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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

Direct stimulation of EBNA1-specific CTL responses in vitro using LCL stimulators. CTL bulk cultures were generated from the HLA B*3501+ EBV-seropositive donors MW, CS, TK, and TC by incubating PBMCs with irradiated LCLs (responder/stimulator ratio of 20:1). CTL cultures were split and restimulated with additional irradiated LCLs on day 7. Stimulator cells were the class I− LCL 721.221, HLA B*3501-transfected 721.221 cells, the autologous LCL for each donor, or an LCL from the B*3501− donor DM. CTL cultures were also generated from donor TK after stimulation with the T2 cell line or B*3501-transfected T2 cells. On day 10, each CTL bulk culture was screened in chromium release assays for lysis of HLA B*3501+ PHA blasts that had been pretreated with 1 μM of the HPV peptide for 1 h or left untreated. An E/T ratio of 20:1 was used in each of these assays. These data are a representation of two separate experiments.
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fig3: Direct stimulation of EBNA1-specific CTL responses in vitro using LCL stimulators. CTL bulk cultures were generated from the HLA B*3501+ EBV-seropositive donors MW, CS, TK, and TC by incubating PBMCs with irradiated LCLs (responder/stimulator ratio of 20:1). CTL cultures were split and restimulated with additional irradiated LCLs on day 7. Stimulator cells were the class I− LCL 721.221, HLA B*3501-transfected 721.221 cells, the autologous LCL for each donor, or an LCL from the B*3501− donor DM. CTL cultures were also generated from donor TK after stimulation with the T2 cell line or B*3501-transfected T2 cells. On day 10, each CTL bulk culture was screened in chromium release assays for lysis of HLA B*3501+ PHA blasts that had been pretreated with 1 μM of the HPV peptide for 1 h or left untreated. An E/T ratio of 20:1 was used in each of these assays. These data are a representation of two separate experiments.

Mentions: To delineate the possible mechanisms involved in the induction of EBNA1-specific T cell responses, PBMCs from four HLA B*3501+ EBV-exposed individuals (MW, CS, TK, and TC) were stimulated with either B*3501+ or B*3501− LCLs to generate CTL cultures that were then tested for reactivity with the HPV epitope. These stimulator cells included the autologous LCLs, an HLA-mismatched LCL, the class I− LCL 721.221, or the 721.221 cell line that had been transfected with the HLA B*3501 gene. In all cases, the HLA B*3501+ stimulator cells were shown to be highly efficient at stimulating EBNA1-specific CTLs, whereas the CTL cultures stimulated with B*3501− cell lines showed negligible peptide-specific cytotoxicity (Fig. 3). This stimulation of an HPV-specific memory response was clearly dependent on the expression of TAP in the stimulator cells as TAP 1– and TAP 2–deficient T2 cells transfected with B*3501 were unable to stimulate a significant HPV-specific response (Fig. 3 C). These data suggest that the HPV-specific T cell response can be directly activated by EBV-infected B cells and this EBNA1 epitope is efficiently processed through the 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)

Direct stimulation of EBNA1-specific CTL responses in vitro using LCL stimulators. CTL bulk cultures were generated from the HLA B*3501+ EBV-seropositive donors MW, CS, TK, and TC by incubating PBMCs with irradiated LCLs (responder/stimulator ratio of 20:1). CTL cultures were split and restimulated with additional irradiated LCLs on day 7. Stimulator cells were the class I− LCL 721.221, HLA B*3501-transfected 721.221 cells, the autologous LCL for each donor, or an LCL from the B*3501− donor DM. CTL cultures were also generated from donor TK after stimulation with the T2 cell line or B*3501-transfected T2 cells. On day 10, each CTL bulk culture was screened in chromium release assays for lysis of HLA B*3501+ PHA blasts that had been pretreated with 1 μM of the HPV peptide for 1 h or left untreated. An E/T ratio of 20:1 was used in each of these assays. These data are a representation of two separate experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Direct stimulation of EBNA1-specific CTL responses in vitro using LCL stimulators. CTL bulk cultures were generated from the HLA B*3501+ EBV-seropositive donors MW, CS, TK, and TC by incubating PBMCs with irradiated LCLs (responder/stimulator ratio of 20:1). CTL cultures were split and restimulated with additional irradiated LCLs on day 7. Stimulator cells were the class I− LCL 721.221, HLA B*3501-transfected 721.221 cells, the autologous LCL for each donor, or an LCL from the B*3501− donor DM. CTL cultures were also generated from donor TK after stimulation with the T2 cell line or B*3501-transfected T2 cells. On day 10, each CTL bulk culture was screened in chromium release assays for lysis of HLA B*3501+ PHA blasts that had been pretreated with 1 μM of the HPV peptide for 1 h or left untreated. An E/T ratio of 20:1 was used in each of these assays. These data are a representation of two separate experiments.
Mentions: To delineate the possible mechanisms involved in the induction of EBNA1-specific T cell responses, PBMCs from four HLA B*3501+ EBV-exposed individuals (MW, CS, TK, and TC) were stimulated with either B*3501+ or B*3501− LCLs to generate CTL cultures that were then tested for reactivity with the HPV epitope. These stimulator cells included the autologous LCLs, an HLA-mismatched LCL, the class I− LCL 721.221, or the 721.221 cell line that had been transfected with the HLA B*3501 gene. In all cases, the HLA B*3501+ stimulator cells were shown to be highly efficient at stimulating EBNA1-specific CTLs, whereas the CTL cultures stimulated with B*3501− cell lines showed negligible peptide-specific cytotoxicity (Fig. 3). This stimulation of an HPV-specific memory response was clearly dependent on the expression of TAP in the stimulator cells as TAP 1– and TAP 2–deficient T2 cells transfected with B*3501 were unable to stimulate a significant HPV-specific response (Fig. 3 C). These data suggest that the HPV-specific T cell response can be directly activated by EBV-infected B cells and this EBNA1 epitope is efficiently processed through the 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