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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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Characterization of EBNA1-specific T cells. (A) Recognition of LCL 111 by T cell clones derived from the M3-W1 T cell line. (B) FACS® analysis of M3-W1-B9 T cells for CD8 expression. T cells were stained with anti–CD4-PE or anti–CD8-FITC. Positive staining for CD8 T cells is shown as an open histogram and control antibody staining is represented as a shaded histogram. (C) Identification of minimal EBNA1 T cell epitope for MHC class I binding. Four different peptides were made and pulsed onto 1359mel cells at 10 μM concentration. After washing, the peptide-pulsed cells were cocultured with T cells overnight. IFN-γ release was determined from culture supernatants. (D) EBNA1-P518–526 peptide titration experiment for M3W1-B9 T cell recognition. EBNA1-P518–526 peptide at various concentrations were pulsed on 1359mel cells and used as target cells to stimulate T cells. A control peptide EBNA1-P 572–584 was also used at various concentrations. Experiments were repeated twice with similar results.
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fig2: Characterization of EBNA1-specific T cells. (A) Recognition of LCL 111 by T cell clones derived from the M3-W1 T cell line. (B) FACS® analysis of M3-W1-B9 T cells for CD8 expression. T cells were stained with anti–CD4-PE or anti–CD8-FITC. Positive staining for CD8 T cells is shown as an open histogram and control antibody staining is represented as a shaded histogram. (C) Identification of minimal EBNA1 T cell epitope for MHC class I binding. Four different peptides were made and pulsed onto 1359mel cells at 10 μM concentration. After washing, the peptide-pulsed cells were cocultured with T cells overnight. IFN-γ release was determined from culture supernatants. (D) EBNA1-P518–526 peptide titration experiment for M3W1-B9 T cell recognition. EBNA1-P518–526 peptide at various concentrations were pulsed on 1359mel cells and used as target cells to stimulate T cells. A control peptide EBNA1-P 572–584 was also used at various concentrations. Experiments were repeated twice with similar results.

Mentions: To further characterize the T cell line M3-W1, we generated 18 T cell clones by limiting dilution methods. Recognition of LCL 111 by different T cell clones is presented in Fig. 2 A. One of the T cell clones (designated M3W1-B9) with strong T cell reactivity was chosen for further study. FACS® analysis revealed that the M3W1-B9 T cells were CD8+ (Fig. 2 B). Recognition of the EBNA1-P518–530 peptide by M3W1-B9 CD8+ T cells was also blocked by antibody against MHC class I, but not by MHC class II or control antibodies, suggesting that these T cell clones resemble the bulk CD8+ T cell line (not depicted).


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)

Characterization of EBNA1-specific T cells. (A) Recognition of LCL 111 by T cell clones derived from the M3-W1 T cell line. (B) FACS® analysis of M3-W1-B9 T cells for CD8 expression. T cells were stained with anti–CD4-PE or anti–CD8-FITC. Positive staining for CD8 T cells is shown as an open histogram and control antibody staining is represented as a shaded histogram. (C) Identification of minimal EBNA1 T cell epitope for MHC class I binding. Four different peptides were made and pulsed onto 1359mel cells at 10 μM concentration. After washing, the peptide-pulsed cells were cocultured with T cells overnight. IFN-γ release was determined from culture supernatants. (D) EBNA1-P518–526 peptide titration experiment for M3W1-B9 T cell recognition. EBNA1-P518–526 peptide at various concentrations were pulsed on 1359mel cells and used as target cells to stimulate T cells. A control peptide EBNA1-P 572–584 was also used at various concentrations. Experiments were repeated twice with similar results.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Characterization of EBNA1-specific T cells. (A) Recognition of LCL 111 by T cell clones derived from the M3-W1 T cell line. (B) FACS® analysis of M3-W1-B9 T cells for CD8 expression. T cells were stained with anti–CD4-PE or anti–CD8-FITC. Positive staining for CD8 T cells is shown as an open histogram and control antibody staining is represented as a shaded histogram. (C) Identification of minimal EBNA1 T cell epitope for MHC class I binding. Four different peptides were made and pulsed onto 1359mel cells at 10 μM concentration. After washing, the peptide-pulsed cells were cocultured with T cells overnight. IFN-γ release was determined from culture supernatants. (D) EBNA1-P518–526 peptide titration experiment for M3W1-B9 T cell recognition. EBNA1-P518–526 peptide at various concentrations were pulsed on 1359mel cells and used as target cells to stimulate T cells. A control peptide EBNA1-P 572–584 was also used at various concentrations. Experiments were repeated twice with similar results.
Mentions: To further characterize the T cell line M3-W1, we generated 18 T cell clones by limiting dilution methods. Recognition of LCL 111 by different T cell clones is presented in Fig. 2 A. One of the T cell clones (designated M3W1-B9) with strong T cell reactivity was chosen for further study. FACS® analysis revealed that the M3W1-B9 T cells were CD8+ (Fig. 2 B). Recognition of the EBNA1-P518–530 peptide by M3W1-B9 CD8+ T cells was also blocked by antibody against MHC class I, but not by MHC class II or control antibodies, suggesting that these T cell clones resemble the bulk CD8+ T cell line (not depicted).

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