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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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Related in: MedlinePlus

CTL recognition of endogenously processed EBNA1 epitopes. (A) HLA B35+ and HLA B35− LCLs were used as targets in a standard 51Cr-release assay to assess endogenous processing of EBNA1. (A) A CTL clone specific for the HLA B35-binding HPVGEADYFEY epitope was added to target cells at the E/T ratios indicated. (B) An HLA B35+ LCL, 721.221 LCLs, 721.221 LCLs transfected with HLA B*3501, T2 LCLs, and T2 LCLs transfected with HLA B*3501 were used as targets in a standard 51Cr-release assay to assess CTL activity to an HPVGEADYFEY-specific CTL clone at an E/T ratio of 5:1. These data are a representation of three separate experiments.
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fig5: CTL recognition of endogenously processed EBNA1 epitopes. (A) HLA B35+ and HLA B35− LCLs were used as targets in a standard 51Cr-release assay to assess endogenous processing of EBNA1. (A) A CTL clone specific for the HLA B35-binding HPVGEADYFEY epitope was added to target cells at the E/T ratios indicated. (B) An HLA B35+ LCL, 721.221 LCLs, 721.221 LCLs transfected with HLA B*3501, T2 LCLs, and T2 LCLs transfected with HLA B*3501 were used as targets in a standard 51Cr-release assay to assess CTL activity to an HPVGEADYFEY-specific CTL clone at an E/T ratio of 5:1. These data are a representation of three separate experiments.

Mentions: In the next set of experiments, we further assessed the endogenous processing of EBNA1 CTL epitopes by different cell types using in vitro cytotoxicity assays. In the first instance, we used a CTL clone(s) specific for the HLA B35–binding HPV epitope to assess CTL lysis of B cells expressing EBNA1. Blake et al. (13) have previously shown that T cells specific for this epitope are unable to recognize HLA-matched EBV-transformed LCLs. Contrary to these observations, we found that HPV-specific CTL clones do recognize HLA B35+ LCLs (Fig. 5 A), although there was some degree of variation in the level of CTL lysis amongst different HLA B35+ LCLs. It is important to mention here that the level of lysis observed for EBNA1-specific CTLs is quite comparable to that seen for CTL clones specific for other latent antigens (EBNA2-6; references 5 and 23). The fine specificity of this CTL recognition was further confirmed by the recognition of 721.221 LCLs transfected with HLA B35 (referred to as 0.221.B35 LCL), whereas no CTL lysis was observed for 721.221 LCLs or other LCLs that were negative for HLA B35 (Fig. 5, A and B). We have also demonstrated that the endogenous presentation of the HPV epitope was clearly dependent on the expression of TAP as T2 cells expressing HLA B35 were not recognized by these CTL clones (Fig. 5 B). In addition, the killing of HLA B35 LCLs was completely blocked by the pretreatment of these cells with BFA (not depicted). Taken together, these results suggest that in spite of the highly stable expression of EBNA1 in B cells, CTL epitopes within this protein can be directly presented by EBV-transformed LCLs, and this presentation proceeds 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)

CTL recognition of endogenously processed EBNA1 epitopes. (A) HLA B35+ and HLA B35− LCLs were used as targets in a standard 51Cr-release assay to assess endogenous processing of EBNA1. (A) A CTL clone specific for the HLA B35-binding HPVGEADYFEY epitope was added to target cells at the E/T ratios indicated. (B) An HLA B35+ LCL, 721.221 LCLs, 721.221 LCLs transfected with HLA B*3501, T2 LCLs, and T2 LCLs transfected with HLA B*3501 were used as targets in a standard 51Cr-release assay to assess CTL activity to an HPVGEADYFEY-specific CTL clone at an E/T ratio of 5:1. These data are a representation of three separate experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211806&req=5

fig5: CTL recognition of endogenously processed EBNA1 epitopes. (A) HLA B35+ and HLA B35− LCLs were used as targets in a standard 51Cr-release assay to assess endogenous processing of EBNA1. (A) A CTL clone specific for the HLA B35-binding HPVGEADYFEY epitope was added to target cells at the E/T ratios indicated. (B) An HLA B35+ LCL, 721.221 LCLs, 721.221 LCLs transfected with HLA B*3501, T2 LCLs, and T2 LCLs transfected with HLA B*3501 were used as targets in a standard 51Cr-release assay to assess CTL activity to an HPVGEADYFEY-specific CTL clone at an E/T ratio of 5:1. These data are a representation of three separate experiments.
Mentions: In the next set of experiments, we further assessed the endogenous processing of EBNA1 CTL epitopes by different cell types using in vitro cytotoxicity assays. In the first instance, we used a CTL clone(s) specific for the HLA B35–binding HPV epitope to assess CTL lysis of B cells expressing EBNA1. Blake et al. (13) have previously shown that T cells specific for this epitope are unable to recognize HLA-matched EBV-transformed LCLs. Contrary to these observations, we found that HPV-specific CTL clones do recognize HLA B35+ LCLs (Fig. 5 A), although there was some degree of variation in the level of CTL lysis amongst different HLA B35+ LCLs. It is important to mention here that the level of lysis observed for EBNA1-specific CTLs is quite comparable to that seen for CTL clones specific for other latent antigens (EBNA2-6; references 5 and 23). The fine specificity of this CTL recognition was further confirmed by the recognition of 721.221 LCLs transfected with HLA B35 (referred to as 0.221.B35 LCL), whereas no CTL lysis was observed for 721.221 LCLs or other LCLs that were negative for HLA B35 (Fig. 5, A and B). We have also demonstrated that the endogenous presentation of the HPV epitope was clearly dependent on the expression of TAP as T2 cells expressing HLA B35 were not recognized by these CTL clones (Fig. 5 B). In addition, the killing of HLA B35 LCLs was completely blocked by the pretreatment of these cells with BFA (not depicted). Taken together, these results suggest that in spite of the highly stable expression of EBNA1 in B cells, CTL epitopes within this protein can be directly presented by EBV-transformed LCLs, and this presentation proceeds 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