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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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Presentation of EBNA1 to CD8+ T cells does not occur in cells with defects in the proteasome/TAP-dependent processing pathway. (A) Top: EBNA1 immunoblot (as in Fig. 1 B) of protein extracts from the Sal-BL/Sal-LCL and Chep-BL/Chep-LCL pairings. X50-7 is a standard B95.8 virus-transformed LCL. Middle: Three CD8+ T cell clones specific for the HPV/B*3501 epitope were tested against the B*3501+ Sal-BL/Sal-LCL pair of targets and against Sal-BL cells precoated with HPV peptide. Control targets were the B*3501+ RT-LCL (B95) and an HLA-mismatched LCL precoated with HPV peptide. Bottom: Three T cell clones specific for the RPQ/B*0701 epitope were tested against the B*0701+ Chep-BL/Chep-LCL pair of targets and against Chep-BL cells precoated with RPQ peptide. Control targets were the B*0701+ GT-LCL (B95) and an HLA-mismatched LCL precoated with RPQ peptide. (B) Top panels: CD8+ T cell clones specific for the B*3501-restricted epitopes HPV (EBNA1) or YPL (EBNA3A) were tested against the Sal-BL/Sal-LCL pair of targets and against Sal-BL cells preexposed for 2 d to CD40 ligand–expressing mouse L cells (L+CD40L). Control targets included the Chep-BL/Chep-LCL pair, Chep-BL cells similarly exposed to L+CD40L cells, and L+CD40L cells themselves. Bottom: CD8+ T cell clones specific for the RPQ/B*0701 epitope were tested against the Chep-BL/Chep-LCL pair and against Chep-BL cells preexposed as described above to L+CD40L cells. Control targets included the HLA-mismatched Akata BL line, with and without preexposure to L+CD40L cells, the HLA-mismatched RT-LCL (Ak) transformed with the Akata virus strain, and L+CD40L cells themselves. (C) CD8+ T cell clones against the HPV and YPL epitopes were tested against the TAP− T2:B35 LCL target line precoated or not with the relevant epitope peptide. Control targets included the B*3501+ RT-LCL (B95) and an HLA-mismatched LCL precoated with the relevant peptide. In all the above assays, T cells were tested at 1,000–10,000 cells per well and targets at 25,000 cells per well. Results expressed as in Fig. 2.
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fig4: Presentation of EBNA1 to CD8+ T cells does not occur in cells with defects in the proteasome/TAP-dependent processing pathway. (A) Top: EBNA1 immunoblot (as in Fig. 1 B) of protein extracts from the Sal-BL/Sal-LCL and Chep-BL/Chep-LCL pairings. X50-7 is a standard B95.8 virus-transformed LCL. Middle: Three CD8+ T cell clones specific for the HPV/B*3501 epitope were tested against the B*3501+ Sal-BL/Sal-LCL pair of targets and against Sal-BL cells precoated with HPV peptide. Control targets were the B*3501+ RT-LCL (B95) and an HLA-mismatched LCL precoated with HPV peptide. Bottom: Three T cell clones specific for the RPQ/B*0701 epitope were tested against the B*0701+ Chep-BL/Chep-LCL pair of targets and against Chep-BL cells precoated with RPQ peptide. Control targets were the B*0701+ GT-LCL (B95) and an HLA-mismatched LCL precoated with RPQ peptide. (B) Top panels: CD8+ T cell clones specific for the B*3501-restricted epitopes HPV (EBNA1) or YPL (EBNA3A) were tested against the Sal-BL/Sal-LCL pair of targets and against Sal-BL cells preexposed for 2 d to CD40 ligand–expressing mouse L cells (L+CD40L). Control targets included the Chep-BL/Chep-LCL pair, Chep-BL cells similarly exposed to L+CD40L cells, and L+CD40L cells themselves. Bottom: CD8+ T cell clones specific for the RPQ/B*0701 epitope were tested against the Chep-BL/Chep-LCL pair and against Chep-BL cells preexposed as described above to L+CD40L cells. Control targets included the HLA-mismatched Akata BL line, with and without preexposure to L+CD40L cells, the HLA-mismatched RT-LCL (Ak) transformed with the Akata virus strain, and L+CD40L cells themselves. (C) CD8+ T cell clones against the HPV and YPL epitopes were tested against the TAP− T2:B35 LCL target line precoated or not with the relevant epitope peptide. Control targets included the B*3501+ RT-LCL (B95) and an HLA-mismatched LCL precoated with the relevant peptide. In all the above assays, T cells were tested at 1,000–10,000 cells per well and targets at 25,000 cells per well. Results expressed as in Fig. 2.

Mentions: Subsequent experiments aimed to identify the pathway whereby endogenously expressed wild-type EBNA1 was presented to CD8 T cells. We first examined the situation in EBV+ BL cell lines that have retained the original tumor-like “group I” phenotype in vitro. Compared with LCLs, such lines show more limited patterns of EBV-latent antigen expression usually restricted to EBNA1 only and, most importantly, are deficient in their capacity to process endogenously expressed antigen via the conventional proteasome/TAP-dependent pathway (22). Here we used two EBV+ BL cell lines that are known to have this processing deficiency, the B*3501+ Sal-BL line and the B*0701+ Chep-BL line. As controls, we used LCLs generated from the normal B cells of these same patients by in vitro transformation either with their own resident EBV strain (in the case of Sal-LCL) or with the B95.8 strain (in the case of Chep-LCL). The EBNA1 immunoblot of protein extracts from these BL/LCL pairs is shown in Fig. 4 A, confirming that the BL lines express the native EBNA1 protein at levels at least equal to those seen in the matching LCLs. Furthermore, sequencing the EBNA1 gene in the Sal-BL and Chep-BL virus strains confirmed that the relevant epitope sequences, HPV and RPQ, respectively, were conserved (not depicted). As shown in Fig. 4 A, three CD8+ T cell clones specific for the HPV/B*3501 epitope recognized the Sal-LCL as expected, but did not recognize the EBNA1-expressing Sal-BL target unless this was exogenously coated with synthetic epitope peptide. Likewise, three clones specific for the RPQ/B*0701 epitope recognized the Chep-LCL but not Chep-BL cells unless the latter were peptide coated. As a specificity control, in each case there was no recognition of a peptide-coated HLA-mismatched BL or LCL target.


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)

Presentation of EBNA1 to CD8+ T cells does not occur in cells with defects in the proteasome/TAP-dependent processing pathway. (A) Top: EBNA1 immunoblot (as in Fig. 1 B) of protein extracts from the Sal-BL/Sal-LCL and Chep-BL/Chep-LCL pairings. X50-7 is a standard B95.8 virus-transformed LCL. Middle: Three CD8+ T cell clones specific for the HPV/B*3501 epitope were tested against the B*3501+ Sal-BL/Sal-LCL pair of targets and against Sal-BL cells precoated with HPV peptide. Control targets were the B*3501+ RT-LCL (B95) and an HLA-mismatched LCL precoated with HPV peptide. Bottom: Three T cell clones specific for the RPQ/B*0701 epitope were tested against the B*0701+ Chep-BL/Chep-LCL pair of targets and against Chep-BL cells precoated with RPQ peptide. Control targets were the B*0701+ GT-LCL (B95) and an HLA-mismatched LCL precoated with RPQ peptide. (B) Top panels: CD8+ T cell clones specific for the B*3501-restricted epitopes HPV (EBNA1) or YPL (EBNA3A) were tested against the Sal-BL/Sal-LCL pair of targets and against Sal-BL cells preexposed for 2 d to CD40 ligand–expressing mouse L cells (L+CD40L). Control targets included the Chep-BL/Chep-LCL pair, Chep-BL cells similarly exposed to L+CD40L cells, and L+CD40L cells themselves. Bottom: CD8+ T cell clones specific for the RPQ/B*0701 epitope were tested against the Chep-BL/Chep-LCL pair and against Chep-BL cells preexposed as described above to L+CD40L cells. Control targets included the HLA-mismatched Akata BL line, with and without preexposure to L+CD40L cells, the HLA-mismatched RT-LCL (Ak) transformed with the Akata virus strain, and L+CD40L cells themselves. (C) CD8+ T cell clones against the HPV and YPL epitopes were tested against the TAP− T2:B35 LCL target line precoated or not with the relevant epitope peptide. Control targets included the B*3501+ RT-LCL (B95) and an HLA-mismatched LCL precoated with the relevant peptide. In all the above assays, T cells were tested at 1,000–10,000 cells per well and targets at 25,000 cells per well. Results expressed as in Fig. 2.
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Related In: Results  -  Collection

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fig4: Presentation of EBNA1 to CD8+ T cells does not occur in cells with defects in the proteasome/TAP-dependent processing pathway. (A) Top: EBNA1 immunoblot (as in Fig. 1 B) of protein extracts from the Sal-BL/Sal-LCL and Chep-BL/Chep-LCL pairings. X50-7 is a standard B95.8 virus-transformed LCL. Middle: Three CD8+ T cell clones specific for the HPV/B*3501 epitope were tested against the B*3501+ Sal-BL/Sal-LCL pair of targets and against Sal-BL cells precoated with HPV peptide. Control targets were the B*3501+ RT-LCL (B95) and an HLA-mismatched LCL precoated with HPV peptide. Bottom: Three T cell clones specific for the RPQ/B*0701 epitope were tested against the B*0701+ Chep-BL/Chep-LCL pair of targets and against Chep-BL cells precoated with RPQ peptide. Control targets were the B*0701+ GT-LCL (B95) and an HLA-mismatched LCL precoated with RPQ peptide. (B) Top panels: CD8+ T cell clones specific for the B*3501-restricted epitopes HPV (EBNA1) or YPL (EBNA3A) were tested against the Sal-BL/Sal-LCL pair of targets and against Sal-BL cells preexposed for 2 d to CD40 ligand–expressing mouse L cells (L+CD40L). Control targets included the Chep-BL/Chep-LCL pair, Chep-BL cells similarly exposed to L+CD40L cells, and L+CD40L cells themselves. Bottom: CD8+ T cell clones specific for the RPQ/B*0701 epitope were tested against the Chep-BL/Chep-LCL pair and against Chep-BL cells preexposed as described above to L+CD40L cells. Control targets included the HLA-mismatched Akata BL line, with and without preexposure to L+CD40L cells, the HLA-mismatched RT-LCL (Ak) transformed with the Akata virus strain, and L+CD40L cells themselves. (C) CD8+ T cell clones against the HPV and YPL epitopes were tested against the TAP− T2:B35 LCL target line precoated or not with the relevant epitope peptide. Control targets included the B*3501+ RT-LCL (B95) and an HLA-mismatched LCL precoated with the relevant peptide. In all the above assays, T cells were tested at 1,000–10,000 cells per well and targets at 25,000 cells per well. Results expressed as in Fig. 2.
Mentions: Subsequent experiments aimed to identify the pathway whereby endogenously expressed wild-type EBNA1 was presented to CD8 T cells. We first examined the situation in EBV+ BL cell lines that have retained the original tumor-like “group I” phenotype in vitro. Compared with LCLs, such lines show more limited patterns of EBV-latent antigen expression usually restricted to EBNA1 only and, most importantly, are deficient in their capacity to process endogenously expressed antigen via the conventional proteasome/TAP-dependent pathway (22). Here we used two EBV+ BL cell lines that are known to have this processing deficiency, the B*3501+ Sal-BL line and the B*0701+ Chep-BL line. As controls, we used LCLs generated from the normal B cells of these same patients by in vitro transformation either with their own resident EBV strain (in the case of Sal-LCL) or with the B95.8 strain (in the case of Chep-LCL). The EBNA1 immunoblot of protein extracts from these BL/LCL pairs is shown in Fig. 4 A, confirming that the BL lines express the native EBNA1 protein at levels at least equal to those seen in the matching LCLs. Furthermore, sequencing the EBNA1 gene in the Sal-BL and Chep-BL virus strains confirmed that the relevant epitope sequences, HPV and RPQ, respectively, were conserved (not depicted). As shown in Fig. 4 A, three CD8+ T cell clones specific for the HPV/B*3501 epitope recognized the Sal-LCL as expected, but did not recognize the EBNA1-expressing Sal-BL target unless this was exogenously coated with synthetic epitope peptide. Likewise, three clones specific for the RPQ/B*0701 epitope recognized the Chep-LCL but not Chep-BL cells unless the latter were peptide coated. As a specificity control, in each case there was no recognition of a peptide-coated HLA-mismatched BL or LCL target.

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