Ann L. N. Chapman ¹, Alan B. Rickinson ¹, Wendy A. Thomas ¹, Ruth F. Jarrett ², John Crocker ³, and Steven P. Lee ^{1, @}

¹ CRC Institute for Cancer Studies, University of Birmingham, Vincent Drive, Birmingham, B15 2TT, U.K.,
² Leukemia Research Fund Virus Centre, Department of Veterinary Pathology, Veterinary School, Bearsden Road, Glasgow G61 1QH U. K., and
³ Department of Cellular Pathology, Birmingham Heartlands Hospital, Birmingham B9 5SS, U.K.

^@ To whom requests for reprints should be addressed at CRC Institute for Cancer Studies, University of Birmingham, Vincent Drive, Edgbaston, Birmingham, B15 2TT, U.K.,
Phone:  0121-414-2803;  Fax: 0121-414-4486;  E-mail: s.p.lee@bham.ac.uk

Approximately 40 % of Hodgkin's Disease (HD) cases carry Epstein-Barr Virus (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 Cytotoxic T Lymphocytes (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. 

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; CTL, cytotoxic T lymphocytes.

Introduction:

It is now well established that 40% of HD 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-ß (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.
...
Discussion:

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 (E1GA) 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- 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  chain (52) , and/or because of the action of immunosuppressive cytokines, such as IL-10 and transforming growth factor-ß (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) .

Footnotes:

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.).

2. To whom requests for reprints should be addressed, at CRC Institute for Cancer Studies, University of Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TT, United Kingdom. Phone: 0121-414-2803; Fax: 0121-414-4486; E-mail: s.p.lee@bham.ac.uk

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; CTL, cytotoxic T lymphocytes.

1. Glaser S. L., Lin R. J., Stewart S. L., Ambinder R. F., Jarrett R. F., Brousset P., Pallesen G., Gulley M. L., Khan G., O’Grady J., Hummel M., Preciado M. V., Knecht H., Chan J. C., Claviez A. Epstein-Barr virus-associated Hodgkin’s disease. Epidemiologic characteristics in international data. Int. J. Cancer, 70: 375-382, 1997.

2. Ambinder R. F., Browning P. J., Lorenzana I., Leventhal B. G., Cosenza H., Mann R. B., MacMahon E. M., Medina R., Cardona V., Grufferman S. Epstein-Barr virus and childhood Hodgkin’s disease in Honduras and the United States. Blood, 81: 462-467, 1993.

3. Gulley M. L., Eagan P. A., Quintanilla-Martinez L., Picado A. L., Smir B. N., Childs C., Dunn C. D., Craig F. E., Williams J. W. J., Banks P. M. Epstein-Barr virus DNA is abundant and monoclonal in the Reed-Sternberg cells of Hodgkin’s disease: association with mixed cellularity subtype and Hispanic American ethnicity. Blood, 83: 1595-1602, 1994.

4. Rickinson A. B., Kieff E. Epstein-Barr virus Fields B. N. Knipe D. M. Howley P. M. eds. . Fields Virology, 2397-2446, Lippincott-Raven Publishers Philadelphia 1996.

5. Rowlands D. C., Ito M., Mangham D. C., Reynolds G., Herbst H., Hallissey M. T., Fielding J. W., Newbold K. M., Jones E. L., Young L. S. Epstein-Barr virus and carcinomas: rare association of the virus with gastric adenocarcinomas. Br. J. Cancer, 68: 1014-1019, 1993.

6. Rickinson A. B., Moss D. J. Human cytotoxic T lymphocyte responses to Epstein-Barr virus infection. Annu. Rev. Immunol., 15: 405-431, 1997.

7. Rooney C. M., Smith C. A., Ng C. C., Loftin S., Li C. F., Krance R. A., Brenner M. K., Heslop H. E. Use of gene modified virus-specific T lymphocytes to control Epstein-Barr virus-related lymphoproliferation. Lancet, 345: 9-13, 1995.

8. Rooney C. M., Smith C. A., Ng C. C., Loftin S. K., Sixbey J. W., Gan Y. J., Srivastava D. K., Bowman L. C., Krance R. A., Brenner M. K., Heslop H. E. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood, 92: 1549-1555, 1998.

9. Khanna R., Bell S., Sherritt M., Galbraith A., Burrows S. R., Rafter L., Clarke B., Slaughter R., Falk M. C., Douglass J., Williams T., Elliott S. L., Moss D. J. Activation and adoptive transfer of Epstein-Barr
virus-specific cytotoxic T cells in solid organ transplant patients with posttransplant lymphoproliferative disease. Proc. Natl. Acad. Sci. USA, 96: 10391-10396, 1999.

10. Henry-Amar M., Joly F. Late complications after Hodgkin’s disease. Ann Oncol, 7 (Suppl. 4): 115-126,
1996.

11. Hancock S. L., Hoppe R. T. Long-term complications of treatment and causes of mortality after Hodgkin’s disease. Semin. Radiat. Oncol., 6: 225-242, 1996.

12. Brooks L. A., Lear A. L., Young L. S., Rickinson A. B. Transcripts from the Epstein-Barr virus BamHI A fragment are detectable in all three forms of virus latency. J. Virol., 67: 3182-3190, 1993.

13. Murray R. J., Kurilla M. G., Brooks J. M., Thomas W. A., Rowe M., Kieff E., Rickinson A. B. Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV)-implications for the immune control of EBV-positive malignancies. J. Exp. Med., 176: 157-168, 1992.

14. Khanna R., Burrows S. R., Kurilla M. G., Jacob C. A., Misko I. S., Sculley T. B., Kieff E., Moss D. J.
Localization of Epstein-Barr virus cytotoxic T cell epitopes using recombinant vaccinia-implications for vaccine development. J. Exp. Med., 176: 169-176, 1992.

15. Levitskaya J., Coram M., Levitsky V., Imreh S., Steigerwald-Mullen P. M., Klein G., Kurilla M. G., Masucci M. G. Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen 1. Nature (Lond.), 375: 685-688, 1995.

16. Levitskaya J., Shapiro A., Leonchiks A., Ciechanover A., Masucci M. G. Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein-Barr virus
nuclear antigen 1. Proc. Natl. Acad. Sci. USA, 94: 12616-12621, 1997.

17. Blake N., Lee S., Redchenko I., Thomas W., Steven N., Leese A., Steigerwald-Mullen P., Kurilla M. G., Frappier L., Rickinson A. Human CD8+ T cell responses to EBV EBNA1: HLA class I presentation of the (Gly-Ala)-containing protein requires exogenous processing. Immunity, 7: 791-802, 1997.

18. Kienzle N., Sculley T. B., Poulsen L., Buck M., Cross S., Raab Traub N., Khanna R. Identification of a
cytotoxic T-lymphocyte response to the novel BARF0 protein of Epstein-Barr virus: a critical role for antigen expression. J. Virol., 72: 6614-6620, 1998.

19. Kienzle N., Sculley T. B., Greco S., Khanna R. Cutting edge: silencing virus-specific cytotoxic T cell-mediated immune recognition by differential splicing: a novel implication of RNA processing for antigen presentation. J. Immunol., 162: 6963-6966, 1999.

20. Pallesen G., Hamilton Dutoit S. J., Rowe M., Young L. S. Expression of Epstein-Barr virus latent gene
products in tumor cells of Hodgkin’s disease. Lancet, 337: 320-322, 1991.

21. Herbst H., Dallenbach F., Hummel M., Niedobitek G., Pileri S., Muller-Lantzsch N., Stein H. Epstein-Barr virus latent membrane protein expression in Hodgkin and Reed-Sternberg cells. Proc. Natl. Acad. Sci. USA, 88: 4766-4770, 1991.

22. Grasser F. A., Murray P. G., Kremmer E., Klein K., Remberger K., Feiden W., Reynolds G., Niedobitek G., Young L. S., Muellerlantzsch N. Monoclonal antibodies directed against the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1)-immunohistologic detection of EBNA1 in the malignant cells of Hodgkin’s disease.Blood, 84: 3792-3798, 1994.

23. Niedobitek G., Kremmer E., Herbst H., Whitehead L., Dawson C. W., Niedobitek E., von Ostau C., Rooney N., Grasser F. A., Young L. S. Immunohistochemical detection of the Epstein-Barr virus-encoded latent membrane protein 2A in Hodgkin’s disease and infectious mononucleosis. Blood, 90: 1664-1672, 1997.

24. Deacon E. M., Pallesen G., Niedobitek G., Crocker J., Brooks L., Rickinson A. B., Young L. S. Epstein-Barr virus and Hodgkin’s disease-transcriptional analysis of virus latency in the malignant cells. J. Exp. Med., 177: 339-349, 1993.

25. Lee S. P., Thomas W. A., Murray R. J., Khanim F., Kaur S., Young L. S., Rowe M., Kurilla M., Rickinson A. B. HLA A2.1-restricted cytotoxic T cells recognizing a range of Epstein-Barr virus isolates through a defined epitope in latent membrane protein LMP2. J. Virol., 67: 7428-7435, 1993.

26. Lee S. P., Tierney R. J., Thomas W. A., Brooks J. M., Rickinson A. B. Conserved CTL epitopes within EBV latent membrane protein 2-a potential target for CTL-based tumor therapy. J. Immunol., 158: 3325-3334, 1997.

27. Khanna R., Burrows S. R., Nicholls J., Poulsen L. M. Identification of cytotoxic T cell epitopes within
Epstein-Barr virus (EBV) oncogene latent membrane protein 1 (LMP1): evidence for HLA A2
supertype-restricted immune recognition of EBV-infected cells by LMP1-specific cytotoxic T lymphocytes.
Eur. J. Immunol., 28: 451-458, 1998.

28. Lee S. P., Constandinou C. M., Thomas W. A., Croom-Carter D., Blake N. W., Murray P. G., Crocker J., Rickinson A. B. Antigen presenting phenotype of Hodgkin Reed-Sternberg cells: analysis of the HLA class I processing pathway and the effects of interleukin 10 on Epstein-Barr virus-specific cytotoxic T cell recognition. Blood, 92: 1020-1030, 1998.

29. Murray P. G., Constandinou C. M., Crocker J., Young L. S., Ambinder R. F. Analysis of major
histocompatibility complex class I, TAP expression, and LMP2 epitope sequence in Epstein-Barr virus-positive Hodgkin’s disease. Blood, 92: 2477-2483, 1998.

30. Oudejans J. J., Jiwa N. M., Kummer J. A., Horstman A., Vos W., Baak J. A., Kluin P. M., Vandervalk P., Walboomers J. M., Meijer C. M. Analysis of major histocompatibility complex class I expression on Reed-Sternberg cells in relation to the cytotoxic T cell response in Epstein-Barr virus-positive and Epstein-Barr virus-negative Hodgkin’s disease. Blood, 87: 3844-3851, 1996.

31. Sing A. P., Ambinder R. F., Hong D. J., Jensen M., Batten W., Petersdorf E., Greenberg P. D. Isolation of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes that lyse Reed-Sternberg cells: implications for immune-mediated therapy of EBV(+) Hodgkin’s disease. Blood, 89: 1978-1986, 1997.

32. Frisan T., Sjoberg J., Dolcetti R., Boiocchi M., Dere V., Carbone A., Brautbar C., Battat S., Biberfeld P., Eckman M., Ost O., Christensson B., Sundstrom C., Bjorkholm M., Pisa P., Masucci M. G. Local
suppression of Epstein-Barr virus (EBV)-specific cytotoxicity in biopsies of EBV-positive Hodgkin’s disease. Blood, 86: 1493-1501, 1995.

33. Roskrow M. A., Suzuki N., Gan Y. J., Sixbey J. W., Ng C. C., Kimbrough S., Hudson M., Brenner M. K., Heslop H. E., Rooney C. M. Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes for the treatment of patients with EBV-positive relapsed Hodgkin’s disease. Blood, 91: 2925-2934, 1998.

34. Herbst H., Foss H. D., Samol J., Araujo I., Klotzbach H., Krause H., Agathanggelou A., Niedobitek G., Stein H. Frequent expression of interleukin 10 by Epstein-Barr virus-harboring tumor cells of Hodgkin’s disease. Blood, 87: 2918-2929, 1996.

35. Hsu S. M., Lin J., Xie S. S., Hsu P. L., Rich S. Abundant expression of transforming growth factor-ß1 and -ß2 by Hodgkin’s Reed-Sternberg cells and by reactive T lymphocytes in Hodgkin’s disease. Hum. Pathol., 24: 249-255, 1993.

36. Bunce M., O’Neill C. M., Barnardo M. C., Krausa P., Browning M. J., Morris P. J., Welsh K. I.
Phototyping. Comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR
with 144 primer mixes utilizing sequence-specific primers (PCR-SSP). Tissue Antigens, 46: 355-367, 1995.

37. Miller G., Lipman M. Release of infectious Epstein-Barr virus by transformed marmoset leukocytes. Proc. Natl. Acad. Sci. USA, 70: 190-194, 1973.

38. Lee S. P., Chan A. T., Cheung S. T., Thomas W. A., Croomcarter D., Dawson C. W., Tsai C. H., Leung S. F., Johnson P. J., Huang D. P. CTL control of EBV in nasopharyngeal carcinoma (NPC): EBV-specific CTL responses in the blood and tumors of NPC patients and the antigen-processing function of the tumor cells. J. Immunol., 165: 573-582, 2000.

39. Hill A. B., Lee S. P., Haurum J. S., Murray N., Yao Q. Y., Rowe M., Signoret N., Rickinson A. B.,
McMichael A. J. Class I major histocompatibility complex-restricted cytotoxic T lymphocytes specific for
Epstein-Barr virus (EBV) nuclear antigens fail to lyse the EBV-transformed B lymphoblastoid lines against
which they were raised. J. Exp. Med., 181: 2221-2228, 1995.

40. Schmidt C., Burrows S. R., Sculley T. B., Moss D. J., Misko I. S. Non-responsiveness to an immunodominant Epstein-Barr virus-encoded cytotoxic T-lymphocyte epitope in nuclear antigen 3A: implications for vaccine strategies. Proc. Natl. Acad. Sci. USA, 88: 9478-9482, 1991.

41. Burrows S. R., Sculley T. B., Misko I. S., Schmidt C., Moss D. J. An Epstein-Barr virus-specific cytotoxic T cell epitope in EBNA3. J. Exp. Med., 171: 345-350, 1990.

42. Burrows S. R., Gardner J., Khanna R., Steward T., Moss D. J., Rodda S., Suhrbier A. Five new cytotoxic T-cell epitopes identified within Epstein-Barr virus nuclear antigen 3. J. Gen. Virol., 75: 2489-2493, 1994.

43. Gavioli R., Kurilla M. G., de Campos-Lima P. O., Wallace L. E., Dolcetti R., Murray R. J., Rickinson A. B., Masucci M. G. Multiple HLA-A11-restricted cytotoxic T-lymphocyte epitopes of different immunogenicities in the Epstein-Barr virus-encoded nuclear antigen 4. J. Virol., 67: 1572-1578, 1993.

44. Morgan S. M., Wilkinson G. W. G., Floettmann E., Blake N., Rickinson A. B. A recombinant adenovirus expressing an Epstein-Barr virus (EBV) target antigen can selectively reactivate rare components of EBV cytotoxic T-lymphocyte memory in vitro. J. Virol., 70: 2394-2402, 1996.

45. Burrows S. R., Misko I. S., Sculley T. B., Schmidt D., Moss D. J. An Epstein-Barr virus-specific cytotoxic T cell epitope present on A- and B-type transformants. J. Virol., 64: 3974-3976, 1990.

46. Brooks J. M., Murray R. J., Thomas W. A., Kurilla M. G., Rickinson A. B. Different HLA-B27 subtypes present the same immunodominant Epstein-Barr virus peptide. J. Exp. Med., 178: 879-887, 1993.

47. Steven N. M., Annels N. E., Kumar A., Leese A. M., Kurilla M. G., Rickinson A. B. Immediate early and early lytic cycle proteins are frequent targets of the Epstein-Barr virus-induced cytotoxic T cell response. J. Exp. Med., 185: 1605-1617, 1997.

48. Bogedain C., Wolf H., Modrow S., Stuber G., Jilg W. Specific cytotoxic T-lymphocytes recognize the
immediate-early transactivator ZTA of Epstein-Barr virus. J. Virol., 69: 4872-4879, 1995.

49. Rammensee H. G., Friede T., Stevanoviic S. MHC ligands and peptide motifs: first listing. Immunogenetics, 41: 178-228, 1995.

50. Tan L. C., Gudgeon N., Annels N. E., Hansasuta P., O’Callaghan C. A., Rowland Jones S., McMichael A. J., Rickinson A. B., Callan M. A re-evaluation of the frequency of CD8(+) T cells specific for EBV in healthy virus carriers. J. Immunol., 162: 1827-1835, 1999.

51. Slivnick D. J., Ellis T. M., Nawrocki J. F., Fisher R. I. The impact of Hodgkin’s disease on the immune system. Semin. Oncol., 17: 673-682, 1990.

52. Renner C., Ohnesorge S., Held G., Bauer S., Jung W., Pfitzenmeier J. P., Pfreundschuh M. T cells from patients with Hodgkin’s disease have a defective T-cell receptor  chain expression that is reversible by T-cell stimulation with CD3 and CD28. Blood, 88: 236-241, 1996.

53. van den Berg A., Visser L., Poppema S. High expression of the CC Chemokine TARC in Reed-Sternberg cells: a possible explanation for the characteristic T-cell infiltrate in Hodgkin’s lymphoma. Am. J. Pathol., 154: 1685-1691, 1999.

54. Bryden H., MacKenzie J., Andrew L., Alexander F. E., Angus B., Krajewski A. S., Armstrong A. A., Gray D., Cartwright R. A., Kane E., Wright D. H., Taylor P., Jarrett R. F. Determination of HLA-A*02 antigen status in Hodgkin’s disease and analysis of an HLA-A*02-restricted epitope of the Epstein-Barr virus LMP-2 protein. Int. J. Cancer, 72: 614-618, 1997.

55. Redchenko I., Rickinson A. B. Accessing Epstein-Barr virus-Specific T-cell memory with peptide-loaded dendritic cells. J. Virol., 73: 334-342, 1999.

1. Frenster JH, "Phytohemagglutinin-Activated Autochthonous Lymphocytes for Systemic Immunotherapy of Human Neoplasms", Ann. N. Y. Acad. Sci. 277, 45-51 (1976).

2. Frenster  JH, Papalian  MM, Masek MA,  and Frenster JA., "Electron Microscopic Analysis of Lymph Node Cellular Activity in Hodgkin's Disease", J. Natl. Cancer Inst., Vol. 63, pp. 331-335, Aug. 1979.