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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 EBNA1 CTL epitopes in epithelial cells and the effect of MHC class I and II inhibitors on endogenous processing of EBNA1 epitopes. (A) SVMR6 cells were infected with a recombinant adenovirus expressing either full-length EBNA1-FLR or EBNA1ΔGA-FLR. These cells were used as targets in a standard 51Cr-release assay to assess endogenous processing of an HLA B8–restricted FLR epitope encoded within EBNA1. The FLR-specific CTL clone LC13 was used as effector cells in the assay. An E/T ratio of 5:1 was used in the assay. The inset gel photo shows relative expression levels of full-length AdEBNA1 and AdEBNA1ΔGA after infection of SVMR6 cells. (B) SVMR6 keratinocytes were pretreated with either class I inhibitors, 10 μg/ml lactacystin, 1 μg/ml BFA, and 10 μg/ml Cbz-L3, or class II inhibitors, 80 μM chloroquine, 100 μM leupeptin, and 50 μM pepstatin, for 45 min. The cells were then infected with a recombinant adenovirus expressing full-length EBNA1 (Ad EBNA1-FLR). At 18 h after infection, the cells were used as targets in a standard 51Cr-release assay to assess endogenous presentation of the FLR epitope. FLR-specific CTL clone LC13 was used as effector cells in the assay. An E/T ratio of 5:1 was used in the assay. This data is a representation of three separate experiments. (C) CTL recognition of EBNA1-expressing SVMR6 cells and LCLs (HLA B35+, MW LCL; or HLA B35−, AS LCL) by HLA B35–restricted HPV-specific CTL clone (DY1). SVMR6 cells were transfected with an expression vector encoding the HLA B*3501 allele. Target cells were either pretreated with 100 μM leupeptin or left untreated. An E/T ratio of 5:1 was used in the assay.
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fig7: Endogenous processing of EBNA1 CTL epitopes in epithelial cells and the effect of MHC class I and II inhibitors on endogenous processing of EBNA1 epitopes. (A) SVMR6 cells were infected with a recombinant adenovirus expressing either full-length EBNA1-FLR or EBNA1ΔGA-FLR. These cells were used as targets in a standard 51Cr-release assay to assess endogenous processing of an HLA B8–restricted FLR epitope encoded within EBNA1. The FLR-specific CTL clone LC13 was used as effector cells in the assay. An E/T ratio of 5:1 was used in the assay. The inset gel photo shows relative expression levels of full-length AdEBNA1 and AdEBNA1ΔGA after infection of SVMR6 cells. (B) SVMR6 keratinocytes were pretreated with either class I inhibitors, 10 μg/ml lactacystin, 1 μg/ml BFA, and 10 μg/ml Cbz-L3, or class II inhibitors, 80 μM chloroquine, 100 μM leupeptin, and 50 μM pepstatin, for 45 min. The cells were then infected with a recombinant adenovirus expressing full-length EBNA1 (Ad EBNA1-FLR). At 18 h after infection, the cells were used as targets in a standard 51Cr-release assay to assess endogenous presentation of the FLR epitope. FLR-specific CTL clone LC13 was used as effector cells in the assay. An E/T ratio of 5:1 was used in the assay. This data is a representation of three separate experiments. (C) CTL recognition of EBNA1-expressing SVMR6 cells and LCLs (HLA B35+, MW LCL; or HLA B35−, AS LCL) by HLA B35–restricted HPV-specific CTL clone (DY1). SVMR6 cells were transfected with an expression vector encoding the HLA B*3501 allele. Target cells were either pretreated with 100 μM leupeptin or left untreated. An E/T ratio of 5:1 was used in the assay.

Mentions: Our intracellular kinetic studies had shown that the EBNA1 protein was highly unstable in epithelial cells. To test the hypothesis that the rapid degradation of EBNA1 in these cells may enhance CTL recognition in vitro, SVMR6 cells were infected with a recombinant adenovirus expressing either full-length EBNA1-FLR or EBNA1ΔGA-FLR, and then exposed to the CTL clone (LC13) specific for the FLR epitope. Cells infected with these recombinant adenoviruses showed similar levels of protein expression (Fig. 7 A, inset). The data presented in Fig. 7 A shows that SVMR6 cells expressing either full-length EBNA1-FLR or EBNA1ΔGA-FLR were efficiently recognized by an LC13 CTL clone and the level of lysis was comparable to that seen in LCLs expressing EBNA1 constructs (Fig. 6). As with the endogenous processing of the EBNA1 epitopes in B cells, the CTL lysis of SVMR6 cells was also blocked in the presence of BFA. However, in contrast to the degradation of full-length EBNA1, the presentation of the FLR CTL epitope in SVMR6 cells was blocked by the proteasome inhibitors, lactacystin and Cbz-L3 (Fig. 7 B). These observations strongly suggest that the full-length EBNA1 protein is unlikely to be the primary source of endogenously processed epitopes. We hypothesize that EBNA1 CTL epitopes are primarily derived from DRiPs and this presentation is dependent on the proteasomal pathway. The endogenous presentation of the FLR epitope within EBNA1 was not affected by the addition of chloroquine or pepstatin. Unexpectedly, pretreatment of target cells with leupeptin significantly enhanced the CTL recognition of the FLR epitope within the COOH terminus of EBNA1 (Fig. 7 B). This increase in CTL lysis was not observed when leupeptin-treated target cells were presensitized with synthetic FLR peptide (not depicted). To determine whether this enhanced killing of target cells after leupeptin treatment occurred for another EBNA1 epitope, HLA B*3501-transfected SVMR6 cells and LCLs were infected with AdEBNA1 and then exposed to the HPV-specific CTL clone, DY1. Data presented in Fig. 7 C shows that leupeptin treatment of these cells had no effect on the CTL recognition by an HPV-specific CTL clone.


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 EBNA1 CTL epitopes in epithelial cells and the effect of MHC class I and II inhibitors on endogenous processing of EBNA1 epitopes. (A) SVMR6 cells were infected with a recombinant adenovirus expressing either full-length EBNA1-FLR or EBNA1ΔGA-FLR. These cells were used as targets in a standard 51Cr-release assay to assess endogenous processing of an HLA B8–restricted FLR epitope encoded within EBNA1. The FLR-specific CTL clone LC13 was used as effector cells in the assay. An E/T ratio of 5:1 was used in the assay. The inset gel photo shows relative expression levels of full-length AdEBNA1 and AdEBNA1ΔGA after infection of SVMR6 cells. (B) SVMR6 keratinocytes were pretreated with either class I inhibitors, 10 μg/ml lactacystin, 1 μg/ml BFA, and 10 μg/ml Cbz-L3, or class II inhibitors, 80 μM chloroquine, 100 μM leupeptin, and 50 μM pepstatin, for 45 min. The cells were then infected with a recombinant adenovirus expressing full-length EBNA1 (Ad EBNA1-FLR). At 18 h after infection, the cells were used as targets in a standard 51Cr-release assay to assess endogenous presentation of the FLR epitope. FLR-specific CTL clone LC13 was used as effector cells in the assay. An E/T ratio of 5:1 was used in the assay. This data is a representation of three separate experiments. (C) CTL recognition of EBNA1-expressing SVMR6 cells and LCLs (HLA B35+, MW LCL; or HLA B35−, AS LCL) by HLA B35–restricted HPV-specific CTL clone (DY1). SVMR6 cells were transfected with an expression vector encoding the HLA B*3501 allele. Target cells were either pretreated with 100 μM leupeptin or left untreated. An E/T ratio of 5:1 was used in the assay.
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fig7: Endogenous processing of EBNA1 CTL epitopes in epithelial cells and the effect of MHC class I and II inhibitors on endogenous processing of EBNA1 epitopes. (A) SVMR6 cells were infected with a recombinant adenovirus expressing either full-length EBNA1-FLR or EBNA1ΔGA-FLR. These cells were used as targets in a standard 51Cr-release assay to assess endogenous processing of an HLA B8–restricted FLR epitope encoded within EBNA1. The FLR-specific CTL clone LC13 was used as effector cells in the assay. An E/T ratio of 5:1 was used in the assay. The inset gel photo shows relative expression levels of full-length AdEBNA1 and AdEBNA1ΔGA after infection of SVMR6 cells. (B) SVMR6 keratinocytes were pretreated with either class I inhibitors, 10 μg/ml lactacystin, 1 μg/ml BFA, and 10 μg/ml Cbz-L3, or class II inhibitors, 80 μM chloroquine, 100 μM leupeptin, and 50 μM pepstatin, for 45 min. The cells were then infected with a recombinant adenovirus expressing full-length EBNA1 (Ad EBNA1-FLR). At 18 h after infection, the cells were used as targets in a standard 51Cr-release assay to assess endogenous presentation of the FLR epitope. FLR-specific CTL clone LC13 was used as effector cells in the assay. An E/T ratio of 5:1 was used in the assay. This data is a representation of three separate experiments. (C) CTL recognition of EBNA1-expressing SVMR6 cells and LCLs (HLA B35+, MW LCL; or HLA B35−, AS LCL) by HLA B35–restricted HPV-specific CTL clone (DY1). SVMR6 cells were transfected with an expression vector encoding the HLA B*3501 allele. Target cells were either pretreated with 100 μM leupeptin or left untreated. An E/T ratio of 5:1 was used in the assay.
Mentions: Our intracellular kinetic studies had shown that the EBNA1 protein was highly unstable in epithelial cells. To test the hypothesis that the rapid degradation of EBNA1 in these cells may enhance CTL recognition in vitro, SVMR6 cells were infected with a recombinant adenovirus expressing either full-length EBNA1-FLR or EBNA1ΔGA-FLR, and then exposed to the CTL clone (LC13) specific for the FLR epitope. Cells infected with these recombinant adenoviruses showed similar levels of protein expression (Fig. 7 A, inset). The data presented in Fig. 7 A shows that SVMR6 cells expressing either full-length EBNA1-FLR or EBNA1ΔGA-FLR were efficiently recognized by an LC13 CTL clone and the level of lysis was comparable to that seen in LCLs expressing EBNA1 constructs (Fig. 6). As with the endogenous processing of the EBNA1 epitopes in B cells, the CTL lysis of SVMR6 cells was also blocked in the presence of BFA. However, in contrast to the degradation of full-length EBNA1, the presentation of the FLR CTL epitope in SVMR6 cells was blocked by the proteasome inhibitors, lactacystin and Cbz-L3 (Fig. 7 B). These observations strongly suggest that the full-length EBNA1 protein is unlikely to be the primary source of endogenously processed epitopes. We hypothesize that EBNA1 CTL epitopes are primarily derived from DRiPs and this presentation is dependent on the proteasomal pathway. The endogenous presentation of the FLR epitope within EBNA1 was not affected by the addition of chloroquine or pepstatin. Unexpectedly, pretreatment of target cells with leupeptin significantly enhanced the CTL recognition of the FLR epitope within the COOH terminus of EBNA1 (Fig. 7 B). This increase in CTL lysis was not observed when leupeptin-treated target cells were presensitized with synthetic FLR peptide (not depicted). To determine whether this enhanced killing of target cells after leupeptin treatment occurred for another EBNA1 epitope, HLA B*3501-transfected SVMR6 cells and LCLs were infected with AdEBNA1 and then exposed to the HPV-specific CTL clone, DY1. Data presented in Fig. 7 C shows that leupeptin treatment of these cells had no effect on the CTL recognition by an HPV-specific CTL clone.

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