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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 cells inhibit the in vitro outgrowth of EBV-transformed LCLs of the appropriate HLA type. EBV+ target B cell lines were seeded in microtest plate wells at a range of input cell numbers (20,000–313 per well), either alone or in the presence of added CD8+ T cell clones (10,000 per well) specific for the HPV/B*3501 or YPL/B*3501 epitopes. For each target, results are expressed as the minimum target cell seeding required for successful outgrowth from the different types of coculture. The corresponding value for target cells in the absence of T cells is shown as a dotted horizontal line. Targets included B*3501+ and B*3501− LCLs transformed either with B95.8 virus or with the dl7 strain as well as the TAP− LCL T2:B35 and the processing-deficient B*3501+ Sal-BL line, both either pulsed or not pulsed with the appropriate epitope peptide HPV or YPL. Cultures were assessed for cell growth after 4 wk. Control cultures set up with T cells alone contained no viable cells by that stage.
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fig6: EBNA1-specific CD8+ T cells inhibit the in vitro outgrowth of EBV-transformed LCLs of the appropriate HLA type. EBV+ target B cell lines were seeded in microtest plate wells at a range of input cell numbers (20,000–313 per well), either alone or in the presence of added CD8+ T cell clones (10,000 per well) specific for the HPV/B*3501 or YPL/B*3501 epitopes. For each target, results are expressed as the minimum target cell seeding required for successful outgrowth from the different types of coculture. The corresponding value for target cells in the absence of T cells is shown as a dotted horizontal line. Targets included B*3501+ and B*3501− LCLs transformed either with B95.8 virus or with the dl7 strain as well as the TAP− LCL T2:B35 and the processing-deficient B*3501+ Sal-BL line, both either pulsed or not pulsed with the appropriate epitope peptide HPV or YPL. Cultures were assessed for cell growth after 4 wk. Control cultures set up with T cells alone contained no viable cells by that stage.

Mentions: Fig. 6 presents the results of one such experiment involving a variety of target B cell lines each seeded alone and in cocultures with three CD8+ T cell clones specific for the B*3501-restricted EBNA1 epitope HPV and with two clones specific for the B*3501-restricted EBNA3A epitope YPL. Results are expressed as the minimum target cell seeding giving successful outgrowth in each case compared with the growth of target cells alone (dotted line) showing the effects of T cell addition. The key findings are shown in Fig. 6 A, where the three HPV-specific clones all significantly inhibited outgrowth not just of the B*3501+ LCL, RT (dl7a), expressing a GAr-deleted EBNA1 protein, but also of two B*3501+ LCLs, RT (B95) and PB (B95), expressing wild-type EBNA1. Indeed, the degree of inhibition seen in these cocultures was similar to that mediated in the same assay by the two YPL-specific clones. Note that in all cocultures where the addition of T cells had prevented B cell outgrowth, there were no viable cells of either type detectable at 4 wk. These effects were not nonspecific consequences of coculture because three B*3501− LCLs, SC (B95), YC (B95), and CM (B95), showed no significant inhibition of outgrowth from either HPV- or YPL-specific clones. Indeed, with some of these HLA-mismatched combinations we even observed a slight enhancement of LCL outgrowth from low target cell seedings. Fig. 6 B shows outgrowth data from processing-deficient target lines that again parallel the IFN-γ results seen earlier with these same lines (Fig. 4). Thus, the T2:B35-LCL was not growth inhibited by HPV- or YPL-specific effectors, whereas the same targets were strongly inhibited if preexposed to the relevant epitope peptide. Likewise, both HPV- and YPL-specific effectors did not affect outgrowth of the B*3501+ Sal-BL line, unless these cells were first epitope loaded.


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 cells inhibit the in vitro outgrowth of EBV-transformed LCLs of the appropriate HLA type. EBV+ target B cell lines were seeded in microtest plate wells at a range of input cell numbers (20,000–313 per well), either alone or in the presence of added CD8+ T cell clones (10,000 per well) specific for the HPV/B*3501 or YPL/B*3501 epitopes. For each target, results are expressed as the minimum target cell seeding required for successful outgrowth from the different types of coculture. The corresponding value for target cells in the absence of T cells is shown as a dotted horizontal line. Targets included B*3501+ and B*3501− LCLs transformed either with B95.8 virus or with the dl7 strain as well as the TAP− LCL T2:B35 and the processing-deficient B*3501+ Sal-BL line, both either pulsed or not pulsed with the appropriate epitope peptide HPV or YPL. Cultures were assessed for cell growth after 4 wk. Control cultures set up with T cells alone contained no viable cells by that stage.
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Related In: Results  -  Collection

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

fig6: EBNA1-specific CD8+ T cells inhibit the in vitro outgrowth of EBV-transformed LCLs of the appropriate HLA type. EBV+ target B cell lines were seeded in microtest plate wells at a range of input cell numbers (20,000–313 per well), either alone or in the presence of added CD8+ T cell clones (10,000 per well) specific for the HPV/B*3501 or YPL/B*3501 epitopes. For each target, results are expressed as the minimum target cell seeding required for successful outgrowth from the different types of coculture. The corresponding value for target cells in the absence of T cells is shown as a dotted horizontal line. Targets included B*3501+ and B*3501− LCLs transformed either with B95.8 virus or with the dl7 strain as well as the TAP− LCL T2:B35 and the processing-deficient B*3501+ Sal-BL line, both either pulsed or not pulsed with the appropriate epitope peptide HPV or YPL. Cultures were assessed for cell growth after 4 wk. Control cultures set up with T cells alone contained no viable cells by that stage.
Mentions: Fig. 6 presents the results of one such experiment involving a variety of target B cell lines each seeded alone and in cocultures with three CD8+ T cell clones specific for the B*3501-restricted EBNA1 epitope HPV and with two clones specific for the B*3501-restricted EBNA3A epitope YPL. Results are expressed as the minimum target cell seeding giving successful outgrowth in each case compared with the growth of target cells alone (dotted line) showing the effects of T cell addition. The key findings are shown in Fig. 6 A, where the three HPV-specific clones all significantly inhibited outgrowth not just of the B*3501+ LCL, RT (dl7a), expressing a GAr-deleted EBNA1 protein, but also of two B*3501+ LCLs, RT (B95) and PB (B95), expressing wild-type EBNA1. Indeed, the degree of inhibition seen in these cocultures was similar to that mediated in the same assay by the two YPL-specific clones. Note that in all cocultures where the addition of T cells had prevented B cell outgrowth, there were no viable cells of either type detectable at 4 wk. These effects were not nonspecific consequences of coculture because three B*3501− LCLs, SC (B95), YC (B95), and CM (B95), showed no significant inhibition of outgrowth from either HPV- or YPL-specific clones. Indeed, with some of these HLA-mismatched combinations we even observed a slight enhancement of LCL outgrowth from low target cell seedings. Fig. 6 B shows outgrowth data from processing-deficient target lines that again parallel the IFN-γ results seen earlier with these same lines (Fig. 4). Thus, the T2:B35-LCL was not growth inhibited by HPV- or YPL-specific effectors, whereas the same targets were strongly inhibited if preexposed to the relevant epitope peptide. Likewise, both HPV- and YPL-specific effectors did not affect outgrowth of the B*3501+ Sal-BL line, unless these cells were first epitope loaded.

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