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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-specific T cells. (A) T cells were generated from HLA-B8–expressing PBMCs of donor M after in vitro stimulation with synthetic peptides from EBNA1. 1.5 × 105 cells per well of PBMCs were used to generate EBNA1-P518–530 peptide–specific T cells. Peptides other than those used for repeated stimulations served as negative controls. For T cell recognition assays, peptides were pulsed onto 1359mel target cells and cocultured with T cells overnight. Cytokine release assays were performed as previously described (reference 11). (B) T cell recognition (T cell line M3-W1 from donor M) of 1359mel cells pulsed with EBNA1-P518–530 peptide was specifically inhibited by antibody against MHC class I molecules. T cell recognition assays were performed at an E/T ratio of 1:1. All results are expressed as IFN-γ release in picogram/milliliter and are the averages of duplicate values. All antibodies were used at a final concentration of 20 μg/ml each.
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fig1: Generation of EBNA1-specific T cells. (A) T cells were generated from HLA-B8–expressing PBMCs of donor M after in vitro stimulation with synthetic peptides from EBNA1. 1.5 × 105 cells per well of PBMCs were used to generate EBNA1-P518–530 peptide–specific T cells. Peptides other than those used for repeated stimulations served as negative controls. For T cell recognition assays, peptides were pulsed onto 1359mel target cells and cocultured with T cells overnight. Cytokine release assays were performed as previously described (reference 11). (B) T cell recognition (T cell line M3-W1 from donor M) of 1359mel cells pulsed with EBNA1-P518–530 peptide was specifically inhibited by antibody against MHC class I molecules. T cell recognition assays were performed at an E/T ratio of 1:1. All results are expressed as IFN-γ release in picogram/milliliter and are the averages of duplicate values. All antibodies were used at a final concentration of 20 μg/ml each.

Mentions: Recent studies demonstrated that human CD4+ T cells consistently and predominantly respond to MHC class II–restricted peptides derived from EBNA1 (9–11, 18), suggesting that EBNA1 can be processed through the MHC class II pathway for T cell recognition. During the course of our work on the identification of MHC class II–restricted EBNA1 peptides, we generated several T cell lines that are capable of stimulating T cells after coculturing with EBNA1-P518–530 (YNLRRGTALAIPQ) peptide-pulsed 1359mel cells. Representative data from one of these cell lines, designated M3-W1, is shown in Fig. 1 A. To determine the restriction element for T cell recognition, we tested M3-W1 T cell activity in response to the peptide-pulsed target cells in the presence of anti–MHC class I (HLA-A, HLA-B, and HLA-C), anti–HLA-DR, anti–HLA-DP, and anti–HLA-DQ or isotype control antibodies. To our surprise, T cell recognition of the EBNA1-P518–530 peptide by T cell line M3-W1 was specifically blocked by an anti–MHC class I monoclonal antibody, but not by anti–HLA-DP, anti-DQ, anti-DR, or isotype control antibodies (Fig. 1 B). These results suggest that human M3-W1 T cells recognize a peptide derived from EBNA1 presented by HLA class I molecules.


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-specific T cells. (A) T cells were generated from HLA-B8–expressing PBMCs of donor M after in vitro stimulation with synthetic peptides from EBNA1. 1.5 × 105 cells per well of PBMCs were used to generate EBNA1-P518–530 peptide–specific T cells. Peptides other than those used for repeated stimulations served as negative controls. For T cell recognition assays, peptides were pulsed onto 1359mel target cells and cocultured with T cells overnight. Cytokine release assays were performed as previously described (reference 11). (B) T cell recognition (T cell line M3-W1 from donor M) of 1359mel cells pulsed with EBNA1-P518–530 peptide was specifically inhibited by antibody against MHC class I molecules. T cell recognition assays were performed at an E/T ratio of 1:1. All results are expressed as IFN-γ release in picogram/milliliter and are the averages of duplicate values. All antibodies were used at a final concentration of 20 μg/ml each.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Generation of EBNA1-specific T cells. (A) T cells were generated from HLA-B8–expressing PBMCs of donor M after in vitro stimulation with synthetic peptides from EBNA1. 1.5 × 105 cells per well of PBMCs were used to generate EBNA1-P518–530 peptide–specific T cells. Peptides other than those used for repeated stimulations served as negative controls. For T cell recognition assays, peptides were pulsed onto 1359mel target cells and cocultured with T cells overnight. Cytokine release assays were performed as previously described (reference 11). (B) T cell recognition (T cell line M3-W1 from donor M) of 1359mel cells pulsed with EBNA1-P518–530 peptide was specifically inhibited by antibody against MHC class I molecules. T cell recognition assays were performed at an E/T ratio of 1:1. All results are expressed as IFN-γ release in picogram/milliliter and are the averages of duplicate values. All antibodies were used at a final concentration of 20 μg/ml each.
Mentions: Recent studies demonstrated that human CD4+ T cells consistently and predominantly respond to MHC class II–restricted peptides derived from EBNA1 (9–11, 18), suggesting that EBNA1 can be processed through the MHC class II pathway for T cell recognition. During the course of our work on the identification of MHC class II–restricted EBNA1 peptides, we generated several T cell lines that are capable of stimulating T cells after coculturing with EBNA1-P518–530 (YNLRRGTALAIPQ) peptide-pulsed 1359mel cells. Representative data from one of these cell lines, designated M3-W1, is shown in Fig. 1 A. To determine the restriction element for T cell recognition, we tested M3-W1 T cell activity in response to the peptide-pulsed target cells in the presence of anti–MHC class I (HLA-A, HLA-B, and HLA-C), anti–HLA-DR, anti–HLA-DP, and anti–HLA-DQ or isotype control antibodies. To our surprise, T cell recognition of the EBNA1-P518–530 peptide by T cell line M3-W1 was specifically blocked by an anti–MHC class I monoclonal antibody, but not by anti–HLA-DP, anti-DQ, anti-DR, or isotype control antibodies (Fig. 1 B). These results suggest that human M3-W1 T cells recognize a peptide derived from EBNA1 presented by HLA class I molecules.

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