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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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B*0701- and A*0203-restricted CD8+ T cell recognition of endogenously expressed EBNA1. (A) Three CD8+ T cell clones specific for the RPQ/B*0701 epitope were tested against a panel of B*0701+ and B*0701− LCL targets transformed with the wild-type EBV strain B95.8, against the B*0701+ NA-LCL (dl7) carrying an EBNA1 gene deleted for the GAr-coding sequence, or against the B*0701+ MD-LCL (dE1) carrying an EBV genome from which the EBNA1 gene has been completely deleted. As a positive control, T cells were tested against MD-LCL (dE1) cells that had been precoated with the target epitope peptide RPQ. (B) Three CD8+ T cell clones specific for the VLK/A*0203 epitope were tested against a panel of HLA-matched and -mismatched LCL targets transformed with the wild-type EBV strain B95.8. As a positive control, T cells were tested with 10−7 M VLK epitope peptide alone. T cells were tested at 104 cells/well and LCL targets were tested at 2 × 104 cells/well. Results expressed as in Fig. 2.
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fig3: B*0701- and A*0203-restricted CD8+ T cell recognition of endogenously expressed EBNA1. (A) Three CD8+ T cell clones specific for the RPQ/B*0701 epitope were tested against a panel of B*0701+ and B*0701− LCL targets transformed with the wild-type EBV strain B95.8, against the B*0701+ NA-LCL (dl7) carrying an EBNA1 gene deleted for the GAr-coding sequence, or against the B*0701+ MD-LCL (dE1) carrying an EBV genome from which the EBNA1 gene has been completely deleted. As a positive control, T cells were tested against MD-LCL (dE1) cells that had been precoated with the target epitope peptide RPQ. (B) Three CD8+ T cell clones specific for the VLK/A*0203 epitope were tested against a panel of HLA-matched and -mismatched LCL targets transformed with the wild-type EBV strain B95.8. As a positive control, T cells were tested with 10−7 M VLK epitope peptide alone. T cells were tested at 104 cells/well and LCL targets were tested at 2 × 104 cells/well. Results expressed as in Fig. 2.

Mentions: The greater sensitivity of IFN-γ release assays now allowed us to examine target cell recognition by CD8+ T cell clones to the other EBNA1 epitopes. As shown in Fig. 3 A, three clones specific for the B*0701-restricted RPQ epitope, though giving different absolute levels of response, all showed reproducible recognition of the wild-type LCL from a B*0701+ donor NA, again at levels around 50–70% of that seen against the paired dl7 transformant and clearly well above the background shown by HLA-mismatched LCLs. This assay also includes another pair of target LCLs established from a B*0701+ donor MD, one (MD-B95) transformed with recombinant virus containing the complete B95.8 genomic sequence, the other (MD-dE1) transformed with a recombinant from which the entire EBNA1 gene has been deleted, the genome maintenance function of EBNA1 having been rendered redundant by integration of the virus genome into host cell DNA (16). There was clear recognition of the wild-type but not the EBNA1-deleted LCL, further confirming that recognition is absolutely dependent upon EBNA1 being expressed in the target cells. Similar results to the above were observed with the one available CD8+ T cell clone specific for a second B*0701-restricted EBNA1 epitope, IPQ (unpublished data). Fig. 3 B shows data from three representative clones specific for the A*0203-restricted EBNA1 epitope, VLK. Though there were no dl7 transformants available as internal standards in this case, all three VLK-specific clones clearly recognized wild-type EBNA1-expressing LCLs from A*0203+ donors but not from HLA-mismatched donors, even from donors with other A*02 subtype alleles.


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)

B*0701- and A*0203-restricted CD8+ T cell recognition of endogenously expressed EBNA1. (A) Three CD8+ T cell clones specific for the RPQ/B*0701 epitope were tested against a panel of B*0701+ and B*0701− LCL targets transformed with the wild-type EBV strain B95.8, against the B*0701+ NA-LCL (dl7) carrying an EBNA1 gene deleted for the GAr-coding sequence, or against the B*0701+ MD-LCL (dE1) carrying an EBV genome from which the EBNA1 gene has been completely deleted. As a positive control, T cells were tested against MD-LCL (dE1) cells that had been precoated with the target epitope peptide RPQ. (B) Three CD8+ T cell clones specific for the VLK/A*0203 epitope were tested against a panel of HLA-matched and -mismatched LCL targets transformed with the wild-type EBV strain B95.8. As a positive control, T cells were tested with 10−7 M VLK epitope peptide alone. T cells were tested at 104 cells/well and LCL targets were tested at 2 × 104 cells/well. Results expressed as in Fig. 2.
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Related In: Results  -  Collection

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fig3: B*0701- and A*0203-restricted CD8+ T cell recognition of endogenously expressed EBNA1. (A) Three CD8+ T cell clones specific for the RPQ/B*0701 epitope were tested against a panel of B*0701+ and B*0701− LCL targets transformed with the wild-type EBV strain B95.8, against the B*0701+ NA-LCL (dl7) carrying an EBNA1 gene deleted for the GAr-coding sequence, or against the B*0701+ MD-LCL (dE1) carrying an EBV genome from which the EBNA1 gene has been completely deleted. As a positive control, T cells were tested against MD-LCL (dE1) cells that had been precoated with the target epitope peptide RPQ. (B) Three CD8+ T cell clones specific for the VLK/A*0203 epitope were tested against a panel of HLA-matched and -mismatched LCL targets transformed with the wild-type EBV strain B95.8. As a positive control, T cells were tested with 10−7 M VLK epitope peptide alone. T cells were tested at 104 cells/well and LCL targets were tested at 2 × 104 cells/well. Results expressed as in Fig. 2.
Mentions: The greater sensitivity of IFN-γ release assays now allowed us to examine target cell recognition by CD8+ T cell clones to the other EBNA1 epitopes. As shown in Fig. 3 A, three clones specific for the B*0701-restricted RPQ epitope, though giving different absolute levels of response, all showed reproducible recognition of the wild-type LCL from a B*0701+ donor NA, again at levels around 50–70% of that seen against the paired dl7 transformant and clearly well above the background shown by HLA-mismatched LCLs. This assay also includes another pair of target LCLs established from a B*0701+ donor MD, one (MD-B95) transformed with recombinant virus containing the complete B95.8 genomic sequence, the other (MD-dE1) transformed with a recombinant from which the entire EBNA1 gene has been deleted, the genome maintenance function of EBNA1 having been rendered redundant by integration of the virus genome into host cell DNA (16). There was clear recognition of the wild-type but not the EBNA1-deleted LCL, further confirming that recognition is absolutely dependent upon EBNA1 being expressed in the target cells. Similar results to the above were observed with the one available CD8+ T cell clone specific for a second B*0701-restricted EBNA1 epitope, IPQ (unpublished data). Fig. 3 B shows data from three representative clones specific for the A*0203-restricted EBNA1 epitope, VLK. Though there were no dl7 transformants available as internal standards in this case, all three VLK-specific clones clearly recognized wild-type EBNA1-expressing LCLs from A*0203+ donors but not from HLA-mismatched donors, even from donors with other A*02 subtype alleles.

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