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Endogenous presentation of CD8+ T cell epitopes from Epstein-Barr virus-encoded nuclear antigen 1.

Tellam J, Connolly G, Green KJ, Miles JJ, Moss DJ, Burrows SR, Khanna R - J. Exp. Med. (2004)

Bottom Line: However, these different degradation rates do not correspond to the level of major histocompatibility complex class I-restricted presentation of EBNA1 epitopes.Functional assays revealed that processing of these EBNA1 epitopes is proteasome and transporter associated with antigen processing dependent.Based on these observations, we propose that defective ribosomal products, not the full-length antigen, are the primary source of endogenously processed CD8+ T cell epitopes from EBNA1.

View Article: PubMed Central - PubMed

Affiliation: EBV Unit, Tumour Immunology Laboratory, Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, 300 Herston Road, Brisbane (Qld) 4006, Australia.

ABSTRACT
Epstein-Barr virus (EBV)-encoded nuclear antigen (EBNA)1 is thought to escape cytotoxic T lymphocyte (CTL) recognition through either self-inhibition of synthesis or by blockade of proteasomal degradation by the glycine-alanine repeat (GAr) domain. Here we show that EBNA1 has a remarkably varied cell type-dependent stability. However, these different degradation rates do not correspond to the level of major histocompatibility complex class I-restricted presentation of EBNA1 epitopes. In spite of the highly stable expression of EBNA1 in B cells, CTL epitopes derived from this protein are efficiently processed and presented to CD8+ T cells. Furthermore, we show that EBV-infected B cells can readily activate EBNA1-specific memory T cell responses from healthy virus carriers. Functional assays revealed that processing of these EBNA1 epitopes is proteasome and transporter associated with antigen processing dependent. We also show that the endogenous presentation of these epitopes is dependent on the newly synthesized protein rather than the long-lived stable EBNA1. Based on these observations, we propose that defective ribosomal products, not the full-length antigen, are the primary source of endogenously processed CD8+ T cell epitopes from EBNA1.

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Endogenous processing of an inserted HLA B8–restricted CTL epitope within EBNA1. (A) Two HLA B8+ LCLs and the same LCLs transfected with either the EBNA1-FLR-GFP or EBNA1ΔGA-FLR-GFP expression constructs were used as targets in a standard 51Cr-release assay to assess CTL activity to a B8-specific CTL clone, LC13. An E/T ratio of 5:1 was used in this assay. (B) C1R.B8 LCLs, C1R.B8.ICP47 LCLs, and the same LCLs infected with a recombinant adenovirus encoding full-length EBNA1 (Ad-EBNA1-FLR) were used as targets in a standard 51Cr-release assay to assess CTL activity. An HLA B8–restricted FLR-specific CTL clone, LC13, was used as an effector in this assay. An E/T ratio of 5:1 was used in this assay. These data are a representation of two separate experiments.
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fig6: Endogenous processing of an inserted HLA B8–restricted CTL epitope within EBNA1. (A) Two HLA B8+ LCLs and the same LCLs transfected with either the EBNA1-FLR-GFP or EBNA1ΔGA-FLR-GFP expression constructs were used as targets in a standard 51Cr-release assay to assess CTL activity to a B8-specific CTL clone, LC13. An E/T ratio of 5:1 was used in this assay. (B) C1R.B8 LCLs, C1R.B8.ICP47 LCLs, and the same LCLs infected with a recombinant adenovirus encoding full-length EBNA1 (Ad-EBNA1-FLR) were used as targets in a standard 51Cr-release assay to assess CTL activity. An HLA B8–restricted FLR-specific CTL clone, LC13, was used as an effector in this assay. An E/T ratio of 5:1 was used in this assay. These data are a representation of two separate experiments.

Mentions: Although data presented in Fig. 5 clearly showed that EBNA1 epitopes can be endogenously processed through the class I pathway, it was important to further confirm these observations by using another human class I–restricted epitope. To address this issue, we tested the endogenous presentation of an inserted HLA B8–restricted CTL epitope, FLRGRAYGL (referred to as FLR), from an EBV-encoded EBNA3 protein within the EBNA1 COOH-terminal sequence (referred to as EBNA1-FLR), which allowed the study of endogenous processing of EBNA1 using an FLR-specific human CTL clone (11). The target cells were either transfected with expression vectors (EBNA1-FLR-GFP or EBNA1ΔGA-FLR-GFP) or infected with adenovirus encoding full-length EBNA1-FLR protein. It should be noted that target LCLs used in these assays were transformed with the B95.8 strain of EBV, which carries a mutation within the FLR epitope (29). As in the case of the HPV epitope, data presented in Fig. 6 shows that this epitope was endogenously processed by some HLA B8+ cells (AS LCL and C1R.B8) expressing EBNA1-FLR, whereas other HLA B8+ cells (LC LCLs) failed to present this epitope. The specificity of this recognition was confirmed by the inhibition of CTL killing of target cells coexpressing on HSV-1–encoded TAP inhibitor ICP47 (Fig. 6 B). These observations further confirm that CTL epitopes within EBNA1 can be endogenously processed and directly presented to CD8+ T cells through a TAP-dependent pathway.


Endogenous presentation of CD8+ T cell epitopes from Epstein-Barr virus-encoded nuclear antigen 1.

Tellam J, Connolly G, Green KJ, Miles JJ, Moss DJ, Burrows SR, Khanna R - J. Exp. Med. (2004)

Endogenous processing of an inserted HLA B8–restricted CTL epitope within EBNA1. (A) Two HLA B8+ LCLs and the same LCLs transfected with either the EBNA1-FLR-GFP or EBNA1ΔGA-FLR-GFP expression constructs were used as targets in a standard 51Cr-release assay to assess CTL activity to a B8-specific CTL clone, LC13. An E/T ratio of 5:1 was used in this assay. (B) C1R.B8 LCLs, C1R.B8.ICP47 LCLs, and the same LCLs infected with a recombinant adenovirus encoding full-length EBNA1 (Ad-EBNA1-FLR) were used as targets in a standard 51Cr-release assay to assess CTL activity. An HLA B8–restricted FLR-specific CTL clone, LC13, was used as an effector in this assay. An E/T ratio of 5:1 was used in this assay. These data are a representation of two separate experiments.
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Related In: Results  -  Collection

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fig6: Endogenous processing of an inserted HLA B8–restricted CTL epitope within EBNA1. (A) Two HLA B8+ LCLs and the same LCLs transfected with either the EBNA1-FLR-GFP or EBNA1ΔGA-FLR-GFP expression constructs were used as targets in a standard 51Cr-release assay to assess CTL activity to a B8-specific CTL clone, LC13. An E/T ratio of 5:1 was used in this assay. (B) C1R.B8 LCLs, C1R.B8.ICP47 LCLs, and the same LCLs infected with a recombinant adenovirus encoding full-length EBNA1 (Ad-EBNA1-FLR) were used as targets in a standard 51Cr-release assay to assess CTL activity. An HLA B8–restricted FLR-specific CTL clone, LC13, was used as an effector in this assay. An E/T ratio of 5:1 was used in this assay. These data are a representation of two separate experiments.
Mentions: Although data presented in Fig. 5 clearly showed that EBNA1 epitopes can be endogenously processed through the class I pathway, it was important to further confirm these observations by using another human class I–restricted epitope. To address this issue, we tested the endogenous presentation of an inserted HLA B8–restricted CTL epitope, FLRGRAYGL (referred to as FLR), from an EBV-encoded EBNA3 protein within the EBNA1 COOH-terminal sequence (referred to as EBNA1-FLR), which allowed the study of endogenous processing of EBNA1 using an FLR-specific human CTL clone (11). The target cells were either transfected with expression vectors (EBNA1-FLR-GFP or EBNA1ΔGA-FLR-GFP) or infected with adenovirus encoding full-length EBNA1-FLR protein. It should be noted that target LCLs used in these assays were transformed with the B95.8 strain of EBV, which carries a mutation within the FLR epitope (29). As in the case of the HPV epitope, data presented in Fig. 6 shows that this epitope was endogenously processed by some HLA B8+ cells (AS LCL and C1R.B8) expressing EBNA1-FLR, whereas other HLA B8+ cells (LC LCLs) failed to present this epitope. The specificity of this recognition was confirmed by the inhibition of CTL killing of target cells coexpressing on HSV-1–encoded TAP inhibitor ICP47 (Fig. 6 B). These observations further confirm that CTL epitopes within EBNA1 can be endogenously processed and directly presented to CD8+ T cells through a TAP-dependent pathway.

Bottom Line: However, these different degradation rates do not correspond to the level of major histocompatibility complex class I-restricted presentation of EBNA1 epitopes.Functional assays revealed that processing of these EBNA1 epitopes is proteasome and transporter associated with antigen processing dependent.Based on these observations, we propose that defective ribosomal products, not the full-length antigen, are the primary source of endogenously processed CD8+ T cell epitopes from EBNA1.

View Article: PubMed Central - PubMed

Affiliation: EBV Unit, Tumour Immunology Laboratory, Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, 300 Herston Road, Brisbane (Qld) 4006, Australia.

ABSTRACT
Epstein-Barr virus (EBV)-encoded nuclear antigen (EBNA)1 is thought to escape cytotoxic T lymphocyte (CTL) recognition through either self-inhibition of synthesis or by blockade of proteasomal degradation by the glycine-alanine repeat (GAr) domain. Here we show that EBNA1 has a remarkably varied cell type-dependent stability. However, these different degradation rates do not correspond to the level of major histocompatibility complex class I-restricted presentation of EBNA1 epitopes. In spite of the highly stable expression of EBNA1 in B cells, CTL epitopes derived from this protein are efficiently processed and presented to CD8+ T cells. Furthermore, we show that EBV-infected B cells can readily activate EBNA1-specific memory T cell responses from healthy virus carriers. Functional assays revealed that processing of these EBNA1 epitopes is proteasome and transporter associated with antigen processing dependent. We also show that the endogenous presentation of these epitopes is dependent on the newly synthesized protein rather than the long-lived stable EBNA1. Based on these observations, we propose that defective ribosomal products, not the full-length antigen, are the primary source of endogenously processed CD8+ T cell epitopes from EBNA1.

Show MeSH
Related in: MedlinePlus