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CD8 T cell recognition of endogenously expressed epstein-barr virus nuclear antigen 1.

Lee SP, Brooks JM, Al-Jarrah H, Thomas WA, Haigh TA, Taylor GS, Humme S, Schepers A, Hammerschmidt W, Yates JL, Rickinson AB, Blake NW - J. Exp. Med. (2004)

Bottom Line: Here, using CD8+ T cell clones to native EBNA1 epitopes upstream and downstream of the GAr domain and assaying recognition by interferon gamma release, we show that the EBNA1 naturally expressed in EBV-transformed lymphoblastoid cell lines (LCLs) is in fact presented to CD8+ T cells via a proteasome/peptide transporter-dependent pathway.Furthermore, LCL recognition by such CD8+ T cells, although slightly lower than seen with paired lines expressing a GAr-deleted EBNA1 protein, leads to strong and specific inhibition of LCL outgrowth in vitro.Endogenously expressed EBNA1 is therefore accessible to the MHC class I pathway despite GAr-mediated stabilization of the mature protein.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cancer Studies, University of Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TT, UK. s.p.lee@bham.ac.uk

ABSTRACT
The Epstein-Barr virus (EBV) nuclear antigen (EBNA)1 contains a glycine-alanine repeat (GAr) domain that appears to protect the antigen from proteasomal breakdown and, as measured in cytotoxicity assays, from major histocompatibility complex (MHC) class I-restricted presentation to CD8+ T cells. This led to the concept of EBNA1 as an immunologically silent protein that although unique in being expressed in all EBV malignancies, could not be exploited as a CD8 target. Here, using CD8+ T cell clones to native EBNA1 epitopes upstream and downstream of the GAr domain and assaying recognition by interferon gamma release, we show that the EBNA1 naturally expressed in EBV-transformed lymphoblastoid cell lines (LCLs) is in fact presented to CD8+ T cells via a proteasome/peptide transporter-dependent pathway. Furthermore, LCL recognition by such CD8+ T cells, although slightly lower than seen with paired lines expressing a GAr-deleted EBNA1 protein, leads to strong and specific inhibition of LCL outgrowth in vitro. Endogenously expressed EBNA1 is therefore accessible to the MHC class I pathway despite GAr-mediated stabilization of the mature protein. We infer that EBNA1-specific CD8+ T cells do play a role in control of EBV infection in vivo and might be exploitable in the control of EBV+ malignancies.

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EBNA1-specific CD8+ T cell recognition of LCLs measured by cytotoxicity. (A) Schematic diagram of wild-type EBNA1 protein showing the sequence of CD8+ epitopes. Numbers refer to coordinates in the 641–amino acid sequence of the B95.8 strain EBNA1 protein. (B) Immunoblot of SDS-PAGE–separated protein extracts from the B95.8 cell line, B95.8 virus-transformed LCLs, RT (B95), and NA (B95), expressing wild-type EBNA1 and from the corresponding dl7 virus transformants, RT (dl7a) and NA (dl7), expressing GAr-deleted EBNA1. BJAB is an EBV− B lymphoma cell line. The blot is probed with the EBNA1-specific monoclonal antibody IH4. (C) Results of 5-h chromium release assays in which B*3501-restricted CD8+ T cell clones specific for the HPV (EBNA1) epitope or for the YPL (EBNA3A) epitope were used as effectors on a common panel of LCL targets. Targets included two dl7 virus transformants from B*3501+ donor RT, RT-LCL (dl7a), and RT-LCL (dl7b) expressing GAr-deleted EBNA1, three B*3501+ LCLs from donors RT, GT, and IM transformed either with B95.8 virus (for RT and GT) or by spontaneous outgrowth with the donor's own EBV strain (for IM), and two B95.8 LCLs from B*3501− donors CM and DH. Results are shown as percent-specific lysis at effector/target ratios of 5:1 and 2.5:1.
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fig1: EBNA1-specific CD8+ T cell recognition of LCLs measured by cytotoxicity. (A) Schematic diagram of wild-type EBNA1 protein showing the sequence of CD8+ epitopes. Numbers refer to coordinates in the 641–amino acid sequence of the B95.8 strain EBNA1 protein. (B) Immunoblot of SDS-PAGE–separated protein extracts from the B95.8 cell line, B95.8 virus-transformed LCLs, RT (B95), and NA (B95), expressing wild-type EBNA1 and from the corresponding dl7 virus transformants, RT (dl7a) and NA (dl7), expressing GAr-deleted EBNA1. BJAB is an EBV− B lymphoma cell line. The blot is probed with the EBNA1-specific monoclonal antibody IH4. (C) Results of 5-h chromium release assays in which B*3501-restricted CD8+ T cell clones specific for the HPV (EBNA1) epitope or for the YPL (EBNA3A) epitope were used as effectors on a common panel of LCL targets. Targets included two dl7 virus transformants from B*3501+ donor RT, RT-LCL (dl7a), and RT-LCL (dl7b) expressing GAr-deleted EBNA1, three B*3501+ LCLs from donors RT, GT, and IM transformed either with B95.8 virus (for RT and GT) or by spontaneous outgrowth with the donor's own EBV strain (for IM), and two B95.8 LCLs from B*3501− donors CM and DH. Results are shown as percent-specific lysis at effector/target ratios of 5:1 and 2.5:1.

Mentions: This work used CD8+ effector T cell clones against the four EBNA1 epitopes shown in Fig. 1 A and referred to as HPV (B*3501-restricted), RPQ and IPQ (both B*0701-restricted), and VLK (A*0203-restricted). The EBNA1 specificity of these CD8+ clones was in each case confirmed in cytotoxicity assays against targets exogenously loaded with epitope peptide and targets infected with a vaccinia expressing GAr-deleted EBNA1 (14; not depicted). Target LCLs were generated from selected donors by transformation with the reference B95.8 EBV strain and with a recombinant B95.8 virus, dl7, carrying a GAr-deleted EBNA1 gene (4). Fig. 1 B shows an immunoblot of SDS-PAGE–separated proteins from two such LCL pairs, probed with the IH4 monoclonal antibody against the COOH-terminal region of EBNA1 downstream of the GAr domain. Both wild-type transformants express the standard 80-kD EBNA1 protein as does the reference B95.8 cell line itself, whereas the dl7 transformants express the 52-kD GAr-deleted protein. These LCLs are identical in their expression of the other EBV-latent cycle proteins (unpublished data).


CD8 T cell recognition of endogenously expressed epstein-barr virus nuclear antigen 1.

Lee SP, Brooks JM, Al-Jarrah H, Thomas WA, Haigh TA, Taylor GS, Humme S, Schepers A, Hammerschmidt W, Yates JL, Rickinson AB, Blake NW - J. Exp. Med. (2004)

EBNA1-specific CD8+ T cell recognition of LCLs measured by cytotoxicity. (A) Schematic diagram of wild-type EBNA1 protein showing the sequence of CD8+ epitopes. Numbers refer to coordinates in the 641–amino acid sequence of the B95.8 strain EBNA1 protein. (B) Immunoblot of SDS-PAGE–separated protein extracts from the B95.8 cell line, B95.8 virus-transformed LCLs, RT (B95), and NA (B95), expressing wild-type EBNA1 and from the corresponding dl7 virus transformants, RT (dl7a) and NA (dl7), expressing GAr-deleted EBNA1. BJAB is an EBV− B lymphoma cell line. The blot is probed with the EBNA1-specific monoclonal antibody IH4. (C) Results of 5-h chromium release assays in which B*3501-restricted CD8+ T cell clones specific for the HPV (EBNA1) epitope or for the YPL (EBNA3A) epitope were used as effectors on a common panel of LCL targets. Targets included two dl7 virus transformants from B*3501+ donor RT, RT-LCL (dl7a), and RT-LCL (dl7b) expressing GAr-deleted EBNA1, three B*3501+ LCLs from donors RT, GT, and IM transformed either with B95.8 virus (for RT and GT) or by spontaneous outgrowth with the donor's own EBV strain (for IM), and two B95.8 LCLs from B*3501− donors CM and DH. Results are shown as percent-specific lysis at effector/target ratios of 5:1 and 2.5:1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211813&req=5

fig1: EBNA1-specific CD8+ T cell recognition of LCLs measured by cytotoxicity. (A) Schematic diagram of wild-type EBNA1 protein showing the sequence of CD8+ epitopes. Numbers refer to coordinates in the 641–amino acid sequence of the B95.8 strain EBNA1 protein. (B) Immunoblot of SDS-PAGE–separated protein extracts from the B95.8 cell line, B95.8 virus-transformed LCLs, RT (B95), and NA (B95), expressing wild-type EBNA1 and from the corresponding dl7 virus transformants, RT (dl7a) and NA (dl7), expressing GAr-deleted EBNA1. BJAB is an EBV− B lymphoma cell line. The blot is probed with the EBNA1-specific monoclonal antibody IH4. (C) Results of 5-h chromium release assays in which B*3501-restricted CD8+ T cell clones specific for the HPV (EBNA1) epitope or for the YPL (EBNA3A) epitope were used as effectors on a common panel of LCL targets. Targets included two dl7 virus transformants from B*3501+ donor RT, RT-LCL (dl7a), and RT-LCL (dl7b) expressing GAr-deleted EBNA1, three B*3501+ LCLs from donors RT, GT, and IM transformed either with B95.8 virus (for RT and GT) or by spontaneous outgrowth with the donor's own EBV strain (for IM), and two B95.8 LCLs from B*3501− donors CM and DH. Results are shown as percent-specific lysis at effector/target ratios of 5:1 and 2.5:1.
Mentions: This work used CD8+ effector T cell clones against the four EBNA1 epitopes shown in Fig. 1 A and referred to as HPV (B*3501-restricted), RPQ and IPQ (both B*0701-restricted), and VLK (A*0203-restricted). The EBNA1 specificity of these CD8+ clones was in each case confirmed in cytotoxicity assays against targets exogenously loaded with epitope peptide and targets infected with a vaccinia expressing GAr-deleted EBNA1 (14; not depicted). Target LCLs were generated from selected donors by transformation with the reference B95.8 EBV strain and with a recombinant B95.8 virus, dl7, carrying a GAr-deleted EBNA1 gene (4). Fig. 1 B shows an immunoblot of SDS-PAGE–separated proteins from two such LCL pairs, probed with the IH4 monoclonal antibody against the COOH-terminal region of EBNA1 downstream of the GAr domain. Both wild-type transformants express the standard 80-kD EBNA1 protein as does the reference B95.8 cell line itself, whereas the dl7 transformants express the 52-kD GAr-deleted protein. These LCLs are identical in their expression of the other EBV-latent cycle proteins (unpublished data).

Bottom Line: Here, using CD8+ T cell clones to native EBNA1 epitopes upstream and downstream of the GAr domain and assaying recognition by interferon gamma release, we show that the EBNA1 naturally expressed in EBV-transformed lymphoblastoid cell lines (LCLs) is in fact presented to CD8+ T cells via a proteasome/peptide transporter-dependent pathway.Furthermore, LCL recognition by such CD8+ T cells, although slightly lower than seen with paired lines expressing a GAr-deleted EBNA1 protein, leads to strong and specific inhibition of LCL outgrowth in vitro.Endogenously expressed EBNA1 is therefore accessible to the MHC class I pathway despite GAr-mediated stabilization of the mature protein.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cancer Studies, University of Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TT, UK. s.p.lee@bham.ac.uk

ABSTRACT
The Epstein-Barr virus (EBV) nuclear antigen (EBNA)1 contains a glycine-alanine repeat (GAr) domain that appears to protect the antigen from proteasomal breakdown and, as measured in cytotoxicity assays, from major histocompatibility complex (MHC) class I-restricted presentation to CD8+ T cells. This led to the concept of EBNA1 as an immunologically silent protein that although unique in being expressed in all EBV malignancies, could not be exploited as a CD8 target. Here, using CD8+ T cell clones to native EBNA1 epitopes upstream and downstream of the GAr domain and assaying recognition by interferon gamma release, we show that the EBNA1 naturally expressed in EBV-transformed lymphoblastoid cell lines (LCLs) is in fact presented to CD8+ T cells via a proteasome/peptide transporter-dependent pathway. Furthermore, LCL recognition by such CD8+ T cells, although slightly lower than seen with paired lines expressing a GAr-deleted EBNA1 protein, leads to strong and specific inhibition of LCL outgrowth in vitro. Endogenously expressed EBNA1 is therefore accessible to the MHC class I pathway despite GAr-mediated stabilization of the mature protein. We infer that EBNA1-specific CD8+ T cells do play a role in control of EBV infection in vivo and might be exploitable in the control of EBV+ malignancies.

Show MeSH
Related in: MedlinePlus