K. For six NPC patients, tumor biopsy material was collected, rinsed with RPMI 1640 (Life Technologies, Paisley, Scotland) to remove any traces of blood, and then finely minced using a scalpel. Cell suspensions were frozen and transported to the U.K. for analysis. All donors were HLA typed by PCR-based DNA typing.

Cell lines

LCLs were generated in vitro by transformation of B cells using the standard EBV isolate B95.8 (25) and were cultured in RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 IU/ml penicillin (growth medium). The cell lines C15 and c666.1 were derived from NPC biopsies and carry the EBV genome; both have been described previously (26, 27). C15 was passaged in nude mice, whereas c666.1 was cultured in vitro using growth medium containing only 1% FCS. Both lines were HLA typed by PCR-based DNA typing (C15: HLA A3, A11, B49; c666.1: HLA B58.01 (no A alleles detected)).

Reactivation of EBV-specific CTLs from blood and tumor biopsy material

PBMC from healthy virus carriers and NPC patients were stimulated in vitro with the autologous LCL (irradiated at 4000 rad) at a responder to stimulator ratio of 40:1. Cells were cultured in T cell medium (growth medium containing 1% pooled human AB serum, Sigma, Poole, U.K.). After 7 days, fresh medium and autologous LCL (irradiated) were added to the culture, and on day 14 the cells were cloned by limiting dilution to 0.3 and 3 cells/well (five 96-well plates for each cell dilution). The cloning procedure and subsequent expansion of clones in IL-2-conditioned medium have been described previously (28). In an attempt to isolate preactivated EBV-specific CTLs from the NPC biopsy, tumor cell suspensions from patients NPC3, -5 and -10 were cultured in a 96-well round-bottom plate using four different protocols. In protocol 1 cells were cultured in T cell medium containing IL-2 (10 IU/ml) and refed with this medium on day 7. On day 14 cells were fed with T cell medium containing IL-2 (100 IU/ml) plus 30% culture supernatant from the MLA 144 cell line (a gibbon lymphosarcoma-derived cell line that secretes IL-2, European Collection of Animal Cell Cultures, Public Health Laboratory Service (Salisbury, U.K.). Protocol 2 was the same as protocol 1, except that cultures were fed with T cell medium containing IL-2 (100 IU/ml) plus 30% MLA 144 supernatant on days 7 and 14. In protocol 3 cells were cultured in T cell medium containing IL-2 (100 IU/ml) plus 30% MLA 144 supernatant and refed with this medium on days 7 and 14. Protocol 4 was the same as protocol 3, except that cells were stimulated on day 7 with autologous LCL (irradiated; responder:stimulator ratio, 40:1). On day 21, all cultures were cloned by limiting dilution as described above. Tumor cell suspensions from patients NPC4, -7, and -8 were cultured using protocol 4 only.

Chromium release assays

Clones derived from healthy Chinese donors and NPC patients were screened for EBV specificity using a standard 4- to 5-h chromium release assay as described previously (28). Target cells were preinfected with recombinant vaccinia virus for 2 h at a multiplicity of infection of 10, followed by 16 h of incubation in growth medium. Targets were then labeled with [51Cr]O4 and used in the assay. Supernatants were harvested into 1% formaldehyde solution before counting to inactivate the vaccinia. The vaccinia constructs used have all been described previously (2) with the exception of vacc BARFO (a gift from Dr. M. G. Kurilla, DuPont, Wilmington, DE), which was constructed in the same manner using cDNA from the BARFO open reading frame (29). The target cells employed for this study were the autologous LCL that expresses the full panel of EBV latent proteins. As reported previously, many EBV-specific clones generated in vitro can lyse an LCL coated with the cognate viral peptide or expressing the target EBV Ag from a vaccinia vector, but they mediate little or no lysis of the LCL alone, even though the CTLs were initially reactivated using this EBV-positive cell line (30). Clones that mediated high background levels of lysis of the LCL alone upon initial screening were retested after several weeks of culture, by which time killing of the LCL alone had been sufficiently reduced to allow identification of the target EBV Ag. For the purposes of this study the definition of EBV target Ag specificity required that the percent specific lysis of a target cell expressing one EBV Ag be at least double that observed with targets expressing the other EBV Ags and that this value exceed all others by at least 15% specific lysis. All Ag-specific responses were confirmed in at least two replicate assays.

In some cases after having identified the target viral protein, clones were tested for their ability to recognize peptide epitopes previously defined in this protein that were appropriate for the HLA type of the donor (see Table IGo). Where assays involved the use of synthetic peptides (peptide sensitization assays), [51Cr]O4-labeled targets were plated out in growth medium (100 µl/well) containing a known concentration of peptide. The cells were then incubated for 1 h before the addition of CTLs (100 µl/well). Recorded peptide concentrations refer to those in the final 200-µl volume. Peptides were synthesized using fluorenylmethoxycarbonyl chemistry by J. Fox (Alta Bioscience, University of Birmingham, Birmingham, U.K.). They were dissolved in DMSO, and protein concentrations were measured using a modified Biuret assay. None of the peptides used in this study mediated target cell lysis in the absence of CTLs.


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  		Table I. Epitope peptides used to screen EBV-specific clones

 
When testing the Ag-processing function of NPC cells, NPC cell lines and control LCL targets were preinfected with recombinant vaccinia virus for 1 h (multiplicity of infection, 10) in the presence of 20% FCS and then incubated in growth medium for a further 3–4 h before labeling with [51Cr]O4. This was designed to minimize the effect of spontaneous cell death in the C15 line, which does not survive >24 h ex vivo. Having excised the C15 tumor from the mouse, surrounding mouse tissue was removed with a scalpel; the tumor cells were then washed four times with RPMI and tested in a chromium release assay without delay. As expected, levels of spontaneous release seen with this tumor line were higher than those found with other target cells, but in all cases they were <=35% of maximum cell lysis.

Direct visualization of CTL-CTL killing

This technique has been described previously (38). Briefly, CTL clones were incubated overnight in a 96-well round-bottom plate (300 cells/well) in T cell medium (100 µl/well) containing peptide at a concentration of 10 µM. Specific recognition of the peptide results in T cell-T cell killing, and cell viability is assessed the following day using an inverted phase microscope.

Western blotting

Cell lines were lysed by sonication in 8 M urea/10 mM Tris buffer, pH 7.0, and the protein concentration was determined using a Bio-Rad DC protein assay kit (Richmond, CA). The lysate was diluted in electrophoresis sample buffer, and 50 µg protein was loaded per track of a discontinuous SDS-polyacrylamide gel with a 7.5% acrylamide resolving gel. The electrophoresed proteins were transferred to a nitrocellulose membrane, and TAP1 and TAP2 proteins were detected using anti-C-terminal peptide immune rabbit sera (39) (diluted 1/500) and a chemiluminescence detection protocol.

Immunofluorescence

Surface expression of HLA class I molecules on c666.1 cell lines was measured by incubating viable cells with mAb W6/32 (40) for 30 min on ice. After washing with PBS containing 10% (v/v) normal goat serum (NGS), bound Ab was detected by incubation with FITC-labeled goat anti-mouse IgG (Sigma-Aldrich, St. Louis, MO) for 20 min on ice. After three washes in PBS and 10% NGS, cells were analyzed using a FACScan (Becton Dickinson, Mountain View, CA). W6/32 (culture supernatant) and FITC-labeled goat anti-mouse IgG were diluted 1/1 and 1/50, respectively, using PBS and 10% NGS.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EBV-specific CTL responses in healthy Chinese virus carriers

To date, characterizing EBV-specific CTL responses in healthy virus carriers has largely focussed on Caucasian donors. However, given that NPC is predominantly found among the Chinese population, we began by characterizing the EBV-specific CTL response in 21 healthy donors of Chinese origin. PBMCs were stimulated in vitro using the autologous LCL, and responding cells were cloned by limiting dilution and then screened for EBV specificity using a panel of autologous LCL targets expressing individual EBV latent proteins (including a GAr-deleted form of EBNA1 (E1{Delta}GA) and the putative BARFO protein product) from recombinant vaccinia vectors. Representative data obtained with four CTL clones from donor C3 are illustrated in Fig. 1Go. Clones 41 and 89 are clearly specific for an epitope in EBNA3B, whereas clones 40 and 52 recognize epitopes in EBNA3C and LMP2, respectively. The results obtained from screening clones from all 21 donors are summarized in Table IIGo. The numbers of clones available for testing varied considerably between donors, but evidence for an EBV-specific CTL response was detected in 14 of 21 (67%) cases. For 9 of these 14 donors the majority of EBV-specific clones targeted one or another of the EBNA3 family of proteins, suggesting that within the Chinese population the EBNA3 proteins are again the favored targets of the CTL response. However, importantly with 11 of 21 (52%) donors a CTL response specific for the NPC-associated EBV protein LMP2 was detected. These included three cases where clones targeted the previously defined HLA A11-restricted LMP2 epitope SSC (see Table IGo) and two cases where clones specific for the B40-restricted epitope IED were identified. LMP2-specific CTLs were also identified that targeted the TYG or LTA epitopes (restricted through A24 and A2.06, respectively; see Table IGo). Nevertheless, 7 of the 11 donors carried an LMP2-specific CTL response that failed to recognize any of the previously defined LMP2 epitopes. CTL responses to the other NPC-associated proteins, EBNA1, LMP1, and BARFO, were not detected in any donor.



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  		FIGURE 1. Screening LCL-reactivated CTL clones for EBV-specific responses. The figure shows four selected clones from donor C3 screened in a cytotoxicity assay against a panel of target cells expressing individual EBV latent proteins from vaccinia vectors. E:T cell ratio = 5:1. The data shown are representative of several repeated assays.

 

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  		Table II. EBV-specific CTL responses reactivated from the blood of healthy SE Asian donors

 
During this phase of the work we investigated further some of the EBV-specific responses identified. Three donors (C3, C10, and C14), who were all HLA A33 and B58 positive, yielded a relatively large number of clones specific for EBNA3B, suggesting the presence of an immunodominant epitope in this protein. Focusing initially on clones from donor C3, the HLA restriction of this response was determined in a cytotoxicity assay using a panel of HLA-matched and -mismatched targets infected with vaccinia vectors expressing EBNA3B (vE3B) or LMP2 (vLMP2) as a control. Clear lysis was observed only with the B58-matched targets infected with vE3B (Fig. 2GoA), demonstrating an EBNA3B-specific B58-restricted CTL response. To define the target epitope, these clones were then tested in a visual assay of CTL-CTL killing against a series of overlapping synthetic peptides (14- and 15-mer peptides overlapping by 10 aa) representing the entire EBNA3B sequence. This identified a single peptide representing aa 276–290 in the EBNA3B sequence (data not shown). To define the target epitope more precisely, a panel of shorter peptides spanning this region was tested over a wide range of concentrations in a peptide sensitization assay (Fig. 2GoB). When tested at higher concentrations (10-6 and 10-7 M), clear lysis of targets was seen with several of the peptides, but when the concentration was reduced to 10-9 M, only peptides representing residues 278–287 and 279–287 sensitized targets to lysis, indicating that the minimal target epitope is located at residues 279–287 (sequence VSFIEFVGW). HLA B58 was carried by 4 of 21 of the healthy Chinese virus carriers analyzed in this study, and in all cases EBNA3B-specific CTL clones were identified. A selection of clones was available from only three of these donors for testing with the EBNA3B 279–287 epitope (referred to as VSF). Peptide sensitization assays revealed that seven of eight clones from donor C3 and five of five clones from donor C14 mediated clear lysis of targets coated with this peptide. Only two clones from donor C10 were available for testing with the VSF peptide epitope, and neither recognized it. Thus, it appears that at least in a proportion of B58-positive Chinese donors, the VSF epitope represents a dominant target for the CTL response.



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  		FIGURE 2. HLA B58-restricted CTL recognition of an EBNA3B epitope located at residues 279–287. A, A CTL clone from donor C3 (HLA A24, 33; B27.04/6, 58) was tested in a chromium release assay against a panel of HLA-matched and -mismatched LCL targets expressing EBNA3B from a recombinant vaccinia vector (vE3B). As a control, targets were infected with a vaccinia vector expressing LMP2 (vLMP2). B, The same clone was subsequently tested against autologous LCL targets preincubated with overlapping synthetic peptides from EBNA3B region 271–295 or with an equivalent dilution of DMSO solvent (no peptide, control). E:T cell ratio = 3:1. All results are expressed as a percentage of specific lysis and are representative of those from several repeated assays.

 
We were also interested to explore the CTL response specific for EBNA-LP identified in donor C8 (HLA type A2.03, 11; B35, 40), because no target epitopes had previously been defined in this viral protein. Using a panel of HLA-matched and -mismatched targets infected with a vaccinia vector expressing EBNA-LP (vLP) or the vector alone (vTK-), the response was clearly shown to be HLA A*0203 restricted (Fig. 3GoA). To map the target epitope we tested the same clone against a panel of overlapping synthetic peptides (14- and 15-mer peptides overlapping by 10 aa) representing the entire EBNA-LP sequence and identified a single peptide (representing residues 280–294) that mediated CTL-CTL lysis (data not shown). Peptide sensitization experiments with a series of shorter peptides tested over a wide range of concentrations identified the minimal epitope as a 9-mer peptide (residues 284–292, sequence SLREWLLRI; Fig. 3GoB).



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  		FIGURE 3. HLA A2.03-restricted CTL recognition of an EBNA-LP epitope located at residues 284–292. A, A CTL clone from donor C8 (HLA type A2.03, 11; B35, 40) was tested in a chromium release assay against a panel of HLA-matched and -mismatched LCL targets expressing EBNA-LP from a recombinant vaccinia vector (vLP). As a control, targets were infected with the vaccinia vector alone (vTK-). B, The same clone was subsequently tested against autologous LCL targets preincubated with overlapping synthetic peptides from EBNA-LP region 275–299 or with an equivalent dilution of DMSO solvent (no peptide, control). E:T cell ratio = 2:1. All results are expressed as a percentage of specific lysis and are representative of those from several repeated assays.

 
EBV-specific CTL responses in untreated NPC patients

Reactivating CTL responses from the peripheral blood. EBV-specific CTL responses in 10 newly diagnosed NPC patients were analyzed in the same manner as the healthy virus carriers using LCL reactivation of PBMCs followed by limiting dilution cloning. Again, the numbers of clones available for testing varied considerably between donors, but evidence for an EBV-specific CTL response was detected in 6 of 10 (60%) cases (Table IIIGo). Although the total number of these specific clones was relatively small, a large proportion of them again targeted the EBNA3 family of proteins. Note that for NPC6 this included a response to the B58-restricted EBNA3B epitope VSF described above; an EBNA3B-specific CTL clone was also isolated from the only other B58-positive NPC patient tested, although this clone was not available for testing with the VSF peptide epitope. CTL responses to the NPC-associated EBV proteins LMP1 and BARFO were not detected in any patient. CTLs specific for the EBNA1 epitope VLK (see Table IGo) were isolated from NPC1 and NPC3. However, these were unable to recognize cells expressing the full-length EBNA1 molecule (data not shown) and were only detectable using a deleted form of EBNA1 lacking the GAr domain. Nevertheless, responses to the NPC-associated LMP2 protein were detected in 3 of 10 patients. An LMP2-specific CTL clone isolated from NPC10 was shown to recognize the A24-restricted epitope TYG; however, LMP2-specific responses from NPC1 and NPC9 did not map to any of the previously defined LMP2 epitopes. The clones from NPC1 (A2.03, 24; B40.03, 40.09) were analyzed for HLA restriction using a panel of HLA-matched and -mismatched LCL targets infected with a vaccinia vector expressing LMP2 (vLMP2) or the vector alone (vTK-). Representative results from one of these clones are shown in Fig. 4GoA and clearly demonstrate an A2.03-restricted LMP2-specific response. HLA A2.03 is carried by >10% of the Chinese population (41, 42), and therefore, this novel LMP2-specific response may be of therapeutic benefit to a significant number of NPC cases. Accordingly, we attempted to map the target epitope by initially screening clones against a series of overlapping peptides (14 and 15 mer, overlapping by 10 aa) representing the entire LMP2 protein. CTL-CTL killing was observed with two overlapping peptides representing aa 441–454 and 445–458. Peptide sensitization assays using shorter peptides from this region over a range of concentrations defined the minimal epitope as a 9 mer located at residues 447–455 (sequence LLSAWILTA; Fig. 4GoB).


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  		Table III. EBV-specific CTL responses reactivated from the blood of untreated NPC patients

 


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  		FIGURE 4. HLA A2.03-restricted CTL recognition of an LMP2 epitope located at residues 447–455. A, A CTL clone from donor NPC1 (HLA type A2.03, 24 B40.03, 40.09) was tested in a chromium release assay against a panel of HLA-matched and -mismatched LCL targets expressing LMP2 from a recombinant vaccinia vector (vLMP2). As a control, targets were infected with the vaccinia vector alone (vTK-). B, The same clone was subsequently tested against autologous LCL targets preincubated with overlapping synthetic peptides from LMP2 region 437–462 or with an equivalent dilution of DMSO solvent (no peptide, control). E:T cell ratio = 2:1. All results are expressed as a percentage of specific lysis and are representative of those from several repeated assays.

 
Reactivating CTL responses from the NPC tumor. Having characterized the EBV-specific CTL response in the blood of NPC patients, it was important to determine whether such effectors could traffic to and retain function at the tumor site. A tumor biopsy, taken at the same time as the blood sample, was collected from 6 of the 10 NPC patients studied above. The biopsy was finely minced with a scalpel, and the cell suspension was frozen and returned to the U.K. for analysis. In an attempt to expand preactivated as well as resting CTLs, four different protocols for CTL outgrowth were applied to tumor material from the first three patients studied (NPC4, NPC6, and NPC10; see Materials and Methods). Protocols 1 and 2 yielded relatively few growing clones, none of which recognized any of the EBV latent proteins (data not shown). Using protocols 3 and 4, the numbers of clones isolated was increased, but EBV-specific CTLs were only detected using protocol 4. Subsequently, reactivations with tumor material from NPC5, -7, and -8 used protocol 4 alone. Clones were screened in cytotoxicity assays using targets expressing individual EBV latent proteins from vaccinia vectors, and the results are summarized in Table IVGo. In most cases relatively few clones were available for testing, but EBV-specific CTL responses were detected in half the tumors studied. Tumor-infiltrating lymphocytes from NPC3 appeared to contain a relatively strong response to the GAr-deleted EBNA1 protein. These clones were shown to be specific for the A2.03-restricted VLK epitope and were not able to recognize targets expressing the full-length EBNA1 protein (data not shown). Note that the same response was detected in the peripheral blood of this patient (Table IIIGo). Tumor-infiltrating lymphocytes from NPC4 included a weak CTL response to EBNA3A and -3B, whereas we were unable to detect any EBV-specific CTLs in the blood from this patient. Tumor material from NPC10 yielded clones specific for EBNA3A or -3C, which correlated well with findings from the blood of this donor (although it was not known whether responses in the tumor and blood targeted the same epitopes). No EBV-specific CTLs were detected in the tumor from NPC5, -7, and -8; however, no such responses were identified in the blood from these patients. Given that EBNA1 is protected from processing, there was therefore no evidence for a CTL response within the tumor that might target the EBV-positive malignant cells.


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  		Table IV. EBV-specific CTL responses reactivated from the tumor biopsy of untreated NPC

 
Ag-processing function of NPC cell lines

The ability of NPC cells to process and present endogenously synthesized Ag to HLA class I-restricted CTLs is of great importance to the possibility of a T cell-based therapy for this disease. However, to date this issue has been addressed in only a single study in which the TAP-dependent HLA class I processing pathway of the north African-derived NPC cell line C15 was shown to be intact (43). The results of this earlier study are clearly of great significance to the present work, and thus we sought to confirm these findings and to extend them to an NPC cell line (c666.1) derived from the population most at risk for this disease, namely the Chinese.

The C15 cell line was passaged in nude mice, and the tumor was harvested for in vitro analysis. Previous studies have demonstrated that this line expresses high levels of HLA class I on the cell surface (26), and HLA typing revealed this line to be A11 positive, so to assess the Ag-processing function we tested two A11-restricted CTL clones specific for either the IVT or AVF epitopes in EBNA3B (see Table IGo). Fig. 5GoA shows the results of a cytotoxicity assay in which these clones were tested for recognition of the C15 line or an A11-matched LCL target. Target cells were infected with a vaccinia vector expressing EBNA3B (vE3B) or with the vector alone (vTK-). Alternatively, the target cells were preloaded with the IVT or AVF epitope peptides or with DMSO alone (no peptide control). Clear lysis was observed with the C15 tumor cells expressing EBNA3B, whereas targets infected with the vaccinia vector alone were not lysed. Note that the level of lysis was comparable with that achieved by preloading the C15 line directly with the target peptide epitope. The same pattern of results was observed when the A11-matched LCL was used as the target cell.



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  		FIGURE 5. Ag-presenting function in NPC cell lines C15 and c666.1. A, Two A11-restricted EBNA3B-specific CTL clones (CMc23 and CMc21) tested against C15 and an A11-matched LCL target. All targets had either been preincubated with the cognate peptide epitope or preinfected with a recombinant vaccinia vector expressing the target EBV protein. As controls, targets were preincubated with an equivalent dilution of DMSO solvent (no peptide) or with the vaccinia vector alone (vTK-). E:T cell ratio = 5:1. B, Viable cells from c666.1 and an LCL were analyzed by FACScan for surface expression of HLA class I using the mAb W6/32. Dotted line, staining with second step Ab alone. Solid line, W6/32 staining. C, c666.1, a BL cell line and an LCL were analyzed by Western blotting for expression of TAP1 and TAP2. D, A B58-restricted EBNA3B-specific CTL clone was tested against c666.1 and a panel of B58-matched and -mismatched LCL targets. All targets had been preinfected with a recombinant vaccinia vector expressing EBNA3B (vE3B). As controls, targets were preinfected with the vaccinia vector alone (vTK-). All data shown are representative of those from several repeated assays.

 
c666.1 is a newly described NPC cell line derived from an NPC tumor of southern Chinese origin (27). HLA typing of this line revealed that it carries HLA B58, but no other HLA A or B alleles. Immunofluorescence studies using the HLA class I-specific mAb W632 demonstrated that HLA class I molecules are expressed on the surface of the c666.1 line, but at a lower level than that seen with an LCL (Fig. 5GoB). Expression of the TAP1 and TAP2 proteins in c666.1 was examined by Western blot analysis and compared with a Burkitt tumor cell line and an LCL. The Burkitt line expressed little or no TAP1 and TAP2 (as reported previously (23)), whereas c666.1 expressed TAP1 and TAP2 at a similar level to that seen in the LCL (Fig. 5CGo). Having identified a B58-restricted CTL response to EBNA3B during the course of the present study (Fig. 2Go) we were able to test the ability of the c666.1 line to present endogenously synthesized Ag through a naturally expressed HLA allele. As shown in Fig. 5GoD, a B58-restricted CTL clone specific for the EBNA3B epitope VSF mediated clear lysis of the c666.1 line expressing EBNA3B from a vaccinia vector (vE3B), but not when this line was infected with the vector alone (vTK-). Levels of lysis were similar to those obtained with a vE3B-infected B58-matched LCL target. HLA-mismatched LCL targets infected with vE3B were not lysed.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies of EBV-specific CTL responses in healthy virus carriers have focused on donors of Caucasian origin (1, 2), yet NPC is primarily a disease that affects the Chinese population. Therefore, we began the present study by attempting to reactivate EBV-specific CTLs from the peripheral blood of 21 healthy Chinese virus carriers using the autologous EBV-transformed LCL. As reported previously with Caucasian donors, we again observed a hierarchy of immunodominance among the EBV latent Ags; the EBNA3 family of proteins represented dominant targets for many of the healthy Chinese donors studied, whereas little or no response was identified for EBNA1, EBNA2, EBNA-LP, LMP1, and BARFO. Importantly, however, more than half the Chinese virus carriers possessed a detectable response to the NPC-associated viral protein LMP2, including responses to previously defined epitopes presented through HLA alleles present at high frequency in this population (e.g., HLA A11, A24, B40) (28). In fact, the number of EBV-specific CTL responses identified in these Chinese donors may be an underestimate, because they were reactivated in vitro using an LCL transformed with the EBV strain B95.8 and were screened using vaccinia recombinants expressing B95.8-derived EBV latent genes. Given that B95.8 was originally isolated from a Caucasian donor (44), it is possible that some EBV-specific CTL responses in the Chinese donors may have gone undetected if they targeted viral epitopes not conserved in the B95.8 strain. However, recent work on a Chinese-derived EBV isolate has sequenced the full length of the EBNA3A, -3B, and -3C genes and demonstrated a high degree of amino acid sequence conservation (98, 97.5, and 99%, respectively) compared with the B95.8 strain (R. S. Midgley and A. B. Rickinson, manuscript in preparation). Furthermore, sequencing an EBV isolate derived from a Chinese NPC patient has demonstrated 98% amino acid sequence identity in the LMP2 gene (Y.-S. Chang, personal communication). Nevertheless, we are currently extending the present study using a Chinese-derived strain of EBV to identify any additional virus-specific CTL responses in the Chinese population.

In 7 of 21 Chinese virus carriers we failed to detect any EBV-specific CTL clones. Possible explanations for this finding include a very low frequency of EBV-specific CTL precursors present in the blood of these donors, the use of a Caucasian-derived EBV strain or that these donors were not infected with EBV. Only one of these seven donors had been tested serologically for EBV infection (and was positive), yet given the very high frequency of EBV infection in the adult Chinese population (45), the latter explanation seems unlikely at least for the majority of these donors.

Having demonstrated that many healthy Chinese virus carriers mount a CTL response to at least one NPC-associated EBV Ag, namely LMP2, it was important to determine whether such a response could also be detected in NPC patients. The majority of EBV-specific clones isolated from these patients again targeted the EBNA3 proteins, whereas no responses were detected to the NPC-associated EBV proteins LMP1 or BARFO. Two patients mounted a detectable response to the VLK epitope in EBNA1. CTL responses to this epitope have been described previously and are possibly a result of cross-priming, whereby APCs have taken up exogenous EBNA1 protein released from dying EBV-infected cells and processed it for presentation on HLA class I molecules (31). However, because endogenously expressed EBNA1 protein is protected from processing via the conventional HLA class I processing pathway, these CTL responses observed in NPC patients are unlikely to be of therapeutic value. Nevertheless, CTL clones specific for LMP2 were isolated from 3 of 10 NPC cases. NPC1 yielded a relatively high number of LMP2-specific clones, and further analysis revealed that each of these was HLA A2.03 restricted and targeted a novel epitope located at aa residues 447–455 in the LMP2 sequence. However, for both NPC9 and NPC10, only a single LMP2-specific CTL clone was isolated. Thus, although LMP2-specific CTL were detected in some NPC patients, these responses were often relatively weak. The observation that NPC patients mount little or no response to LMP2 may explain how an LMP2-positive tumor cell can escape immune surveillance, and suggests that boosting such a weak response may have therapeutic benefit. It remains to be determined whether those healthy Chinese virus carriers who mount a relatively strong CTL response to LMP2 will be protected from developing NPC. It should be noted, however, that although several studies have demonstrated LMP2 gene transcripts in NPC (16, 17), and NPC patients, unlike most infected individuals, show detectable serum Ab responses to LMP2 (46), to date there is no direct evidence of LMP2 protein expression in this tumor. This may well reflect the fact that currently available serological reagents are of insufficient sensitivity to detect LMP2 protein even in some LCLs (47). Nevertheless, our results highlight the importance of developing improved reagents with which to address this issue.

The numbers of EBV-specific CTL clones identified in the present study varied considerably between donors, but overall fewer were isolated from NPC patients (mean, 3.2 ± 4.3 clones/donor) than from the healthy controls (mean, 8.9 ± 9.7 clones/donor). Although this difference was not statistically significant (p = 0.17, by Wilcoxon two-sample test), it correlates with previous reports of generalized immunosuppression in NPC patients (20) and of reduced EBV-specific T cell immunity as measured by a regression assay (21). Note that the average age of NPC patients was higher than that of healthy controls, which might also explain this difference, although some of the oldest NPC patients actually generated the largest number of EBV-specific clones.

Analyzing EBV-specific CTL responses in NPC tumor biopsy material, we successfully isolated virus-specific clones from the tumors of three of six patients, each clone targeting one of the EBNA3 proteins or the VLK epitope in EBNA1 (which again could only recognize the GAr-deleted form of this protein). Similar work on EBV-positive tumor biopsy material from Hodgkin’s disease patients suggested that EBV-specific CTL responses may be repressed at the tumor site (48). One possible mechanism for this could be through the action of cytokines such as IL-10, which is known to suppress cellular immune responses (49). IL-10 is also expressed in NPC tumors (50), yet our findings demonstrate that EBV-specific CTLs can be cultured from NPC biopsies and display Ag-specific cytolytic activity in vitro. However, we did not detect any responses to LMP2 or any other NPC-associated viral protein. This may reflect the fact that such responses were also undetectable even in the blood of five of six of the NPC patients from whom tumor biopsy material was available, and the remaining patient possessed only a very weak LMP2-specific response (see Table IIIGo). Thus, a further study of more patients is required to determine whether these responses are indeed excluded from the tumor site. Nevertheless, the fact that NPC10 possessed a weak LMP2-specific CTL response in the blood but we were unable to detect such effectors in the biopsy, indicates that these CTL do not accumulate and/or expand at the tumor site. Thus, another explanation for the persistence of EBV-positive tumor cells in an NPC patient is that the appropriate CTL effectors never reach the tumor site or, alternatively, that they are selectively rendered nonfunctional at this location.

HLA A11 is carried at high frequency in the Chinese population (41), and 9 of 21 of the Chinese virus carriers and 3 of 10 NPC patients studied in this work were A11 positive. The majority of A11-positive Caucasian individuals mount a dominant CTL response to one or two defined epitopes in EBNA3B, namely IVT and AVF (see Table IGo) (34). However, no such responses were identified among the A11-positive Chinese donors. This is in agreement with previous work showing that the IVT and AVF epitope sequences are altered in Chinese virus strains such that they do not bind the A11 molecule and are therefore not recognized by CTL (51). Several of the defined CTL target epitopes in LMP2 are also altered in Chinese virus strains compared with B95.8 and other Caucasian virus isolates. These include the epitopes IED, which carries an L->I change at position 9 of the amino acid sequence, and TYG, which carries a C->S change at position 8 (28). Our previous work showed that these changes do not affect the antigenicity of these epitope sequences (28), and the present study indicates that it does not affect their immunogenicity either, since several Chinese donors (who are likely to carry these altered sequences) mounted detectable CTL responses to one or another of these epitopes. All EBV-specific CTL clones generated from the healthy Chinese donors and NPC patients were tested for recognition of previously defined epitopes, and in many cases they failed to recognize these. For three EBV-specific CTL responses we were able to define novel epitopes restricted through HLA alleles present at significant frequency in the Chinese population (HLA A2.03, >10%; HLA B58, 13%) (41, 42); nevertheless, our findings indicate the presence of several more epitopes within the EBV latent proteins.

Analyzing the NPC cell lines C15 and c666.1 indicated that the TAP-dependent HLA class I processing pathway is intact in NPC and that the tumor cells can be lysed by CTL. Our data with C15, an NPC line derived from a North African patient (26), support the findings of an earlier study (43), while the c666.1 line demonstrates that the Ag-processing function is also intact in NPC tumor cells of Chinese origin.

What, then, are the prospects for a CTL-based therapy for NPC? To date, a number of potential target Ags have been identified, namely LMP1, BARFO, and in particular, LMP2. Several target epitopes have been defined in these proteins that are presented through HLA alleles present at high frequency in the Chinese population. Furthermore, these CTL target epitope sequences appear to be antigenically and immunogenically conserved in Chinese virus strains, including those present in NPC tumors. Despite a general immunosuppressed state in NPC patients, the present work suggests that weak LMP2-specific CTL responses can be detected in at least some cases; hence, boosting these responses could be of therapeutic benefit. Functional CTL are present at the tumor site, although to date no CTL responses specific for tumor-associated Ags have been detected there. Finally, studies of NPC cell lines indicate that the tumor is capable of processing endogenously expressed EBV Ags for recognition by HLA class I-restricted CTL and that this results in lysis of the malignant cell. Therefore, there is now a sound theoretical basis for attempting to treat NPC by boosting or eliciting LMP-specific CTL responses, either through immunization or adoptive transfer of in vitro expanded CTLs. However, it remains to be determined whether such CTLs will traffic to and remain functional at the tumor site.


   Footnotes
 
1 This work was supported by a Medical Research Council Career Development Award (to S.P.L.), a UICC (International Union Against Cancer) International Cancer Technology Transfer Fellowships Award (to S.T.C.), the Hong Kong Research Grants Council (CUHK 395/95 M), and Research Grants Council Direct Grant (1998/99). Back

2 Address correspondence and reprint requests to Dr. S. P. Lee, Clinical Research Center Institute for Cancer Studies, University of Birmingham, Vincent Drive, Edgbaston, Birmingham, United Kingdom B15 2TA. Back

3 Current address: Department of Surgery, Queen Mary Hospital, University of Hong Kong, Pok Fu Lam Road, Hong Kong, People’s Republic of China. Back

4 Abbreviations used in this paper: NPC, nasopharyngeal carcinoma; EBNA, EBV nuclear Ag; LCL, lymphoblastoid cell line; LMP, latent membrane protein; GAr, glycine-alanine repeat; NGS, normal goat serum. Back

Received for publication January 24, 2000. Accepted for publication April 18, 2000.


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K. C. M. Straathof, C. M. Bollard, U. Popat, M. H. Huls, T. Lopez, M. C. Morriss, M. V. Gresik, A. P. Gee, H. V. Russell, M. K. Brenner, et al.
Treatment of nasopharyngeal carcinoma with Epstein-Barr virus-specific T lymphocytes
Blood, March 1, 2005; 105(5): 1898 - 1904.
[Abstract] [Full Text] [PDF]

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S. P. Lee, J. M. Brooks, H. Al-Jarrah, W. A. Thomas, T. A. Haigh, G. S. Taylor, S. Humme, A. Schepers, W. Hammerschmidt, J. L. Yates, et al.
CD8 T Cell Recognition of Endogenously Expressed Epstein-Barr Virus Nuclear A