Approximately 40% of Hodgkin’s disease (HD) cases carry EBV in the malignant Hodgkin-Reed Sternberg (H-RS) cells, with expression of viral latent membrane proteins (LMPs) 1 and 2. These viral proteins are targets for CTLs in healthy EBV carriers, and their expression in EBV-associated HD raises the possibility of targeting them for a CTL-based immunotherapy. Here we characterize the CTL response to EBV latent antigens in both the blood and tumor-infiltrating lymphocytes of HD patients using two approaches: (a) in vitro reactivation of CTLs by stimulation with the autologous EBV-transformed lymphoblastoid cell line; and (b) an enzyme-linked immunospot assay to quantify frequencies of CTLs specific for known LMP1/2 epitopes. We detected EBV-specific CTLs in blood and biopsy samples from both EBV-negative and EBV-positive HD patients. However, as in healthy EBV carriers, LMP-specific CTL precursors occurred only at low frequency in the blood of HD patients, and with the exception of one EBV-negative HD case, were undetectable in the tumor. These data give rise to two considerations: (a) they may explain why EBV-positive tumor cells persist in the presence of an existing EBV-specific immune response; and (b) they provide a rationale for selectively boosting/eliciting LMP-specific CTL responses as a therapy for EBV-positive HD.

It is now well established that ∼40% of HD3 cases in Western countries and a far higher proportion in some developing regions are associated with EBV infection of the malignant H-RS cells (1, 2, 3). The role of EBV in the etiology of HD remains unclear, but its oncogenic potential is apparent from its ability to transform human B cells in vitro into permanently growing LCLs and its association with a number of other human malignancies (4, 5).

Despite its oncogenic potential, EBV is widespread in the human population, where it persists as an asymptomatic infection of B cells. The evidence suggests that HLA class I-restricted CTLs play a key role in controlling EBV in healthy virus carriers (reviewed in Ref. 6). Given the importance of CTLs in the control of EBV infection, clinical studies have explored the possibility of infusing EBV-specific T-cell lines to treat or prevent the outgrowth of EBV-positive lymphomas that can occur in transplant recipients (7, 8, 9). The results of these studies not only support the role of CD8+ CTLs in controlling EBV infection but also demonstrate the efficacy of adoptive T-cell therapy for treating EBV-positive lymphomas in immunosuppressed individuals. Therefore, there is now considerable interest in the possibility of extending this approach to treat other EBV-positive malignancies. A T-cell-based therapy for EBV-positive cases of HD may be of particular value for treating advanced stage or relapsed disease, in which conventional therapeutic strategies are rarely curative, and would also avoid the late complications seen with radiotherapy and chemotherapy, such as myocardial damage and the development of secondary tumors (reviewed Refs. 10, 11).

EBV-specific CTL responses in healthy virus carriers have been studied extensively by cocultivating PBMCs in vitro with the autologous EBV-transformed LCL. Within an LCL, EBV establishes a predominantly latent infection with the expression of at least eight viral proteins, i.e., six nuclear antigens (EBNAs 1, 2, 3A, 3B, 3C, and LP) and two LMPs (LMP1 and LMP2; Ref. 4). RNA transcripts from the BARF0 open reading frame in the BamHI A region of the viral genome have also been detected (12). Studies on T-cell responses in healthy virus carriers have demonstrated a hierarchy of immunodominance among EBV latent antigens. Thus, for most donors, the T-cell response generated in vitro after LCL stimulation is dominated by cells specific for the EBNA3 family of proteins (EBNA3A, EBNA3B, and EBNA3C) with subdominant reactivities detectable to EBNA2, LP, LMP1, and/or LMP2 (13, 14). EBNA1 is protected from processing by the classical HLA class I route, because of the presence of an internal GAr region (15, 16). However, using targets expressing a GAr-deleted EBNA1 molecule, CTLs specific for this protein have been identified in several donors (17). CTLs that recognize an HLA-A2-restricted epitope within BARF0 have also been described (18, 19).

In contrast to an LCL, EBV-infected H-RS cells display a more restricted pattern of viral latent gene expression. Thus, immunohistochemical studies have demonstrated the expression of EBNA1, LMP1, and LMP2 proteins in H-RS cells (20, 21, 23), and transcriptional studies indicate that a BARF0 protein product may also be expressed (24). However, the immunodominant EBNA3 family of proteins is not present. Nevertheless, several CTL target epitopes have now been defined in LMP1 and particularly in LMP2, many of which are restricted through common HLA alleles (e.g., HLA-A2; Refs. 25, 26, 27).

The expression of known CTL target antigens in H-RS cells offers the potential for a CTL-based therapy for HD. However, to develop an effective therapy we must first determine why the host’s immune response has failed to clear the tumor. One possibility is that H-RS cells cannot be recognized by CTLs because of a defect in the HLA class I antigen processing pathway. However, most EBV-positive Hodgkin’s tumors have high levels of expression of HLA class I molecules and of the transporters associated with antigen processing (TAPs 1 and 2; Refs. 28, 29, 30). Furthermore, HD-derived cell lines can process and present vector-introduced EBV antigens to specific HLA class I-restricted CTLs with resultant killing of the H-RS cell (28, 31).

Another possibility to explain the persistence of EBV-positive H-RS cells is that the host fails to mount a CTL response to those viral proteins present in the tumor. A few reports have attempted to study EBV-specific CTL responses in the blood of HD patients with EBV-positive disease, but these have largely analyzed polyclonal T-cell populations generated after stimulation of PBMCs with the autologous LCL (32, 33). Such polyclonal populations are usually dominated by reactivities to the EBNA3 antigens, and thus weaker responses to tumor-associated viral antigens, such as the LMPs, may have been masked. In one study, an LCL-reactivated polyclonal line from a single HD patient was cloned by limiting dilution, and a single LMP2-specific clone identified; however, the EBV status of this patient’s tumor was not reported (31).

A third possibility is that LMP-specific CTLs fail to access the tumor site or fail to function in the tumor microenvironment. Currently, there is little information available on this issue, although one study reported that EBV-specific CTLs are suppressed in EBV-positive tumors while still present in the circulating lymphocyte pool of the same patient (32). Furthermore, EBV-positive H-RS cells produce a number of immunosuppressive cytokines including IL-10 and transforming growth factor-β (34, 35).

In the present study, we have addressed some of these issues by conducting a detailed analysis of the EBV-specific CTL response in HD patients: (a) EBV-specific CTLs were reactivated from peripheral blood using the autologous LCL, followed by limiting dilution cloning of responder cells to enable detection of subdominant responses. The same approach was also used to analyze EBV-specific responses in TILs from EBV-positive and -negative Hodgkin’s tumors; and (b) an Elispot assay was used to quantitate CTL responses to defined LMP1 and LMP2 epitopes in PBMCs and TILs from HD patients.

Patient Samples.

Pretreatment blood and/or biopsy samples were obtained from 24 newly diagnosed HD patients (see Table 1), having previously obtained informed consent. Blood was fractionated on Ficoll density gradients, and PBMCs were cryopreserved. Tumor biopsy material was collected, rinsed with RPMI 1640 to remove any traces of blood, and then disaggregated using a scalpel or Medimachine (Dako, Ltd.). Cell suspensions were centrifuged on Ficoll density gradients to purify mononuclear cells and stored frozen.

Donors were HLA typed by PCR-based DNA typing (36) and tested for EBV infection by immunofluorescence to detect serum antibodies to the viral capsid antigen. The EBV status of HD biopsies was determined by in situ hybridization as described previously (28).

Cell Lines.

LCLs were generated in vitro by transformation of B cells using the EBV isolate B95.8 (37) and cultured in RPMI 1640 containing 10% FCS, 2 mml-glutamine, 100 μg/ml streptomycin, and 100 IU/ml penicillin (growth medium). PHA-activated T-cell blasts were generated by stimulation of PBMCs with PHA (10 μg/ml; Murex Biotek, Chattillon, France).

Reactivation of EBV-specific CTLs from Blood and Tumor Biopsy Material.

PBMCs and TILs from HD patients were stimulated in vitro with the autologous LCL (irradiated at 4000 rads) at a responder:stimulator ratio of 40:1. Cells were cultured in T-cell medium (growth medium containing 1% pooled human AB serum; Sigma Chemical Co., Poole, United Kingdom). After 7 days, fresh medium and autologous LCL (irradiated) were added to the culture. On day 14, cells were cloned by limiting dilution to 0.3 and 3 cells/well (five 96-well plates for each cell dilution) and maintained in IL-2-conditioned medium by intermittent restimulation with irradiated autologous LCL, as described previously (26).

Chromium Release Assays.

Clones derived from PBMCs and TILs were screened using a standard 4-h chromium release assay for EBV specificity. Clones were tested against a panel of target cells expressing individual EBV antigens from recombinant vaccinia vectors as described previously (26). The vaccinia constructs used in this study have all been described previously (13, 38). Clones were screened for EBNA1 specificity using a truncated form of EBNA1 (termed E1ΔGA) lacking the GAr region. In most cases, the target cell used for this study was the autologous LCL that expresses all EBV latent proteins. As reported previously, many EBV-specific clones generated in vitro from healthy virus carriers can lyse an LCL coated with the cognate viral peptide or expressing the target EBV antigen from a vaccinia vector but mediate little or no lysis of the LCL alone, although the CTLs were initially reactivated using this EBV-positive cell line (39). Clones that mediated high background levels of lysis of the LCL alone on initial screening were retested after 7–14 days of culture, by which time killing of the LCL alone had reduced sufficiently to identify the target EBV antigen. For the purposes of this study, the definition of EBV target specificity required that the percentage of specific lysis of a target cell expressing one EBV antigen be at least double that observed with targets expressing the other EBV antigens, and that this value exceeded all others by at least 15% specific lysis. All antigen-specific responses were confirmed in at least two replicate assays.

In some cases, having identified the target viral protein, clones were tested for recognition of peptide epitopes defined previously in this molecule and that were appropriate for the HLA type of the donor (see Tables 1 and 2). Where assays involved the use of synthetic peptides (peptide sensitization assays) 51CrO4-labeled targets were plated out in growth medium (100 μl/well) containing a known concentration of peptide. 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 Dr. John Fox (Alta Bioscience, University of Birmingham, Birmingham, United Kingdom). They were dissolved in DMSO, and protein concentrations were measured using a modified Biuret assay. None of the peptides mediated target cell lysis in the absence of CTLs.

Elispot Assay.

Ninety-six-well polyvinylidene difluoride-backed plates (Millipore, Bedford, MA) were precoated with 15 μg/ml of an anti-IFN-γ monoclonal antibody 1-DIK (Mabtech, Stockholm, Sweden) for 3 h at room temperature. Plates were then washed six times with RPMI 1640 and blocked with RPMI 1640 + 10% FCS for 1 h. PBMCs or TILs were added in duplicate wells in 100-μl volumes at 105, 2 × 105, and 4 × 105 cells/well (cell input number) in the presence of 2 μg/ml peptide. Wells containing PHA (100 μg/ml) or an equivalent dilution of DMSO solvent were used as positive and negative controls, respectively. Plates were incubated overnight at 37°C with 5% CO2. Cells were discarded the next day, and plates were washed six times with 0.05% Tween 20 diluted in PBS. A biotinylated anti-IFN-γ monoclonal antibody 7-B6-1 (Mabtech) was added at 1 μg/ml and left for 3 h at room temperature. After 6 additional washes, a 1:1000 dilution of streptavidin-conjugated alkaline phosphatase was added for 2 h. Plates were washed again six times. Individual cytokine-producing cells were detected as dark spots after a 30-min reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium using an alkaline phosphatase-conjugate substrate kit (Bio-Rad, Richmond, CA). Spots were counted under a dissection microscope. The number of specific T-cell responders was calculated by taking the mean number of spots from duplicate wells and subtracting the mean number of spots in negative control wells with the same cell input number. Where more than one input cell number was used, mean corrected values were plotted on a graph, and a line of best fit was drawn through the points to obtain the frequency of specific precursors. Results are expressed as number of SFCs/106 PBMCs or TILs.

CTL Responses to EBV Latent Antigens in Peripheral Blood from HD Patients.

EBV-specific CTL clones were reactivated from peripheral blood samples of 11 untreated HD patients, 7 with EBV-negative disease and 4 with EBV-positive tumors. Clones were screened for antigen specificity against a panel of autologous LCL targets preinfected with recombinant vaccinia viruses expressing individual EBV latent antigens. In many cases, the target peptide epitope was subsequently identified by peptide sensitization assays. Representative results from one donor with EBV-positive HD, HD15, are shown in Fig. 1, where clones demonstrate clear specificity for GAr-deleted EBNA1, EBNA3A, EBNA3C or LMP2; results from all donors are summarized in Table 3.

EBV-specific CTL clones were obtained from all EBV-seropositive HD patients studied. In the majority of cases, the strongest CTL responses were directed against one or other of the EBNA3 family of latent antigens, which are known to be immunodominant in healthy EBV carriers (13, 14). Responses to E1ΔGA, LMP2, and EBNA2 were seen less frequently, whereas CTLs specific for LP, LMP1, or BARF0 were not detected in this patient cohort. A similar pattern of responses was seen in patients with EBV-negative and -positive HD. Donor HD1 was EBV-seronegative but was included to investigate whether LCL reactivation of PBMCs was capable of eliciting a primary EBV-specific response in vitro. Two hundred and forty clones were generated from the blood of this donor, but none was specific for any of the viral antigens tested.

Two individuals in each group showed relatively weak reactivity to LMP2, one of the antigens expressed in EBV-positive HD. In two cases (HD4 and HD14), responses were HLA-A2 restricted (data not shown), and the target epitopes were defined as LLW and CLG, respectively (Fig. 2). Responses to the GAr-deleted form of EBNA1 (E1ΔGA) were seen in three individuals, and in one case (HD5) mapped to the HLA B35-restricted epitope HPV.

Patient HD6 demonstrated an unusually dominant response to EBNA2, with 217 of 223 clones targeting this EBV protein. To date, only a single HLA class I-restricted epitope has been described in this normally subdominant antigen. Therefore, we decided to explore this response further using a panel of HLA-matched and -mismatched targets expressing EBNA2 from a recombinant vaccinia vector. As shown in Fig. 3,A, the response was clearly restricted through HLA B38, and although the donor carries the B38.01 subtype, clones recognized EBNA2 on a background of either B38.01 or B38.02 subtypes. Using peptide sensitization assays, the minimal target epitope was mapped to residues 14–23 (sequence YHLIVDTDSL; Fig. 3 B). This epitope sequence accords well with the predicted HLA B38.01 peptide binding motif (49), having a histidine residue at position 2 and leucine at the COOH terminus.

CTL Responses to EBV Latent Antigens in HD Biopsies.

Biopsies were available from 3 EBV-negative and 3 EBV-positive HD tumors. Reactivations were performed using the autologous LCL as the stimulator cell. CTL lines derived from biopsy material grew more slowly than blood-derived lines and required additional cycles of restimulation with autologous LCL before limiting dilution cloning was performed. Nevertheless, from all five EBV-seropositive cases, we were able to isolate CTL clones targeting the immunodominant EBNA3 proteins, and in almost all cases we identified the target epitopes (Table 4). Representative results of TIL-derived EBNA3A-specific CTL clones from an EBV-positive tumor (HD18) are illustrated in Fig. 4,A. Of 30 such clones derived from this tumor biopsy, 18 were specific for the HLA B8-restricted epitope QAK (Fig. 4 B). In one case (HD5), we identified CTLs specific for the B35-restricted EBNA1 epitope HPV, but LMP1 or LMP2-specific CTLs were not detected in any biopsy. No EBV-specific CTLs were isolated from the biopsy of patient HD2. This donor was EBV seronegative, which again suggests that our reactivation protocol had not generated primary responses in vitro.

Comparison of results from pretreatment blood and biopsy samples was possible for two patients in each group (donors 5, 9, 14, and 17; Tables 3 and 4). In most cases, similar reactivities were seen at both sites, and where the target epitope was determined, this was also conserved. Only one of these patients (HD14) carried a detectable LMP2-specific response in the blood (5 clones of 235 tested). Such a response was not detected in the tumor biopsy from this patient; however, this may reflect the fact that considerably fewer clones were available for testing from the biopsy (n = 67).

Quantitation of Circulating LMP1/2-specific CTL Precursor Frequencies by Elispot Assay.

The above results indicate that CTL precursors specific for the tumor-associated viral antigens LMP1 and LMP2 are reactivated only infrequently from peripheral blood of HD patients after in vitro stimulation with the autologous LCL. However, this approach may underestimate the true number of precursors because it requires that antigen-specific T cells proliferate over at least 5 weeks of in vitro culture and that they mediate cytotoxic function. Therefore, to complement our studies using LCL reactivation, we analyzed EBV-specific T-cell responses in HD patients and healthy EBV carriers using the Elispot assay. This avoids the need for prolonged in vitro culture and provides more quantitative data on the frequency of T cells specific for a given epitope.

The Elispot assay was applied to blood samples from 11 HD patients and 12 healthy EBV carriers to measure precursor frequencies of T cells specific for known peptide epitopes within LMP1 and LMP2. The epitopes studied included two HLA-A2-restricted epitopes in LMP1 (YLL and YLQ) and four epitopes within LMP2 [CLG, LLW, and FLY (HLA-A2-restricted); TYG (HLA-A24-restricted)]. The immunodominant HLA-A2-restricted GLC epitope from the EBV lytic cycle protein BMLF1 was included as a positive control (50). Eight patients had EBV-negative tumors, whereas three had EBV-positive disease. Assays were performed in duplicate at three input cell numbers for all patients except HD12 (performed at 125,000 cells/well, in duplicate) and HD21 (performed at 170,000 cells/well, in duplicate). Results are shown in Table 5.

Patient HD3 and the healthy EBV carrier C1 are EBV seronegative and had no detectable EBV-specific CTLs, thus acting as negative controls; all other donors studied are EBV seropositive. All EBV-seropositive HD patients had detectable responses to one or more epitopes from LMP2, although these were often very weak in both patient groups (range, 10–180 SFCs/106 PBMCs; mean, 28.1). With the exception of donors C4 and C6, precursor frequencies to LMP2-derived epitopes were also low in healthy EBV carriers (range, 0–640 SFCs/106 PBMCs; mean, 61.5). Responses to either of the LMP1 epitopes were very weak or undetectable in HLA-A2-positive HD patients and healthy EBV carriers, with the exception of three EBV-negative HD cases (HD5, HD9, and HD13), where precursor frequencies of 70–270 SFCs/106 PBMCs were detected. All HLA-A2-positive individuals showed reactivity to the epitope GLC, although precursor frequencies varied considerably between donors. For EBV-negative HD patients, the GLC-specific response ranged from 100 to 1360 SFCs/106 PBMCs (mean, 431.4). Only two HLA-A2-positive HD patients with EBV-positive disease were available for study, both of whom had very low precursor frequencies to GLC (mean, 35.0); however, a similar low precursor frequency was also observed in some healthy EBV carriers (range, 20–700 SFCs/106 PBMCs; mean, 212.5).

Quantitation of EBV-specific CTL Precursor Frequencies in TILs from HD Biopsies by Elispot Assay.

Cryopreserved TILs were available from six HD patients, two with EBV-negative tumors, and four with EBV-positive tumors. TILs were thawed, purified on a Ficoll density gradient, and used directly in Elispot assays. Epitopes used in this study were selected for each individual according to their HLA type and in some cases also on the reactivities detected previously by LCL stimulation (Table 4). Results are summarized in Table 6 and, where possible, are compared with data obtained from the patients’ blood samples.

EBV-specific T-cell precursors were detected by Elispot in TILs from both EBV-negative tumors studied. TILs from donor HD5 (EBV-negative disease) showed the strongest responses to the HPV, EGG, and AVF epitopes, which compared well with the results observed with PBMCs from this patient (Table 6) and also with data obtained by LCL reactivation of PBMCs and TILs (Tables 3 and 4). It should be noted, however, that the frequency of responses in TILs was almost always significantly lower than that observed in PBMCs for this patient; indeed, the relatively strong responses to the LMP1 epitopes YLL and YLQ were almost undetectable in the TIL population. In contrast, Elispot results from donor HD9 (EBV-negative HD) showed an increased response to the LMP1 epitope YLL in TILs compared with PBMCs. HD9 also showed a strong response both in TILs and PBMCs to the EBNA3A epitope RPP, which confirmed results obtained by LCL reactivation (see Table 3 and 4).

TILs from four EBV-positive tumors were examined, and EBV-specific T-cell precursors were detected in all cases. The HLA types of these patients meant that in only one case (HD24) was it possible to screen for a known epitope in LMP2. T cells specific for the HLA-A24-restricted TYG epitope were detected at a frequency of 35 SFCs/106 TILs from this patient.

In the present study, we have conducted a detailed analysis of the EBV-specific CTL response in HD patients in an attempt to explain the persistence of EBV-positive H-RS cells and to explore the possibility of a CTL-based therapy for virus-positive cases of this disease. Using LCL reactivation of PBMCs from EBV-positive and -negative HD patients followed by limiting dilution cloning, we identified the target antigens of the EBV-specific response in peripheral blood samples. Note that LCL-reactivated polyclonal lines from each patient were also analyzed (data not shown), but from these we were only able to detect immunodominant EBV-specific responses (e.g., the EBNA2-specific response in patient HD6; Table 3); minor responses were only detectable after cloning. Immunohistochemical studies on HD biopsies and in vitro analysis of H-RS cell lines suggest that H-RS cells are capable of presenting endogenously synthesized EBV proteins. Therefore, because of an increased antigenic load, it was conceivable that CTL responses to viral proteins expressed in the tumor might be increased in EBV-positive HD patients. However, the results summarized in Table 3 indicated that HD patients display a similar pattern of immunodominance to that observed in healthy EBV carriers (13, 14, 38). Thus, in the majority of cases, the dominant response was to one or other of the EBNA3 family of proteins. Responses to LMP2 were relatively weak, and responses to LMP1 and BARF0 were undetectable. Note that CTLs specific for the GAr-deleted form of EBNA1 (E1ΔGA) were detected in blood samples from some HD patients, but these effectors are unlikely to target an EBV-positive H-RS cell because full-length EBNA1 protein expressed in the tumor will not be processed and presented to HLA class I-restricted T cells (15, 16). Although numbers were small, there was no obvious difference between patients with EBV-negative and -positive HD in the frequency and pattern of CTL responses reactivated using this protocol.

Using the more sensitive Elispot assay, we measured circulating precursor frequencies to predefined A2- and A24-restricted CTL target epitopes in LMP1 and LMP2. Responses to at least one LMP-derived epitope were detectable in the blood of all EBV-seropositive HD patients; however, precursor frequencies were generally low when compared with the immunodominant EBV lytic cycle epitope GLC. These results were again comparable with those seen in healthy EBV carriers (Table 5). Previous reports have claimed that HD patients often possess a generalized defect in cell-mediated immunity, including an impaired response to T-cell mitogens and a decreased capacity of T cells to respond in a mixed lymphocyte response (51). However, using both LCL reactivation and Elispot assays, we observed no obvious suppression of the EBV-specific CTL response in HD patients when compared with healthy EBV carriers.

Having observed a weak LMP-specific CTL response in the blood of EBV-positive HD cases, it was important to determine whether such responses could also be detected at the tumor site. The only other study to examine virus-specific CTL responses at the tumor site of EBV-positive HD patients was reported by Frisan et al.(32) and revealed evidence for local suppression of virus-specific CTLs. Thus, by culturing TILs in IL-2-conditioned medium, they were able to isolate EBV-specific polyclonal CTL lines from three of three EBV-negative HD biopsies but none of six EBV-positive tumors. Furthermore, by studying a single patient with EBV-positive disease, they were able to use the autologous LCL to reactivate an EBV-specific polyclonal CTL line from the blood but not from the tumor biopsy. However, again using the autologous LCL, we were able to reactivate EBV-specific CTL clones not only from the biopsy of EBV-negative tumors but also from three of three EBV-positive tumors (Table 4 and Fig. 4). The difference between our findings and those of Frisan et al.(32) may be explained by the fact that in our study tumor-derived EBV-specific responses were often weak and therefore may have gone undetected in a T-cell line without limiting dilution cloning. Our results therefore demonstrate that EBV-specific effectors are present in at least some EBV-positive HD tumors, and that given the appropriate stimulus, they can be reactivated and expanded in vitro. Nevertheless, it should be noted that none of the tumor-derived clones targeted EBV proteins known to be expressed in H-RS cells. In one case (HD14), the donor was known to possess a relatively weak LMP2-specific response in their blood (Table 3); yet no such response could be identified in their tumor. This may simply reflect the fact that fewer clones were isolated from the tumor than from the blood of this patient, but it demonstrates that LMP2-specific CTLs have not accumulated and/or expanded at the tumor site, despite the presence of their target antigen.

Using the Elispot assay, we were able to demonstrate that EBV-specific T cells are not only present in EBV-positive tumors but they are active directly ex vivo, releasing IFN-γ in response to antigenic stimulation (Table 6). Only one EBV-positive HD tumor (HD24) was available for study that carried an appropriate HLA type to examine responses to predefined LMP-derived epitopes. A clear response to an EBV lytic cycle epitope was detected in this tumor, but only a very weak response was detected to the LMP2-derived epitope TYG. In contrast, a clear LMP1-specific response was detected in TILs from one of two EBV-negative HD cases studied.

The failure of LMP-specific CTLs to accumulate and/or expand within an EBV-positive tumor may be explained if they are functionally impaired in vivo, e.g., by down-regulation of the T-cell receptor ζ chain (52), and/or because of the action of immunosuppressive cytokines, such as IL-10 and transforming growth factor-β (34, 35). Furthermore, H-RS cells secrete the chemokine TARC, which may cause an influx of activated T cells with a Th2 phenotype that prevents the generation of an effective cell-mediated immune response (53). If there is some degree of inactivation of circulating CTLs in vivo, it is clearly possible, as demonstrated here, to overcome this by a period of in vitro culture. It remains to be seen, however, if patient-derived T cells activated in vitro and then returned to the donor can retain their function when they enter the tumor site.

The present study represents a first step in the detailed analysis of EBV-specific responses in the blood and tumor sites of HD patients and points the way to further studies involving larger numbers of patients. Nevertheless, our data suggest that EBV-positive H-RS cells may persist despite an existing EBV-specific CTL response in the blood and the tumor of HD patients because of the low frequency of CTL precursors that target EBV proteins expressed in H-RS cells. This may also explain why the HLA-A2 allele, through which many LMP-specific CTL responses are mediated, is not associated with increased protection from HD (54). Furthermore, our findings have important clinical implications in that they suggest that boosting/eliciting the relevant component of the EBV-specific CTL response, either by immunization or adoptive transfer of T cells expanded in vitro, may prove an effective therapy for EBV-positive HD. A recent clinical study has attempted to treat three patients with EBV-positive HD by adoptive transfer of virus-specific polyclonal CTL lines (33). After infusion of T cells, some patients showed an improvement of stage B symptoms with stabilization of disease and/or a decrease in EBV load. The lack of a complete response after T-cell infusions may partly be explained by the use of CTL lines reactivated in vitro using the autologous LCL. As mentioned above, one might expect only a minor component of the total EBV-specific response in such lines to target proteins expressed in H-RS cells. Therefore, the relevant EBV-specific CTL response may not have been boosted sufficiently. Reactivating PBMCs with the autologous LCL, followed by limiting dilution cloning, we succeeded in isolating functional LMP-specific CTLs from two of four EBV-positive HD patients. However, in many such patients, LMP-specific precursor frequencies may be too low to isolate potentially therapeutic T cells using this method. Strategies that selectively reactivate LMP-specific CTLs are likely to yield T-cell populations with improved therapeutic potential. One approach would be the use of autologous dendritic cells pulsed with LMP-derived peptide epitopes, a strategy that has been successful previously in reactivating LMP-specific responses from healthy EBV carriers (55).

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

This work was supported by a Medical Research Council (MRC) Clinical Training Fellowship Award (to A. L. N. C.) and a MRC Career Development Award (to S. P. L.).

            
3

The abbreviations used are: HD, Hodgkin’s disease; H-RS, Hodgkin-Reed-Sternberg; LCL, lymphoblastoid cell line; PBMC, peripheral blood mononuclear cell; LMP, latent membrane protein; GAr, glycine-alanine repeat; IL, interleukin; TIL, tumor-infiltrating lymphocyte; Elispot, enzyme-linked immunospot; PHA, phytohemagglutinin; SFC, spot-forming cell; HLA, human leukocyte antigen.

Fig. 1.

Screening of T-cell clones derived from the blood of a HD patient for EBV-specific responses. Selected clones derived by LCL stimulation of PBMCs from donor HD15 were screened in a cytotoxicity assay against a panel of autologous LCL targets expressing individual EBV latent proteins from recombinant vaccinia vectors. E:T ratio, 3:1. The data shown are representative of several repeated assays.

Fig. 1.

Screening of T-cell clones derived from the blood of a HD patient for EBV-specific responses. Selected clones derived by LCL stimulation of PBMCs from donor HD15 were screened in a cytotoxicity assay against a panel of autologous LCL targets expressing individual EBV latent proteins from recombinant vaccinia vectors. E:T ratio, 3:1. The data shown are representative of several repeated assays.

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Fig. 2.

CTL clones derived from two HD patients (HD14 and HD4) recognize the LMP2 epitopes CLG and LLW, respectively. Selected CTL clones derived by LCL stimulation of PBMCs from donors HD14 (A) and HD4 (B) were tested against autologous LCL targets preincubated with 2 μg/ml of the peptide epitopes CLG or LLW, or with an equivalent dilution of DMSO solvent (no peptide, control). E:T ratio, 5:1. The data shown are representative of several repeated assays.

Fig. 2.

CTL clones derived from two HD patients (HD14 and HD4) recognize the LMP2 epitopes CLG and LLW, respectively. Selected CTL clones derived by LCL stimulation of PBMCs from donors HD14 (A) and HD4 (B) were tested against autologous LCL targets preincubated with 2 μg/ml of the peptide epitopes CLG or LLW, or with an equivalent dilution of DMSO solvent (no peptide, control). E:T ratio, 5:1. The data shown are representative of several repeated assays.

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Fig. 3.

HLA-B38-restricted CTL recognition of an EBNA2 epitope located at residues 14–23. A, a representative CTL clone derived by LCL stimulation of PBMCs from donor HD6 (HLA A1, 2 B8, 38.01) was tested in a cytotoxicity assay against a panel of HLA-matched and -mismatched LCL targets expressing EBNA2 from a recombinant vaccinia vector (vE2). As a control, targets were infected with the vaccinia vector alone (vTK−). E:T ratio, 5:1. B, the same clone was subsequently tested against autologous LCL targets preincubated with overlapping synthetic peptides from EBNA2 region 10–27 or with an equivalent dilution of DMSO solvent (No peptide). E:T ratio, 3:1. All results are expressed as a percentage of specific lysis and are representative of several repeated assays.

Fig. 3.

HLA-B38-restricted CTL recognition of an EBNA2 epitope located at residues 14–23. A, a representative CTL clone derived by LCL stimulation of PBMCs from donor HD6 (HLA A1, 2 B8, 38.01) was tested in a cytotoxicity assay against a panel of HLA-matched and -mismatched LCL targets expressing EBNA2 from a recombinant vaccinia vector (vE2). As a control, targets were infected with the vaccinia vector alone (vTK−). E:T ratio, 5:1. B, the same clone was subsequently tested against autologous LCL targets preincubated with overlapping synthetic peptides from EBNA2 region 10–27 or with an equivalent dilution of DMSO solvent (No peptide). E:T ratio, 3:1. All results are expressed as a percentage of specific lysis and are representative of several repeated assays.

Close modal
Fig. 4.

Screening LCL-reactivated, TIL-derived CTL clones for EBV-specific responses. A, two representative clones derived by LCL stimulation of TILs from donor HD18 (EBV-positive tumor) were screened in a cytotoxicity assay against a panel of autologous LCL targets expressing individual EBV latent proteins from vaccinia vectors. E:T ratio, 3:1. B, the same clones were subsequently tested against autologous PHA blast targets preincubated with 2 μg/ml of synthetic peptide epitopes from EBNA3A: FLR (residues 325–333), QAK (158–166), and VFS (491–499), or with an equivalent dilution of DMSO solvent (no peptide, control). E:T ratio, 5:1. The data shown are representative of several repeated assays.

Fig. 4.

Screening LCL-reactivated, TIL-derived CTL clones for EBV-specific responses. A, two representative clones derived by LCL stimulation of TILs from donor HD18 (EBV-positive tumor) were screened in a cytotoxicity assay against a panel of autologous LCL targets expressing individual EBV latent proteins from vaccinia vectors. E:T ratio, 3:1. B, the same clones were subsequently tested against autologous PHA blast targets preincubated with 2 μg/ml of synthetic peptide epitopes from EBNA3A: FLR (residues 325–333), QAK (158–166), and VFS (491–499), or with an equivalent dilution of DMSO solvent (no peptide, control). E:T ratio, 5:1. The data shown are representative of several repeated assays.

Close modal
Table 1

Patient details

PatientSex/AgeClinical stageHD subtypeEBV statusaEBV serologyHLA type
HD1 M/36 2B NS − − A3 B7, 38 
HD2 M/34 2A NS − − A24 B7, 35 
HD3 M/26 2A NS − − A2, 24 B7 
HD4 M/43 3B LP − A1, 2 B8, 44 
HD5 F/38 1A NS − A2, 11 B35, 44 
HD6 F/27 2A NS − A1, 2 B8, 38 
HD7 F/76 1A NS − A1 B8 
HD8 F/28 2A MC − A11, 29 B35, 44 
HD9 F/33 1A NS − A2, 3 B7 
HD10 M/32 2A NS − A2, 32 B44 
HD11 F/17 4A MC/NS − A2, 30 B13, 40 
HD12 M/18 2B NS − A2, 68 B13, 44 
HD13 M/25 2A NS − A2, 24 B35, 40 
HD14 M/25 1A LD A2, 29 B15, 44 
HD15 M/46 1A NS A1, 68 B8, 51 
HD16 F/48 1A MC A1 B8 
HD17 M/52 1A NS A1, 3 B37, 44 
HD18 M/57 4B MC A1, 29 B8 
HD19 M/29 2B MC A3, 24 B44, 51 
HD20 M/28 1A NS A1, 3 B37, 51 
HD21 M/37 3B MC A2, 24 B8, 60 
HD22 M/66 4B MC A3, 30 B14, 27 
HD23 F/56 4B NS A1, 3 B8 
HD24 M/20 2A MC A1, 24 B8, 51 
PatientSex/AgeClinical stageHD subtypeEBV statusaEBV serologyHLA type
HD1 M/36 2B NS − − A3 B7, 38 
HD2 M/34 2A NS − − A24 B7, 35 
HD3 M/26 2A NS − − A2, 24 B7 
HD4 M/43 3B LP − A1, 2 B8, 44 
HD5 F/38 1A NS − A2, 11 B35, 44 
HD6 F/27 2A NS − A1, 2 B8, 38 
HD7 F/76 1A NS − A1 B8 
HD8 F/28 2A MC − A11, 29 B35, 44 
HD9 F/33 1A NS − A2, 3 B7 
HD10 M/32 2A NS − A2, 32 B44 
HD11 F/17 4A MC/NS − A2, 30 B13, 40 
HD12 M/18 2B NS − A2, 68 B13, 44 
HD13 M/25 2A NS − A2, 24 B35, 40 
HD14 M/25 1A LD A2, 29 B15, 44 
HD15 M/46 1A NS A1, 68 B8, 51 
HD16 F/48 1A MC A1 B8 
HD17 M/52 1A NS A1, 3 B37, 44 
HD18 M/57 4B MC A1, 29 B8 
HD19 M/29 2B MC A3, 24 B44, 51 
HD20 M/28 1A NS A1, 3 B37, 51 
HD21 M/37 3B MC A2, 24 B8, 60 
HD22 M/66 4B MC A3, 30 B14, 27 
HD23 F/56 4B NS A1, 3 B8 
HD24 M/20 2A MC A1, 24 B8, 51 
a

EBV status of tumor determined by in situ hybridization for EBER1 expression.

Table 2

Epitope peptides used to screen EBV-specific clones

Peptide nameProteinAmino acid residuesHLA restrictionRef.
HPV EBNA1 407–417 B35.01  17  
VLK EBNA1 574–582 A2.03  17  
DTP EBNA2 42–51 B51  40  
YHL EBNA2 14–23 B38 a 
FLR EBNA3A 325–333 B8  41  
QAK EBNA3A 158–166 B8  42  
RPP EBNA3A 379–387 B7  39  
RLR EBNA3A 603–611 A3  39  
VFS EBNA3A 491–499 A29  6  
AVF EBNA3B 399–408 A11  43  
IVT EBNA3B 416–424 A11  43  
EGG EBNA3C 163–171 B44  44  
EEN EBNA3C 281–290 B44  45  
RRIY EBNA3C 258–266 B27  46  
YLL LMP1 125–133 A2  27  
YLQ LMP1 159–167 A2  27  
CLG LMP2 426–434 A2  25  
LLW LMP2 329–337 A2  26  
FLY LMP2 356–364 A2 b 
TYG LMP2 419–427 A24  26  
GLC BMLF1 280–288 A2  47  
RAK BZLF1 190–197 B8  48  
Peptide nameProteinAmino acid residuesHLA restrictionRef.
HPV EBNA1 407–417 B35.01  17  
VLK EBNA1 574–582 A2.03  17  
DTP EBNA2 42–51 B51  40  
YHL EBNA2 14–23 B38 a 
FLR EBNA3A 325–333 B8  41  
QAK EBNA3A 158–166 B8  42  
RPP EBNA3A 379–387 B7  39  
RLR EBNA3A 603–611 A3  39  
VFS EBNA3A 491–499 A29  6  
AVF EBNA3B 399–408 A11  43  
IVT EBNA3B 416–424 A11  43  
EGG EBNA3C 163–171 B44  44  
EEN EBNA3C 281–290 B44  45  
RRIY EBNA3C 258–266 B27  46  
YLL LMP1 125–133 A2  27  
YLQ LMP1 159–167 A2  27  
CLG LMP2 426–434 A2  25  
LLW LMP2 329–337 A2  26  
FLY LMP2 356–364 A2 b 
TYG LMP2 419–427 A24  26  
GLC BMLF1 280–288 A2  47  
RAK BZLF1 190–197 B8  48  
a

Defined in present study.

b

A. B. Rickinson et al., unpublished data.

Table 3

EBV-specific CTL responses reactivated from the blood of HD patients

DonorHLA typeNo. of clones testedTarget antigena
E1ΔGAEBNA2EBNA3AEBNA3BEBNA3CLPLMP1LMP2BARF0
EBV-negative HD             
 HD1b A3 B7, 38 240          
 HD4 A1, 2 B8, 44 250   30 (QAK)   9 (EGG/EEN)c   3 (LLW)  
 HD5 A2, 11 B35, 44 68 10 (HPV)     2 (EGG)     
 HD6 A1, 2 B8, 38 223  217 (YHL)        
 HD7 A1 B8 221         
 HD8 A11, 29 B35, 44 200      2     
 HD9 A2, 3 B7 20    4 (RPP)       
EBV-positive HD             
 HD14 A2, 29 B15, 44 235   16 10   5 (CLG)  
 HD15 A1, 68 B8, 51 200  10   3    
 HD16 A1 B8 200   43       
 HD17 A1, 3 B37, 44 200      2     
DonorHLA typeNo. of clones testedTarget antigena
E1ΔGAEBNA2EBNA3AEBNA3BEBNA3CLPLMP1LMP2BARF0
EBV-negative HD             
 HD1b A3 B7, 38 240          
 HD4 A1, 2 B8, 44 250   30 (QAK)   9 (EGG/EEN)c   3 (LLW)  
 HD5 A2, 11 B35, 44 68 10 (HPV)     2 (EGG)     
 HD6 A1, 2 B8, 38 223  217 (YHL)        
 HD7 A1 B8 221         
 HD8 A11, 29 B35, 44 200      2     
 HD9 A2, 3 B7 20    4 (RPP)       
EBV-positive HD             
 HD14 A2, 29 B15, 44 235   16 10   5 (CLG)  
 HD15 A1, 68 B8, 51 200  10   3    
 HD16 A1 B8 200   43       
 HD17 A1, 3 B37, 44 200      2     
a

Number of clones that were specific for a defined EBV latent protein. The target epitope, where defined, is shown as a three-letter code (see Table 2). Unless otherwise stated, this epitope was recognized by all of the clones indicated.

b

EBV-seronegative.

c

Four of nine clones were specific for EGG; one of nine was specific for EEN.

Table 4

EBV-specific CTL responses reactivated from the tumor biopsy of untreated HD patients

DonorHLA typeNo. of clones testedTarget antigena
E1ΔGAEBNA2EBNA3AEBNA3BEBNA3CLPLMP1LMP2BARF0
EBV-negative HD             
 HD2b A24 B7, 35 147          
 HD5 A2, 11 B35, 44 69 7 (HPV)   6 (AVF)c 14 (EGG)     
 HD9 A2, 3 B7 56    1 (RPP)       
EBV-positive HD             
 HD14 A1, 68 B8, 51 67      2     
 HD17 A1, 3 B37, 44 226      1     
 HD18 A1, 29 B8 273   30 (QAK/FLR)d       
DonorHLA typeNo. of clones testedTarget antigena
E1ΔGAEBNA2EBNA3AEBNA3BEBNA3CLPLMP1LMP2BARF0
EBV-negative HD             
 HD2b A24 B7, 35 147          
 HD5 A2, 11 B35, 44 69 7 (HPV)   6 (AVF)c 14 (EGG)     
 HD9 A2, 3 B7 56    1 (RPP)       
EBV-positive HD             
 HD14 A1, 68 B8, 51 67      2     
 HD17 A1, 3 B37, 44 226      1     
 HD18 A1, 29 B8 273   30 (QAK/FLR)d       
a

Number of clones that were specific for a defined EBV latent protein (target epitope, where defined, indicated with three-letter code).

b

EBV-seronegative.

c

Five of six clones were specific for AVF.

d

Eighteen of 30 clones were specific for QAK; 1 of 30 clones was specific for FLR.

Table 5

Circulating CTL precursor frequency to LMP1/2 epitopes in HD patients and healthy control donors

DonorHLA typeEBV serologyLMP2aLMP1aBMLF1a
CLGLLWFLYTYGYLLYLQGLC
EBV-negative HD           
 HD3 A2, 24 B7 − 0b 
 HD4 A1, 2 B8, 44 50 40 40 10 530 
 HD5 A2, 11 B35, 44 100 270 100 100 
 HD9 A2, 3 B7 20 70 20 220 
 HD10 A2, 32 B44 40 20 20 460 
 HD11 A2, 30 B13, 40 50 20 120 1360 
 HD12 A2, 68 B13, 44 10 30 10 100 
 HD13 A2, 24 B35, 40 180 260 250 
EBV-positive HD           
 HD14 A2, 29 B15, 44 40 40 10 10 40 
 HD19 A3, 24 B44, 51 50 
 HD21 A2, 24 B8, 60 10 30 10 30 
Healthy control donors           
 c1 A2 B40, 44 − 
 c2 A3, 24 B7, 37 50 
 c3 A1, 2 B17, 44 20 
 c4 A2 B14, 15 510 70 80 330 
 c5 A2, 32 B44 100 50 130 10 30 280 
 c6 A2 B27, 44 640 40 20 700 
 c7 A1, 2 B39, 40 50 15 150 
 c8 A2, 11 B16 10 40 15 10 20 270 
 c9 A2 B15, 40 15 30 
 c10 A2, 11 B8, 44 50 50 95 
 c11 A2 B47, 60 95 20 25 170 
 c12 A2, 24 B27, 35 10 15 80 
DonorHLA typeEBV serologyLMP2aLMP1aBMLF1a
CLGLLWFLYTYGYLLYLQGLC
EBV-negative HD           
 HD3 A2, 24 B7 − 0b 
 HD4 A1, 2 B8, 44 50 40 40 10 530 
 HD5 A2, 11 B35, 44 100 270 100 100 
 HD9 A2, 3 B7 20 70 20 220 
 HD10 A2, 32 B44 40 20 20 460 
 HD11 A2, 30 B13, 40 50 20 120 1360 
 HD12 A2, 68 B13, 44 10 30 10 100 
 HD13 A2, 24 B35, 40 180 260 250 
EBV-positive HD           
 HD14 A2, 29 B15, 44 40 40 10 10 40 
 HD19 A3, 24 B44, 51 50 
 HD21 A2, 24 B8, 60 10 30 10 30 
Healthy control donors           
 c1 A2 B40, 44 − 
 c2 A3, 24 B7, 37 50 
 c3 A1, 2 B17, 44 20 
 c4 A2 B14, 15 510 70 80 330 
 c5 A2, 32 B44 100 50 130 10 30 280 
 c6 A2 B27, 44 640 40 20 700 
 c7 A1, 2 B39, 40 50 15 150 
 c8 A2, 11 B16 10 40 15 10 20 270 
 c9 A2 B15, 40 15 30 
 c10 A2, 11 B8, 44 50 50 95 
 c11 A2 B47, 60 95 20 25 170 
 c12 A2, 24 B27, 35 10 15 80 
a

-, not tested.

b

Number of SFCs/106 PBMCs (having first subtracted the number of spots obtained in control wells without antigen).

Table 6

EBV-specific T-cell precursor frequencies in TIL populations compared with PBMCs

DonorSampleLMP2aLMP1aEBNA1aEBNA2aEBNA3AaEBNA3BaEBNA3CaBMLF1aBZLF1a
TYGLLWFLYYLLYLQHPVDTPRPPRLRFLRQAKAVFEGGRRIYGLCRAK
EBV-negative HD                  
 HD5 Blood – 100b 270 100 910 – – – – – 50 680 – 100 – 
 HD5 Biopsy – – 20 160 – – – – – 40 240 – 10 – 
 HD9 Blood – 20 70 20 – – 570 – – – – – – 220 – 
 HD9 Biopsy – – 15 200 20 – – 610 – – – – – – 50 – 
EBV-positive HD                  
 HD20 Blood – – – – – – 180 – 10 – – – – – – – 
 HD20 Biopsy – – – – – – 160 – 75 – – – – – – – 
 HD22 Blood – – – – – – – – – – – – 50 – – 
 HD22 Biopsy – – – – – – – – – – – – 45 – – 
 HD23c Biopsy – – – – – – – – 30 50 – – – – 195 
 HD24c Biopsy 35 – – – – – 60 – – 45 30 – – – – 120 
DonorSampleLMP2aLMP1aEBNA1aEBNA2aEBNA3AaEBNA3BaEBNA3CaBMLF1aBZLF1a
TYGLLWFLYYLLYLQHPVDTPRPPRLRFLRQAKAVFEGGRRIYGLCRAK
EBV-negative HD                  
 HD5 Blood – 100b 270 100 910 – – – – – 50 680 – 100 – 
 HD5 Biopsy – – 20 160 – – – – – 40 240 – 10 – 
 HD9 Blood – 20 70 20 – – 570 – – – – – – 220 – 
 HD9 Biopsy – – 15 200 20 – – 610 – – – – – – 50 – 
EBV-positive HD                  
 HD20 Blood – – – – – – 180 – 10 – – – – – – – 
 HD20 Biopsy – – – – – – 160 – 75 – – – – – – – 
 HD22 Blood – – – – – – – – – – – – 50 – – 
 HD22 Biopsy – – – – – – – – – – – – 45 – – 
 HD23c Biopsy – – – – – – – – 30 50 – – – – 195 
 HD24c Biopsy 35 – – – – – 60 – – 45 30 – – – – 120 
a

-, not tested.

b

Number of SFCs/106 PBMCs or TILs (having first subtracted the number of spots obtained in control wells without antigen).

c

Blood samples not available.

We thank Faz Khan and June Freeland for help with collection of samples and clinical data and Keely Jenner for help with in situ hybridization.

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