Specific Cytotoxic T Lymphocytes Recognize the Immediate ... - NCBI

JOURNAL OF VIROLOGY, Aug. 1995, p. 4872–4879 0022-538X/95/$04.0010 Copyright 1995, American Society for Microbiology

Vol. 69, No. 8

Specific Cytotoxic T Lymphocytes Recognize the Immediate-Early Transactivator Zta of Epstein-Barr Virus ¨ RGY STUBER,2 CHRISTOPH BOGEDAIN,1* HANS WOLF,1 SUSANNE MODROW,1 GYO 1 AND WOLFGANG JILG Institut fu ¨r Medizinische Mikrobiologie und Hygiene, Universita ¨t Regensburg, D-93053 Regensburg, Germany,1 and Microbiology and Tumor Biology Center, Karolinska Institutet, S-171 77 Stockholm, Sweden2 Received 6 February 1995/Accepted 28 April 1995

We identified the immediate-early transactivator Zta of Epstein-Barr virus as a target for specific cytotoxic T lymphocytes (CTL). Cells pulsed with overlapping synthetic peptides representing the entire amino acid sequence of Zta proved to be efficient for the in vitro stimulation of Zta-specific CTL in several donors. With peptide-pulsed target cells, we found that CTL from several donors recognize a peptide comprising 15 amino acids. The immune response against this peptide exerted by CTL lines from different donors was found to be restricted by two different molecules of the major histocompatibility complex: HLA-B8 and HLA-Cw6. The latter molecule could for the first time be identified as a restricting element for a CTL response. The epitope of the HLA-B8-restricted CTL could be mapped to an octameric sequence between amino acid positions 190 and 197 of the Zta protein, whereas the minimal epitope of HLA-Cw6-restricted CTL consists of 11 to 15 residues between positions 187 and 201. Thus, the HLA-B8 and HLA-Cw6 epitopes widely overlap but are not completely identical. In vitro stimulation of blood lymphocytes from a panel of HLA-B8-positive or HLA-Cw6positive virus carriers, using autologous cells pulsed with the Zta peptides comprising the HLA-B8 or HLACw6 epitope, respectively, revealed in both cases that most of these donors developed a Zta-specific cytotoxic activity. These data, as well as the high spread of the major histocompatibility complex molecules HLA-B8 and HLA-Cw6 in most populations, suggest that an efficient CTL response directed against gene products of the immediate-early group of the lytic cycle exists in vivo in a considerable portion of virus carriers. A CTL response against proteins expressed immediately after the switch into the lytic cycle could eliminate lytically activated cells at an early stage and would thus efficiently prevent the production and release of progeny virions.

uncontrolled proliferation of immortalized cells seems to be efficiently prevented by a multicomponent CTL response. However, the lack of a detectable release of virus from the B-cell reservoir of immunocompetent carriers suggests additional control mechanisms which render a full lytic cycle more difficult. The control may be exerted at multiple levels such as transcription (6, 14, 30, 40), translation, and immune elimination of infected cells. There is indeed some evidence for immune surveillance mechanisms directed against products of the lytic cycle. The resting B cells representing the reservoir of the virus are presumed to express only EBNA 1 (22). According to our present knowledge, this protein cannot be recognized by CTL. These cells, therefore, will not be attacked by the immune system. On the other hand, cells expressing only EBNA 1 are able to switch directly into the lytic cycle without passing through the other stages of latency (36), i.e., without synthesizing proteins which could serve as targets for specific CTL. Production and release of infectious virus by these cells could therefore be inhibited only by immune mechanisms directed against gene products of the lytic cycle. Several lines of evidence suggest the existence of CTL directed against this group of proteins: in an extensive evaluation of the specificity of CTL clones directed against EBV-infected cells, Khanna et al. (20) found several clones which were obviously EBV specific but did not recognize proteins of the latent state; Pothen and coworkers (35) showed that purified viral proteins of the lytic cycle were able to induce a proliferative T-cell response, whereas Schendel et al. (38) observed a CTL response against B cells infected with the predominantly lytic EBV strain P3-HR1 which was possibly

Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus. Primary infection may cause infectious mononucleosis; more often, it remains clinically silent. In any case, the first contact with EBV inevitably results in a lifelong latent infection of a certain subpopulation of resting B cells (16, 22). EBV has two notable biological characteristics: the virus is able to immortalize B cells in vivo and in vitro, and it is associated with three human malignancies, Burkitt’s lymphoma, nasopharyngeal carcinoma, and malignant lymphomas, in immunosuppressed individuals. Development of these tumors, however, is a very rare event, despite the immortalizing potential of EBV and its high spread in the human population. In a healthy individual, highly efficient immunological control mechanisms exist which inhibit viral replication and the proliferation of immortalized EBV-infected cells. The best-characterized components of these mechanisms are specific cytotoxic T cells (CTL) directed against viral gene products of the latent state, which include the EBV nuclear antigens (EBNAs) 1 to 6 and the latent membrane proteins 1 and 2. Depending on their phenotypes, cells latently infected by EBV express these proteins, following one of three different patterns (22, 37) due to transcription from different promoters and differential splicing. With the exception of EBNA 1, all of these gene products can be recognized by EBV-specific CTL (15, 20, 24, 31, 32); in most cases, CTL responses in one individual are directed against at least two different epitopes of these latent proteins (31). Thus,

* Corresponding author. Present address: MediGene GmbH, Lochhamer Str. 11, D-82152 Martinsried/Munich, Germany. Phone: 4989-89 56 32 16. Fax: 49-89-89 56 32 20. 4872

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CTL DIRECTED AGAINST Zta OF EBV

directed against products of the lytic cycle. Furthermore, immediate-early proteins of the lytic cycle are known to serve as CTL targets in other herpesvirus infections: CTL responses against immediate-early gene products of murine (23) and human (7) cytomegalovirus as well as of varizella-zoster virus (1) have been described. For EBV, however, a direct proof that CTL are active against viral products of the lytic cycle and a precise identification of possible target proteins are lacking. In this study, we addressed the question of whether EBVencoded proteins expressed during the lytic cycle may serve as target structures for EBV-specific CTL. Since an immune response at a very early stage after the switch from latency to virus replication would efficiently eliminate lytically infected cells and thus prevent the release of infectious virus, we focused on the immediate-early protein Zta, also termed Z, ZEBRA, or EB1, a viral transactivator encoded by the open reading frame BZLF1. This protein is the first or among the first proteins synthesized when the virus enters the lytic cycle and plays a crucial role in the activation of other genes involved in the process of ongoing viral replication (reviewed in reference 21). We were able to show that this protein can indeed be recognized by EBV-specific CTL, that the recognition site is located to a 15-amino-acid-long sequence within the C-terminal part of the molecule, and that the CTL response is restricted by two different class I major histocompatibility complex (MHC) molecules.

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labelled for 1 h with 100 mCi of Na51CrO4 (Du Pont, Bad Homburg, Germany) and coincubated with the effector cells at known effector/target (E/T) ratios in triplicate for 4 h in V-bottom 96-well plates in standard chromium release assays. When vaccinia virus-infected target cells were used, the target cells were infected 12 h prior to labelling at a multiplicity of infection of 10. For peptide pulsing of target cells, the chromium-labelled cells were coincubated for 1 h with each peptide in a concentration of 4 3 1027 M at 378C and in a volume of 25 ml prior to addition of CTL to a final volume of 125 ml. If the target cells were pulsed with peptide pools, the indicated concentrations again refer to each single peptide in the cocktail. In the inhibition experiments, monoclonal antibodies W6-32, specific for class I molecules, and L243, specific for class II molecules (2), were added to the target cells 1 h prior to coincubation with the effector cells (1 mg of monoclonal antibody per ml). Peptide-induced class I MHC reconstitution assay. Flow cytometric detection of the peptides’ capacity to reconstitute acid-denatured MHC molecules was performed as described earlier (44). Briefly, 2 3 106 cells of the HLA-B8-positive LCL GSB1 were washed twice, and the pellet was treated with citrate buffer (pH 3.3) for 1 min. After neutralization with serum free AIM-V medium (Gibco/ BRL, Paisley, Scotland), the cells were centrifuged and resuspended in AIM-V medium to a cell density of 106/ml. Aliquots of 200 ml were transferred into U-bottom 96-well plates, and human b2-microglobulin was added to a final concentration of 100 nM. The synthetic peptides were added to a final concentration of 100 mM. Acid-treated controls received b2-microglobulin only. The HLA-A2-binding peptide M58-66 was included as a negative control. After 20 min, the HLA-B8-specific monoclonal antibody GSP 8.1 (Genetic Systems, Seattle, Wash.) was added, and the cells were incubated at room temperature for 4 h. After two washings, the cells were incubated with saturating amounts of fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse antibody for 30 min at 48C and analyzed on a FacScan flow cytometer (Becton Dickinson, Mountain View, Calif.). Each peptide’s capacity to reconstitute HLA-B8 was calculated as mean fluorescence intensity of the sample minus its FITC conjugate control divided by that of the acid-treated negative control with b2-microglobulin alone minus its FITC conjugate control. The results are given as the mean values of four independent experiments and include standard deviations.

MATERIALS AND METHODS Cells and viruses. Cells were cultivated in RPMI 1640 growth medium (Gibco/ BRL, Eggenstein, Germany) containing 10% heat-inactivated fetal calf serum, 2 mM glutamine, and 100 g of kanamycin or gentamycin per ml. Phytohemagglutinin (PHA)-activated blasts (PHA blasts) were generated by incubation of peripheral blood lymphocytes (PBL) for 2 to 6 days in the presence of 25 mg of PHA C (Boehringer Mannheim, Mannheim, Germany) per ml. For the determination of the restricting MHC molecules of the CTL, the lymphoblastoid cell lines (LCLs) 9007, 9009, 9011, 9018, 9019, 9049, and 9060 from the 10th International Histocompatibility Workshop Panel, with known HLA types, were used as allogenic targets (12). HLA types from donor cells were determined by standard serological methods. For the class I MHC reconstitution assays, the HLA-B8-positive LCL GSB1 was used (41a). The recombinant vaccinia virus Z-Vac used for the expression of Zta was described previously (6). Synthetic peptides. A complete set of peptides comprising between 13 and 15 amino acids, overlapping by 5 to 9 amino acids, and encompassing the entire amino acid sequence of the Zta protein derived from EBV strain B95-8, as well as the 8-, 9-, or 10-amino-acid-long variants of the immunogenic peptide Zta187– 201 and peptides containing sequences from the HIV envelope protein (HIV-env, showing the sequence RIQGRPGRAFVTIG) and from the influenza virus matrix protein (M58-66, showing the sequence GILGFVTL), were synthesized on a model 9050 synthesizer (Milligen, Eschborn, Germany) by using fluorenylmethyloxycarbonyl chemistry as described elsewhere (29). CTL lines. CTL lines were derived from EBV-positive healthy adults, as determined by detection of immunoglobulin G antibodies against EBNA and virus capsid antigen, using standard serological methods. PBL were purified from peripheral blood by density gradient centrifugation using Ficoll Histopaque (Sigma, Deisenhofen, Germany) and cultivated in RPMI 1640 growth medium with 10% heat-inactivated human serum, 2 mM glutamine, nonessential amino acids, 2 mM sodium pyruvate, and 100 mg of gentamycin or kanamycin per ml (T-cell medium). T cells were stimulated with PHA blasts pulsed with synthetic peptides for the generation of specific CTL lines. Recombinant human interleukin 2 (Boehringer Mannheim) was added to the cells after 1 week of culture to a concentration of 20 U/ml. For stimulation with peptides, fresh PBL were incubated in the presence of 1026 M each peptide for 16 h in a small volume prior to addition of 3 volumes of T-cell medium. One and two weeks later, autologous PHA blasts were coincubated for 2 h at 378C in T-cell medium containing 2 3 1026 M each peptide, irradiated with 5,000 rad, and added to the CTL in a ratio of 1:10 in a small volume without washing away the peptides (so that the concentration of the peptides was approximately 4 3 1027 M). After 6 h of incubation in a small volume, 3 volumes of T-cell medium containing 20 U of interleukin 2 per ml was added. Stimulation was repeated weekly, but in the following stimulations the peptides were washed away from the stimulator cells to avoid peptide binding to and lysis of the effector cells. If peptide pools were used for the stimulation, the indicated concentrations refer to each single peptide in the cocktail. Cytotoxicity assays. One million autologous or allogenic target cells were

RESULTS Identification of a peptide derived from Zta as a target for specific CTL. A set of 30 overlapping synthetic peptides representing the entire amino acid sequence of Zta was used for preparation of cells for in vitro stimulation of CTL. The peptides were divided into three pools of 10 peptides each representing the N-terminal (pool 1), middle (pool 2), and C-terminal (pool 3) parts of the protein. PBL from eight healthy donors were stimulated with each of these pools for 2 to 3 weeks before being tested for cytotoxicity. As targets, autologous PHA blasts which had been infected with the recombinant vaccinia virus containing the complete Zta gene (Z-Vac) were used. Cells infected with wild-type vaccinia virus served as control targets. PBL lines of two donors, both stimulated with pool 3 peptides, showed specific CTL responses against autologous cells infected with Z-Vac of 14% (donor 1) and 43% (donor 2), both after subtraction of the value for the wild-type vaccinia virus control, using an E/T ratio of 30:1. Retesting of these two CTL lines against autologous PHA blasts pulsed with pool 3 peptides resulted in specific lysis rates of 9% (donor 1) and 37% (donor 2), both after subtraction of the lysis obtained with the control peptide HIV-env, at an E/T ratio of 30:1. Subsequently the CTL line from donor 2 was tested for recognition of autologous PHA blasts pulsed with individual peptides of pool 3; only blasts pulsed with a peptide containing amino acids 187 to 201 of Zta (peptide Zta187–201) were specifically recognized (Fig. 1). These experiments were repeated with eight additional donors, using peptide pools 1 to 3 and peptide Zta187–201 for stimulation. Autologous blasts pulsed with the three pools and Zta187–201 served as targets. Two additional individuals (donor 3 and donor 4) whose PBL specifically recognized peptide Zta187–201 were found. No other peptides were recognized by these two donors, nor did any of the other donors show specific reactions, suggesting that peptide Zta187–201 contains the dominant epitope of Zta recognized by specific CTL.

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FIG. 1. Recognition of peptide Zta187–201 by a CTL line from donor 2 stimulated with pool 3 peptides. Autologous PHA blasts were pulsed with the single peptides from pool 3. The E/T ratio was 6:1.

Identification of HLA-B8 and HLA-Cw6 as restricting elements for the recognition of Zta187–201. CTL lines from donors 2, 3, and 4 were used for the following experiments. The observed CTL response against peptide Zta187–201 was restricted by class I MHC antigens. This was shown by blocking experiments using monoclonal antibodies directed against class I and class II MHC antigens (Fig. 2A); furthermore, the CTL lines from these donors specifically lysed target cells with the recombinant vaccinia virus construct Z-Vac (Fig. 2B), indicating that they recognize peptides derived from endogenously synthesized proteins. HLA typing of the three donors revealed that donors 2 and 3 shared the class I molecules HLA-B13 and HLA-Cw6, donors 3 and 4 shared HLA-Cw7, but donor 4 shared no class I MHC molecules with donor 2. Testing the three CTL lines specific for Zta187–201 against peptide Zta187– 201-pulsed target cells of each of the three donors showed that CTL of donors 2 and 3 recognized target cells of both of these donors but not cells of donor 4, whereas the CTL from donor 4 recognized only the autologous targets (Fig. 3). Thus, the CTL response to peptide Zta187–201 was obviously restricted by two different MHC class I molecules. To identify the restricting MHC antigens, CTL lines of donors 2, 3, and 4 were tested against a panel of allogenic target cells (PHA blasts and LCLs pulsed with peptide Zta187–201) showing different HLA types but sharing at least one MHC class I molecule with donors 2, 3, and 4. In these experiments, CTL derived from donors 2 and 3 recognized exclusively targets expressing HLA-Cw6 (Table 1), whereas the CTL from donor 4 specifically lysed HLA-B8positive cells only (Table 2). Moreover, CTL obtained from donor 2 recognized the class I MHC null mutant LCL 721.221 transfected with an expression plasmid containing the HLACw6 gene (41) but not the untransfected parent cells, thus confirming the role of HLA-Cw6 as the restricting element for this CTL line (Table 1). Determination of the minimal lengths of Zta peptides bound to HLA-B8 and HLA-Cw6. We synthesized six peptides with a length of 10 amino acids which spanned the region between amino acid positions 187 and 201 of Zta and overlapped by 9 amino acids. The CTL lines from donors 4 (HLA-B8 restricted) and 2 (HLA-Cw6 restricted) were tested against HLA-B8- and HLA-Cw6-positive cells (from donors 7 and 2), respectively, pulsed with these six peptides. Only the two adjacent peptides Zta189–198 and Zta190–199 mediated specific lysis of the HLA-B8-positive target cells by the HLA-B8-restricted CTL line (Table 3). Both peptides have an eight- or nine-amino-acid-long binding motif specific for HLA-B8 (42). Subsequently a nonameric peptide (Zta190–198) and an oc-

FIG. 2. (A) Inhibition of reactivity of Zta187–201-specific CTL from donors 2 and 4 by preincubation of target cells with monoclonal antibodies against MHC class I (hatched bars) and class II (empty bars) molecules, respectively. Autologous target cells were pulsed with Zta187–201. (B) Recognition of autologous PHA blasts expressing the entire Zta protein by using the recombinant vaccinia virus construct Z-Vac (filled circles) by CTL from donors 2 and 4. Wild-type vaccinia virus-infected target cells served as negative controls (open circles).

tameric peptide (Zta190–197) showing this motif were synthesized. Patterns of recognition of these peptides, as well as peptides Zta189–198 and Zta190–199 and the parent peptide Zta187–201, were compared in different dilutions, using an HLA-B8-restricted CTL line stimulated with the peptide Zta190–199 (Fig. 4). Cells loaded with the octamer Zta190–197 were lysed with the highest efficiency, as indicated by a specific lysis of 68% and a half-maximal lysis point at a peptide concentration of approximately 1.7 3 1028 M, followed by target cells pulsed with Zta190–198 and Zta190–199, with maximal specific lysis rates of 55 and 48% (all after subtraction of the lysis obtained with the control peptide HIV-env, and at an E/T ratio of 20:1) as well as half-maximal lysis points of 3 3 1028 and 6 3 1028 M, respectively. Thus, peptide Zta190–197 with the sequence RAKFKQLL was recognized best in connection with HLA-B8. Moreover, experiments assessing peptide binding to HLA-B8 by using the immunofluorescence reconstitution assay confirmed that peptides Zta187–201, Zta190–198, and Zta190–197 were all efficiently bound to HLA-B8 (Fig. 5). Peptide Zta187–201 showed the lowest binding, most probably because of its suboptimal length. Besides the HLA-B8 motif, the 15-amino-acid-long sequence represented by peptide Zta187–201 also contains a bind-

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FIG. 3. Cross-reactivity of Zta187–201-specific CTL lines from donors 2, 3, and 4 against autologous targets and cells of the two other donors. Filled circles, target cells (PHA blasts) pulsed with peptide Zta187–201; open circles, target cells (PHA blasts) pulsed with peptide HIV-env as negative controls.

ing motif for HLA-Cw6 (13). Surprisingly, none of the decapeptides derived from this sequence were recognized by the HLA-Cw6-restricted CTL lines from donors 2 and 3, suggesting that the minimal epitope recognized by HLA-Cw6-restricted T cells consists of more than 10 amino acids. Induction of Zta-specific CTL in HLA-B8- and HLA-Cw6positive donors. Six of twenty HLA-typed donors were positive for HLA-B8, and five were positive for HLA-Cw6. PBL from the donors positive for HLA-B8 were stimulated by using cells pulsed with the well-recognized peptide Zta190–198, whereas in the case of the PBL from HLA-Cw6-positive donors, peptide Zta187–201 was used for stimulation. The reactivity of the resulting CTL lines against autologous target cells pulsed with the same peptides was then investigated. Highly specific CTL could be demonstrated after two to three rounds of stimulation in four of the six HLA-B8-positive donors (Fig. 6A). Three of the five HLA-Cw6-positive donors clearly developed Zta-specific CTL against peptide Zta187–201 (Fig. 6B). In both groups, one additional donor showed a slight but significant and reproducible specific lysis above the background level. Possible influence of sequence variation in peptide Zta190–198 on CTL recognition. Recently, a point mutation in the Zta gene resulting in an amino acid change at position 195 from glutamine in the B95-8 sequence to histidine was described (8, 34). This position is located within the epitopes of the HLA-

TABLE 1. Reactivities of CTL lines specific for peptide Zta187–201 against a panel of allogenic target cells HLA class I typea Target cells

Donor 2c A

Donor 2 Donor 4 Donor 5 Donor 6 9007 9009 9049 721.221g 721.221-Cw6h a

% Specific lysisb with effector cells from:

10/32 1/2 2/3 1/3 2 1 33

B

13/37 8/44 57/60 7/8 57 37 65

Cw

6 5/7 3/6 7 6 6 8 6

Donor 3d

30:1e

6:1

30:1

6:1

29 1 19 0 30 29 0 0 25

19 2 10 1 13 14 0 0 24

21 3 20 2 NDf ND ND ND ND

12 1 9 0 ND ND ND ND ND

Restricting elements are underlined. Specific lysis was obtained by subtracting the lysis of target cells pulsed with the control peptide HIV-env from the lysis of target cells pulsed with peptide Zta187–201. Values above 5% were considered positive. c HLA-A10, 32; B13, 37; Cw6. d HLA-A3, 30; B7, 13; Cw6, 7. e E/T ratio. f ND, not determined. g An HLA-A, -B, and -C null mutant LCL. h 721.221 cells transfected with an expression vector for HLA-Cw6 (41). b

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TABLE 2. Reactivities of the CTL line from donor 4a specific for peptide Zta187–201 against a panel of allogenic target cells HLA class I typeb

Target cells

Donor Donor Donor Donor Donor Donor 9011 9018 9019 9060

3 5 6 7 8 9

% Specific lysisc

A

B

Cw

30:1d

6:1

3/30 2/3 1/3 1/3 1/32 2/25 1 3/24 30 1

7/13 57/60 7/8 7/8 7/8 8/44 52 18 18 62

6/7 3/6 7 4/7 7 5/7 NDe 5 5 9

0 0 18 31 26 23 0 0 0 0

1 0 13 22 14 13 0 0 0 0

a

HLA-A1, 2; B8, 44; Cw5, 7. b Restricting elements are underlined. c Specific lysis was obtained by subtracting the lysis of target cells pulsed with the control peptide HIV-env from the lysis of target cells pulsed with peptide Zta187–201. Values above 5% were considered positive. d E/T ratio. e ND, not determined.

B8- and HLA-Cw6-restricted CTL. To address the questions of whether this amino acid exchange alters the binding of the peptide to the HLA-B8 molecule or recognition of target cells by an HLA-B8-restricted CTL and whether specific CTL against this sequence can be induced in HLA-B8-positive donors, a nonameric peptide with a histidine at position 195 showing the sequence RAKFKHLLQ (Zta190–198His) was synthesized. Two HLA-B8-restricted CTL lines derived from donor 7 which were stimulated by cells pulsed either with peptide Zta190–198, showing the B95-8 sequence, or with peptide Zta190–198His were able to recognize target cells pulsed with both peptides. However, the lysis of target cells pulsed with the peptide used for the stimulation was in most experiments higher than that of target cells pulsed with the other peptide (Fig. 7). Evaluation of peptide binding to HLA-B8 indeed showed that peptides Zta190–198 and Zta190–198His both efficiently bound HLA-B8, as reflected by reconstitution of HLA-B8 molecules on the surface of acid-stripped GSB1 cells (data not shown). DISCUSSION The immediate-early transactivator Zta of EBV plays a key role in the activation of viral replication: in some cell lines, this

FIG. 4. Influence of peptide length on the recognition of HLA-B8-positive target cells by an HLA-B8-restricted CTL line. The CTL line derived from donor 7 was stimulated with peptide Zta190–198 for 3 weeks. Autologous PHA blasts were pulsed with peptides Zta187–201, Zta190–199, Zta190–198, and Zta190–197 in different concentrations. The E/T ratio was 20:1.

protein alone is sufficient to trigger the complete lytic cycle (10, 17). Zta activates transcription from a variety of promoters, inducing a cascade of gene activations as well as viral DNA replication during the lytic cycle (21, 39). Thus, CTL directed against this protein should be able to prevent replication and release of infectious virus by eliminating EBV-infected cells at a very early stage of the lytic cycle. This could be essential for cells which switch directly from EBNA 1-positive latency to the lytic cycle without prior expression of other latent proteins (36) and which therefore cannot be attacked by CTL directed against these antigens. Generally, a cellular immune response against Zta, as well as against other proteins expressed during the lytic cycle, adds an additional degree of safety to the immune surveillance mechanisms based on CTL specific for the proteins of latency, coming into action whenever EBV-infected cells are able to escape the immune attacks during the latent state and start to produce virus. Possible target cells in vivo may be the EBV-infected B cells, which were shown to be able to switch into the lytic cycle and to release infectious virus (25), but also infected epithelial cells, e.g., of salivary glands, which tend to favor the lytic cycle and thus enable virus shedding into the saliva. Cyclic elimination of EBV-infected epithelial cells by CTL and their reinfection could explain the discontinuous shedding of virus into the saliva observed in many EBV carriers (43). Moreover, EBV replication found in epithelial cells of

TABLE 3. Screening of amino acid positions 187 to 201 of Zta for the HLA-B8 epitope % Specific lysisa Peptide

Amino acid sequence

Zta187–201

RKCRAKFKQLLQHYR

22

4

Zta187–196 Zta188–197 Zta189–198 Zta190–199 Zta191–200 Zta192–201

RKCRAKFKQL KCRAKFKQLL CRAKFKQLLQ RAKFKQLLQH AKFKQLLQHY KFKQLLQHYR

0 1 33 57 0 0

0 0 3 18 0 0

27

4 3 10

Mb

4 3 1028 M

a Specific lysis was calculated by subtracting the lysis of control cells (pulsed with peptide HIV-env) from the lysis of target cells pulsed with the different peptides. Effector cells were derived from donor 4; target cells were HLA-B8matched PHA blasts derived from donor 7. b Peptide concentration.

FIG. 5. Flow cytometric comparison of the HLA-B8-binding capacities of peptides Zta187–201, Zta190–198, and Zta190–197 as assessed by reconstitution of HLA-B8 on the surface of acid-stripped GSB1 cells. Peptide M58-66 served as the negative control.

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FIG. 6. Activities of Zta-specific CTL lines derived from different EBV carriers positive for HLA-B8 (A) or HLA-Cw6 (B). CTL were stimulated for 2 to 3 weeks with either peptide Zta190–198 (HLA-B8-positive cells) or peptide Zta187–201 (HLA-Cw6-positive cells). Target cells were autologous PHA blasts pulsed with the same peptides (hatched bars); controls were autologous PHA blasts pulsed with peptide HIV-env (open bars). The E/T ratio was 30:1.

the tongues of individuals with oral hairy leukoplakia (4) points to a role of specific CTL against viral products of the lytic cycle, since this phenomenon primarily occurs in immunodeficient patients. Looking for possible target sequences for Zta-specific CTL, we found surprisingly two epitopes within a 15-amino-acidlong sequence recognized by CTL restricted by either HLA-B8 or HLA-Cw6. These epitopes widely overlap but are not completely identical. Restriction of overlapping or even identical CTL epitopes by different class I MHC molecules has been described in other viral systems such as human immunodeficiency virus type 1 (18, 33), influenza A virus (5), and hepatitis B virus (28). Sequence characteristics of the octameric epitope RAKFK QLL recognized by the HLA-B8-restricted CTL lines and showing an efficient HLA-B8-binding capacity accord well with previous investigations identifying the sequence requirements for HLA-B8-binding peptides (x-x-K/R-x-K/R-x-x-(x)-L/I) (42). The Zta epitope has positively charged lysine residues at positions 3 and 5 of the nonamer peptide. Positively charged residues at these anchor positions were found to be necessary for optimal fitting into the peptide-binding groove of HLA-B8 (42). The leucine residue present at position 8 in the Zta epitope is consistent with the observation that HLA-B8-binding peptides frequently have leucine or isoleucine residues at position 8 or 9. Only a few examples of HLA-C-restricted CTL have been reported so far: HLA-Cw7-restricted CTL possibly directed


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FIG. 7. Influence of an amino acid change at position 195 on the recognition by HLA-B8-restricted CTL lines derived from donor 7. One CTL line was stimulated with peptide Zta190–198-pulsed cells (A), and another CTL line was stimulated with peptide Zta190–198His-pulsed cells (B), for 2 to 3 weeks. Autologous PHA blasts as target cells were pulsed with peptides Zta190–198, Zta190– 198His, and HIV-env in different concentrations and tested at an E/T ratio of 20:1.

against EBV proteins of the lytic cycle (38), HLA-Cw3 (26)and HLA-Cw4 (19)-restricted CTL directed against epitopes of the human immunodeficiency virus envelope protein, and HLA-Cw3-restricted CTL specific for epitopes of influenza virus and Sendai virus proteins (11). The present data provide additional evidence for a further viral epitope which is restricted by an MHC molecule of the HLA-C locus and therefore again confirm suggestions of Falk and coworkers that a major role of the HLA-C loci is the presentation of foreign epitopes to CTL (13). In our study, the MHC molecule HLACw6 could for the first time be identified as a restricting element for CTL responses. The minimal epitope recognized by the HLA-Cw6-restricted CTL is located between amino acid positions 187 and 201 of the Zta protein and must have a length of between 11 and 15 amino acids, since the HLA-Cw6restricted CTL failed to recognize target cells pulsed with the 10-amino-acid-long overlapping peptides of this region. Interestingly, in the region of the Zta gene in which the CTL epitopes are localized, three functionally important elements can be found: the DNA binding domain, the DNA dimerization domain (9), and the nuclear targeting signal (27). Thus, this region should be highly conserved, and extensive sequence variations in the HLA-B8- and HLA-Cw6-restricted epitopes are not a likely prerequisite for a functionally important CTL epitope. A recently reported sequence variation in some EBV strains at amino acid position 195 (8, 34), just within the recognition sequence for HLA-B8- and HLA-Cw6-restricted CTL, did not crucially influence peptide binding to HLA-B8, recognition by HLA-B8-restricted CTL, or induction of specific CTL. This is most likely because the amino acid change is not located at one of the anchor positions for peptide binding to HLA-B8 molecules (42). Hence, Zta-specific CTL responses

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can probably be active against a broad range of different EBV strains. The majority of our donors revealing HLA-B8 or HLA-Cw6 were able to mount a CTL response to Zta. This finding, as well as the wide spread of these HLA molecules, at least in the Caucasian and black populations (gene frequencies for HLA-B8 and HLA-Cw6 are 9.6 and 8.6% in Caucasians, 0.2 and 3.8% in Orientals, and 5.5 and 12.9% in blacks [3]), leads one to suppose that immune surveillance mechanisms controlling EBV-infected cells by means of Zta-specific CTL are operative in large parts of the population. Individuals who are negative for HLA-B8 or HLA-Cw6 could either have Ztaspecific CTL restricted by other HLA molecules which have not yet been discovered because of the necessarily limited number of donors screened in our experiments or have CTL directed at other proteins of the lytic cycle, especially proteins of the immediate-early or early group. The first reason may be of relevance in populations in which these two haplotypes are less frequent. Regarding the second point, preliminary experiments in our laboratory did indeed show that the immediateearly transactivator protein Rta, which also has a key role in activation of the lytic cycle, can also serve as a target for specific CTL (6a). The stimulation of EBV-specific CTL with peptide-pulsed autologous cells provided reliable and highly reproducible data similarly as shown by Missale and colleagues (28) for hepatitis B virus-specific CTL. Although it would have been desirable to use PBL from EBV-negative donors of HLA-B8 or HLA-Cw6 haplotype as negative controls, no donor showing these characteristics was found because of the low proportion of seronegative individuals among adults. Nevertheless, there is no obvious reason to doubt the specificity of our results. In summary, in this study we identified, for the first time, CTL directed against a lytic cycle product of EBV. Zta-specific CTL turned out to be restricted by one of two MHC class I molecules, either HLA-B8 or HLA-Cw6, and recognized an epitope within a 15-amino-acid-long sequence localized in the C-terminal part of the protein. A high proportion of individuals who are positive for HLA-B8 or HLA-Cw6 seem to be able to mount a cellular immune response against this protein, since the majority of our donors showing these MHC molecules developed CTL after in vitro stimulation of their PBL. Thus, in addition to CTL which eliminate EBV-positive cells expressing gene products of latency associated with the immortalized phenotype, there seems to be a second level of immunological control of EBV based on CTL-destroying B lymphocytes which are starting to produce virus. This could be a reason why free virus is very rarely detected in the serum samples of healthy EBV carriers. ACKNOWLEDGMENTS This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 217, project B3). We are grateful to Sabine Rosshuber, Andreas Reis, Fritz Schwarzmann, Hans-Helmut Niller, and Gerald Glaser for repeated blood donations. We thank Astrid Brunner for peptide synthesis, I. Kratochwill and K. Lackner (Institut fu ¨r Klinische Chemie, Klinikum der Universita¨t Regensburg) for HLA typing, and David Bradley for reading the manuscript. We are especially grateful to Alexander Steinle and Dolores Schendel (Institut fu ¨r Immunolgie, University of Munich) for much helpful discussion and for providing the Workshop cell lines as well as the HLA-Cw6 transfectant 721.221-Cw6. REFERENCES 1. Arvin, A. M., M. Sharp, S. Smith, C. M. Koropchak, P. S. Diaz, P. Kinchington, W. Ruyechan, and J. Hay. 1991. Equivalent recognition of a varizellazoster virus immediate early protein (IE62) and glycoprotein I by cytotoxic

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