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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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Requirement of serine proteases for the generation of EBNA1 T cell epitope. (A) Specific inhibition of M3-W1-B9 CD8+ T cell recognition of HEK293/B8/EBNA1-GFP target cells by protease inhibitors. Target cells were incubated with various protease inhibitors for 2 h, washed, and cocultured with CD8+ T cells overnight for IFN-γ release assays. Solvents used to solubilize inhibitors were also used as controls. Experiments were performed in triplicate wells. CD8+ T cell recognition of target cells was inhibited by treatment with TPCK and AEBSF inhibitors. (B) Effect of protease inhibitors on recognition of 1359mel cells by 1359mel-specific CD8+ T cells. (C) Dose-dependent inhibition of M3-W1-B9 CD8+ T cell recognition of HEK293/B8/EBNA1-GFP target cells by protease inhibitors. Target cells were treated with different concentrations of protease inhibitors. After washes, the treated cells were cocultured with M3-W1-B9 CD8+ T cells overnight for IFN-γ release assays.
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fig10: Requirement of serine proteases for the generation of EBNA1 T cell epitope. (A) Specific inhibition of M3-W1-B9 CD8+ T cell recognition of HEK293/B8/EBNA1-GFP target cells by protease inhibitors. Target cells were incubated with various protease inhibitors for 2 h, washed, and cocultured with CD8+ T cells overnight for IFN-γ release assays. Solvents used to solubilize inhibitors were also used as controls. Experiments were performed in triplicate wells. CD8+ T cell recognition of target cells was inhibited by treatment with TPCK and AEBSF inhibitors. (B) Effect of protease inhibitors on recognition of 1359mel cells by 1359mel-specific CD8+ T cells. (C) Dose-dependent inhibition of M3-W1-B9 CD8+ T cell recognition of HEK293/B8/EBNA1-GFP target cells by protease inhibitors. Target cells were treated with different concentrations of protease inhibitors. After washes, the treated cells were cocultured with M3-W1-B9 CD8+ T cells overnight for IFN-γ release assays.

Mentions: Next, we sought to determine if other proteases are involved in the degradation of short-lived DRiPs of EBNA1 because the GAr domain may need to be removed before they are further degraded by proteasomes. To identify individual protease inhibitors that might inhibit the presentation of the HLA-B8–restricted epitope to T cells, we selected seven protease inhibitors for further testing. Fig. 10 A shows that two such protease inhibitors, TPCK and AEBSF, significantly blocked CD8+ T cell recognition of the HLA-B8–expressing HEK293/EBNA1-GFP target cells when treated for 2 h at the lowest effective concentrations suggested by the manufacturer. None of the other protease inhibitors were effective, even after treatment for 2 or 8 h. To test whether TPCK or AEBSF affected T cell recognition of other antigens, we treated 1359mel with the same concentrations of TPCK or AEBSF as shown in Fig. 10 A. No inhibition was observed for recognition of 1359mel cells by 1359mel-specific CD8+ T cells (Fig. 10 B), suggesting that both inhibitors are specific for processing and presentation of the HLA-B8–restricted EBNA1 epitope. TPCK or AEBSF inhibited T cell recognition of target cells in a dose-dependent manner (Fig. 10 C), whereas TLCK produced a partial inhibitory effect when used at a high concentration. Although TLCK and TPCK have similar specificities, TLCK is unstable in solution according to the manufacturer. AEBSF is an irreversible, specific inhibitor of serine proteases, whereas TPCK is an irreversible inhibitor of chymotrypsin and many other serine and cysteine proteases. These results suggest that serine proteases are also required for the processing of the HLA-B8–restricted T cell epitope.


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)

Requirement of serine proteases for the generation of EBNA1 T cell epitope. (A) Specific inhibition of M3-W1-B9 CD8+ T cell recognition of HEK293/B8/EBNA1-GFP target cells by protease inhibitors. Target cells were incubated with various protease inhibitors for 2 h, washed, and cocultured with CD8+ T cells overnight for IFN-γ release assays. Solvents used to solubilize inhibitors were also used as controls. Experiments were performed in triplicate wells. CD8+ T cell recognition of target cells was inhibited by treatment with TPCK and AEBSF inhibitors. (B) Effect of protease inhibitors on recognition of 1359mel cells by 1359mel-specific CD8+ T cells. (C) Dose-dependent inhibition of M3-W1-B9 CD8+ T cell recognition of HEK293/B8/EBNA1-GFP target cells by protease inhibitors. Target cells were treated with different concentrations of protease inhibitors. After washes, the treated cells were cocultured with M3-W1-B9 CD8+ T cells overnight for IFN-γ release assays.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211826&req=5

fig10: Requirement of serine proteases for the generation of EBNA1 T cell epitope. (A) Specific inhibition of M3-W1-B9 CD8+ T cell recognition of HEK293/B8/EBNA1-GFP target cells by protease inhibitors. Target cells were incubated with various protease inhibitors for 2 h, washed, and cocultured with CD8+ T cells overnight for IFN-γ release assays. Solvents used to solubilize inhibitors were also used as controls. Experiments were performed in triplicate wells. CD8+ T cell recognition of target cells was inhibited by treatment with TPCK and AEBSF inhibitors. (B) Effect of protease inhibitors on recognition of 1359mel cells by 1359mel-specific CD8+ T cells. (C) Dose-dependent inhibition of M3-W1-B9 CD8+ T cell recognition of HEK293/B8/EBNA1-GFP target cells by protease inhibitors. Target cells were treated with different concentrations of protease inhibitors. After washes, the treated cells were cocultured with M3-W1-B9 CD8+ T cells overnight for IFN-γ release assays.
Mentions: Next, we sought to determine if other proteases are involved in the degradation of short-lived DRiPs of EBNA1 because the GAr domain may need to be removed before they are further degraded by proteasomes. To identify individual protease inhibitors that might inhibit the presentation of the HLA-B8–restricted epitope to T cells, we selected seven protease inhibitors for further testing. Fig. 10 A shows that two such protease inhibitors, TPCK and AEBSF, significantly blocked CD8+ T cell recognition of the HLA-B8–expressing HEK293/EBNA1-GFP target cells when treated for 2 h at the lowest effective concentrations suggested by the manufacturer. None of the other protease inhibitors were effective, even after treatment for 2 or 8 h. To test whether TPCK or AEBSF affected T cell recognition of other antigens, we treated 1359mel with the same concentrations of TPCK or AEBSF as shown in Fig. 10 A. No inhibition was observed for recognition of 1359mel cells by 1359mel-specific CD8+ T cells (Fig. 10 B), suggesting that both inhibitors are specific for processing and presentation of the HLA-B8–restricted EBNA1 epitope. TPCK or AEBSF inhibited T cell recognition of target cells in a dose-dependent manner (Fig. 10 C), whereas TLCK produced a partial inhibitory effect when used at a high concentration. Although TLCK and TPCK have similar specificities, TLCK is unstable in solution according to the manufacturer. AEBSF is an irreversible, specific inhibitor of serine proteases, whereas TPCK is an irreversible inhibitor of chymotrypsin and many other serine and cysteine proteases. These results suggest that serine proteases are also required for the processing of the HLA-B8–restricted T cell epitope.

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