This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fogg, M. H.
Right arrow Articles by Wang, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fogg, M. H.
Right arrow Articles by Wang, F.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*(L)-ALANINE
*GLYCINE

 Previous Article  |  Next Article 

Journal of Virology, October 2005, p. 12681-12691, Vol. 79, No. 20
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.20.12681-12691.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The CD8+ T-Cell Response to an Epstein-Barr Virus-Related Gammaherpesvirus Infecting Rhesus Macaques Provides Evidence for Immune Evasion by the EBNA-1 Homologue

Mark H. Fogg,1 Amitinder Kaur,2 Young-Gyu Cho,1,{dagger} and Fred Wang1*

Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115,1 Department of Immunology, New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts 017712

Received 26 May 2005/ Accepted 22 July 2005


arrow
ABSTRACT
 
Epstein-Barr virus (EBV) infection persists for life in humans, similar to other gammaherpesviruses in the same lymphocryptovirus (LCV) genus that naturally infect Old World nonhuman primates. The specific immune elements required for control of EBV infection and potential immune evasion strategies essential for persistent EBV infection are not well defined. We evaluated the cellular immune response to latent infection proteins in rhesus macaques with naturally and experimentally acquired rhesus LCV (rhLCV) infection. RhLCV EBNA-1 (rhEBNA-1) was the most frequently targeted latent infection protein and induced the most robust responses by peripheral blood mononuclear cells tested ex vivo using the gamma interferon ELISPOT assay. In contrast, although in vitro stimulation and expansion of rhLCV-specific T lymphocytes demonstrated cytotoxic T-lymphocyte (CTL) activity against autologous rhLCV-infected B cells, rhEBNA-1-specific CTL activity could not be detected. rhEBNA-1 CTL epitopes were identified and demonstrated that rhEBNA-1-specific CTL were stimulated and expanded in vitro but did not lyse targets expressing rhEBNA-1. Similarly, rhEBNA-1-specific CTL clones were able to lyse targets pulsed with rhEBNA-1 peptides or expressing rhEBNA-1 deleted for the glycine-alanine repeat (GAR) but not full-length rhEBNA-1 or rhLCV-infected B cells. These studies show that the rhLCV-specific immune response to latent infection proteins is similar to the EBV response in humans, and a potential immune evasion mechanism for EBNA-1 has been conserved in rhLCV. Thus, the rhLCV animal model can be used to analyze the immune responses important for control of persistent LCV infection and the role of the EBNA-1 GAR for immune evasion in vivo.


arrow
INTRODUCTION
 
Epstein-Barr virus (EBV) is a gamma-1 herpesvirus that infects nearly all adult humans and maintains an asymptomatic, life-long latent virus infection in peripheral blood B cells (24). EBV infection can immortalize the growth of peripheral blood B cells in tissue culture, but in vivo the proliferation of EBV-infected B cells is controlled by the host immune response. The cellular immune response is believed to be important for control of EBV infection in vivo because T lymphocytes can prevent the outgrowth of EBV-infected B cells in tissue culture (18), immunosuppression associated with transplantation or human immunodeficiency virus infection can result in EBV-driven lymphoproliferation (6), and adoptive immunotherapy with EBV-stimulated T lymphocytes can be effective treatment for EBV-associated posttransplant lymphoproliferative syndrome (28).

The specificity and restriction of the T lymphocytes important for control of EBV infection in vivo remains to be determined. CD8+ cytotoxic T lymphocytes (CTL) specific for epitopes in the six latent infection nuclear proteins (EBNA-1, -2, -3A, -3B, -3C, and -LP) and three latent infection membrane proteins (LMP1, -2A, and -2B) have been detected in EBV-infected individuals (4, 9, 11, 19, 30) and are presumed to be important for control of persistent, latent EBV infection in vivo. CTL specific for epitopes in EBNA-3A, -3B, and -3C frequently dominate the repertoire of CTL specific for EBV latent infection proteins, and it is hypothesized that the immunodominance of the EBNA-3 response may also correlate with protection against latent EBV infection. The success of adoptive immunotherapy with in vitro expanded populations of EBV-specific T lymphocytes (28) that are frequently enriched for EBNA-3-specific CTL is consistent with this hypothesis, but does not exclude other important immune components that might be present in these therapeutic cell populations.

It is unclear why the EBV immune response and the repertoire of CTL fail to completely eliminate EBV infection. Down-regulation of latent EBV gene expression to an EBNA-1-restricted pattern of expression, i.e., type III to type I latency, may be one mechanism EBV uses to evade the immunodominant EBNA-3-specific CTL response (29). A more restricted pattern of latent infection gene expression may also be advantageous for the virus if EBNA-1-specific CTL do not effectively kill EBV infected B cells. The glycine-alanine repeat (GAR) domain of EBNA-1 inhibits proteosomal degradation of GAR containing proteins, i.e., in cis, and thereby inhibits antigen processing and presentation of EBNA-1 peptides to CD8+ CTL (13, 14). Thus, EBNA-1-specific CTL typically do not recognize and effectively kill EBV infected B cells endogenously expressing EBNA-1 in tissue culture, but this can be overcome by expressing EBNA-1 deleted for the GAR or coating EBV-infected B cells with EBNA-1 peptide. The potential importance for this EBNA-1 GAR-dependent mechanism of immune evasion has recently been challenged by three groups showing various degrees of EBNA-1-specific CTL recognition or killing of EBV-infected B cells in tissue culture, putatively resulting from presentation of EBNA-1 peptides from incompletely translated EBNA-1, or defective ribosomal products (12, 31, 32). Thus, it remains unclear to what extent persistent, latent EBV infection in humans is dependent on an EBNA-1 GAR-mediated mechanism for immune evasion in vivo.

EBV-related herpesviruses in the same lymphocryptovirus (LCV) genus infect most Old World nonhuman primate species and have a biology similar to EBV's. They immortalize simian B cells in tissue culture, they infect most of the population by adulthood, virus infection persists for life as a latent infection in the peripheral blood and as a lytic infection in the oropharynx, and virus-positive B cell lymphomas occur in immunosuppressed hosts (reviewed in reference 33). Experimental rhesus LCV (rhLCV) infection in immunocompetent and immunosuppressed rhesus macaques can reproduce the acute, persistent, and lymphomagenic phenotypes of EBV infection in humans (17, 25). The identical repertoire of lytic and latent infection viral genes in the rhLCV and EBV genomes provides important genetic validation that rhLCV is an accurate animal model for EBV infection (26). Not only is there a rhLCV homologue for every EBV gene, but the rhLCV homologue has also been able to functionally substitute for a given EBV gene in virtually every in vitro assay tested to date (7, 8, 10, 16, 20, 27, 35). Thus, rhLCV can provide a highly accurate biologic and genetic animal model for studying EBV pathogenesis.

The only instance where a rhLCV homologue has failed to reproduce EBV gene function in vitro has been the inability of the rhLCV EBNA-1 (rhEBNA-1) GAR to inhibit antigen presentation (5). In these experiments, a simian immunodeficiency virus (SIV) Gag peptide sequence recognized by a SIV Gag-specific CTL clone was cloned into the rhEBNA-1 gene. A similar approach, insertion of an EBV EBNA-3B CTL epitope into EBV EBNA-1, was originally used by Levitskaya et al. (13) to demonstrate that the EBNA-1 GAR could inhibit the processing and presentation of the EBNA-1-encoded EBNA-3B epitope to EBNA-3B-specific CTL, i.e., the EBNA-1 GAR inhibited antigen processing and presentation in cis when the CTL epitope and GAR were in the same molecule.

In contrast to the EBV EBNA-1 studies, the rhEBNA-1 protein containing the SIV Gag epitope was processed and the Gag epitope was efficiently presented to a Gag-specific rhesus CTL clone after expression in rhesus B cells by infection with recombinant vaccinia virus or in fibroblasts by infection with recombinant adenovirus. Thus, there was no evidence of a cis-inhibitory effect of the rhEBNA-1 GAR on antigen presentation in these studies with a heterologous CTL epitope expressed within a recombinant rhEBNA-1. These tissue culture assays with heterologous epitopes and recombinant proteins suggested that an EBNA-1 GAR-mediated immune evasion strategy might not have been conserved in rhesus macaques.

In the current study, we examined the repertoire of latent infection LCV proteins recognized by CTL in naturally and experimentally infected rhesus macaques as a model for better understanding the immune components important for control of EBV infection. The presence or absence of effective killing by rhEBNA-1-specific CTL might provide more direct evidence for whether an EBNA-1 GAR-mediated immune evasion strategy is important or essential for persistent LCV infection in vivo.


arrow
MATERIALS AND METHODS
 
Animals. Rhesus macaques (Macaca mulatta) enrolled in the study were housed at the New England Primate Research Center in accordance with institutional and federal guidelines of animal care (1). Nineteen rhesus macaques were randomly selected from the out-bred conventional colony and naturally acquired rhLCV infection was confirmed by serologic testing for viral capsid antibodies as previously described (23). Four LCV-naïve rhesus macaques randomly selected from the specific-pathogen-free colony were experimentally infected by oral inoculation with rhLCV 24 to 36 months prior to study (25). Persistent rhLCV infection after experimental inoculation was confirmed in these animals by reverse transcription-PCR detection of rhLCV EBERs (EBNA-encoded RNAs) expression in peripheral blood mononuclear cells (PBMC) and persistent viral capsid antibody titers.

Recombinant vaccinia viruses. Recombinant vaccinia viruses expressing the rhLCV latent infection proteins rhEBNA-1, -2, -3A, -3B, -3C and -LP, truncated rhLMP1 (amino acids 212 to 589) and rhLMP2A (amino acids 1 to 309) were generated as described (15). A recombinant vaccinia virus expressing a mutant rhEBNA-1 deleted for amino acids 41 to 270 (vvrhEBNA-1dGAR) was generated using plasmid templates as previously described (5).

rhEBNA-1 peptides. We synthesized 69 peptides (15-mers overlapping by 10) spanning the N-terminal 100 and C-terminal 260 residues of the rhEBNA-1 protein, i.e., excluding the GAR, by F-moc chemistry at the Massachusetts General Hospital peptide core facility (Charlestown, MA) using an automated peptide synthesizer (MBS 396, Advanced Chemtech, Louisville, KY). Lyophilized peptides were dissolved in dimethyl sulfoxide at a concentration of 100 mg/ml.

Isolation of peripheral blood mononuclear cells. PBMC were isolated from heparinized blood by density gradient centrifugation over Ficoll-Hypaque and suspended at 2 x 106 cells per ml in RPMI 1640 medium containing 2 mM L-glutamine, supplemented with 10% fetal bovine serum, and penicillin and streptomycin, both at 50 IU/ml (R-10 medium).

Generation of LCV-immortalized B-lymphoblastoid cell lines. LCV-immortalized B lymphoblastoid cell lines (LCL) were generated by incubating PBMC with viral supernatants and culturing the infected PBMC in R-10 medium with 0.5 µg/ml cyclosporine A at 37°C in a 5% CO2 incubator for 4 to 6 weeks. LCL were expanded and maintained in R-10 medium. Viral supernatants containing the baboon LCV (baLCV; cercopithicine herpesvirus 12) were derived from S594 cells (21), and viral supernatants containing the rhesus LCV (rhLCV; cercopithicine herpesvirus 15) were derived from LCL8664 cells (22).

Fractionation of T lymphocytes. CD4+ or CD8+ T lymphocytes were selectively depleted from PBMC by immunomagnetic selection (StemCell Technologies). Briefly, 107 PBMC in 1 ml of phosphate-buffered saline were incubated at room temperature for 15 min with 20 µl of either anti-dextran/anti-CD4 or anti-dextran/anti-CD8 tetrameric antibody cocktail followed by a 15-min incubation at room temperature with 60 µl of dextran coated magnetic nanoparticle colloid. The suspension was then passed through a 0.3-in. high-gradient magnetic column of stainless steel mesh resulting in the depletion of either CD4+ or CD8+ T lymphocytes from the flow through fraction which was used in subsequent experiments. Flow cytometry analysis demonstrated the removal of >95% of CD4+ or CD8+ T lymphocytes.

In vitro stimulation of rhLCV-specific CTL. To expand rhLCV-specific CTL, PBMC were stimulated weekly for 3 weeks with autologous, {gamma}-irradiated (100 Gy) rhLCV-LCL in R-10 medium at 37°C in a 5% CO2 incubator. Initial stimulation was at an effector-stimulator ratio of 50:1, followed by two restimulations at 30:1 in the presence of recombinant human interleukin-2 (kindly donated by M. Gately, Hoffmann-La Roche) at 10 IU/ml final concentration if required. Peptide-specific CTL were expanded by incubating one-third of PBMC with peptide for 1 h at 37°C to generate stimulator cells. After washing once in R-10 medium, stimulator cells were added to the remaining two thirds of PBMC in a 24-well plate; 5 to 10 IU/ml of recombinant human interleukin-2 was added to the cultures 4 to 7 days after stimulation and twice a week thereafter. The proportion of CD3+, CD4+ and CD8+ cells present following stimulation was determined by flow cytometry analysis. CTL assays were performed 14 days after antigen-specific stimulation.

rhEBNA-1-specific CD8+ T-lymphocyte CTL clones. Clones were derived from rhLCV-LCL stimulated cultures by limiting dilution after CD4+ T-lymphocyte depletion by immunomagnetic selection. CD4+ T-cell-depleted cultures were plated into 96-well U-bottomed plates in R-10 medium containing 50 IU/ml human interleukin-2 and 5 µg/ml concanavalin A. A total of 96 replicates containing 10, 3, and 1 cells/well with 2 x 105/ml {gamma}-irradiated (100 Gy) autologous rhLCV-LCL and 106/ml {gamma}-irradiated (30 Gy) human feeder PBMC were generated. Concanavalin A was removed after 3 to 4 days, and clones were subsequently maintained in culture with R-10 medium containing 50 IU/ml interleukin-2. Wells with growing cells were screened for antigen specificity by CTL assays using peptide-pulsed baLCV-LCL targets. rhEBNA-1-specific CTL clones were restimulated with autologous rhLCV-LCL and feeder PBMC every 2 weeks.

Enzyme linked immunospot assay for gamma interferon release. Gamma interferon (IFN-{gamma}) ELISPOT assays were carried out using a commercially available kit (MAbtech). PBMC or T-lymphocyte-depleted populations in R-10 medium were infected with recombinant vaccinia viruses at a multiplicity of infection of 10 PFU for 90 min at 37°C, and vaccinia virus-infected cell populations were then placed at various concentrations into a 96-well microtiter plate coated with anti-human IFN-{gamma} monoclonal antibody clone GZ-4. Alternatively, PBMC were incubated with rhEBNA-1 peptides at 1 µg/ml final concentration directly in the microtiter plate. Following overnight incubation, cells were removed by extensive washing with phosphate-buffered saline. Wells were serially incubated with the biotinylated anti-human IFN-{gamma} monoclonal antibody clone 7-B6-1 followed by streptavidin/alkaline phosphatase. Spots were visualized by the use of an alkaline phosphatase conjugation substrate kit (Bio-Rad, CA). Spots were counted by a KS ELISPOT Automated Reader (Carl Zeiss Inc., NY) using KS ELISPOT software 4.5 (Zellnet, N.J.). Results for antigen-specific responses were calculated by subtracting background values of cells infected with control vaccinia virus or dimethyl sulfoxide with no peptide and reported as spot-forming cells (SFC) per 106 cells. ELISPOT assays using in vitro-stimulated T-lymphocyte cultures were carried out essentially as described above with the exception that dilutions of effector cells began at 2 x 104 per well and target cells were 104 autologous baLCV-LCL infected overnight with recombinant vaccinia viruses.

Chromium release assays. Target cells consisted of autologous rhLCV-LCL or autologous baLCV-LCL infected overnight with recombinant vaccinia viruses expressing individual rhLCV proteins (multiplicity of infection, 10 PFU) or pulsed with individual rhEBNA-1 peptides. Target cells were labeled with 51chromium (50 µCi) for 1 h at 37°C and were dispensed at 104 cells/well with effector cells at various effector-to-target cell (E:T) ratios in 96-well U-bottomed plates for 5 h at 37°C. Plates were spun at 1,000 rpm for 10 min at 4°C, 30 µl of supernatant was harvested from each well onto wells of a LumaPlate-96 (Becton Dickinson, CA), and the amount of 51Cr released into the supernatant was assayed in a 1450 MicroBeta Plus liquid scintillation counter (Wallac, Finland). Spontaneous lysis was calculated by the incubation of targets in R-10 medium alone, and maximal lysis was determined by the addition of 5% Triton X-100 (Sigma). The % lysis was calculated by [(test cpm -spontaneous cpm)/(max cpm -spontaneous cpm)] x 100. Spontaneous release of target cells was <25% in all assays.

Interferon ELISA. IFN-{gamma} enzyme-linked immunosorbent assays (ELISAs) were carried out using a commercially available kit from MAbtech. Briefly, 104 rhEBNA-1-specific CTL clones were incubated in wells of a 96-well plate with target cells at a ratio of 2:1 in 200 µl of R-10 medium containing 10 IU/ml IL-2. After 48 h, supernatants were harvested and 100 µl was assayed for the presence of IFN-{gamma} by ELISA by incubation in wells of a 96-well plate coated with the anti-human IFN-{gamma} monoclonal antibody clone GZ-4. After 1 h, wells were extensively washed with phosphate-buffered saline and serially incubated with biotinylated anti-human IFN-{gamma} monoclonal antibody clone 7-B6-1 followed by streptavidin/alkaline phosphatase. Peroxidase activity was measured by addition of O-phenylenediamine dihydrochloride (Sigma-Aldrich). The absorbance was measured at 450 nm after 30 min using a Bio-Rad microplate reader. Standards of a known concentration were used to establish a standard curve from which the concentration of IFN-{gamma} in unknown samples was calculated.


arrow
RESULTS
 
Repertoire of latent infection rhLCV proteins recognized by ex vivo analysis of PBMC from naturally and experimentally infected rhesus macaques. Twenty-three healthy, rhLCV-seropositive rhesus macaques at the New England Primate Research Center were studied. Nineteen randomly selected animals had been raised in the conventional colony and had acquired rhLCV infection naturally. Four LCV-naïve animals were raised in a specific-pathogen-free colony and had been experimentally infected by oral inoculation with rhLCV 2 to 3 years prior to analysis (25).

Recombinant vaccinia viruses expressing full-length rhEBNA-1, -2, -3A, -3B, -3C, and -LP were constructed. Full-length rhLMP1 and rhLMP2A could not be effectively expressed from recombinant vaccinia viruses, so vaccinia viruses expressing a truncated rhLMP1 (amino acids 212 to 589) and rhLMP2A (amino acids 1 to 309) were used for these studies.

Ex vivo T-lymphocyte responses to specific rhLCV proteins were detected by IFN-{gamma} ELISPOT analysis of PBMC incubated overnight with different recombinant vaccinia viruses. Responses were calculated as the number of IFN-{gamma} spot-forming cells per million PBMC (SFC/106 PBMC) minus background control values (PBMC infected with vector control vaccinia virus). Responses greater than 50 SFC/106 PBMC and at least twice the number of spots compared to those obtained with vector control vaccinia virus were considered positive. Six rhLCV-seronegative animals had no significant ex vivo IFN-{gamma} ELISPOT response to any of the rhLCV latent infection proteins, consistent with their rhLCV-naïve status. In contrast, 17 of 23 rhLCV-seropositive animals had a significant ex vivo IFN-{gamma} ELISPOT response to at least one rhLCV latent infection protein (Table 1), and each of the rhLCV proteins was recognized by at least one animal among the cohort of rhLCV-seropositive animals.


View this table:
[in this window]
[in a new window]
  		TABLE 1. IFN-{gamma} ELISPOT responses (SFC/106 PBMC) to rhLCV latent infection proteins by ex vivo analysis of PBMC from 23 rhLCV-seropositive macaquesb

The repertoire of rhLCV proteins recognized by a given animal varied. For example, rhEBNA-1, -3B, and -3A stimulated a significant IFN-{gamma} ELISPOT response in PBMC from Mm144-97, with the strongest responses to rhEBNA-1 followed by rhEBNA-3B and -3A (240, 145, and 96 SFC/106 PBMC, respectively) (Table 1). A negative response in ex vivo testing of PBMC was consistent with either an absent response or a precursor frequency of responding T lymphocytes below the level of detection without in vitro stimulation and expansion. The average number of rhLCV latent proteins recognized among seropositive animals was 1.56 (range, 0 to 6; Table 1), and there was no significant difference between the responses in naturally and experimentally infected animals.

The rhLCV latent infection protein most often recognized among seropositive animals was rhEBNA-1 (11 of 23 seropositive animals, 48%; Fig. 1A). The rest of the rhLCV latent proteins were recognized by less than a quarter of the animals (Fig. 1A). There was no obvious dominance of a single rhEBNA-3 protein in this cohort as rhEBNA-LP was the next most frequently recognized rhLCV latent protein followed by rhLMP2A and rhEBNA-3C. rhEBNA-1 responses were not only the most frequent, but also represented the T-lymphocyte response with the greatest magnitude. In every animal recognizing rhEBNA-1, the rhEBNA-1 response was greater than the response to any other latent infection protein (Table 1), and in the 11 rhEBNA-1 responders the average number of spot-forming cells was 208 SFC/106 PBMC (Fig. 1B). The next most robust average response was to rhEBNA-3C and rhEBNA-LP (average, 152 and 114 SFC/106 PBMC, respectively).



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 1. IFN-{gamma} ELISPOT responses to individual rhLCV latent proteins by ex vivo analysis of PBMC from rhLCV-seropositive rhesus macaques. (A) Number of animals with a positive IFN-{gamma} ELISPOT response to a specific rhLCV latent infection protein by ex vivo PBMC analysis from a total of 23 rhLCV-seropositive macaques. (B) Average magnitude of IFN-{gamma} ELISPOT responses to a specific rhLCV latent infection protein by ex vivo analysis of PBMC. Values represent averages of positive responses to a given latent infection protein.

In order to determine whether IFN-{gamma} ELISPOT responses present in PBMC ex vivo were from CD4+ or CD8+ T lymphocytes, we selectively depleted these T-lymphocyte subsets from PBMC prior to analysis. As shown in Fig. 2, depletion of CD8+ T lymphocytes abrogated the IFN-{gamma} ELISPOT response to rhEBNA-1 by PBMC from Mm141-97 and Mm144-97, whereas depletion of CD4+ T lymphocytes had little or no effect. In 5 additional animals, CD4+ T-lymphocyte depletion had no significant effect on the ex vivo IFN-{gamma} ELISPOT response to rhEBNA-1 consistent with a CD8+ T-cell mediated response (data not shown). Similarly, CD8+ T-lymphocyte depletion eliminated the IFN-{gamma} ELISPOT response to rhEBNA-2 observed with PBMC from Mm309-98 whereas CD4+ T-lymphocyte depletion had no effect (Fig. 2). Using recombinant vaccinia viruses to express rhLCV proteins should bias responses towards CD8+ T cells, and the depletion studies indicate that these ex vivo responses to rhLCV latent infection proteins were dominated by CD8+ T-cell responses. However, the presence of some CD4+ T-cell responses cannot be ruled out.



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 2. CD8+ T-lymphocyte-restricted IFN-{gamma} ELISPOT responses to rhLCV latent infection proteins by ex vivo analysis of PBMC. Unfractionated, CD4+ or CD8+ T-lymphocyte-depleted PBMC from three rhesus macaques (Mm141-97, Mm144-97, and Mm309-98) were screened by overnight infection with recombinant vaccinia viruses expressing (A) rhEBNA-1 or (B) rhEBNA-2. IFN-{gamma} ELISPOT responses after subtraction of negative control values from cultures infected with vector control vaccinia viruses are shown.

In vitro stimulation and expansion of rhLCV latent protein-specific CTL. In order to test for cytolytic activity, rhLCV-specific T lymphocytes were expanded in vitro by stimulation with irradiated, autologous rhLCV immortalized B cells (rhLCV-LCL) for three weeks. The expanded cell lines were predominantly T lymphocytes (>85% of cells were CD3+) of which between 87 to 95% were CD8+ and 3 to 11% were CD4+ (data not shown). The expanded polyclonal T-lymphocyte lines were used as effectors against rhLCV-LCL targets in chromium release assays. In vitro-stimulated T-lymphocyte lines effectively lysed autologous rhLCV-LCL targets (Fig. 3, solid symbols). In order to test whether the effector cells were specific for rhLCV antigens and not cell-derived antigens, the T-lymphocyte lines were tested against B cells from the same animal immortalized with the baboon LCV (baLCV) as a rhLCV negative, major histocompatibility complex class I identical target.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 3. Cytolytic activity of T-lymphocyte lines after in vitro stimulation with rhLCV-LCL. PBMC from four rhesus macaques (Mm127-86, squares; Mm141-97, circles; Mm161-85, diamonds; and Mm309-98, triangles) were stimulated in vitro for 3 weeks using autologous rhLCV-LCL. The cytotoxicity of the T-lymphocyte line against autologous rhLCV-LCL targets (solid lines and symbols) or baLCV-LCL targets (dashed lines and open symbols) was determined in a 5-h chromium release assay. Results are presented as the percent lysis at the indicated effector-to-target ratio (E:T).

BaLCV is useful for generating autologous negative control target cells because baLCV is able to immortalize rhesus macaque B cells and rhLCV-specific CTL are unlikely to recognize baLCV latent infection proteins because of significant sequence divergence between rhLCV and baLCV latent infection genes. The effectiveness of autologous baLCV-LCL as negative control targets was demonstrated by the low-level or absent lysis of baLCV-LCL targets incubated with rhLCV-LCL-stimulated T-lymphocyte lines from four rhesus macaques (Fig. 3, open symbols). These results suggested that polyclonal T-lymphocyte lines expanded in vitro by stimulation with autologous rhLCV-LCL contained CTL specific for rhLCV latent infection proteins.

In order to determine which rhLCV latent infection proteins were recognized by the CTL lines, autologous baLCV-LCL infected with vaccinia viruses expressing either vector control or single rhLCV latent infection proteins were used as targets in chromium release assays. Polyclonal CTL lines were derived from eight rhesus macaques (Table 2), and lines from all animals effectively lysed autologous rhLCV-LCL targets (Table 2, rhLCV-LCL). CTL lines from five animals lysed baLCV-LCL targets expressing rhEBNA-3C, four lines lysed targets expressing rhEBNA-2, and three lines lysed targets expressing rhEBNA-3A or 3B. None of the CTL lines lysed targets expressing rhEBNA-1, rhEBNA-LP, rhLMP1, or rhLMP2.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Cytolytic activity of rhLCV-LCL-stimulated T-lymphocyte lines against autologous rhLCV-LCL targets or autologous baLCV-LCL targets expressing individual rhLCV latent infection proteins

In the case of rhLMP1 and rhLMP2, none of the animals studied had a detectable IFN-{gamma} ELISPOT response by ex vivo analysis of PBMC, and only one animal had an rhEBNA-LP response based on ex vivo IFN-{gamma} ELISPOT assays (Mm309-98, Tables 1 and 2). In the case of rhEBNA-2 and the rhEBNA-3s, there were a total of seven ex vivo IFN-{gamma} ELISPOT responses in PBMC from these animals, and five out of seven targets were recapitulated by the in vitro-stimulated CTL lines (Table 2, italic values). Nine of the cytotoxic responses to rhEBNA-2 and rhEBNA-3-expressing targets by in vitro-stimulated CTL were not predicted from ex vivo IFN-{gamma} ELISPOT assays. The detection of rhLCV-specific responses in the cytotoxic assays but not in ex vivo IFN-{gamma} ELISPOT assays was consistent with a precursor frequency below the level of detection in IFN-{gamma} ELISPOT assays and increased detection sensitivity after in vitro stimulation and expansion.

Five animals in Table 2 were predicted to have rhEBNA-1 responses based on ex vivo PBMC testing. The lack of rhEBNA-1-specific cytolytic activity by in vitro-stimulated CTL lines in chromium release assays was in marked contrast to the dominant rhEBNA-1 ex vivo response detected by IFN-{gamma} ELISPOT analysis. In order to determine whether the lack of rhEBNA-1-specific cytotoxic responses was due to the lack of in vitro expansion of the rhEBNA-1-specific T lymphocytes, a defect in cytolytic activity, or a defect in rhEBNA-1 presentation by the target cell, IFN-{gamma} ELISPOT analysis was performed on the in vitro-stimulated T-lymphocyte lines from three animals using baLCV-LCL infected with recombinant vaccinia viruses as targets. In the case of rhEBNA-1, there was neither significant cytolytic activity nor IFN-{gamma} ELISPOT response in any of the three CTL lines (Fig. 4A, B, and C) even though all three animals had significant rhEBNA-1 responses in PBMC ex vivo. This was in contrast to the other latent infection proteins, where strong cytolytic activity was associated with a significant IFN-{gamma} ELISPOT response in most instances (e.g., rhEBNA-2 in Fig. 4A, rhEBNA-3B in Fig. 4B, and rhEBNA-3A and -3B in Fig. 4C). These results suggested that rhEBNA-1-specific T lymphocytes either had not been expanded in vitro by stimulation with autologous rhLCV-LCL or were present but unable to generate an effective cytotoxic or IFN-{gamma} response against rhEBNA-1-expressing target cells.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 4. Comparison of cytolytic and IFN-{gamma}-secreting activity of in vitro-stimulated T-lymphocyte lines. T-lymphocyte lines from three animals (A: Mm211-98, B: Mm161-85, and C: Mm141-97) were tested for cytotoxic activity by chromium release assay (solid bars, left axis) or IFN-{gamma} ELISPOT response (lines, right axis) using baLCV-LCL targets infected with recombinant vaccinia viruses expressing individual rhLCV latent infection proteins. Results are presented as the percent specific lysis at E:T ratios ranging from 30:1 to 16:1. Nonradioactive infected targets were used in an overnight IFN-{gamma} ELISPOT assay, and the number of spot-forming cells per 106 T lymphocytes following subtraction of ELISPOT responses with baLCV-LCL targets infected with control vaccinia viruses is shown.

Identification of CD8+ T-lymphocyte epitopes within the rhEBNA-1 protein. In order to develop a more sensitive means of detecting and stimulating rhEBNA-1-specific CD8+ T lymphocytes, we identified regions containing rhEBNA-1 epitopes recognized by rhesus CD8+ T lymphocytes in two animals. We synthesized a series of overlapping 15-mer peptides spanning the entire unique sequence of the rhEBNA-1 protein and assembled the individual peptides into 17 pools containing seven, eight, or nine peptides each. The pools were constructed such that each peptide was uniquely represented in two different pools, thereby allowing easy identification of individual peptides, i.e., positive results from two pools would identify the target since only one peptide would be common to both pools. PBMC from one experimentally (Mm141-97) and one naturally infected (Mm543-91) rhesus macaque, who were known to have an ex vivo IFN-{gamma} rhEBNA-1 response, were incubated overnight with the peptide pools in IFN-{gamma} ELISPOT assays. Both animals had a predominant positive response to two peptide pools each (data not shown), and the identity of the pools suggested that PBMC from Mm141-97 recognized an epitope contained within peptide 67 (amino acids 331 to 345, GGRGFKKFENMAKNL) and that PBMC from Mm543 to 91 recognized an epitope contained within peptide 82 (amino acids 406 to 420, PGPQPGPMRESTDCY).

CD4+ and CD8+ T-lymphocyte-depleted PBMC were then screened by IFN-{gamma} ELISPOT assays with these individual peptides. Unfractionated PBMC from Mm141-97 and Mm543-91 recognized peptides 67 and 82, respectively, confirming that these rhEBNA-1 peptides contained the epitopes recognized in the rhEBNA-1 peptide pools (Fig. 5). In addition, the IFN-{gamma} ELISPOT response to the respective peptides was ablated from both animals when CD8+ T lymphocytes were depleted and increased slightly when CD8+ T lymphocytes were enriched (Fig. 5). When the IFN-{gamma} ELISPOT response in PBMC from Mm141-97 to peptide 67 was compared with the response to a pool containing all of the rhEBNA-1 peptides, the number of spot-forming cells per 106 PBMC to peptide 67 was equal to or greater than the response to all rhEBNA-1 peptides (Fig. 5A). There was no CD4+ T-lymphocyte response to peptide 67 or the whole pool of rhEBNA-1 peptides, suggesting that a CD8+ T-lymphocyte epitope within peptide 67 was the predominant target of the rhEBNA-1 response in this animal.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 5. rhEBNA-1 peptide-specific IFN-{gamma} ELISPOT responses in PBMC, CD4+ and CD8+ T-lymphocyte-enriched populations. Peptide 67 and peptide 82 were identified as potential rhEBNA-1 epitope-containing peptides for Mm141-97 (peptide 67) and Mm543-91 (peptide 82) as described in the text. Unfractionated, CD4+ or CD8+ T-lymphocyte-depleted PBMC from (A) Mm141-97 and (B) Mm543-91 were stimulated overnight with a pool of all rhEBNA-1 peptides, the specified rhEBNA-1 peptide, or dimethyl sulfoxide only in medium and assayed by IFN-{gamma} ELISPOT. Results are shown as the total number of spot-forming cells per 106 PBMC with no background subtraction.

In Mm543-91 peptide 82 was a dominant target of the CD8+ T-lymphocyte response, but there was also a significant response using the whole peptide pool with CD4+-enriched T lymphocytes (Fig. 5B), suggesting the presence of CD4+ T-lymphocyte epitopes within the subdominant, positive peptide pools. In summary, epitope containing peptides representing the dominant rhEBNA-1-specific CD8+ T-lymphocyte response from two rhLCV-infected animals were defined.

In vitro expansion of rhEBNA-1-specific CD8+ T lymphocytes using rhEBNA-1 peptides. The rhEBNA-1 peptides 67 and 82 were used to stimulate and expand T lymphocytes from PBMC of Mm141-97 and Mm543-91, respectively, in order to determine whether rhEBNA-1-specific CD8+ T lymphocytes were capable of recognizing and killing rhLCV-LCL. Peptide 67- and 82-specific T-lymphocyte lines were generated by a 2-week in vitro stimulation with the respective rhEBNA-1 peptide. The lines were then tested for their ability to lyse autologous rhLCV-LCL targets in a chromium release assay. As shown in Fig. 6, neither peptide 67 (Fig. 6A) nor peptide 82 (Fig. 6B)-specific T-lymphocyte lines lysed autologous rhLCV-LCL (Fig. 6, open triangles) even though these T-lymphocyte populations had cytolytic activity against peptide-pulsed, autologous baLCV-LCL (Fig. 6, solid squares) or peptide-pulsed autologous rhLCV-LCL (Fig. 6, solid triangles) targets. The ability of the peptide-stimulated rhEBNA-1-specific CTL lines to lyse peptide-loaded targets but not rhLCV-LCL endogenously expressing rhEBNA-1 suggested a defect in antigen presentation of endogenous rhEBNA-1 by the rhLCV-LCL targets.



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 6. Cytolytic activity of rhEBNA-1 peptide-stimulated CTL lines. CTL lines from (A) Mm141-97 and (B) Mm543-91 derived by in vitro stimulation of PBMC with rhEBNA-1 peptide 67 and 82, respectively, for 2 weeks were tested for cytolytic activity against baLCV-LCL (squares) or rhLCV-LCL (triangles) targets pulsed with either peptide (solid symbols) or dimethyl sulfoxide alone (open symbols). Results are presented as percent lysis at the E:T ratios shown.

rhEBNA-1 peptide-specific CTL are present in polyclonal T-lymphocyte lines stimulated in vitro with autologous rhLCV-LCL. We used the rhEBNA-1 epitope containing peptides to revisit the question of whether rhEBNA-1-specific T lymphocytes were absent versus present, but undetectable in rhLCV-LCL-stimulated T-lymphocyte lines. As previously seen, the CTL line established from Mm141-97 PBMC by in vitro stimulation for 3 weeks with autologous rhLCV-LCL had no cytolytic activity against autologous baLCV-LCL targets alone (Fig. 7, open square), infected with control vaccinia virus (Fig. 7, open circle), or infected with rhEBNA-1-expressing vaccinia virus (Fig. 7, solid circle). However, when autologous baLCV-LCL targets were pulsed with peptide 67, the rhLCV-LCL-stimulated CTL line efficiently lysed peptide-pulsed targets (Fig. 7, solid square) indicating that rhEBNA-1-specific CTL were present in the T-lymphocyte line and were capable of cytotoxic activity. Thus, the lack of cytotoxic activity against rhEBNA-1-expressing targets was most likely due to a defect in antigen presentation of endogenously expressed rhEBNA-1 in the target cells.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 7. Cytolytic activity of rhLCV-LCL-stimulated CTL lines against targets pulsed with rhEBNA-1 peptide or expressing wild-type or GAR-deleted rhEBNA-1. The rhLCV-LCL-stimulated CTL line from Mm141-97 was tested for cytolytic activity against baLCV-LCL targets with (solid squares) and without (open squares) exposure to peptide 67 or after infection with vaccinia virus expressing full-length rhEBNA-1 (solid circles), control vaccinia virus (open circles), or a GAR-deleted rhEBNA-1 (shaded circles). Representative results from one of three experiments are presented as the percent lysis at the E:T ratios shown.

Since this phenotype was similar to the inhibition of antigen presentation associated with the EBV EBNA-1 GAR, we tested whether expression of a GAR-deleted rhEBNA-1 would result in lysis of target cells expressing the mutant rhEBNA-1. The rhLCV-stimulated CTL line showed modest but significantly more lysis of baLCV-LCL targets infected with the GAR-deleted rhEBNA-1 vaccinia virus (Fig. 7, shaded circle) versus wild-type rhEBNA-1 vaccinia virus (Fig. 7, solid circle), indicating that a domain within the rhEBNA-1 deletion was capable of inhibiting processing and presentation of rhEBNA-1 peptide epitopes in cis. The functional similarity to the EBV EBNA-1 GAR strongly implicates the rhEBNA-1 GAR as the domain responsible for the cis-acting inhibition of antigen presentation.

rhEBNA-1-specific CD8+ T-lymphocyte clones do not lyse rhEBNA-1 expressing cells but can lyse cells expressing GAR-deleted rhEBNA-1. Since the rhLCV-LCL-stimulated CTL lines were polyclonal, we could not test the activity of the rhEBNA-1-specific CTL against the autologous rhLCV-LCL without first generating rhEBNA-1-specific CTL clones, i.e., CTL specific for other latent infection antigens such as rhEBNA-2 and rhEBNA-3s would lyse the targets. rhEBNA1-specific CTL clones were generated by CD4+ T-lymphocyte depletion and limiting dilution of rhLCV-LCL-stimulated T-lymphocyte lines from Mm141-97. These clones were generated using only rhLCV-LCL for stimulation and were not stimulated with rhEBNA-1 peptides. CTL clones were screened for cytolytic activity against rhEBNA-1 peptide 67-pulsed baLCV-LCL, and eight rhEBNA-1-specific clones were identified (two representative clones shown in Fig. 8, solid triangle).



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 8. Cytolytic and IFN-{gamma} secreting activity of rhEBNA-1-specific CTL clones against rhLCV-LCL. (A) Two representative rhEBNA-1-specific CTL clones from Mm141-97 are shown (left and right panels). Targets used in the 5-h chromium release assay were rhLCV-LCL (squares), baLCV-LCL with (solid triangles) and without (open triangles) peptide 67, and baLCV-LCL infected with recombinant vaccinia viruses expressing either full-length rhEBNA-1 (open circles) or GAR-deleted rhEBNA-1 (solid circles). Representative results from one of three experiments are presented as the percent lysis at the E:T ratios shown. (B) Identical, nonradioactive targets were used in an overnight IFN-{gamma} assay at an E:T ratio of 1:1, and supernatants were analyzed for the presence of IFN-{gamma} by ELISA. Results represent the mean of duplicate cultures.

All rhEBNA-1-specific clones were unable to lyse autologous rhLCV-LCL (Fig. 8, open square), but were able to recognize and lyse peptide-pulsed rhLCV-LCL (data not shown). Similarly, all clones failed to lyse autologous baLCV-LCL expressing rhEBNA-1 after recombinant vaccinia virus infection (Fig. 8, open circle). There was modest but significant lysis of baLCV-LCL expressing a GAR-deleted rhEBNA-1 (Fig. 8, solid circle) by all clones, indicating that the intermediate lysis of the GAR-deleted rhEBNA-1 targets compared to peptide-loaded targets was a uniform property of the rhEBNA-1-specific CTL. Thus, the autologous rhLCV-LCL can stimulate the expansion of rhEBNA-1-specific T lymphocytes in vitro, but these rhEBNA-1-specific T lymphocytes are unable to lyse the autologous rhLCV-LCL in vitro, consistent with immune evasion through a rhEBNA-1 GAR-mediated inhibition of antigen presentation in cis.

We tested the rhEBNA-1 CTL clones for increased IFN-{gamma} production in the supernatants, since Lee et al. (12) recently described EBV EBNA-1-specific CTL that could respond to full-length EBNA-1 expressing targets by increasing IFN-{gamma} production in the absence of cytolytic activity. Negative control supernatants from two rhEBNA-1-specific CTL clones cocultured with baLCV-LCL contained no detectable IFN-{gamma}, whereas positive control supernatants from CTL clones cultured with peptide 67-loaded baLCV-LCL contained 600 pg/ml IFN-{gamma} (Fig. 8b). Supernatants from wells containing baLCV-LCL infected with GAR-deleted rhEBNA-1 expressing vaccinia viruses contained detectable, but 10-fold lower levels of IFN-{gamma} (50 pg/ml), consistent with the intermediate cytotoxic activity against GAR-deleted rhEBNA-1 versus peptide-loaded target cells. However, there was no detectable IFN-{gamma} in supernatants from wells containing rhLCV-LCL or baLCV-LCL expressing wild-type rhEBNA-1, suggesting that in these studies, the rhEBNA-1 GAR inhibition of antigen presentation extends to IFN-{gamma} production as well as cytotoxic responses of rhEBNA-1-specific CTL.


arrow
DISCUSSION
 
We set out to characterize the CD8+ T-lymphocyte response to rhLCV infection in rhesus macaques to better understand the repertoire of immune responses important for controlling LCV infection and the immune evasion mechanisms that might contribute to persistent LCV infection. We analyzed the immune responses to individual rhLCV latent infection proteins using two different, but complementary assays. The IFN-{gamma} ELISPOT assay provides direct quantitation of CD8+ T-lymphocyte responses in the peripheral blood without bias from in vitro stimulation and expansion. However, low-frequency responses may not be detected, functional killing of virus-infected cells is not demonstrated, and without depletion or blocking studies, the contribution of CD4+ T lymphocytes to the response cannot be excluded. The presence of a vaccinia virus-encoded soluble IFN-{gamma} receptor could also potentially reduce the ELISPOT response by competing for the detection of IFN-{gamma}. In vitro stimulation and expansion of LCV-specific T lymphocytes provides an opportunity to assess killing of virus infected cells using the chromium release assay and can enhance detection of CTL present at a lower precursor frequency, but may introduce bias by selecting for cells that most successfully expand in tissue culture. Therefore, we felt that the use of both approaches were complementary and important for this initial evaluation of the rhLCV cellular immune response.

The repertoire of CD8+ T lymphocytes to rhLCV latent infection proteins appeared broader compared to the more focused human CD8+ T-lymphocyte response to EBV EBNA-3 latent proteins (9, 11, 19, 30), although the use of slightly different assays for rhesus and human responses makes direct comparisons difficult. Ex vivo IFN-{gamma} ELISPOT analysis of rhesus PBMC demonstrated a broad response directed towards all latent infection rhLCV proteins, with rhEBNA-1, rhEBNA-3C, rhEBNA-LP, and rhLMP2 being recognized most frequently by this cohort of animals. In vitro stimulation of rhLCV-specific T lymphocytes resulted in an enrichment for rhEBNA-3 responses, with rhEBNA-3C/rhEBNA-2/rhEBNA-3A, and -3B becoming the most frequently recognized latent infection proteins. Thus, the type of assay used to assess CD8+ T-lymphocyte activity can have a significant impact on the interpretation of which targets "dominate" the response.

Both assays suggest that rhEBNA-3C is frequently targeted by CD8+ T lymphocytes in infected rhesus macaques, but individually the responses to rhEBNA-3A, -B, and -3C are not strikingly greater than responses to other latent infection proteins such as rhLMP2 or rhEBNA-LP. Thus, we conclude that all three rhEBNA-3s are immunogenic in LCV-infected rhesus macaques, and rhEBNA-3C-specific CTL are frequently present at various precursor frequencies in infected animals. However, a variety of other latent infection proteins were also recognized in many animals so that there was no obvious immunodominance by the rhEBNA-3 proteins

The current results also show that targets expressing full-length rhEBNA-1 are not effectively killed by rhEBNA-1-specific CTL, but expression of a GAR-deleted rhEBNA-1 allows more efficient processing and presentation of rhEBNA-1 peptide epitopes. rhLCV-LCL-stimulated T-lymphocyte lines failed to elicit a cytotoxic or IFN-{gamma} ELISPOT response in assays using targets expressing full-length rhEBNA-1. The mapping of-specific rhEBNA-1 epitopes from two animals showed that rhEBNA-1-specific T lymphocytes were present in the rhLCV-stimulated cultures and that these rhEBNA-1-specific T lymphocytes had cytotoxic activity. Similarly, rhEBNA-1-specific T-lymphocyte clones retained cytotoxic activity against peptide-loaded targets, but failed to lyse targets expressing full-length rhEBNA-1 and, most importantly rhLCV-infected targets. The ability to lyse targets expressing a GAR-deleted rhEBNA-1 indicated that a cis-acting block in rhEBNA-1 antigen presentation was operative in rhLCV-infected B cells, similar to EBV-infected B cells.

It is not clear why rhEBNA-1 can block presentation of native rhEBNA-1 peptide epitopes, but failed to block presentation of a heterologous SIV epitope inserted into a recombinant rhEBNA-1 (5). It is possible that overexpression of the SIV Gag/rhEBNA-1 chimera by recombinant vaccinia virus or adenovirus infection overwhelmed the inhibitory activity of the rhEBNA-1 GAR. Since the rhEBNA-1 GAR is much shorter than the EBNA-1 GAR, seven glycine/alanine-rich repeats over 47 amino acids versus 84 G1-3A repeats over 252 amino acids, it may not be as potent an inhibitor of antigen presentation as the EBNA-1 GAR. In any case, the rhEBNA-1 expression level in an rhLCV-LCL is probably the most relevant in vitro scenario, and the current studies indicate that a cis-acting block in antigen presentation has been conserved in rhLCV.

The high precursor frequency of rhEBNA-1-specific T lymphocytes detected by ex vivo analysis of PBMC was also striking: 48% of seropositive animals had IFN-{gamma} ELISPOT responses in PBMC infected with rhEBNA-1 vaccinia virus, and rhEBNA-1 also elicited the most potent IFN-{gamma} response in PBMC among all latent infection proteins. In contrast, no IFN-{gamma} ELISPOT response to rhEBNA-1 vaccinia virus-infected targets was detected in rhLCV-stimulated CTL lines, despite the presence of rhEBNA-1-specific CTL documented by the use of rhEBNA-1 peptides. This difference may be explained by the presence of professional antigen presenting cells in PBMC that were subsequently lost in the stimulated CTL lines. The professional antigen-presenting cells in PBMC may lead to the detection of rhEBNA-1-specific IFN-{gamma} responses through the uptake of exogenous antigen, i.e., a cross-presentation mechanism similar to the cross-priming mechanism proposed for generating EBV EBNA-1-specific CTL (3).

The high precursor frequency of rhEBNA-1-specific T lymphocytes in PBMC is not inconsistent with a biologically significant GAR-mediated inhibition of antigen presentation. The high precursor frequency indicates efficient in vivo priming of naïve T lymphocytes and their subsequent differentiation into effector cells. This priming in vivo probably occurs by dendritic cells processing and presenting exogenously derived rhEBNA-1 from dead or dying rhLCV-infected cells. The rhEBNA-1 GAR may not significantly influence the priming of rhEBNA-1-specific T lymphocytes in vivo because the inhibitory activity of the GAR is not believed to have a major effect on the ability of dendritic cells to process and present exogenous EBV EBNA-1 to both naïve T lymphocytes (2) and EBNA-1-specific CTL clones (3).

The high precursor frequency of rhEBNA-1-specific T lymphocytes in persistently infected macaques may also be due in part to expansion and maintenance of memory CTL. This may be due to continued stimulation from cross-presentation of exogenous rhEBNA-1 from dying cells, where the GAR may exert little influence. Alternatively, there could be direct peptide presentation by rhLCV-infected B cells, where the GAR could be inhibiting efficient processing of rhEBNA-1. In this case, small amounts of rhEBNA-1 peptides sufficient for T-cell stimulation may be derived from full-length rhEBNA-1 or come from defective ribosomal products (28). Whether from cross- or direct presentation, the maintenance of a high precursor frequency in persistently infected animals suggests the presence of continued stimulation of rhEBNA-1-specific T lymphocytes in vivo.

The key question is whether there is sufficient rhEBNA-1 peptide presentation by target cells in vivo to activate effector function, i.e., killing of the rhLCV-infected cell. The GAR may be important for preventing sufficient peptide presentation to activate CTL killing in vivo, but sufficient peptide presentation for T-lymphocyte stimulation may still occur by the mechanisms described above. The ability to present sufficient peptides for stimulation but not killing was reproduced in these studies by the rhLCV-LCL-stimulated CTL lines and clones, where rhEBNA-1 CTL were expanded and capable of lysing peptide loaded targets but were unable to lyse targets expressing full-length rhEBNA-1. Functional conservation of this potential immune evasion mechanism in rhLCV suggests that GAR-mediated inhibition of antigen presentation is biologically important for the virus.

How does this scenario for the rhEBNA-1 GAR and rhEBNA-1 CTL compare to EBV infection in humans? The rhEBNA-1 block for antigen presentation in cis is comparable to the classic paradigm for EBNA-1 GAR-mediated immune evasion. However, this model has recently been challenged by three different observations suggesting that EBV EBNA-1-specific CTL may recognize EBV-infected B cells more effectively than previously believed. Voo et al. (32) identified an EBNA-1-specific CTL clone that had modest killing activity. Lee et al. (12) found that even though their EBNA-1-specific CTL clones failed to lyse EBV-infected B cells, they responded with increased IFN-{gamma} secretion and could control the outgrowth of EBV-infected cells. Finally, Tellam et al. (31) reported that they were able to frequently and reproducibly stimulate EBNA-1-specific CTL that could lyse EBV-infected B cells. Thus, the degree of EBNA-1 immune evasion evidenced in vitro remains an evolving story.

All three groups suggested that newly synthesized protein was required for EBNA-1 recognition through defective ribosomal products and not stable, long-lived EBNA-1 protein. This model still implicates an inhibitory effect of the EBNA-1 GAR on antigen presentation of wild type EBNA-1 protein, and the critical question is what mechanisms are operative in vivo. Defective ribosomal products may be a source for presentation of EBNA-1 peptides to various degrees in actively growing EBV- or rhLCV-infected B cells in tissue culture, but whether this mechanism is limited to tissue culture and whether EBNA-1 GAR-mediated immune evasion is required for persistent EBV infection in vivo remain to be determined.

The precursor frequency of EBNA-1-specific CTL in EBV-infected humans is also not well established, since ex vivo responses have not been extensively studied. Subklewe et al. (30) reported that only 2 of 13 random donors had a significant IFN-{gamma} ELISPOT response when PBMC were exposed to a recombinant vaccinia virus expressing EBNA-1-deleted for the GAR, suggesting that the frequency of EBNA-1 recognition in a random population is low. However, HLA type can have a strong influence, as shown by Blake et al. (3), where PBMC from 15 out of 15 HLA-B*3501 individuals had an ex vivo IFN-{gamma} ELISPOT response to an HLA-B*3501 restricted EBNA-1 epitope. Thus, it remains to be determined whether the frequency and prevalence of EBNA-1-specific CD8+ T lymphocytes in the peripheral blood is truly higher in macaques than that reported for randomly selected humans.

The conservation of a potential immune evasion mechanism in the rhEBNA-1 GAR resurrects the question of whether the ability to evade cytolysis by EBNA-1-specific CTL is fundamentally important for persistent LCV infection in human and non human primate hosts. The fact that rhEBNA-1 peptides are not efficiently presented by rhLCV-infected cells provides a rationale for constructing a mutant rhLCV with an rhEBNA-1 deleted for the GAR to determine its effect on LCV infection in vivo. If GAR-mediated immune evasion is required for persistent infection, the host immune response may be able to successfully eliminate this mutant virus after experimental infection. On the other hand, the GAR may be important for providing stable, long-lived EBNA-1 in latently infected cells. Potential effects of the GAR on EBNA-1 stability and translation may impact episomal maintenance in vivo despite the lack of such an effect in vitro, thereby complicating the interpretation of why a mutant virus containing a GAR-deleted rhEBNA-1 may not persist. Determining whether the mutant virus is able to establish persistent infection in the setting of immunosuppression may be useful for differentiating a dominant effect of the GAR on immune evasion versus episomal maintenance in vivo.


arrow
ACKNOWLEDGMENTS
 
This work was supported by grants from the U.S. Public Health Service (DE14388 and CA68051). Services from the New England Primate Research Center were supported by a base grant to the institution (USPHS P51RR00168).

We thank Ashok Khatri for assistance with peptide synthesis, Therion Inc. for providing the recombinant vaccinia virus vector and assistance with the construction of recombinant vaccinia viruses, Angela Carville for expert assistance with animal resources, and Corrina Hale and Deirdre Garry for technical assistance.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Channing Laboratory, 181 Longwood Ave., Boston, MA 02115. Phone: (617) 525-4258. Fax: (617) 525-4257. E-mail:fwang{at}rics.bwh.harvard.edu. Back

{dagger} Present address: Catholic Research Institutes of Medical Science, Catholic University of Korea, 505 Banpo-Dong, Seocho-Ku, Seoul, 137-701, Korea. Back


arrow
REFERENCES
 
    1
  1. Anonymous. 1996. Guide for the Care and Use of Laboratory Animals. p. 86-123. The Institute of Laboratory Animal Resources, National Research Council, Washington, D.C.
    2. 2
  2. Bickham, K., K. Goodman, C. Paludan, S. Nikiforow, M. L. Tsang, R. M. Steinman, and C. Munz. 2003. Dendritic cells initiate immune control of Epstein-Barr virus transformation of B lymphocytes in vitro. J. Exp. Med. 198:1653-1663.[Abstract/Free Full Text]
    3. 3
  3. Blake, N., T. Haigh, G. Shaka'a, D. Croom-Carter, and A. Rickinson. 2000. The importance of exogenous antigen in priming the human CD8+ T-cell response: lessons from the EBV nuclear antigen EBNA1. J. Immunol. 165:7078-7087.[Abstract/Free Full Text]
    4. 4
  4. Blake, N., S. Lee, I. Redchenko, W. Thomas, N. Steven, A. Leese, P. Steigerwald-Mullen, M. G. Kurilla, L. Frappier, and A. Rickinson. 1997. Hum. CD8+ T-cell responses to EBV EBNA1: HLA class I presentation of the (Gly-Ala)-containing protein requires exogenous processing. Immunity 7:791-802.[CrossRef][Medline]
    5. 5
  5. Blake, N. W., A. Moghaddam, P. Rao, A. Kaur, R. Glickman, Y. G. Cho, A. Marchini, T. Haigh, R. P. Johnson, A. B. Rickinson, and F. Wang. 1999. Inhibition of antigen presentation by the glycine/alanine repeat domain is not conserved in simian homologues of Epstein-Barr virus nuclear antigen 1. J. Virol. 73:7381-7389.[Abstract/Free Full Text]
    6. 6
  6. Cleary, M. L., R. F. Dorfman, and J. Sklar. 1986. Failure in immunological control of the virus infection: post-transplant lymphomas, p. 163-181. In M. A. Epstein and B. G. Achong (ed.), The Epstein-Barr virus: recent advances. Heinemann Medical Books, London, England.
    7. 7
  7. Franken, M., O. Devergne, M. Rosenzweig, B. Annis, E. Kieff, and F. Wang. 1996. Comparative analysis identifies conserved tumor necrosis factor receptor-associated factor 3 binding sites in the human and simian Epstein-Barr virus oncogene LMP1. J. Virol. 70:7819-7826.[Abstract]
    8. 8
  8. Fuentes-Panana, E. M., S. Swaminathan, and P. D. Ling. 1999. Transcriptional activation signals found in the Epstein-Barr virus (EBV) latency C promoter are conserved in the latency C promoter sequences from baboon and rhesus monkey EBV-like lymphocryptoviruses (cercopithicine herpesviruses 12 and 15). J. Virol. 73:826-833.[Abstract/Free Full Text]
    9. 9
  9. Gavioli, R., P. O. De Campos-Lima, M. G. Kurilla, E. Kieff, G. Klein, and M. G. Masucci. 1992. Recognition of the Epstein-Barr virus-encoded nuclear antigens EBNA-4 and EBNA-6 by HLA-A11-restricted cytotoxic T lymphocytes: implications for down-regulation of HLA-A11 in Burkitt lymphoma. Proc. Natl. Acad. Sci. USA 89:5862-5866.[Abstract/Free Full Text]
    10. 10
  10. Jiang, H., Y. G. Cho, and F. Wang. 2000. Structural, functional, and genetic comparisons of Epstein-Barr virus nuclear antigen 3A, 3B, and 3C homologues encoded by the rhesus lymphocryptovirus. J. Virol. 74:5921-5932.[Abstract/Free Full Text]
    11. 11
  11. Khanna, R., S. R. Burrows, M. G. Kurilla, C. A. Jacob, I. S. Misko, T. B. Sculley, E. Kieff, and D. J. Moss. 1992. Localization of Epstein-Barr virus cytotoxic T-cell epitopes using recombinant vaccinia: implications for vaccine development. J. Exp. Med. 176:169-176.[Abstract/Free Full Text]
    12. 12
  12. Lee, S. P., J. M. Brooks, H. Al-Jarrah, W. A. Thomas, T. A. Haigh, G. S. Taylor, S. Humme, A. Schepers, W. Hammerschmidt, J. L. Yates, A. B. Rickinson, and N. W. Blake. 2004. CD8 T-cell recognition of endogenously expressed Epstein-Barr virus nuclear antigen 1. J. Exp. Med. 199:1409-1420.[Abstract/Free Full Text]
    13. 13
  13. Levitskaya, J., M. Coram, V. Levitsky, S. Imreh, P. M. Steigerwald-Mullen, G. Klein, M. G. Kurilla, and M. G. Masucci. 1995. Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1. Nature 375:685-688.[CrossRef][Medline]
    14. 14
  14. Levitskaya, J., A. Sharipo, A. Leonchiks, A. Ciechanover, and M. G. Masucci. 1997. 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.[Abstract/Free Full Text]
    15. 15
  15. Mazzara, G. P., A. Destree, and A. Mahr. 1993. Generation and analysis of vaccinia virus recombinants. Methods Enzymol. 217:557-581.[Medline]
    16. 16
  16. McCann, E. M., G. L. Kelly, A. B. Rickinson, and A. I. Bell. 2001. Genetic analysis of the Epstein-Barr virus-coded leader protein EBNA-LP as a co-activator of EBNA2 function. J. Gen. Virol. 82:3067-3079.[Abstract/Free Full Text]
    17. 17
  17. Moghaddam, A., M. Rosenzweig, D. Lee-Parritz, B. Annis, R. P. Johnson, and F. Wang. 1997. An animal model for acute and persistent Epstein-Barr virus infection. Science 276:2030-2033.[Abstract/Free Full Text]
    18. 18
  18. Moss, D. J., A. B. Rickinson, and J. H. Pope. 1978. Long-term T-cell-mediated immunity to Epstein-Barr virus in man. I. Complete regression of virus-induced transformation in cultures of seropositive donor leukocytes. Int. J. Cancer 22:662-668.[Medline]
    19. 19
  19. Murray, R. J., M. G. Kurilla, J. M. Brooks, W. A. Thomas, M. Rowe, E. Kieff, and A. B. Rickinson. 1992. 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.[Abstract/Free Full Text]
    20. 20
  20. Peng, R., A. V. Gordadze, E. M. Fuentes Panana, F. Wang, J. Zong, G. S. Hayward, J. Tan, and P. D. Ling. 2000. Sequence and functional analysis of EBNA-LP and EBNA2 proteins from nonhuman primate lymphocryptoviruses. J. Virol. 74:379-389.[Abstract/Free Full Text]
    21. 21
  21. Rabin, H., R. H. Neubauer, R. F. Hopkins, 3rd, and M. Nonoyama. 1978. Further characterization of a herpesvirus-positive orang-utan cell line and comparative aspects of in vitro transformation with lymphotropic old world primate herpesviruses. Int. J. Cancer 21:762-767.[Medline]
    22. 22
  22. Rangan, S. R., L. N. Martin, B. E. Bozelka, N. Wang, and B. J. Gormus. 1986. Epstein-Barr virus-related herpesvirus from a rhesus monkey (Macaca mulatta) with malignant lymphoma. Int. J. Cancer 38:425-432.[Medline]
    23. 23
  23. Rao, P., H. Jiang, and F. Wang. 2000. Cloning of the rhesus lymphocryptovirus viral capsid antigen and Epstein-Barr virus-encoded small RNA homologues and use in diagnosis of acute and persistent infections. J. Clin. Microbiol. 38:3219-3225.[Abstract/Free Full Text]
    24. 24
  24. Rickinson, A., and E. Kieff. 2001. Epstein-Barr virus, p. 2575-2627. In D. M. Knipe and P. M. Howley (ed.), Fields virology, 4th ed., vol. 2. Lippincott Williams and Wilkins, Philadelphia, Pa.
    25. 25
  25. Rivailler, P., A. Carville, A. Kaur, P. Rao, C. Quink, J. L. Kutok, S. Westmoreland, S. Klumpp, M. Simon, J. C. Aster, and F. Wang. 2004. Experimental rhesus lymphocryptovirus infection in immunosuppressed macaques: an animal model for Epstein-Barr virus pathogenesis in the immunosuppressed host. Blood 104:1482-1489.[Abstract/Free Full Text]
    26. 26
  26. Rivailler, P., H. Jiang, Y. G. Cho, C. Quink, and F. Wang. 2002. Complete nucleotide sequence of the rhesus lymphocryptovirus: genetic validation for an Epstein-Barr virus animal model. J. Virol. 76:421-426.[Abstract/Free Full Text]
    27. 27
  27. Rivailler, P., C. Quink, and F. Wang. 1999. Strong selective pressure for evolution of an Epstein-Barr virus LMP2B homologue in the rhesus lymphocryptovirus. J. Virol. 73:8867-8872.[Abstract/Free Full Text]
    28. 28
  28. Rooney, C. M., C. A. Smith, C. Y. Ng, S. K. Loftin, J. W. Sixbey, Y. Gan, D. K. Srivastava, L. C. Bowman, R. A. Krance, M. K. Brenner, and H. E. Heslop. 1998. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 92:1549-1555.[Abstract/Free Full Text]
    29. 29
  29. Rowe, M., D. T. Rowe, C. D. Gregory, L. S. Young, P. J. Farrell, H. Rupani, and A. B. Rickinson. 1987. Differences in B cell growth phenotype reflect novel patterns of Epstein-Barr virus latent gene expression in Burkitt's lymphoma cells. EMBO J. 6:2743-2751.[Medline]
    30. 30
  30. Subklewe, M., A. Chahroudi, K. Bickham, M. Larsson, M. G. Kurilla, N. Bhardwaj, and R. M. Steinman. 1999. Presentation of Epstein-Barr virus latency antigens to CD8+, interferon-gamma-secreting, T lymphocytes. Eur. J. Immunol. 29:3995-4001.[CrossRef][Medline]
    31. 31
  31. Tellam, J., G. Connolly, K. J. Green, J. J. Miles, D. J. Moss, S. R. Burrows, and R. Khanna. 2004. Endogenous presentation of CD8+ T-cell epitopes from Epstein-Barr virus-encoded nuclear antigen 1. J. Exp. Med. 199:1421-1431.[Abstract/Free Full Text]
    32. 32
  32. Voo, K. S., T. Fu, H. Y. Wang, J. Tellam, H. E. Heslop, M. K. Brenner, C. M. Rooney, and R. F. Wang. 2004. Evidence for the presentation of major histocompatibility complex class I-restricted Epstein-Barr virus nuclear antigen 1 peptides to CD8+ T lymphocytes. J. Exp. Med. 199:459-470.[Abstract/Free Full Text]
    33. 33
  33. Wang, F., P. Rivailler, P. Rao, and Y. Cho. 2001. Simian homologues of Epstein-Barr virus. Phil. Trans. R. Soc. London B Biol. Sci. 356:489-497.[Abstract/Free Full Text]
    34. 34
  34. Yewdell, J. W., L. C. Anton, and J. R. Bennink. 1996. Defective ribosomal products (DRiPs): a major source of antigenic peptides for MHC class I molecules? J. Immunol. 157:1823-1826.[Abstract]
    35. 35
  35. Zhao, B., R. Dalbies-Tran, H. Jiang, I. K. Ruf, J. T. Sample, F. Wang, and C. E. Sample. 2003. Transcriptional regulatory properties of Epstein-Barr virus nuclear antigen 3C are conserved in simian lymphocryptoviruses. J. Virol. 77:5639-5648.[Abstract/Free Full Text]

Journal of Virology, October 2005, p. 12681-12691, Vol. 79, No. 20
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.20.12681-12691.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Strowig, T., Gurer, C., Ploss, A., Liu, Y.-F., Arrey, F., Sashihara, J., Koo, G., Rice, C. M., Young, J. W., Chadburn, A., Cohen, J. I., Munz, C. (2009). Priming of protective T cell responses against virus-induced tumors in mice with human immune system components. JEM 206: 1423-1434 [Abstract] [Full Text]  
  • Song, Y.-J., Izumi, K. M., Shinners, N. P., Gewurz, B. E., Kieff, E. (2008). IRF7 activation by Epstein-Barr virus latent membrane protein 1 requires localization at activation sites and TRAF6, but not TRAF2 or TRAF3. Proc. Natl. Acad. Sci. USA 105: 18448-18453 [Abstract] [Full Text]  
  • Fogg, M. H., Garry, D., Awad, A., Wang, F., Kaur, A. (2006). The BZLF1 Homolog of an Epstein-Barr-Related {gamma}-Herpesvirus Is a Frequent Target of the CTL Response in Persistently Infected Rhesus Macaques. J. Immunol. 176: 3391-3401 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fogg, M. H.
Right arrow Articles by Wang, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fogg, M. H.
Right arrow Articles by Wang, F.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*(L)-ALANINE
*GLYCINE

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»