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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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Inhibition of T cell recognition of EBNA1 by protein synthesis inhibitors. (A) Specific inhibition of M3-W1-B9 CD8+ T cell recognition of HEK293/B8/EBNA1-GFP target cells by an irreversible protein synthesis inhibitor emetine. HLA-B8–expressing HEK293/EBNA1-GFP target cells were treated with an emetine inhibitor at three concentrations for 1 h. After three washes, cells were cocultured with M3-W1-B9 CD8+ T cells overnight for IFN-γ release assays. Similar experiments were performed for the treatment of cells with cycloheximide or puromycin. To determine effect of emetine on recognition of MHC class I/EBNA1 peptide on the cell surface, we also pulsed HLA-B8+ 1359 cells with the EBNA1-P 518–526 peptide after the treatment of 1359mel cells with three different concentration of emetine. (B) Determination of the sensitivity of recognition of TRP2-specific CD8+ T cells to the treatment with emetine. 1363mel cells were treated with three different concentrations of emetine. After three washes, the cells were cocultured with TRP2-specific CD8+ T cells. The treated cells pulsed with a TRP2 peptide were used to examine the effect of emetine on recognition of MHC class I–TRP2 complexes on the cell surface.
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fig9: Inhibition of T cell recognition of EBNA1 by protein synthesis inhibitors. (A) Specific inhibition of M3-W1-B9 CD8+ T cell recognition of HEK293/B8/EBNA1-GFP target cells by an irreversible protein synthesis inhibitor emetine. HLA-B8–expressing HEK293/EBNA1-GFP target cells were treated with an emetine inhibitor at three concentrations for 1 h. After three washes, cells were cocultured with M3-W1-B9 CD8+ T cells overnight for IFN-γ release assays. Similar experiments were performed for the treatment of cells with cycloheximide or puromycin. To determine effect of emetine on recognition of MHC class I/EBNA1 peptide on the cell surface, we also pulsed HLA-B8+ 1359 cells with the EBNA1-P 518–526 peptide after the treatment of 1359mel cells with three different concentration of emetine. (B) Determination of the sensitivity of recognition of TRP2-specific CD8+ T cells to the treatment with emetine. 1363mel cells were treated with three different concentrations of emetine. After three washes, the cells were cocultured with TRP2-specific CD8+ T cells. The treated cells pulsed with a TRP2 peptide were used to examine the effect of emetine on recognition of MHC class I–TRP2 complexes on the cell surface.

Mentions: The GAr domain has been demonstrated to efficiently block its proteasomal degradation of full-length EBNA1, thus inhibiting the generation of MHC class I–restricted peptide for T cell recognition. However, this inhibition may not be absolute, because some T cell epitopes are still processed and presented to T cells. Alternatively, the HLA-B8–restricted T cell epitope might be derived through the rapid degradation of DRiPs (25, 26), which have been estimated to constitute upwards of 30% of newly synthesized proteins and are considered an important source of peptide for presentation by MHC class I molecules (25–27). We reasoned that if the HLA-B8–restricted epitope is generated from proteasomal degradation of full-length EBNA1, the treatment of target cells with protein synthesis inhibitors would not affect T cell recognition because the full-length EBNA1 protein is still present after treatment. Otherwise, it would suggest that the HLA-B8–restricted epitope is derived from DRiPs rather than EBNA1. To test these possibilities, we treated HLA-B8–expressing HEK293/EBNA1-GFP cells with different concentrations of irreversible (emetine) and reversible (puromycin and cycloheximide) protein synthesis inhibitors for 1 h. After washing to remove inhibitors, the treated cells were cocultured with antigen-specific CD8+ T cells. Fig. 9 A shows that recognition of target cells by M3W1-B9 CD8+ T cells was significantly (94%) inhibited at a low concentration (1 μM) and completely inhibited at a 5 μM concentration of the irreversible inhibitor emetine. By contrast, no inhibitory effect was observed when HLA-B8–expressing 293/EBNA1-GFP cells were treated with reversible inhibitors puromycin and cycloheximide, respectively. Because blocking protein synthesis should rapidly decrease the peptide supply required for the export of MHC class I molecules from the ER to the cell surface as well as synthesis of MHC class I molecules (25–27), the treatment of cells with emetine would decrease the overall antigen presentation by MHC class I molecules. To exclude the possibility that inhibition of T cell recognition of the HLA-B8–restricted EBNA1 peptide was due to the expression and export of MHC class I molecules, we pulsed HLA-B8+ 1359mel cells with the EBNA1-P518–526 peptide after the cells were treated with different concentrations of emetine and washed. T cell recognition of peptide-pulsed target cells was slightly inhibited (Fig. 9 A), suggesting that effect of emetine on MHC class I molecules could not account for the inhibition of T cell recognition. To further test the sensitivity of antigen processing of other tumor antigens, such as tyrosinase-related protein 2 (TRP2), to protein synthesis inhibition, we treated 1363mel cells (TRP2+ and HLA-A2+) with different concentrations of emetine and then tested for their ability to stimulate TRP2-specific CD8+ T cells. As shown in Fig. 9 B, the inhibition of recognition of 1363mel target cells by TRP2-specific CD8+ T cells increased with the increasing concentrations of emetine. However, there was only 30% inhibition of T cell recognition at 1 μM emetine treatment compared with >90% inhibition of EBNA1-specific CD8+ T cells at the same concentration of emetine (Fig. 9 A). As expected, T cell recognition of the TRP2 peptide–pulsed target cells after the emetine treatment was slightly inhibited (Fig. 9 B). We also found that the treatment of target cells with emetine at a 1 μM concentration resulted in 20% inhibition of EBNA1-specific P3-B7 CD4+ T cell recognition (not depicted). Taken together, these results indicate that new protein synthesis is necessary and required for the generation of the HLA-B8–restricted epitope for T cell recognition, thus implying that DRiPs are the primary source of CD8+ T cell peptides. On the other hand, the processing and presentation of the TRP2 and CD4+ T cell EBNA1 epitopes are less dependent on the production of short-lived DRiPs.


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)

Inhibition of T cell recognition of EBNA1 by protein synthesis inhibitors. (A) Specific inhibition of M3-W1-B9 CD8+ T cell recognition of HEK293/B8/EBNA1-GFP target cells by an irreversible protein synthesis inhibitor emetine. HLA-B8–expressing HEK293/EBNA1-GFP target cells were treated with an emetine inhibitor at three concentrations for 1 h. After three washes, cells were cocultured with M3-W1-B9 CD8+ T cells overnight for IFN-γ release assays. Similar experiments were performed for the treatment of cells with cycloheximide or puromycin. To determine effect of emetine on recognition of MHC class I/EBNA1 peptide on the cell surface, we also pulsed HLA-B8+ 1359 cells with the EBNA1-P 518–526 peptide after the treatment of 1359mel cells with three different concentration of emetine. (B) Determination of the sensitivity of recognition of TRP2-specific CD8+ T cells to the treatment with emetine. 1363mel cells were treated with three different concentrations of emetine. After three washes, the cells were cocultured with TRP2-specific CD8+ T cells. The treated cells pulsed with a TRP2 peptide were used to examine the effect of emetine on recognition of MHC class I–TRP2 complexes on the cell surface.
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Related In: Results  -  Collection

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

fig9: Inhibition of T cell recognition of EBNA1 by protein synthesis inhibitors. (A) Specific inhibition of M3-W1-B9 CD8+ T cell recognition of HEK293/B8/EBNA1-GFP target cells by an irreversible protein synthesis inhibitor emetine. HLA-B8–expressing HEK293/EBNA1-GFP target cells were treated with an emetine inhibitor at three concentrations for 1 h. After three washes, cells were cocultured with M3-W1-B9 CD8+ T cells overnight for IFN-γ release assays. Similar experiments were performed for the treatment of cells with cycloheximide or puromycin. To determine effect of emetine on recognition of MHC class I/EBNA1 peptide on the cell surface, we also pulsed HLA-B8+ 1359 cells with the EBNA1-P 518–526 peptide after the treatment of 1359mel cells with three different concentration of emetine. (B) Determination of the sensitivity of recognition of TRP2-specific CD8+ T cells to the treatment with emetine. 1363mel cells were treated with three different concentrations of emetine. After three washes, the cells were cocultured with TRP2-specific CD8+ T cells. The treated cells pulsed with a TRP2 peptide were used to examine the effect of emetine on recognition of MHC class I–TRP2 complexes on the cell surface.
Mentions: The GAr domain has been demonstrated to efficiently block its proteasomal degradation of full-length EBNA1, thus inhibiting the generation of MHC class I–restricted peptide for T cell recognition. However, this inhibition may not be absolute, because some T cell epitopes are still processed and presented to T cells. Alternatively, the HLA-B8–restricted T cell epitope might be derived through the rapid degradation of DRiPs (25, 26), which have been estimated to constitute upwards of 30% of newly synthesized proteins and are considered an important source of peptide for presentation by MHC class I molecules (25–27). We reasoned that if the HLA-B8–restricted epitope is generated from proteasomal degradation of full-length EBNA1, the treatment of target cells with protein synthesis inhibitors would not affect T cell recognition because the full-length EBNA1 protein is still present after treatment. Otherwise, it would suggest that the HLA-B8–restricted epitope is derived from DRiPs rather than EBNA1. To test these possibilities, we treated HLA-B8–expressing HEK293/EBNA1-GFP cells with different concentrations of irreversible (emetine) and reversible (puromycin and cycloheximide) protein synthesis inhibitors for 1 h. After washing to remove inhibitors, the treated cells were cocultured with antigen-specific CD8+ T cells. Fig. 9 A shows that recognition of target cells by M3W1-B9 CD8+ T cells was significantly (94%) inhibited at a low concentration (1 μM) and completely inhibited at a 5 μM concentration of the irreversible inhibitor emetine. By contrast, no inhibitory effect was observed when HLA-B8–expressing 293/EBNA1-GFP cells were treated with reversible inhibitors puromycin and cycloheximide, respectively. Because blocking protein synthesis should rapidly decrease the peptide supply required for the export of MHC class I molecules from the ER to the cell surface as well as synthesis of MHC class I molecules (25–27), the treatment of cells with emetine would decrease the overall antigen presentation by MHC class I molecules. To exclude the possibility that inhibition of T cell recognition of the HLA-B8–restricted EBNA1 peptide was due to the expression and export of MHC class I molecules, we pulsed HLA-B8+ 1359mel cells with the EBNA1-P518–526 peptide after the cells were treated with different concentrations of emetine and washed. T cell recognition of peptide-pulsed target cells was slightly inhibited (Fig. 9 A), suggesting that effect of emetine on MHC class I molecules could not account for the inhibition of T cell recognition. To further test the sensitivity of antigen processing of other tumor antigens, such as tyrosinase-related protein 2 (TRP2), to protein synthesis inhibition, we treated 1363mel cells (TRP2+ and HLA-A2+) with different concentrations of emetine and then tested for their ability to stimulate TRP2-specific CD8+ T cells. As shown in Fig. 9 B, the inhibition of recognition of 1363mel target cells by TRP2-specific CD8+ T cells increased with the increasing concentrations of emetine. However, there was only 30% inhibition of T cell recognition at 1 μM emetine treatment compared with >90% inhibition of EBNA1-specific CD8+ T cells at the same concentration of emetine (Fig. 9 A). As expected, T cell recognition of the TRP2 peptide–pulsed target cells after the emetine treatment was slightly inhibited (Fig. 9 B). We also found that the treatment of target cells with emetine at a 1 μM concentration resulted in 20% inhibition of EBNA1-specific P3-B7 CD4+ T cell recognition (not depicted). Taken together, these results indicate that new protein synthesis is necessary and required for the generation of the HLA-B8–restricted epitope for T cell recognition, thus implying that DRiPs are the primary source of CD8+ T cell peptides. On the other hand, the processing and presentation of the TRP2 and CD4+ T cell EBNA1 epitopes are less dependent on the production of short-lived DRiPs.

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