Establishment and Maintenance of CTL Cultures.
Splenocytes were recovered 14 d after priming BALB/c mice with 5 x 10⁶ PFU of vaccinia virus, VVEB1 intravenously. Cells from a nonimmunized BALB/c spleen were coincubated with the V9L peptide (VYGGSKTSL, 1 µM for 1 h), washed extensively, and irradiated. These "stimulator" cells were cocultured with the "effector" spleen cells harvested from the immunized mouse, with 10⁶ stimulators and 2.0 x 10⁷ effectors in the absence of IL-2, in 10 ml. The cells were restimulated weekly by splitting the culture threefold and adding 2 x 10⁶ fresh peptide-pulsed stimulators and IL-2 (10 U/ml; Cetus Corp., Emoryville, CA).

Chromium Release Assays for Cytotoxicity.
⁵¹Cr release cytotoxicity assays were performed as described (10). For peptide-pulsed cells, cells were incubated with peptide (10 µM unless otherwise indicated) for 30 min in RPMI 10% FCS, and then washed thrice. Target cells prepared thus were added at 10,000 cells in 100 µl/well to 96-well plates. Calculations for specific lysis were as previously described (10).

Expression of EBNA-1 and EBNA-1(GA) by Western Blotting and Cell Staining.
Whole cell lysates were made by sonicating 5 x 10⁷ cells suspended in 0.5 ml of PBS, at 4°C, for 2 min. Laemmli sample buffer was added to the lysate, boiled for 2 min, and then loaded on an SDS gel. Lysates were transferred onto nitrocellulose membranes (HiBOND C; Amersham Corp., Arlington Heights, IL) for a total of 1 AmpHour and blocked overnight with 10% milk in PBS. Blots were incubated with the sera indicated, at a dilution of 1:1,000 in 5% nonfat milk for 1 h, washed extensively with PBS/0.2% Tween, and visualized with a horseradish peroxidase–conjugated secondary antibodies.

Cytospin preparations of acetone-fixed cells were incubated with primary antibody for 1 h, washed in PBS, incubated with biotinylated secondary antibodies (Vector Labs., Burlingame, CA) for 1 h, and incubated then with avidin–biotinylated peroxide complex for 40 min followed by reaction with diaminobenzedine/hydrogen peroxide. Cells were stained with 2% methyl green.

	Results

Top Abstract Materials and Methods Results Discussion References

 
The CTL Line V9L Recognizes an Epitope in the COOH-terminal Region of EBNA-1.
CTL against potential epitopes within EBNA-1 were raised using the protocol described in Fig. 1 a. "Candidate" epitopes, octa-, nona-, and decamers that matched the previously characterized consensus motifs for peptides eluted from common mouse MHC alleles (for K^b, K^d, K^k, L^d, and D^d) were identified (13). For instance, residues 509–517 (VYGGSKTSL), which contain a Tyrosine in position 2 and a Leucine in position 9, match the consensus motif for K^d-binding peptides (see Fig. 1 b).

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Figure 1 (a) Scheme for rescue of peptide-specific CTLs by coculturing immunized splenocytes with naive splenocytes pulsed with candidate peptides (see text). (1) Identification of candidate epitopes. (2) Construction of vaccinia virus expressing the EBNA-1 (505–583) fragment (called VVEB-1) containing the candidate epitopes. (3) Immunization of mice and recovery of CTLs. (b) Mouse strains initially immunized with VVEB-1 and the peptides used to restimulate them in vitro. (Column 1) Candidate epitopes derived from the COOH-terminal of EBNA-1 using consensus motifs described in reference 13. (Column 2) Class I molecule for which the candidate peptide in column 1 carries the consensus motif (and strains immunized with VVEB-1). For example, VYGGSKTSL contains a Kd motif (i.e., Y at position 2, and L at position 9, underlined). Splenocytes from BALB/c mice (H2-d) immunized with VVEB-1 were restimulated with splenocytes pulsed with the V9L peptide, as described in a. Cocultures restimulated on peptides (as in column 1) were used as effectors in a Cr–release assay using peptide-pulsed cells as targets (see below for the target cells). (Column 3) Specific lysis above background in 51Cr release assays 5 d after in vitro restimulation with peptide-pulsed cells, was obtained for each of the effector cultures. Target cells used: P815 for all H2-d, L929 for H2-k, and EL-4 for H2-b. (c) V9L CTLs are peptide specific and MHC restricted. P815 cells were pulsed with the peptide dose indicated (in nM) and used as targets in Cr–release assay. K/T ratio was 20:1. Unpulsed cells (0 nM) are not lysed. Hatched bar shows L/Db cells that were pulsed with V9L peptide; these cells do not express Kd and were not lysed. The x-axis represents P815 cells pulsed with peptide at concentration indicated (in nM) and used as target for V9L CTLs.

The EBNA-1 fragment was PCR amplified, with a start Methionine and a stop codon added (Fig. 1 a), and a vaccinia virus (VVEB1) encoding this fragment was created (see Materials and Methods). By DNA sequencing and PCR from the virus, we confirmed that the recombinant virus generated carried the appropriate fragment. Three strains of mice were immunized with a vaccinia virus encoding the EBNA-1 fragment, corresponding to residues 505–583 of EBNA-1 (of the prototypic B-95-8 sequence) (see Fig. 1 b for mouse genotypes and strains). To recover CTLs specific for EBNA-1–derived epitopes, splenocytes derived from the immunized mice (effectors) were cocultured with stimulators, autologous splenocytes pulsed with candidate peptide epitopes (as shown in Fig. 1 a) to restimulate effector cells that were specific for the candidate peptide.

To determine whether CTLs had been generated upon secondary restimulation, a ⁵¹Cr–release assay was used after 5 d of coculture of effectors with stimulators. ⁵¹Cr-labeled target cells expressing the relevant class I MHC molecule (Fig. 1 b) were pulsed with 100 nM of the candidate peptide and the effector cultures tested for the ability to lyse these peptide-pulsed targets. The level of lysis was compared against control targets, in which no peptide was used.

For eight of the nine candidates, no cytotoxicity above background on peptide-pulsed targets was observed (Fig. 1 b, column 3). The ninth candidate, the peptide VYGGSKTSL (V9L), containing a putative K^d-binding motif, showed lysis above background (Fig. 1 b, column 3).

All lines of CTLs were then grown for another 7 d on peptide-pulsed stimulators with IL-2 (100 U/ml) and tested again for cytotoxicity. Again, only the V9L line showed lysis above background. This CTL line grew continuously in culture with weekly restimulation with peptide-pulsed spleen cells supplemented with IL-2. In total, three BALB/c mice immunized at separate times with VVEB1 yielded a V9L-specific CTL response.

The V9L CTL line was unable to lyse K^d-expressing cells unless the V9L peptide was added exogeneously (i.e., it was peptide specific) and was unable to lyse cells that lacked the K^d MHC molecule even in the presence of peptide (i.e., MHC class I restricted) (Fig. 1 c). The dose of peptide required for the recognition of target cells by the V9L CTL line was determined. P815 cells were pulsed with varying doses of peptide (as indicated in Fig. 1 c), washed extensively to remove peptide, and used as targets in a ⁵¹Cr–release assay. The V9L CTL line was capable of recognizing cells pulsed with low doses of peptides (up to 0.005 nM).

Expression of EBNA-1 in Murine Cells.
To determine whether the V9L epitope was presented from murine cells expressing full-length EBNA-1, or EBNA-1 lacking the Gly-Ala region, K^d-expressing murine cells expressing full-length and Gly-Ala–deleted EBNA-1 were generated.

P815 (H2-d, mouse mammary mastocytoma cells) were transfected with an expression vector containing full-length EBNA-1, previously described in reference 7. P815 cells were also transfected with a vector containing an EBNA-1 construct, with the Glycine-Alanine rich region deleted, called EBNA-1, in which residues 93–325 have been deleted (Fig. 2 a and reference 9).

The expression of EBNA-1 and the Gly-Ala–deleted EBNA-1 (EBNA-1) in transfected murine cells were determined by Western blots and immunocytochemistry (Fig. 2, b and c). A rabbit (Rbt) polyclonal serum (11), Rbt--EBNA-1, raised against a fragment of EBNA-1 and an affinity-purified human serum, P-107, with reactivity against the Gly-Ala repeat only (12) were used in two separate Western Blots (Fig. 2, b i and ii). With the Rbt--EBNA-1 serum, a 72-kD band was detected in P815/ EBNA-1 cells, which comigrated with a 72-kD band detected in the EBV-transformed human 721 LCL line, used as a positive control. The 72-kD band was not present in P815 untransfected cells. In P815 cells transfected with EBNA-1 lacking the Gly-Ala repeat (P815/EBNA-1), the size of the mutant protein is expected to be between 40 and 43 kD. Indeed, a band of appropriate size (43 kD) was detected (Fig. 2 b, i). With the P-107 serum, which had been affinity purified to retain reactivity against the Gly-Ala repeat alone, a 72-kD band was detected in P815/ EBNA-1 cells, but no 43-kD band was detected in cells transfected with the Gly-Ala deleted construct.

The intracellular location of EBNA-1 in transfected cells was determined by immunohistochemistry, using the Rbt-EBNA-1 antibody. Cell staining demonstrated that both EBNA-1 and the Gly-Ala–deleted EBNA-1 was located primarily in the nucleus (Fig. 2 c).

The V9L Epitope Is Not Presented to CTLs by Cells Expressing Full-length EBNA-1, whereas Cells Expressing EBNA-1 Are Lysed.
⁵¹Cr–release assays were performed to determine whether the V9L epitope was being presented by murine cells expressing either the full-length EBNA-1, or EBNA-1 lacking the Gly-Ala–rich region. As targets, the transfected P815 cell lines expressing full-length EBNA-1 or EBNA-1 were used. The presence of the MHC restriction element of the V9L CTLs, K^d, in the target cells, had been previously confirmed by using these cells as targets for other K^d-restricted CTLs (not shown).

The results of a representative ⁵¹Cr–release assay using the murine cells appears in Fig. 3. Untransfected P815 cells, used as negative control, were not lysed by V9L CTLs. P815 cells pulsed with the V9L peptide were efficiently lysed. In three separate instances, the P815/EBNA-1 cells were not lysed above background. In contrast, P815/ EBNA-1 cells, which express EBNA-1 lacking the Gly-Ala–rich region, were lysed efficiently.

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Figure 3 V9L CTLs used as effectors in 51Cr–release assays. Targets: P815/EBNA-1 (open circles), P815/EBNA-1 (filled squares), P815 with no peptide (open triangles), and a P815 + V9L peptide pulsed at 10 µm (filled circles).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Polyclonal CTLs or CTL clones derived from EBV carriers by restimulation in vitro with autologous LCLs, are capable of lysing autologous cells expressing most EBV latency-associated antigens (4–6), but not cells expressing EBNA-1. Since several peptides from EBNA-1 are capable of binding human HLA alleles in vitro (15), the nonrecognition of EBNA-1–expressing cells was unlikely to be due to the absence of antigenic epitopes within this protein. Using EBNA-1–EBNA-4 chimeric molecules, Levitskaya et al. demonstrated that the Gly-Ala–rich region within EBNA-1 inhibited the generation of class I–restricted epitopes (9).

We have generated a novel, murine CTL line against an epitope within EBNA-1 itself, to study the processing and presentation of this protein without placing exogenous epitopes within EBNA-1 or creating chimeric molecules. This CTL line, called V9L, was recovered by immunizing mice with a vaccinia virus encoding a short EBNA-1 fragment, followed by restimulation in vitro using candidate epitopes, a method described in Fig. 1 a. The shortest COOH-terminal stretch of EBNA-1 where the largest number of putative epitopes could be identified was expressed through vaccinia and this virus was used to immunize mice.

Cells expressing full-length EBNA-1 were not lysed by the V9L CTL (Fig. 3), whereas cells expressing the Gly-Ala–deleted EBNA-1 (EBNA-1) were effectively lysed. The presentation of EBNA-1–derived epitopes has recently been examined using class I–restricted human CTL clones that recognize peptides from EBNA-1. Like the murine CTL described here, these CTLs were unable to lyse human cells expressing the full-length protein.

To investigate the means by which EBNA-1 is able to resist antigen processing and presentation, the intracellular locations of EBNA-1 and Gly-Ala–deleted EBNA-1 (EBNA-1) was examined by staining transfected P815 cells with the Rbt-EBNA-1 antiserum. P815/EBNA-1 cells and P815/ EBNA-1 cells were both stained prominently in the nucleus (Fig. 2 c). Previously, the nuclear localization sequence in EBNA-1 had been mapped to residues 379–387, outside the region deleted in EBNA-1 (17). Since EBNA-1 and EBNA-1 are both found in the nucleus, it is unlikely that the deletion of the Gly-Ala repeat exposes the protein to more efficient cytosolic proteolysis (18) and thus restores the presentation of the V9L epitope.

Interestingly, the Gly-Ala–rich region is not required for the replication function of EBNA-1, since vectors carrying the EBV-OriP can be maintained episomally in cells expressing the Gly-Ala–deleted EBNA-1. We speculate that this region may have evolved due to the selective advantage it confers on latently infected cells that are known to express EBNA-1 (but not EBNAs 2–6) by protecting them from CTL-mediated rejection.

Since only EBNA-1 is required for the maintenance of the EBV episome in latently infected cells (19), the ability of the Gly-Ala repeat to inhibit the presentation of epitopes derived from this protein may be related to the ability of EBV to maintain a persistent infection of B cells, in the face of a host immune response (2, 9). A subpopulation of latently infected B cells expressing only EBNA-1 (or EBNA-1 in conjunction with LMP-2) have been found in vivo (20). If epitopes derived from EBNA-1 are not presented efficiently, latently infected B cells, in which EBNA-1 is expressed, may be partially protected from elimination by host CTLs. This may allow the reservoir of latently infected cells to seed other cellular compartments, such as epithelial cells, where EBV can replicate and be transmitted. The fact that epitopes from EBNA-1 are not presented effectively to CTLs may also explain why EBV-positive Burkitt's lymphoma cells, which appear to express only EBNA-1 in vivo (21), can survive in immunocompetent hosts, although several other features in these cells may contribute to their lack of immunogenicity for CTLs (20, 22).