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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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HLA-B8 molecule functions as a restriction element for M3-W1-B9 CD8+ T cells. (A) T cell recognition of peptide-pulsed HLA-B8–expressing cell lines. (B) Identification of HLA-B8 molecule as a restriction element for T cell recognition. HEK293 cells cotransfected with HLA-B8 plus full-length EBNA1-GFP or EBNA1-GAr-del-GFP cDNAs (with GAr domain deleted) were tested for recognition by M3-W1-B9 CD8+ T cells. Positive and negative signs indicate cotransfection of target cells in the presence or absence of HLA-B8 cDNA, respectively. (C) Natural processing and presentation of the native form of EBNA1 for T cell recognition. HEK293 cells cotransfected with full-length EBNA1 and HLA-B8 cDNAs were cocultured with M3-W1-B9 CD8+ T cells overnight. IFN-γ secretion from T cells was determined by ELISA. (D) Endogenous generation of HLA-B8–restricted EBNA1 peptide for T cell recognition. HEK293 cells transfected with HLA-B8 cDNA were mixed with HEK293 cells transfected with EBNA1-GFP cDNA at a 1:1 ratio. The mixed cells were then cocultured with M3-W1-B9 CD8+ T cells overnight. IFN-γ release from CD8+ T cells was measured from culture supernatants.
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fig4: HLA-B8 molecule functions as a restriction element for M3-W1-B9 CD8+ T cells. (A) T cell recognition of peptide-pulsed HLA-B8–expressing cell lines. (B) Identification of HLA-B8 molecule as a restriction element for T cell recognition. HEK293 cells cotransfected with HLA-B8 plus full-length EBNA1-GFP or EBNA1-GAr-del-GFP cDNAs (with GAr domain deleted) were tested for recognition by M3-W1-B9 CD8+ T cells. Positive and negative signs indicate cotransfection of target cells in the presence or absence of HLA-B8 cDNA, respectively. (C) Natural processing and presentation of the native form of EBNA1 for T cell recognition. HEK293 cells cotransfected with full-length EBNA1 and HLA-B8 cDNAs were cocultured with M3-W1-B9 CD8+ T cells overnight. IFN-γ secretion from T cells was determined by ELISA. (D) Endogenous generation of HLA-B8–restricted EBNA1 peptide for T cell recognition. HEK293 cells transfected with HLA-B8 cDNA were mixed with HEK293 cells transfected with EBNA1-GFP cDNA at a 1:1 ratio. The mixed cells were then cocultured with M3-W1-B9 CD8+ T cells overnight. IFN-γ release from CD8+ T cells was measured from culture supernatants.

Mentions: To determine the restriction element for M3W1-B9 CD8+ T cells, we pulsed the 9-mer EBNA1-P518–526 peptide onto various MHC class I+ melanoma cell lines and tested for T cell recognition. We found that CD8+ T cells recognized 1359mel cells pulsed with the EBNA1-P518–526 peptide, but not other cells pulsed with the same peptide (Fig. 4 A), suggesting that HLA-B8 is a putative restriction element for T cell recognition. To test this possibility, we cloned the HLA-B8 cDNA and transfected it into HEK293 cells along with EBNA1-GFP or EBNA1-GAr-del-GFP and assessed their ability to present the antigenic peptide to T cells. Although EBNA1-GFP, EBNA1-GAr-del-GFP, or HLA-B8 expressed alone in HEK293 cell did not stimulate T cell responses, HEK293 cells transfected with EBNA1-GFP plus HLA-B8 or EBNA1-GAr-del-GFP plus HLA-B8 cDNAs strongly stimulated IFN-γ release from CD8+ T cells (Fig. 4 B). Although T cell recognition of HEK293 cells transfected with EBNA1-GAr-del-GFP plus HLA-B8 cDNAs was slightly higher than that of HEK293 cells transfected with EBNA1-GFP and HLA-B8 cDNAs, we did not observe any significant inhibitory effect of the GAr domain on T cell responses (Fig. 4 B). These studies indicate that HLA-B8 is an antigen-presenting molecule for M3W1-B9 CD8+ T cells.


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)

HLA-B8 molecule functions as a restriction element for M3-W1-B9 CD8+ T cells. (A) T cell recognition of peptide-pulsed HLA-B8–expressing cell lines. (B) Identification of HLA-B8 molecule as a restriction element for T cell recognition. HEK293 cells cotransfected with HLA-B8 plus full-length EBNA1-GFP or EBNA1-GAr-del-GFP cDNAs (with GAr domain deleted) were tested for recognition by M3-W1-B9 CD8+ T cells. Positive and negative signs indicate cotransfection of target cells in the presence or absence of HLA-B8 cDNA, respectively. (C) Natural processing and presentation of the native form of EBNA1 for T cell recognition. HEK293 cells cotransfected with full-length EBNA1 and HLA-B8 cDNAs were cocultured with M3-W1-B9 CD8+ T cells overnight. IFN-γ secretion from T cells was determined by ELISA. (D) Endogenous generation of HLA-B8–restricted EBNA1 peptide for T cell recognition. HEK293 cells transfected with HLA-B8 cDNA were mixed with HEK293 cells transfected with EBNA1-GFP cDNA at a 1:1 ratio. The mixed cells were then cocultured with M3-W1-B9 CD8+ T cells overnight. IFN-γ release from CD8+ T cells was measured from culture supernatants.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211826&req=5

fig4: HLA-B8 molecule functions as a restriction element for M3-W1-B9 CD8+ T cells. (A) T cell recognition of peptide-pulsed HLA-B8–expressing cell lines. (B) Identification of HLA-B8 molecule as a restriction element for T cell recognition. HEK293 cells cotransfected with HLA-B8 plus full-length EBNA1-GFP or EBNA1-GAr-del-GFP cDNAs (with GAr domain deleted) were tested for recognition by M3-W1-B9 CD8+ T cells. Positive and negative signs indicate cotransfection of target cells in the presence or absence of HLA-B8 cDNA, respectively. (C) Natural processing and presentation of the native form of EBNA1 for T cell recognition. HEK293 cells cotransfected with full-length EBNA1 and HLA-B8 cDNAs were cocultured with M3-W1-B9 CD8+ T cells overnight. IFN-γ secretion from T cells was determined by ELISA. (D) Endogenous generation of HLA-B8–restricted EBNA1 peptide for T cell recognition. HEK293 cells transfected with HLA-B8 cDNA were mixed with HEK293 cells transfected with EBNA1-GFP cDNA at a 1:1 ratio. The mixed cells were then cocultured with M3-W1-B9 CD8+ T cells overnight. IFN-γ release from CD8+ T cells was measured from culture supernatants.
Mentions: To determine the restriction element for M3W1-B9 CD8+ T cells, we pulsed the 9-mer EBNA1-P518–526 peptide onto various MHC class I+ melanoma cell lines and tested for T cell recognition. We found that CD8+ T cells recognized 1359mel cells pulsed with the EBNA1-P518–526 peptide, but not other cells pulsed with the same peptide (Fig. 4 A), suggesting that HLA-B8 is a putative restriction element for T cell recognition. To test this possibility, we cloned the HLA-B8 cDNA and transfected it into HEK293 cells along with EBNA1-GFP or EBNA1-GAr-del-GFP and assessed their ability to present the antigenic peptide to T cells. Although EBNA1-GFP, EBNA1-GAr-del-GFP, or HLA-B8 expressed alone in HEK293 cell did not stimulate T cell responses, HEK293 cells transfected with EBNA1-GFP plus HLA-B8 or EBNA1-GAr-del-GFP plus HLA-B8 cDNAs strongly stimulated IFN-γ release from CD8+ T cells (Fig. 4 B). Although T cell recognition of HEK293 cells transfected with EBNA1-GAr-del-GFP plus HLA-B8 cDNAs was slightly higher than that of HEK293 cells transfected with EBNA1-GFP and HLA-B8 cDNAs, we did not observe any significant inhibitory effect of the GAr domain on T cell responses (Fig. 4 B). These studies indicate that HLA-B8 is an antigen-presenting molecule for M3W1-B9 CD8+ T cells.

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