SHORT COMMUNICATION. RAJIV KHANNA,*,2,3 SCOTT R. BURROWS,*,2 PATTY M. STEIGERWALD-MULLEN, SCOTT A. THOMSON,* MICHAEL G. KURILLA, and DENIS J.

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1 VIROLOGY 214, (1995) SHORT COMMUNICATION Isolation of Cytotoxic T Lymphocytes from Healthy Seropositive Individuals Specific for Peptide Epitopes from Epstein Barr Virus Nuclear Antigen 1: Implications for Viral Persistence and Tumor Surveillance 1 RAJIV KHANNA,*,2,3 SCOTT R. BURROWS,*,2 PATTY M. STEIGERWALD-MULLEN, SCOTT A. THOMSON,* MICHAEL G. KURILLA, and DENIS J. MOSS* *Queensland Institute of Medical Research, The Bancroft Centre, 300 Herston Road, Brisbane, Australia 4029; and Department of Pathology and Microbiology, University of Virginia, Charlottesville, Virginia Received July 24, 1995; accepted October 11, 1995 The question of whether Epstein Barr nuclear antigen 1 (EBNA1) includes cytotoxic T lymphocyte (CTL) epitopes has generated considerable scientific interest, primarily due to its important implications for the overall biology of Epstein Barr virus (EBV). Earlier studies have suggested that EBV-associated malignancies that express only EBNA1 escape virus-specific immune surveillance since this antigen is not a target for CTL recognition. In the present report we have used a modified protocol to demonstrate that EBNA1 includes sequences which can be recognized by both polyclonal and clonal CTLs. CD4 / CTL clones were isolated from a healthy, seropositive donor that recognized the peptide epitope TSLYNLRRGTALA from EBNA1 in association with HLA DR1. Interestingly, these CTLs are unable to lyse EBV-infected B cells suggesting that EBNA1 may not be endogenously processed and/or presented to the host CTL response. Despite recent suggestions that glycine alanine repeat sequences within EBNA1 can inhibit endogenous processing, target cells infected with recombinant vaccinia vectors encoding truncated EBNA1 proteins, without these repeat sequences, were not recognized by this CTL clone. Thus it seems that the presence of glycine alanine repeats is not responsible for inhibiting the processing of the class II-restricted epitope defined in this study. These results substantiate the view that EBV-infected normal and malignant cells, where viral gene expression is limited to EBNA1, can resist CTL-mediated immune surveillance in vivo Academic Press, Inc. Epstein Barr virus (EBV) is a member of the human herpesvirus family that maintains a life-long latent asso- ciation with B lymphocytes and a permissive association with stratified epithelium in the oropharynx (1, 2). Two types of EBV (1 and 2, also referred to as A and B) are recognized that show DNA sequence divergence within the BAM H1 WYH and E regions of the genome (3, 4). There is now convincing evidence that EBV-specific memory CD8 / and CD4 / cytotoxic T lymphocytes (CTL) are responsible for controlling the level of EBV-positive B lymphocytes which all healthy, seropositive individuals carry for life following primary infection with the virus (5 7). Previous studies have defined the relative distribution of CTL epitopes within EBV latent proteins recognized by T cells clones from healthy immune individuals expressing an array of MHC class I alleles (8 10). Using 1 A part of this work was presented at the CIBA Foundation Symposium on Vaccines against Virally Induced Cancers; March 15 17, These two authors contributed equally in this work and their order should be considered arbitrary. 3 To whom reprint requests should be addressed. Fax: ; rajivk@qimr.edu.au. recombinant vaccinia encoding each of the EBV nuclear antigens (EBNA) and latent membrane proteins (LMPs), numerous CTL epitopes were mapped within EBNA2-6 and LMP, while no CTL response was detected against EBNA1. The question of whether EBNA1 includes CTL epitopes has generated considerable scientific interest, primarily due to its important implications for the overall biology of EBV. First, it has been proposed that the EBV-associated tumor, Burkitt s lymphoma (BL), escapes virus-specific immune surveillance by restricting latent gene expression to EBNA1 (11). This view is reinforced by earlier studies which showed that BL patients retain detectable EBV-specific T cell surveillance, indicating that CTL dys- function is an unlikely cause of the outgrowth of these EBV-infected tumors in vivo (12). Second, it has recently been shown that EBV-infected B cells, expressing EBNA1 and LMP2a only, are capable of maintaining the latent viral infection in vivo (13). Third, an earlier study carried out by Trivedi and colleagues clearly showed that mammary carcinoma cells transfected with the EBNA1 gene were not rejected by histocompatible mice, whereas LMP-transfected cells were regularly rejected (14). Thus /95 $12.00 Copyright 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

2 634 SHORT COMMUNICATION ratio of 20:1). These polyclonal CTLs were tested against autologous phytohemagglutinin (PHA) blasts presensitized with an overlapping net of EBNA1 peptides (15-mer overlapping by 10 amino acids) as described earlier (15). Polyclonal CTLs from donors LC and DM, stimulated with autologous LCLs, recognized four different peptides from EBNA1. CTLs from donor LC showed reactivity against peptide 49 (RRGTALAIPQCRLTP), while CTLs from donor DM recognized autologous PHA blasts in the presence of peptides 30 (EPDVPPGAIEQGPAD), 47 (GGSKTSLYNLRRGTA), and 48 (SLYNLRRGTALAIPQ) (Figs. 1a and 1b). In contrast, CTLs from donors CM and NB showed no significant reactivity against any of the EBNA1 peptides (data not shown). These experiments were repeated with two different blood samples and consistently the same set of peptides was recognized by CTLs from donors DM and LC. To confirm the data obtained with polyclonal effectors, CTL clones were generated (16) from donors DM and LC by seeding UM cells or purified CD4 / T cells, after stimulation with either autologous LCLs or EBNA1 peptides (peptides 30, 47, and 48 for donor DM and peptide 49 for donor LC). CD4 / T cells were purified from fresh UM cells using a Lymphokwik reagent (One Lambda Inc., CA). Colonies were harvested after 3 days and amplified in culture with biweekly restimulation with ril-2 and autologous LCL precoated with EBNA1 peptide(s). Two CTL clones were isolated from donor DM (referred to as DM2 and DM6) following stimulation with peptide 48 (SLYNLR- FIG. 1. Identification of CTLs specific for EBNA1 peptides in healthy RGTALAIPQ). Clone DM2 was isolated from UM cells, seropositive individuals. Polyclonal CTLs from donor DM (a) and LC while DM6 was isolated using purified CD4 / T cells. (b) were used as effectors to test for reactivity against autologous These CTL clones showed strong T cell T cell killing PHA blasts presensitized with overlapping EBNA1 peptides. Results are expressed as percentage specific lysis observed in a standard 5- (17) in the presence of peptides GGSKTSLYNLRRGTA hr chromium-release assay. An effector to target ratio of 20:1 was used in the assay. it is plausible that some virus-infected B cells maintain a nonimmunogenic phenotype by down-regulating the critical latent proteins needed for CTL recognition. The present study was specifically designed to consider the possibility of detecting an EBNA1-specific CTL response in healthy EBV-seropositive individuals, since earlier studies have essentially drawn their conclusions on the basis of negative evidence. Unfractionated mononuclear (UM) cells (10 6 /ml) from EBV-seropositive donors LC (HLA A1, B8, B18, DR3, DR11), NB (A2, A24, B7, B35, DR11), CM (HLA A11, A24, B7, B44, DR1, DR4), and DM (A24, A29, B44, Bw47, DR1, DR7) were cultivated with irradiated (8000 R) autologous lymphoblastoid cell lines (LCLs) transformed with the type 1 EBV isolate, B95.8 (responder to stimulator ratio of 50:1) in 2-ml culture wells (Linbro) in RPMI 1640/10% FCS. After 7 days, cultures were restimulated with autolo- gous LCLs and on Day 10 the cells were used as effectors in a standard 51 Cr-release assay (effector:target FIG. 2. MHC restriction analysis for CTL clones. Peptide-coated DM PHA blasts were precoated with peptide 48 (SLYNLRRGTALAIPQ; 1 mg/ml) and then exposed to CTL clones (DM2 and DM6) in the presence of anti-class I (W6/32) or anti-class II (L243) antibody (1:20 diluted ascites). An effector to target ratio of 5:1 was used in the assay. Data from one representative experiment of three are shown.

3 SHORT COMMUNICATION 635 FIG. 3. Minimalizing the EBNA1 epitope. DM anti-cd40 B cell blasts were presensitized with individual overlapping peptides and then exposed to CTL clone DM2. The peptide sequence shown in a box was identified as the minimal epitope sequence required for maximal lysis. An effector to target ratio of 5:1 was used in the assay. Data from one representative experiment of two are shown. and SLYNLRRGTALAIPQ, suggesting that both these were synthesized and screened for recognition by DM2. overlapping sequences might include an EBNA1 epitope. As shown in Fig. 3, 10 different peptides were tested and No CTL clones specific for EBNA1 peptides were iso- compared for the concentration required for half-maximal lated from donors LC or DM following stimulation with lysis of autologous anti-cd40 B cell blasts. These B cell peptides 30 and 49. blasts were generated as described previously (18). Characterization (by FACS analysis) revealed that DM2 Based on this analysis, peptide TSLYNLRRGTALA was and DM6 were ú99.0% CD3 /, CD4 / cells, a phenotype the minimal peptide with maximal activity, inducing halfmaximal suggesting that these clones might be MHC class IIrestricted. lysis at a concentration of mg/ml. To examine this possibility, autologous PHA An important aspect of the characterization of the CTL blasts were presensitized with peptide 48 and then exposed clone was to determine whether DM2 could recognize to the CTL clones in the presence of anti-class I autologous LCLs transformed with a range of virus (W6/32) or anti-class II (L243) antibody. As shown in Fig. strains. These LCLs were established either spontane- 2, recognition of peptide 48-coated PHA blasts was ously (19) or by exogenous virus transformation of peripheral blocked by L243 but was not significantly affected by B cells using type 1 or 2 EBV isolates (16). Surprisblocked W6/32 antibody. This result confirmed that these CTL ingly, no LCLs were recognized by DM2 (Fig. 4). The lack clones are MHC class II-restricted. of CTL recognition by DM2 was not due to any defect in To minimalize this CTL epitope, overlapping peptides the expression of MHC class II alleles, since all LCLs FIG. 4. Recognition of DM LCLs transformed with either type 1 or type 2 EBV isolates by CTL clone DM2. An effector to target ratio of 5:1 was used in the assay. Data from one representative experiment of four are shown.

4 636 SHORT COMMUNICATION FIG. 5. HLA class II restriction analysis for CTL clone DM2. Allogeneic EBV-transformed LCLs, some sharing an MHC class II antigen with donor DM, were exposed to the CTL clone DM2 in the absence or presence of TSLYNLRRGTALA peptide. An effector to target ratio of 5:1 was used in the assay. Data from one representative experiment of two are shown. were efficiently recognized by the clone if presensitized Indeed, the sequence conforms to the previously pubwith the TSLYNLRRGTALA peptide. This result suggests lished HLA DR1 binding motif (20). that LCLs are unable to process and present the EBNA1 To determine whether the lack of CTL lysis of autolo- CTL epitope endogenously. gous LCLs by CTL clone DM2 was due to insufficient To define the HLA restriction of the CTL clone DM2, levels of EBNA1 expressed in these cells, DM/B95.8 a panel of allogeneic LCLs, some sharing an MHC class LCLs and DM CD40 B cell blasts were infected with II allele with donor DM, was presensitized with TSLYNL- recombinant vaccinia constructs encoding the full-length RRGTALA and tested in a standard 51 Cr-release assay. EBNA1 gene (Vacc.EBNA1) and vaccinia encoding T7 As shown in Fig. 5, peptide-sensitized LCLs from RNA polymerase (Vacc.T7) (8). Despite high levels of DR1/ve donors, but not DR10ve donors, were recog- EBNA1 expression in these cells as determined by immunized by CTL clone DM2. These results suggest that noblotting (data not shown), no lysis was seen in the peptide TSLYNLRRGTALA is restricted through HLA DR1. presence of CTL clone DM2 (Fig. 6). These results confirm that, although EBNA1 includes a CTL epitope, this epitope is not processed and presented endogenously by EBV-infected cells. Levitskaya and colleagues have recently demonstrated that repeat sequences of glycine alanine within EBNA1 can inhibit presentation of class I epitopes (21). To explore the possibility that the lack of endogenous processing of the TSLYNLRRGTALA epitope relates to the presence of these repeat sequences, DM target cells were infected with two recombinant vaccinia vectors (Vacc.E1DGA and E1DCGA). Each of these constructs encodes a truncated EBNA1 protein without the glycine alanine repeats but including this epitope (P. M. Steigewald-Mullen and M. G. Kurilla, submitted for publication). Interestingly, target cells expressing these proteins were FIG. 6. Functional analysis of CTL clone DM2 using autologous LCLs or CD40 B cells infected with recombinant vaccinia expressing fullthat the presence of glycine alanine repeats is not re- not recognized by CTL clone DM2 (Fig. 6). Thus it seems length EBNA1 (Vacc.EBNA1), truncated EBNA1 proteins with glycine alanine repeat sequences deleted (Vacc.E1DGA and Vacc.E1DCGA), sponsible for inhibiting the processing of the class IIor with the nuclear localization signal sequence deleted (Vacc.E1DSS), restricted epitope defined in this study. Another possible as target cells. TSLYNLRRGTALA peptide-coated LCLs and CD40 B reason for the inability of infected cells to endogenously cells were used as controls. An effector to target ratio of 5:1 was used in the assay. Data from one representative experiment of four are process this epitope is that EBNA1 localizes to the nu- shown. cleus, thus limiting class II presentation. However, this

5 SHORT COMMUNICATION 637 seems unlikely since target cells were also not recog- REFERENCES nized by DM2 CTLs after infection with a recombinant 1. Yao, Q. Y., Ogan, P., Rowe, M., Wood, M., and Rickinson, A. B., Int. vaccinia (Vacc.E1DSS) encoding the EBNA1 protein with J. Cancer 43, (1989). the nuclear localization signal sequence deleted (Fig. 6). 2. Greenspan, J. S., Greenspan, D., Lennette, E. T., Abrams, D. I., Furthermore, we have recently identified an epitope from Conant, M. A., Petersen, V., and Freese, U. K., N. Engl. J. Med. 313, (1985). another EBV nuclear antigen (EBNA2) which is efficiently 3. Sculley, T. B., Apolloni, A., Stumm, R., Moss, D. J., Mueller-Lantzsch, processed endogenously through the class II pathway N., Misko, I. S., and Cooper, D. A., Virology 171, (1989). (manuscript in preparation). 4. Sample, J., and Kieff, E., J. Virol. 64, (1990). The present study clearly demonstrates that EBNA1 5. Moss, D. J., Rickinson, A. B., and Pope, J. H., Int. J. Cancer 22, 662 includes sequences which can be recognized by both 668 (1978). 6. Moss, D. J., Rickinson, A. B., and Pope, J. H., Int. J. Cancer 23, 618 polyclonal and clonal CTLs (Figs. 1 and 2). Interestingly, 625 (1979). the HLA DR1-restricted CTL clones are unable to recog- 7. Misko, I. S., Soszynski, T. D., Kane, R. G., and Pope, J. H., Clin. nize EBV-infected B cells, suggesting that EBNA1 may Immunol. Immunopathol. 32, (1984). have evolved a mechanism to avoid endogenous pro- 8. Khanna, R., Burrows, S. R., Kurilla, M. G., Jacob, C. A., Misko, I. S., cessing and/or presentation through the class II path- Sculley, T. B., Kieff, E., and Moss, D. J., J. Exp. Med. 176, (1992). way. The presence of memory CTLs specific for EBNA1 9. Murray, R. J., Kurilla, M. G., Brooks, J. M., Thomas, W. A., Rowe, peptide epitopes and their activation in vitro following M., Kieff, E., and Rickinson, A. B., J. Exp. Med. 176, LCL stimulation (Fig. 1), however, suggests that EBNA1 (1992). protein from EBV-infected cells might be exogenously 10. Brooks, J. M., Murray, R. J., Thomas, W. A., Kurilla, M. G., and endocytosed by professional antigen-presenting cells Rickinson, A. B., J. Exp. Med. 178, (1993). 11. Rowe, M., Rowe, D. T., Gregory, C. D., Young, L. S., Farrell, P. J., and presented in association with MHC class II moleand Rupani, H., EMBO J. 6, (1987). cules. Alternatively, epitopes generated via extracellular 12. Rooney, C. M., Rickinson, A. B., Moss, D. J., Lenoir, G. M., and cleavage of EBNA1 by serum proteases might be respon- Epstein, M. A., IARC Sci. Publ (1985). sible for the activation of CTLs. It is also possible that 13. Chen, F., Zou, J. Z., di Renzo, L., Winberg, G., Hu, F., Klein, E., Klein, these CTLs have been activated in vivo by EBV-infected E., and Ernberg, I., J. Virol. 69, (1995). 14. Trivedi, P., Masucci, M. G., Winberg, G., and Klein, G., Int. J. Cancer cells other than B cells. Indeed, earlier studies have 48, (1991). shown that the efficiency of endogenous processing of 15. Burrows, S. R., Gardner, J., Khanna, R., Steward, T., Moss, D. J., class II epitopes is often dependent on the cell lineage Rodda, S., and Suhrbier, A., J. Gen. Virol. 75, (1994). of the antigen-presenting cell (22). The results of the 16. Moss, D. J., Misko, I. S., Burrows, S. R., Burman, K., McCarthy, R., present study substantiate the view that EBV can persist and Sculley, T. B., Nature 331, (1988). 17. Burrows, S. R., Suhrbier, A., Khanna, R., and Moss, D. J., Immunolin normal and malignant cells in vivo in an immunologiogy 76, (1992). cally silent state. 18. Khanna, R., Jacob, C. A., Burrows, S. R., and Moss, D. J., J. Immunol. Methods 164, (1993). 19. Rickinson, A. B., Rowe, M., Hart, I. J., Yao, Q. Y., Henderson, L. E., ACKNOWLEDGMENTS Rabin, H., and Epstein, M. A., Cell Immunol. 87, (1984). 20. Chicz, R. M., Urban, R. G., Lane, W. S., Gorga, J. C., Stern, L. J., We acknowledge the Cetus Corporation for the generous gift of ril- Vignali, D. A., and Strominger, J. L., Nature 358, (1992). 2. This work was supported by grants from Queensland Cancer Fund 21. Levitskaya, J., Coram, M., Levitsky, V., Imreh, S., Steigerwald-Mullen, and the National Cancer Institute (U.S.A.; CA ). M.G.K. is a P. M., Klein, G., Kurilla, M. G., and Masucci, M. G., Nature scholar of the Lucille P. Markey Charitable Trust. P.M.S.M. is supported 375, (1995). by the Medical Scientist Training Program of the University of Virginia 22. Moreno, J., Vignali, D. A., Nadimi, F., Fuchs, S., Adorini, L., and Health Sciences Center. Hammerling, G. J., J. Immunol. 147, (1991).