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Evidence for the presentation of major histocompatibility complex class I-restricted Epstein-Barr virus nuclear antigen 1 peptides to CD8+ T lymphocytes.

Voo KS, Fu T, Wang HY, Tellam J, Heslop HE, Brenner MK, Rooney CM, Wang RF - J. Exp. Med. (2004)

Bottom Line: We also demonstrate that new protein synthesis is required for the generation of the HLA-B8 epitope for T cell recognition, suggesting that defective ribosomal products (DRiPs) are the major source of T cell epitopes.Experiments with protease inhibitors indicate that some serine proteases may participate in the degradation of EBNA1 DRiPs before they are further processed by proteasomes.These findings not only provide the first evidence of the presentation of an MHC class I-restricted EBNA1 epitope to CD8+ T cells, but also offer new insight into the molecular mechanisms involved in the processing and presentation of EBNA1.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
The Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is expressed in all EBV-associated tumors, making it an important target for immunotherapy. However, evidence for major histocompatibility complex (MHC) class I-restricted EBNA1 peptides endogenously presented by EBV-transformed B and tumor cells remains elusive. Here we describe for the first time the identification of an endogenously processed human histocompatibility leukocyte antigen (HLA)-B8-restricted EBNA1 peptide that is recognized by CD8+ T cells. T cell recognition could be inhibited by the treatment of target cells with proteasome inhibitors that block the MHC class I antigen processing pathway, but not by an inhibitor (chloroquine) of MHC class II antigen processing. We also demonstrate that new protein synthesis is required for the generation of the HLA-B8 epitope for T cell recognition, suggesting that defective ribosomal products (DRiPs) are the major source of T cell epitopes. Experiments with protease inhibitors indicate that some serine proteases may participate in the degradation of EBNA1 DRiPs before they are further processed by proteasomes. These findings not only provide the first evidence of the presentation of an MHC class I-restricted EBNA1 epitope to CD8+ T cells, but also offer new insight into the molecular mechanisms involved in the processing and presentation of EBNA1.

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Generation of EBNA1-P518–526 peptide–specific T cells from HLA-B8–expressing PBMCs. (A) Detection of EBNA1-P518–526 peptide–reactive T cells from HLA-B8+ donor PBMCs. 105 PBMCs were seeded per well and experiments were performed in quadruplicate wells. An HLA-A2–restricted NY-ESO-1 peptide served as a control. HLA-mismatched donor 5 and an HLA-B8+ PMBC donor 4 that is seronegative for EBV were also included. (B) Recognition of LCL 1088 by CD8+ T cell clones from the PBMCs of donor 3. T cells generated from PBMCs were stimulated with EBNA1-P518–526 peptide as described in Fig. 1. Six CD8+ T cell clones were generated from two T cell lines and were capable of recognizing HLA-B8–expressing LCL 1088 target cells. (C) D1-B11 CD8+ T cell recognition of HLA-B8–matched LCLs. LCLs were cocultured with T cells at an E/T ratio of 1:1. All experiments were repeated twice with similar results.
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fig7: Generation of EBNA1-P518–526 peptide–specific T cells from HLA-B8–expressing PBMCs. (A) Detection of EBNA1-P518–526 peptide–reactive T cells from HLA-B8+ donor PBMCs. 105 PBMCs were seeded per well and experiments were performed in quadruplicate wells. An HLA-A2–restricted NY-ESO-1 peptide served as a control. HLA-mismatched donor 5 and an HLA-B8+ PMBC donor 4 that is seronegative for EBV were also included. (B) Recognition of LCL 1088 by CD8+ T cell clones from the PBMCs of donor 3. T cells generated from PBMCs were stimulated with EBNA1-P518–526 peptide as described in Fig. 1. Six CD8+ T cell clones were generated from two T cell lines and were capable of recognizing HLA-B8–expressing LCL 1088 target cells. (C) D1-B11 CD8+ T cell recognition of HLA-B8–matched LCLs. LCLs were cocultured with T cells at an E/T ratio of 1:1. All experiments were repeated twice with similar results.

Mentions: Next, we tested whether EBNA1-specific HLA-B8–restricted CD8+ T cells are present in the PBMCs of other donors expressing HLA-B8. Five donor PBMCs were obtained for ELISPOT assays using EBNA1-P518–526 peptide–pulsed target cells. NY-ESO-1 peptide–pulsed target cells served as a specific control. Three of the five donor PBMCs (donors 1, 2, and 3) express HLA-B8 molecules and are serum positive for EBV, whereas donor 4 is positive for HLA-B8 expression but is serum negative for EBV. A mismatched donor PBMC (donor 5) served as a negative control. We found that three of the four HLA-B8+ donor PBMCs specifically responded to the EBNA1-P518–526 peptide (Fig. 7 A). By contrast, neither PBMCs from HLA-B8+ but seronegative nor HLA-B8− donors responded to the EBNA1-P518–526 peptide (Fig. 7 A). These results suggest that EBNA1-specific, HLA-B8–restricted CD8+ T cells are commonly present in HLA-B8+ EBV-infected individuals. To further test whether these EBNA1-specific, HLA-B8–restricted CD8+ T cells are capable of recognizing EBV+ LCL cells, we established T cell clones from the PBMCs of donor 3 by limiting dilution methods. Recognition of HLA-B8–expressing LCL 1088 by these T cell clones is shown in Fig. 7 B. One T clone, D1-B11, was selected for further testing of its ability to recognize HLA-B8+ EBV+ LCL cells. These T cells exhibited strong T cell reactivity against HLA-B8+ LCLs 8 and 1088, but did not respond to HLA-B8− LCL 1 and 2 cells (Fig. 7 C). Taken together, these studies suggest that the PBMCs of EBV-infected donors expressing HLA-B8 molecules contain EBNA1-specific CD8+ T cells that are capable of recognizing HLA-B8+ LCL targets.


Evidence for the presentation of major histocompatibility complex class I-restricted Epstein-Barr virus nuclear antigen 1 peptides to CD8+ T lymphocytes.

Voo KS, Fu T, Wang HY, Tellam J, Heslop HE, Brenner MK, Rooney CM, Wang RF - J. Exp. Med. (2004)

Generation of EBNA1-P518–526 peptide–specific T cells from HLA-B8–expressing PBMCs. (A) Detection of EBNA1-P518–526 peptide–reactive T cells from HLA-B8+ donor PBMCs. 105 PBMCs were seeded per well and experiments were performed in quadruplicate wells. An HLA-A2–restricted NY-ESO-1 peptide served as a control. HLA-mismatched donor 5 and an HLA-B8+ PMBC donor 4 that is seronegative for EBV were also included. (B) Recognition of LCL 1088 by CD8+ T cell clones from the PBMCs of donor 3. T cells generated from PBMCs were stimulated with EBNA1-P518–526 peptide as described in Fig. 1. Six CD8+ T cell clones were generated from two T cell lines and were capable of recognizing HLA-B8–expressing LCL 1088 target cells. (C) D1-B11 CD8+ T cell recognition of HLA-B8–matched LCLs. LCLs were cocultured with T cells at an E/T ratio of 1:1. All experiments were repeated twice with similar results.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211826&req=5

fig7: Generation of EBNA1-P518–526 peptide–specific T cells from HLA-B8–expressing PBMCs. (A) Detection of EBNA1-P518–526 peptide–reactive T cells from HLA-B8+ donor PBMCs. 105 PBMCs were seeded per well and experiments were performed in quadruplicate wells. An HLA-A2–restricted NY-ESO-1 peptide served as a control. HLA-mismatched donor 5 and an HLA-B8+ PMBC donor 4 that is seronegative for EBV were also included. (B) Recognition of LCL 1088 by CD8+ T cell clones from the PBMCs of donor 3. T cells generated from PBMCs were stimulated with EBNA1-P518–526 peptide as described in Fig. 1. Six CD8+ T cell clones were generated from two T cell lines and were capable of recognizing HLA-B8–expressing LCL 1088 target cells. (C) D1-B11 CD8+ T cell recognition of HLA-B8–matched LCLs. LCLs were cocultured with T cells at an E/T ratio of 1:1. All experiments were repeated twice with similar results.
Mentions: Next, we tested whether EBNA1-specific HLA-B8–restricted CD8+ T cells are present in the PBMCs of other donors expressing HLA-B8. Five donor PBMCs were obtained for ELISPOT assays using EBNA1-P518–526 peptide–pulsed target cells. NY-ESO-1 peptide–pulsed target cells served as a specific control. Three of the five donor PBMCs (donors 1, 2, and 3) express HLA-B8 molecules and are serum positive for EBV, whereas donor 4 is positive for HLA-B8 expression but is serum negative for EBV. A mismatched donor PBMC (donor 5) served as a negative control. We found that three of the four HLA-B8+ donor PBMCs specifically responded to the EBNA1-P518–526 peptide (Fig. 7 A). By contrast, neither PBMCs from HLA-B8+ but seronegative nor HLA-B8− donors responded to the EBNA1-P518–526 peptide (Fig. 7 A). These results suggest that EBNA1-specific, HLA-B8–restricted CD8+ T cells are commonly present in HLA-B8+ EBV-infected individuals. To further test whether these EBNA1-specific, HLA-B8–restricted CD8+ T cells are capable of recognizing EBV+ LCL cells, we established T cell clones from the PBMCs of donor 3 by limiting dilution methods. Recognition of HLA-B8–expressing LCL 1088 by these T cell clones is shown in Fig. 7 B. One T clone, D1-B11, was selected for further testing of its ability to recognize HLA-B8+ EBV+ LCL cells. These T cells exhibited strong T cell reactivity against HLA-B8+ LCLs 8 and 1088, but did not respond to HLA-B8− LCL 1 and 2 cells (Fig. 7 C). Taken together, these studies suggest that the PBMCs of EBV-infected donors expressing HLA-B8 molecules contain EBNA1-specific CD8+ T cells that are capable of recognizing HLA-B8+ LCL targets.

Bottom Line: We also demonstrate that new protein synthesis is required for the generation of the HLA-B8 epitope for T cell recognition, suggesting that defective ribosomal products (DRiPs) are the major source of T cell epitopes.Experiments with protease inhibitors indicate that some serine proteases may participate in the degradation of EBNA1 DRiPs before they are further processed by proteasomes.These findings not only provide the first evidence of the presentation of an MHC class I-restricted EBNA1 epitope to CD8+ T cells, but also offer new insight into the molecular mechanisms involved in the processing and presentation of EBNA1.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
The Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is expressed in all EBV-associated tumors, making it an important target for immunotherapy. However, evidence for major histocompatibility complex (MHC) class I-restricted EBNA1 peptides endogenously presented by EBV-transformed B and tumor cells remains elusive. Here we describe for the first time the identification of an endogenously processed human histocompatibility leukocyte antigen (HLA)-B8-restricted EBNA1 peptide that is recognized by CD8+ T cells. T cell recognition could be inhibited by the treatment of target cells with proteasome inhibitors that block the MHC class I antigen processing pathway, but not by an inhibitor (chloroquine) of MHC class II antigen processing. We also demonstrate that new protein synthesis is required for the generation of the HLA-B8 epitope for T cell recognition, suggesting that defective ribosomal products (DRiPs) are the major source of T cell epitopes. Experiments with protease inhibitors indicate that some serine proteases may participate in the degradation of EBNA1 DRiPs before they are further processed by proteasomes. These findings not only provide the first evidence of the presentation of an MHC class I-restricted EBNA1 epitope to CD8+ T cells, but also offer new insight into the molecular mechanisms involved in the processing and presentation of EBNA1.

Show MeSH
Related in: MedlinePlus