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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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Natural processing and presentation of EBNA1 to M3-W1-B9 CD8+ T cells. (A) Recognition of full-length EBNA1-transfected 1359mel cells by M3-W1-B9 T cells. 1359mel cells were transfected with 200 ng EBNA1-GFP or EBNA1-GAr-del-GFP plasmid DNAs using LipofectAMINE. IFN-γ release was determined as described in Fig. 1. (B) Recognition of 1359 fibroblasts transfected with EBNA1-GFP by M3-W1-B9 T cells. 1359 fibroblasts were transfected with EBNA1 plasmid DNA by electroporation. T cell assays were performed at an E/T ratio of 2:1. (C) T cell recognition of autologous PBMCs infected with retroviral/EBNA1-GFP.
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fig3: Natural processing and presentation of EBNA1 to M3-W1-B9 CD8+ T cells. (A) Recognition of full-length EBNA1-transfected 1359mel cells by M3-W1-B9 T cells. 1359mel cells were transfected with 200 ng EBNA1-GFP or EBNA1-GAr-del-GFP plasmid DNAs using LipofectAMINE. IFN-γ release was determined as described in Fig. 1. (B) Recognition of 1359 fibroblasts transfected with EBNA1-GFP by M3-W1-B9 T cells. 1359 fibroblasts were transfected with EBNA1 plasmid DNA by electroporation. T cell assays were performed at an E/T ratio of 2:1. (C) T cell recognition of autologous PBMCs infected with retroviral/EBNA1-GFP.

Mentions: To determine if the T cell peptide derived from the EBNA1 antigen can be endogenously processed and presented to CD8+ T cells, we transfected plasmid DNAs carrying full-length EBNA1-GFP or EBNA1-GAr-del-GFP into 1359mel and 1359 fibroblast cells as targets for T cell recognition. As shown in Fig. 3 A, M3W1-B9 CD8+ T cells recognized 1359mel/EBNA1-GFP and 1359mel/EBNA1-GAr-del-GFP target cells equally well, whereas no T cell reactivity was detected with 1359mel/GFP cells. The expression of EBNA1 as a fusion protein with GFP allowed us to monitor gene expression and transfection efficiency throughout the course of the experiments. CD8+ T cells also recognized 1359 fibroblast cells transfected with full-length EBNA1-GFP cDNA (Fig. 3 B). The transfection efficiency of 1359mel and fibroblasts is ∼10–15%. T cell recognition of 1359mel/EBNA1-GFP was also blocked by antibody against MHC class I, confirming that the recognition is MHC class I restricted (not depicted). To evaluate endogenous processing of EBNA1 in human PBMCs, we constructed a retrovirus-encoding EBNA1-GFP for introducing genes into PBMCs. Although the transduction efficiency of the retrovirus-encoding EBNA1-GFP into PBMCs from donor M was <10%, we found that T cell recognition of PBMCs infected with recombinant retrovirus increased threefold in terms of IFN-γ release from T cells compared with that of uninfected PBMCs (Fig. 3 C). These results indicate that the MHC class I–restricted EBNA1 peptides are endogenously processed and presented to 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)

Natural processing and presentation of EBNA1 to M3-W1-B9 CD8+ T cells. (A) Recognition of full-length EBNA1-transfected 1359mel cells by M3-W1-B9 T cells. 1359mel cells were transfected with 200 ng EBNA1-GFP or EBNA1-GAr-del-GFP plasmid DNAs using LipofectAMINE. IFN-γ release was determined as described in Fig. 1. (B) Recognition of 1359 fibroblasts transfected with EBNA1-GFP by M3-W1-B9 T cells. 1359 fibroblasts were transfected with EBNA1 plasmid DNA by electroporation. T cell assays were performed at an E/T ratio of 2:1. (C) T cell recognition of autologous PBMCs infected with retroviral/EBNA1-GFP.
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Related In: Results  -  Collection

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

fig3: Natural processing and presentation of EBNA1 to M3-W1-B9 CD8+ T cells. (A) Recognition of full-length EBNA1-transfected 1359mel cells by M3-W1-B9 T cells. 1359mel cells were transfected with 200 ng EBNA1-GFP or EBNA1-GAr-del-GFP plasmid DNAs using LipofectAMINE. IFN-γ release was determined as described in Fig. 1. (B) Recognition of 1359 fibroblasts transfected with EBNA1-GFP by M3-W1-B9 T cells. 1359 fibroblasts were transfected with EBNA1 plasmid DNA by electroporation. T cell assays were performed at an E/T ratio of 2:1. (C) T cell recognition of autologous PBMCs infected with retroviral/EBNA1-GFP.
Mentions: To determine if the T cell peptide derived from the EBNA1 antigen can be endogenously processed and presented to CD8+ T cells, we transfected plasmid DNAs carrying full-length EBNA1-GFP or EBNA1-GAr-del-GFP into 1359mel and 1359 fibroblast cells as targets for T cell recognition. As shown in Fig. 3 A, M3W1-B9 CD8+ T cells recognized 1359mel/EBNA1-GFP and 1359mel/EBNA1-GAr-del-GFP target cells equally well, whereas no T cell reactivity was detected with 1359mel/GFP cells. The expression of EBNA1 as a fusion protein with GFP allowed us to monitor gene expression and transfection efficiency throughout the course of the experiments. CD8+ T cells also recognized 1359 fibroblast cells transfected with full-length EBNA1-GFP cDNA (Fig. 3 B). The transfection efficiency of 1359mel and fibroblasts is ∼10–15%. T cell recognition of 1359mel/EBNA1-GFP was also blocked by antibody against MHC class I, confirming that the recognition is MHC class I restricted (not depicted). To evaluate endogenous processing of EBNA1 in human PBMCs, we constructed a retrovirus-encoding EBNA1-GFP for introducing genes into PBMCs. Although the transduction efficiency of the retrovirus-encoding EBNA1-GFP into PBMCs from donor M was <10%, we found that T cell recognition of PBMCs infected with recombinant retrovirus increased threefold in terms of IFN-γ release from T cells compared with that of uninfected PBMCs (Fig. 3 C). These results indicate that the MHC class I–restricted EBNA1 peptides are endogenously processed and presented to 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