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The Journal of Immunology, 2006, 176: 923-930.
Copyright © 2006 by The American Association of Immunologists

Identification of CD8+ T Cell Epitopes within Lytic Antigens of Human Herpes Virus 81

Eliana Ribechini*, Cinzia Fortini*, Mauro Marastoni{dagger}, Serena Traniello*, Susanna Spisani*, Paolo Monini{ddagger} and Riccardo Gavioli2,*

* Department of Biochemistry and Molecular Biology and {dagger} Department of Pharmaceutical Sciences, University of Ferrara, Ferrara, Italy; and {ddagger} Laboratory of Virology, Istituto Superiore di Sanità, Roma, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The human herpesvirus 8 (HHV-8) is a {gamma} herpesvirus with oncogenic potential which establishes a chronic infection that is normally controlled by the immune system of healthy individuals. In particular, CTL responses seem to play a key role in control of the infection. In this study, we characterized epitope-specific CTL responses in healthy HHV-8-seropositive individuals against four HHV-8 lytic Ags: open reading frames (ORF) 26, 70, K3, and K5. We found that the majority of subjects responded to at least one HHV-8 lytic Ag-derived epitope, and some of these epitopes represented dominant targets, suggesting that they could be relevant targets of CTL-mediated immunity in vivo, and may be involved in host control of HHV-8. Specifically, we identified three CTL epitopes from ORF 26, which are presented by HLA-A2, six CTL epitopes from ORF 70 presented by HLA-A2 (three epitopes), -A24 (two epitopes), and -B7 (one epitope), three CTL epitopes from ORF K3 presented by HLA-A2 (two epitopes) and -B7 (one epitope), and one HLA-A2 presented epitope derived from ORF K5. The identified epitopes may be regarded as useful tools for understanding the role of CTL responses to lytic Ags in individuals affected by HHV-8-associated disorders, and for the development of immunotherapies for the treatment/prevention of HHV-8-associated malignancies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human herpesvirus 8 (HHV-8,3 also called Kaposi’s sarcoma (KS)-associated herpesvirus) is a member of the {gamma}2-herpesvirus family (1), and is involved in the pathogenesis of KS, primary effusion lymphomas (PEL), and the immunoblastic variant of Castleman’s disease (2, 3). As with other herpesviruses, such as EBV, HHV-8 can establish a chronic infection which is normally controlled by the immune system of healthy individuals. Cellular immune responses are a critical part of the host defense against herpesviruses, with CTLs playing a key role in recognizing and eliminating infected cells (4). CTLs recognize their target Ags as 8- to 10-aa-long peptides which are derived from intracellular degradation of viral Ags and presented by MHC class I (MHC-I) molecules expressed at the surface of infected cells (5, 6, 7).

The central role of HHV-8-specific CTLs is highlighted by the fact that loss of or decrease in CTL responses, as observed in HIV-infected individuals or organ transplant recipients, results in the development of HHV-8-associated tumors. The presence of CTL responses against both latent and lytic HHV-8 proteins has recently been characterized (8, 9, 10, 11) but, thus far, only a few immunogenic CTL epitopes have been identified within these Ags (12, 13, 14, 15, 16). At present, the role the identified epitope-specific CTL responses play in control of HHV-8-infected cells or HHV-8-positive tumors is not known. Further studies are required to identify protective epitope-specific CTL responses against HHV-8 to characterize new CTL epitopes and determine the presence of epitope-specific responses in carriers or subjects at risk of developing HHV-8-associated tumors.

In this study, to identify novel KS herpesvirus-specific CTL target epitopes, we tested, in healthy HHV-8-infected subjects, CD8+ T cell reactivity to peptides derived from four HHV-8 lytic proteins: open reading frame (ORF) 26 (minor capsid protein), ORF 70 (thymidylate synthases), ORF K3 (MIR I), and ORF K5 (MIR II). In particular, we analyzed CTL responses restricted by three different HLA alleles: A2, A24, and B7. Potential HLA class I-binding peptides were selected using the peptide-binding motif. HLA-A2, the most common HLA class I molecule in humans, presents leucine (L) as the dominant residue at position 2 and valine (V), which prevails at the C terminus (17). The anchor residues for HLA-A24-binding peptides are tyrosine (Y) in position 2 and isoleucine (I) at the C terminus (17); for the B7 allele, dominant residues are proline (P) in position 2 and leucin (L) in position 9 (17). Selected peptides were used to reactivate in vitro cytotoxic T cell responses from HHV-8-seropositive donors. By this approach, we identified and characterized several new CTL epitopes from ORFs 26, 70, K3, and K5 which may be useful in evaluating the presence and role of CTL responses in patients carrying HHV-8-associated malignancies and/or for the development of CTL-based immunotherapeutic strategies against HHV-8-associated tumors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines

The .174/T2 cell line (T2) was obtained by fusion of the peptide transporter mutant .174 LCL with the T cell line CEM (18). T2 cells and PEL cell lines (BC-3 and BCBL-1) were maintained in RPMI 1640 supplemented with 2 mM glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated FCS (HyClone). To induce HHV-8 lytic replication, PEL cell lines were treated for 48 h with 20 ng of 12-O-tetradecanoylphorbol-13-acetate (TPA)/ml (Sigma-Aldrich).

PHA-activated blasts were obtained by stimulation of PBLs with 1 µg/ml purified PHA (Wellcome Diagnostics) for 3 days and expanded in medium supplemented with human rIL-2 (Proleukin; Chiron) as described (19).

Subjects

PBLs from healthy, HHV-8-seropositive individuals were obtained from the Blood Bank of S. Anna Hospital (Ferrara, Italy) and all individuals were HLA-typed by serology (Table I).


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  		Table I. Healthy HHV8-seropositive individuals included in this study

 
Serum Ab titers to latent and lytic Ags of HHV-8 were determined by using two immunofluorescence assays based on BCBL-1 cells, as previously described (20, 21). For immunofluorescence assay of anti-lytic Ags, TPA-induced BCBL-1 cells were used, whereas immunofluorescence assay of anti-latent Ags was performed with isolated nuclei of untreated BCBL-1 cells to avoid aspecific cytosolic signals (20, 21). TPA-induced cells and isolated nuclei were smeared on slides, fixed with a methanol/acetone (1:2; vol/vol) solution, and preblocked by incubation with PBS supplemented with 3% FCS, followed by two incubation steps of 60 min, each at 37°C with diluted (1/10) patient serum and goat fluorescein-conjugated isothiocyanate anti-human Abs, respectively. Titrations were conducted by 4-fold serial dilutions. All microscopic examinations were evaluated in a blinded fashion.

Synthetic peptides

The 9- and 10-mer peptide sequences were obtained using a prediction model which ranks peptides based on a predicted half-life of dissociation to HLA-A2, -A24, and -B7 (Table II). All peptides were synthesized by solid phase method using a continuous flow instrument with online UV monitoring. The stepwise syntheses were conducted by F-moc chemistry as previously described (22). Crude deprotected peptides were purified by HPLC; purity was >98%. Structure verification was achieved by elemental and amino acid analyses and mass spectrometry. Peptide stocks were prepared in DMSO at 10–2 M concentration, kept at –20°C, and diluted in PBS before use.


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  		Table II. HHV-8 lytic proteins and predicted HLA class I-binding peptides

 
Detection of HLA-A2/peptide complex stability

Aliquots of 1 x 106 T2 cells were treated overnight at 26°C in 1 ml of AIM-V medium (Invitrogen Life Technologies) containing increasing concentrations of synthetic peptides (from 10–8 to 10–4 M). After washing, cells were incubated at 37°C for an additional 4 h. Surface expression of HLA class I molecules was detected by indirect immunofluorescence, using MA2.1 mAb specific for HLA-A2 molecules. Mean logarithm fluorescence intensity was determined by FACS analysis (BD Biosciences). The percentage increase in HLA class I expression was calculated over the expression found in untreated T2 cells (23).

Preparation of APCs

T2 cells (2 x 106) were cultured overnight at 26°C in 1 ml of serum-free AIM-V medium. Cells were then washed, treated with mitomycin C to avoid cell proliferation, and pulsed with 10–5 M of the different peptides for 3 h at 37°C in AIM-V medium. After extensive washing, the cells were used as stimulators (24).

Generation of memory CTL cultures

Monocyte-depleted PBLs from HHV-8-seropositive subjects were plated at 3 x 106 cells per well in 24-well plates in RPMI 1640 containing 10% FCS (HyClone) and stimulated with HLA-A2-restricted peptide-pulsed T2 cells at a stimulator-responder ratio of 1:20 or with HLA-A24 and -B7-restricted peptides alone (10 µM). Cultures were restimulated after 7 and 14 days, and the medium was supplemented from day 8 with 10 U/ml rIL-2 (Chiron). On days 14 and 21, T cell cultures were tested for CTL activity using cytotoxicity or ELISPOT assays (25).

Cytotoxicity tests

Cytotoxic activity was tested by a standard 5-h 51Cr-release assay, as previously described (26). Briefly, target cells were labeled with 0.1 µCi/106 cells of Na251CrO4 for 90 min at 37°C and, where indicated, pulsed for 45 min with 10–6 M of the different peptides at 37°C. Cells were then washed, and 4 x 103 cells were used as targets of each CTL at different E:T ratios. Percentage-specific lysis was calculated as 100 x ((cpm sample – cpm medium)/(cpm Triton X-100 – cpm medium)). Spontaneous release was always <20%. None of the tested peptides affected spontaneous release.

ELISPOT assay

To assess production of IFN-{gamma}, nitrocellulose 96-well plates (Whatman) were coated with 10 µg/ml anti-IFN-{gamma} mAb (Pierce) overnight at 4°C. The following day, the plates were washed four times with PBS and then blocked with RPMI 1640 supplemented with 10% FBS for 45 min at 37°C.

CTLs (1 x 105) were added to the wells, stimulated with synthetic peptides, and incubated for 24 h at 37°C. The plates were then washed four times with PBS and four times with PBS containing 0.05% Tween 20, and the secondary biotinylated anti-IFN-{gamma} Ab (Pierce) was added at a concentration of 1 µg/ml for 1 h at 37°C. After washing, streptavidin-conjugated alkaline phosphatase was added at room temperature for 45 min.

The spots were developed using an AEC kit (3-Amino-9-Ethylcarbazole Staining kit; Sigma-Aldrich) for 15 min and counted using an ELISPOT reader (Aelvis). The results were expressed as the number of spots per 106 CTLs minus the value of the negative control, which was always below 40 spots per million CTLs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Selection of HLA-A2, -A24, and -B7-binding peptides derived from HHV-8 lytic proteins

To identify potential CTL epitopes within the HHV-8 lytic Ag ORFs 26, 70, K3, and K5, the amino acid sequences of these proteins were analyzed by a computer program designed to predict HLA-A2, -A24, and -B7-binding peptides (27). This prediction model allows location and ranking of peptides which contain putative peptide-binding motifs for HLA class I molecules, based on an estimation of the half-time dissociation of the HLA-peptide complex. Because the stability of MHC-I/peptide complexes plays an important role in determining CTL responsiveness (25, 26, 28, 29), potential CTL epitopes with high scores were selected. This approach is known to be useful for the identification of new CTL epitopes, although this does not necessarily exclude the fact that other peptides not selected by this approach may represent CTL epitopes.

Selected peptides (Table II) were synthesized and purified (purity >98%), and potential HLA-A2 binders were also tested for HLA-A2 binding (Table II), using their capacity to stabilize HLA-A2 molecules at the cell surface of the mutant T2 cell line. The HLA-A2-restricted EBV-derived CLGCLLTMV (CLG) peptide (30) was used as a positive control in all experiments. Peptides stabilizing HLA-A2 molecules at 10–5 M similarly to the poor HLA-A2 binding CLG epitope were selected as a potential CTL epitope.

Induction of CTL memory responses by selected HHV-8 lytic protein-derived peptides

To determine the immunogenicity of the selected peptides, their capacity to generate epitope-specific CTL cultures was tested. To this end, PBLs obtained from healthy HLA class I-typed, HHV-8-seropositive subjects were stimulated with peptides or with peptide-pulsed T2 cells according to their HLA class I molecules. In particular, HLA-A2-restricted CTL cultures were obtained from PBLs stimulated with HLA-A2-positive T2 cells pulsed with 10–5 M of the different peptides, while HLA-A24- and HLA-B7-restricted CTL cultures were obtained from PBLs stimulated with peptides alone. As a control for memory CTL activation, parallel stimulations of memory precursors specific for the HLA-A2-restricted EBV-derived subdominant CLG epitope (30) were performed. CTL reactivation was tested after two to four stimulations using cytotoxicity and/or IFN-{gamma} ELISPOT assays. All subjects responded to the CLG epitope (data not shown). Fresh ex vivo-isolated PBLs from HHV-8-positive individuals did not respond to any of the tested peptides when assayed by IFN-{gamma} ELISPOT (data not shown). PBLs from five different HLA-A2-positive HHV-8-negative individuals did not respond to any of the HHV-8-derived, HLA-A2-binding peptides when assayed by cytotoxicity or IFN-{gamma} ELISPOT assays after two to four in vitro stimulations (data not shown).

Identification of HLA-A2-presented CTL epitopes within ORF 26 Ag

We selected seven potential HLA-A2-binding peptides within the ORF 26 protein (Table II). These were tested for binding to HLA-A2 molecules using the capacity of exogenous peptides to stabilize HLA-A2 molecules at the cell surface of T2 cells. The T2 cells were treated at 26°C for 18 h in serum-free medium in the presence of peptides. Cells were then kept at 37°C for 4 h, then extensively washed to remove unbound peptides. The surface expression of HLA class I complexes was then evaluated by immunofluorescence, using the mAb MA2.1 specific for HLA-A2 molecules. Peptides stabilizing HLA-A2 molecules (Table II) were selected for immunogenicity studies, while poor binders (RFL, FMG, and YLC) were not used.

The capacity of the selected ORF 26-derived peptides to induce HLA-A2-restricted CTL responses was then assessed by stimulating PBLs from six different healthy HLA-A2-positive donors with peptide-pulsed T2 cells, and CTL cultures were analyzed by cytotoxicity against autologous peptide-pulsed PHA blasts. Only peptides FQW, IVL, and VLD induced peptide-specific CTL cultures (Fig. 1A). The majority of HHV-8-positive individuals (five of six) presented responses directed to at least one peptide from ORF 26, and we found that the VLD peptide is the preferred target epitope of CTL responses against ORF 26. We then amplified a VLD-specific CTL culture from donor 13, and assayed for killing of cells expressing an endogenous ORF 26. To this end, HLA-A2-positive PEL cells were used as targets and, as shown in Fig. 1B, VLD-specific CTLs specifically lysed HLA-A2-positive BC-3 cells induced into the lytic virus cycle, but did not recognize uninduced BC-3 cells or HLA-A2-negative BCBL-1 cells. This demonstrates that VLD-specific cultures obtained by peptide stimulation specifically recognize the endogenously expressed ORF 26 lytic Ag.



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  		FIGURE 1. ORF 26-specific CTL responses in HLA-A2-positive individuals. A, Freshly isolated PBLs from six HHV8- HLA-A2-positive healthy donors were stimulated with T2 cells pulsed with 10–5 M of the indicated peptides. After three consecutive stimulations, cytotoxic activity was tested against HLA-A2 positive PHA blasts, pulsed ({blacksquare}) or not ({square}) with synthetic peptides. Results are expressed as percent-specific lysis recorded at an E:T ratio of 30:1. One representative experiment of two to four different experiments is shown. B, VLD-specific CTL cultures obtained from donor D13 were tested in cytotoxicity assays against HLA-A2-positive (BC-3) and -negative (BCBL-1) cells lines induced or not into the lytic cycle as described in Materials and Methods. Results are expressed as percent-specific lysis recorded at an E:T ratio of 30:1. One representative experiment of two is shown.

 
Identification of HLA-A2, HLA-A24, and HLA-B7 CTL epitopes within ORF 70 Ag

Three ORF 70-derived peptides were selected for potential binding to HLA-A2 molecules, and all three were found to be able to associate with HLA-A2 (Table II). Subsequently, these peptides were evaluated for their capacity to elicit a memory T cell response in five HHV-8-positive donors. CTL cultures were tested for specificity in cytotoxicity assays against autologous peptide-pulsed PHA-blasts, or by IFN-{gamma} ELISPOT. We found that all HHV-8-positive individuals presented CTL responses against the YML epitope (Fig. 2A). Responses to YML were also present in a culture obtained from donor 6, which was assayed by ELISPOT only (data not shown). Two donors also presented responses to the VVQ peptide, and one donor to the SLL peptide, thereby suggesting that these CTL epitopes represent subdominant targets of CTL responses against ORF 70.



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  		FIGURE 2. ORF 70-specific CTL responses in HLA-A2-positive individuals. A, Freshly isolated PBLs obtained from four HLA-A2-positive, HHV-8-positive healthy donors were stimulated with T2 cells pulsed with 10–5 M of the indicated peptides. After three consecutive stimulations, cytotoxic activity was tested against HLA-A2-positive PHA blasts pulsed ({blacksquare}) or not ({square}) with synthetic peptides. Results are expressed as percent-specific lysis recorded at an E:T ratio of 30:1. One representative experiment of two to four different experiments is shown. B, YML-specific CTL cultures obtained from four donors (D5, D11, D13, and D14) were tested in cytotoxicity assays against HLA-A2-positive (BC-3) and -negative (BCBL-1) cell lines, induced or not into the lytic cycle as described in Materials and Methods. Results are expressed as percent-specific lysis recorded at an E:T ratio of 30:1. One representative experiment of two to three experiments is shown.

 
YML-specific cultures were also able to specifically lyse BC-3 cells induced into the lytic virus cycle but not untreated BC-3 cells or HLA-A2-negative BCBL-1 cells, demonstrating that the YML epitope is naturally presented in cells induced into the lytic cycle (Fig. 2B).

Within ORF 70, we also selected seven potential CTL epitopes presented by HLA-A24, and one presented by HLA-B7 (Table II). All peptides, except RYS, that for unknown reasons we were unable to purify, were used to obtain peptide-specific T cell cultures that were tested in cytotoxicity against autologous peptide-pulsed PHA blasts. We found that four of four individuals presented CTL responses against the SYS peptide, and three of them also responded to LYQ (Fig. 3A). Responses to LYQ were also present in a culture obtained from donor 7, which was assayed by ELISPOT only (data not shown and Table III).



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  		FIGURE 3. ORF 70-specific CTL responses in HLA-A24- and HLA-B7-positive individuals. A, Freshly isolated PBLs obtained from four HLA-A24 freshly isolated PBLs obtained from four HLA-A24-positive, HHV-8-positive healthy donors were stimulated with the indicated peptides. After three consecutive stimulations, cytotoxic activity was tested against HLA-A24-positive PHA blasts, pulsed ({blacksquare}) or not with ({square}) synthetic peptides. Results are expressed as percent-specific lysis recorded at an E:T ratio of 30:1. One representative experiment of two to three different experiments is shown. B, Freshly isolated PBLs obtained from the indicated four HLA-B7-positive, HHV-8-positive healthy donors were stimulated with the TPR peptide. After three consecutive stimulations, activity of T cell cultures was assayed by cytotoxicity (left panel) against PHA-blasts pulsed ({blacksquare}) or not ({square}) with synthetic peptides or/and by IFN-{gamma} ELISPOT assay (right panel) using 10 x 104 CTLs stimulated with the TPR peptide. Results are expressed as net spot-forming units per million PBLs. The negative control was always <50 spots/million cells. One representative experiment of two to four different experiments is shown.

 

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  		Table III. Summary of HLA-A2, -A24, and -B7-restricted epitope-specific CTL responses to ORFs 26, 70, K3, and K5 lytic Ags in HHV-8-seropositive individuals

 
As far as the B7-restricted TPR peptide is concerned, we found that it was a target for CTL responses in four of four HLA-B7 individuals (Fig. 3B).

As a whole, these data suggest that ORF 70 is a good target for CTL responses as it contains several epitopes presented by different HLA class I alleles.

Identification of HLA-A2 and HLA-B7 CTL epitopes within ORF K3 and K5 Ags

Within the K3 sequence, we identified three potential HLA-A2-binding peptides, which all associated with HLA-A2 molecules (Table II). Subsequently, the peptides were evaluated for their capacity to induce CTL responses in HHV-8-positive donors. CTL cultures were tested for specificity by IFN-{gamma} ELISPOT. We found that four of eight individuals responded to the GLA peptide and one also responded to FVF. No responses were observed against the YQL peptide (Fig. 4A). Cultures from donors 2 and 6 specifically lysed GLA-pulsed HLA-A2-positive blasts (data not shown).



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  		FIGURE 4. ORF K3-specific CTL responses in HHV-8-positive, HLA-A2-, -B7-positive donors. A, Freshly isolated PBLs obtained from four HLA-A2-positive, HHV-8-positive healthy donors were stimulated with T2 cells pulsed with 10–5 M of the indicated peptides. After three consecutive stimulations, the activity of T cell cultures assayed by IFN-{gamma} ELISPOT using 10 x 104 CTLs stimulated for 24 h with the indicated peptides. Results are expressed as net spot-forming units per million PBLs. The negative control was always <50 spots/million cells. One representative experiment of three to four different experiments is shown. B, Freshly isolated PBLs obtained from four HLA-B7-positive, HHV-8-positive healthy donors were stimulated with the LPR peptide. After three consecutive stimulations, the activity of T cell cultures was assayed by IFN-{gamma} ELISPOT using 10 x 104 CTLs stimulated for 24 h with LPR. Results are expressed as spot-forming units per million PBLs. The negative control was always <50 spots/million cells. One representative experiment of three different experiments is shown.

 
We also assayed two peptides potentially presented by HLA-A24 and HLA-B7 molecules (Table II) and, as shown in Fig. 4B, the HLA-B7 presented LPR peptide but not the HLA-A24 peptide (data not shown), induced peptide-specific responses in three of four HLA-B7-positive donors.

Within the K5 Ag, we identified two potential HLA-A2-binding peptides (Table II), which were assayed for their capacity to induce peptide-specific CTL responses in seven HLA-A2-positive donors. As shown in Fig. 5A, four of seven donors responded to the ALY peptide. No responses were detected against the LLM peptide (data not shown). Furthermore, ALY-specific CTL cultures were able to specifically lyse BC-3 cells induced into the lytic cycle but not untreated cells or BCBL-1 cells (Fig. 5B).



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  		FIGURE 5. ORF K5-specific CTL responses in HHV-8 positive, HLA-A2 positive donors. A, Freshly isolated PBLs obtained from four HLA-A2-positive, HHV-8-positive healthy donors were stimulated with T2 cells pulsed with 10–5 M of the ALY peptide. After three consecutive stimulations, cytotoxic activity was tested against HLA-A2-positive PHA-blasts, pulsed ({blacksquare}) or not ({square}) with synthetic peptides. Results are expressed as percent-specific lysis recorded at an E:T ratio of 30:1. One representative experiment of two is shown. B, ALY-specific CTL cultures obtained from donor 13 was tested in cytotoxicity assays against HLA-A2-positive (BC-3) and -negative (BCBL-1) cell lines, induced or not into the lytic cycle as described in Materials and Methods. Results are expressed as percent-specific lysis recorded at an E:T ratio of 30:1. One representative experiment of two is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study provides the identification of new epitope-specific CTL responses against HHV-8 in healthy HHV-8-positive individuals. In particular, we demonstrated the presence of HLA-A2, -A24, and -B7-restricted CTL responses directed to a panel of epitopes derived from four different HHV-8 lytic Ags (ORFs 26, 70, K3, and K5). Similar to CTL responses directed to EBV lytic Ags, we found a broad reactivity against all four HHV-8 lytic Ags. All subjects, except donor 15, who is HLA-A24-positive, responded to at least one of the selected Ags, a finding that further supports the key role of cell-mediated immune responses in the control of HHV-8 infection and demonstrates that cells producing the virus are subject to direct CTL control in vivo.

Epitope-specific CTL responses were identified using a peptide prediction algorithm and the capacity of selected peptides to induce memory CTL responses in peripheral T cells purified from HHV-8-seropositive individuals. By this approach, we identified three CTL epitopes from ORF 26 which are presented by HLA-A2, six CTL epitopes from ORF 70 presented by HLA-A2 (three epitopes), -A24 (two epitopes), and -B7 (one epitope), three CTL epitopes from ORF K3 presented by HLA-A2 (two epitopes) and -B7 (one epitope), and one HLA-A2-presented epitope derived from ORF K5.

Taken as a whole, these results demonstrate that CTL responses against ORF 70 are present in the large majority of the tested individuals with preferential recognition of the HLA-A2-restricted YML epitope, the HLA-A24-restricted SYS epitope, and the HLA-B7-restricted TPR epitope. Furthermore, HLA-A2-positive individuals presented CTL responses directed to all Ags, with preferential recognition of certain epitopes that may represent immunodominant HLA-A2-restricted targets because they were recognized by the majority of subjects.

We also demonstrated recognition of cells expressing the endogenous Ags for some of the peptide-specific CTL cultures, suggesting that CTLs with these specificities may have a role in controlling HHV-8-infected cells. Further studies are needed to determine whether these CTL responses are functional in preventing HHV-8-related diseases. To this end, these newly identified epitopes may be useful for the evaluation of epitope-specific CTL responses in subjects affected by HHV-8-associated disorders and for the identification of protective epitope-specific CTL responses.

Of note, it has been shown that the early lytic Ags K3 and K5 possess ubiquitin-ligase activity which targets MHC-I molecules expressed at the cell surface to the endocytic compartment (31, 32, 33, 34, 35, 36). This results in down-regulation of MHC-I molecules, which may favor immune escape of HHV-8-infected cells. However, the results from different laboratories, and those presented here, suggest that down-regulation of MHC-I molecules by K3 and K5, if present in vivo, does not completely alter the presentation of immediate early and early lytic Ags. Furthermore, we demonstrated the presence of responses directed to peptides from K3 and K5, although we do not know at present whether K3-expressing cells are recognized by the identified peptide-specific CTLs. CTL responses to K3 would be particularly relevant in control of HHV-8, because K3 is expressed early during lytic infection (37) and killing of K3-expressing cells may lead to abortive infection and reduction of virus spread.

In conclusion, in this study we identify new CTL epitopes derived from lytic Ags of HHV-8. These epitopes will be useful to our understanding of the role of HLA class I-restricted immunity in the development of HHV-8-related diseases. Furthermore, the identified CTL epitopes may be regarded as targets of specific immunotherapies for the treatment/prevention of HHV-8-associated malignancies.


   Acknowledgments
 
We thank the Blood Bank of Ferrara for supplying fresh blood, Teresa Grappa for HLA class I typing, and Anna Forster for the English revision of the text.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Istituto Superiore di Sanità (AIDS Project), the Italian Association for Cancer Research, the Fondazione Cassa di Risparmio di Ferrara, and the Ministero dell’Istruzione dell’Università e della Ricerca Scientifica. Back

2 Address correspondence and reprint requests to Dr. Riccardo Gavioli, Department of Biochemistry and Molecular Biology, Università di Ferrara, Via Luigi Borsari 46, 44100 Ferrara, Italy. E-mail address: r.gavioli{at}unife.it Back

3 Abbreviations used in this paper: HHV-8, human herpesvirus 8; KS, Kaposi’s sarcoma; PEL, primary effusion lymphoma; MHC-I, MHC class I; ORF, open reading frame; TPA, 12-O-tetradecanoylphorbol-13-acetate. Back

Received for publication August 1, 2005. Accepted for publication October 26, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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