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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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(A) Effect of MHC–peptide stripping and cycloheximide treatment on ex vivo intracellular IFN-γ production by EBNA1-specific T cells. PBMCs from an HLA B35+ donor were incubated alone or with either an HLA B*3501+ LCL, an HLA B*3501+ LCL plus 0.01 μM HPV peptide, an HLA B*3501+ LCL treated with citrate buffer and 50 μM cycloheximide, an HLA B*3501+ LCL treated with citrate buffer, or an HLA LHLA B*3501+ LCL treated with cycloheximide. Data shown represents the CD8+ and tetramer+ population (solid bars) and the tetramer+ population producing IFN-γ (shaded bars). This data is a representation of two separate experiments. (B) Surface MHC class I expression on untreated LCLs or LCLs treated with either cycloheximide, citrate buffer alone, or cycloheximide and citrate buffer. LCLs were initially incubated with MHC class I–specific mAb (W6/32) followed by incubation with FITC-labeled anti–mouse Ig. The fluorescence intensity was measured by FACSCalibur™ and data were analyzed by CELLQuest™ software. The results are expressed as mean fluorescence intensity.
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fig8: (A) Effect of MHC–peptide stripping and cycloheximide treatment on ex vivo intracellular IFN-γ production by EBNA1-specific T cells. PBMCs from an HLA B35+ donor were incubated alone or with either an HLA B*3501+ LCL, an HLA B*3501+ LCL plus 0.01 μM HPV peptide, an HLA B*3501+ LCL treated with citrate buffer and 50 μM cycloheximide, an HLA B*3501+ LCL treated with citrate buffer, or an HLA LHLA B*3501+ LCL treated with cycloheximide. Data shown represents the CD8+ and tetramer+ population (solid bars) and the tetramer+ population producing IFN-γ (shaded bars). This data is a representation of two separate experiments. (B) Surface MHC class I expression on untreated LCLs or LCLs treated with either cycloheximide, citrate buffer alone, or cycloheximide and citrate buffer. LCLs were initially incubated with MHC class I–specific mAb (W6/32) followed by incubation with FITC-labeled anti–mouse Ig. The fluorescence intensity was measured by FACSCalibur™ and data were analyzed by CELLQuest™ software. The results are expressed as mean fluorescence intensity.

Mentions: To determine whether the endogenously processed epitopes are derived from newly synthesized protein or from the long-lived stable EBNA1, we used the ex vivo stimulation assay in which PBMCs from an HLA B*3501+ EBV-seropositive individual (MW) were stimulated with MHC–peptide-stripped B*3501+ LCLs that had been incubated in the presence or absence of cycloheximide to block fresh protein synthesis. After overnight incubation, these cells were assessed for binding to an HPV HLA B*3501 tetramer and for intracellular IFN-γ expression. Data presented in Fig. 8 A demonstrate that >50% of HPV HLA B*3501 tetramer+ T cells showed strong intracellular IFN-γ expression after stimulation with untreated HLA B*3501+ LCLs. However, pretreatment of the LCLs with citrate buffer followed by incubation with cycloheximide significantly reduced the proportion of intracellular IFN-γ–expressing T cells within the HPV HLA B*3501 tetramer+ population (18%). On the other hand, LCLs treated with either citrate buffer (34.3%) or cycloheximide (53.1%) alone had a less significant effect on the stimulating capability of these cells. This data is consistent with the levels of surface MHC class I expression on LCLs after each of these treatments (Fig. 8 B). Furthermore, stimulation of PBMCs with PHA in the presence or absence of cycloheximide and/or citrate buffer showed comparable levels of IFN-γ expression (not depicted). These observations indicate that endogenously processed EBNA1 epitopes, which stimulate antigen-specific T cells, are primarily derived from newly synthesized protein rather than the long-lived stable EBNA1.


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)

(A) Effect of MHC–peptide stripping and cycloheximide treatment on ex vivo intracellular IFN-γ production by EBNA1-specific T cells. PBMCs from an HLA B35+ donor were incubated alone or with either an HLA B*3501+ LCL, an HLA B*3501+ LCL plus 0.01 μM HPV peptide, an HLA B*3501+ LCL treated with citrate buffer and 50 μM cycloheximide, an HLA B*3501+ LCL treated with citrate buffer, or an HLA LHLA B*3501+ LCL treated with cycloheximide. Data shown represents the CD8+ and tetramer+ population (solid bars) and the tetramer+ population producing IFN-γ (shaded bars). This data is a representation of two separate experiments. (B) Surface MHC class I expression on untreated LCLs or LCLs treated with either cycloheximide, citrate buffer alone, or cycloheximide and citrate buffer. LCLs were initially incubated with MHC class I–specific mAb (W6/32) followed by incubation with FITC-labeled anti–mouse Ig. The fluorescence intensity was measured by FACSCalibur™ and data were analyzed by CELLQuest™ software. The results are expressed as mean fluorescence intensity.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: (A) Effect of MHC–peptide stripping and cycloheximide treatment on ex vivo intracellular IFN-γ production by EBNA1-specific T cells. PBMCs from an HLA B35+ donor were incubated alone or with either an HLA B*3501+ LCL, an HLA B*3501+ LCL plus 0.01 μM HPV peptide, an HLA B*3501+ LCL treated with citrate buffer and 50 μM cycloheximide, an HLA B*3501+ LCL treated with citrate buffer, or an HLA LHLA B*3501+ LCL treated with cycloheximide. Data shown represents the CD8+ and tetramer+ population (solid bars) and the tetramer+ population producing IFN-γ (shaded bars). This data is a representation of two separate experiments. (B) Surface MHC class I expression on untreated LCLs or LCLs treated with either cycloheximide, citrate buffer alone, or cycloheximide and citrate buffer. LCLs were initially incubated with MHC class I–specific mAb (W6/32) followed by incubation with FITC-labeled anti–mouse Ig. The fluorescence intensity was measured by FACSCalibur™ and data were analyzed by CELLQuest™ software. The results are expressed as mean fluorescence intensity.
Mentions: To determine whether the endogenously processed epitopes are derived from newly synthesized protein or from the long-lived stable EBNA1, we used the ex vivo stimulation assay in which PBMCs from an HLA B*3501+ EBV-seropositive individual (MW) were stimulated with MHC–peptide-stripped B*3501+ LCLs that had been incubated in the presence or absence of cycloheximide to block fresh protein synthesis. After overnight incubation, these cells were assessed for binding to an HPV HLA B*3501 tetramer and for intracellular IFN-γ expression. Data presented in Fig. 8 A demonstrate that >50% of HPV HLA B*3501 tetramer+ T cells showed strong intracellular IFN-γ expression after stimulation with untreated HLA B*3501+ LCLs. However, pretreatment of the LCLs with citrate buffer followed by incubation with cycloheximide significantly reduced the proportion of intracellular IFN-γ–expressing T cells within the HPV HLA B*3501 tetramer+ population (18%). On the other hand, LCLs treated with either citrate buffer (34.3%) or cycloheximide (53.1%) alone had a less significant effect on the stimulating capability of these cells. This data is consistent with the levels of surface MHC class I expression on LCLs after each of these treatments (Fig. 8 B). Furthermore, stimulation of PBMCs with PHA in the presence or absence of cycloheximide and/or citrate buffer showed comparable levels of IFN-γ expression (not depicted). These observations indicate that endogenously processed EBNA1 epitopes, which stimulate antigen-specific T cells, are primarily derived from newly synthesized protein rather than the long-lived stable EBNA1.

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