Limits...
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.

Show MeSH

Related in: MedlinePlus

EBNA1-specific CD8+ T cell recognition of LCLs measured by IFN-γ release. (A) Three CD8+ T cell clones specific for the HPV/B*3501 epitope were tested against a panel of B*3501+ and B*3501− LCL targets transformed either with wild-type B95.8 virus or with the dl7 strain expressing GAr-deleted EBNA1. T cells and LCL targets were both seeded at 104 cells/well. As controls, T cells alone were tested in the presence of 10−7 M HPV epitope peptide or with no peptide. (B) The efficiency of RT-LCL (B95) and RT-LCL (dl7a) target recognition was assessed in titration assays using two HPV (EBNA1)–specific CD8+ T cell clones and one YPL (EBNA3A)–specific clone. The effect of titrating numbers of effector and target cells are shown on the left and right, respectively. When titrating numbers of effectors, LCL targets were added at 104 cells/well, and when titrating numbers of targets, effectors were added at 5 × 103 cells/well. Results represent the mean values of IFN-γ release measured from triplicate cultures (+SD).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211813&req=5

fig2: EBNA1-specific CD8+ T cell recognition of LCLs measured by IFN-γ release. (A) Three CD8+ T cell clones specific for the HPV/B*3501 epitope were tested against a panel of B*3501+ and B*3501− LCL targets transformed either with wild-type B95.8 virus or with the dl7 strain expressing GAr-deleted EBNA1. T cells and LCL targets were both seeded at 104 cells/well. As controls, T cells alone were tested in the presence of 10−7 M HPV epitope peptide or with no peptide. (B) The efficiency of RT-LCL (B95) and RT-LCL (dl7a) target recognition was assessed in titration assays using two HPV (EBNA1)–specific CD8+ T cell clones and one YPL (EBNA3A)–specific clone. The effect of titrating numbers of effector and target cells are shown on the left and right, respectively. When titrating numbers of effectors, LCL targets were added at 104 cells/well, and when titrating numbers of targets, effectors were added at 5 × 103 cells/well. Results represent the mean values of IFN-γ release measured from triplicate cultures (+SD).

Mentions: In earlier work, we described CD8+ T cell clones reactivated in vitro against EBV-latent cycle antigens other than EBNA1, which were clearly EBV epitope and antigen specific in their ability to kill target cells exogenously loaded with synthetic epitope peptide or overexpressing the relevant target antigen from a vaccinia vector, yet which failed to kill naturally infected LCL targets in conventional cytotoxicity assays (20). While studying the basis of this phenomenon, we found that such clones do show specific, HLA class I–restricted recognition of unmanipulated LCL targets when measured by ELISA for IFN-γ release (unpublished data). Fig. 2 A shows data obtained when three HPV-specific CD8+ clones, again representative of the complete range, were retested on similar target cell panels using this more sensitive assay. Now all clones showed significant recognition of three different wild-type B*3501+ LCLs at levels that though below those seen for the RT-LCL (dl7a) transformant, were clearly well above the background shown by HLA-mismatched LCL controls. We went on to compare the efficiency of dl7 and wild-type LCL recognition by representative EBNA1-specific clones in titration assays, varying the numbers of either effector or target cells added. Fig. 2 B shows data from such an experiment assaying two HPV-specific clones, c32 and c33, against the RT (dl7a) and RT (B95) LCL pair. Assaying different numbers of effectors against a standard number of targets (Fig. 2 B, left), levels of wild-type LCL recognition by the HPV-specific clones were consistently 40–70% of those seen against the dl7 transformant. A similar pattern was observed when assaying a standard number of HPV-specific effectors against different numbers of targets (Fig. 2 B, right). Interestingly, when we repeated these experiments using ELISPOT assays (21) as a read out, the different levels of response were apparent not in the numbers of T cells that responded but in the intensity of the IFN-γ produced per responding cell (not depicted). Fig. 2 B also shows the results obtained in the same titration assays with a CD8+ T cell clone (c98) specific for the YPL epitope. Like other YPL-specific effectors tested, this clone did not discriminate between the two types of target LCLs.


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 IFN-γ release. (A) Three CD8+ T cell clones specific for the HPV/B*3501 epitope were tested against a panel of B*3501+ and B*3501− LCL targets transformed either with wild-type B95.8 virus or with the dl7 strain expressing GAr-deleted EBNA1. T cells and LCL targets were both seeded at 104 cells/well. As controls, T cells alone were tested in the presence of 10−7 M HPV epitope peptide or with no peptide. (B) The efficiency of RT-LCL (B95) and RT-LCL (dl7a) target recognition was assessed in titration assays using two HPV (EBNA1)–specific CD8+ T cell clones and one YPL (EBNA3A)–specific clone. The effect of titrating numbers of effector and target cells are shown on the left and right, respectively. When titrating numbers of effectors, LCL targets were added at 104 cells/well, and when titrating numbers of targets, effectors were added at 5 × 103 cells/well. Results represent the mean values of IFN-γ release measured from triplicate cultures (+SD).
© Copyright Policy
Related In: Results  -  Collection

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

fig2: EBNA1-specific CD8+ T cell recognition of LCLs measured by IFN-γ release. (A) Three CD8+ T cell clones specific for the HPV/B*3501 epitope were tested against a panel of B*3501+ and B*3501− LCL targets transformed either with wild-type B95.8 virus or with the dl7 strain expressing GAr-deleted EBNA1. T cells and LCL targets were both seeded at 104 cells/well. As controls, T cells alone were tested in the presence of 10−7 M HPV epitope peptide or with no peptide. (B) The efficiency of RT-LCL (B95) and RT-LCL (dl7a) target recognition was assessed in titration assays using two HPV (EBNA1)–specific CD8+ T cell clones and one YPL (EBNA3A)–specific clone. The effect of titrating numbers of effector and target cells are shown on the left and right, respectively. When titrating numbers of effectors, LCL targets were added at 104 cells/well, and when titrating numbers of targets, effectors were added at 5 × 103 cells/well. Results represent the mean values of IFN-γ release measured from triplicate cultures (+SD).
Mentions: In earlier work, we described CD8+ T cell clones reactivated in vitro against EBV-latent cycle antigens other than EBNA1, which were clearly EBV epitope and antigen specific in their ability to kill target cells exogenously loaded with synthetic epitope peptide or overexpressing the relevant target antigen from a vaccinia vector, yet which failed to kill naturally infected LCL targets in conventional cytotoxicity assays (20). While studying the basis of this phenomenon, we found that such clones do show specific, HLA class I–restricted recognition of unmanipulated LCL targets when measured by ELISA for IFN-γ release (unpublished data). Fig. 2 A shows data obtained when three HPV-specific CD8+ clones, again representative of the complete range, were retested on similar target cell panels using this more sensitive assay. Now all clones showed significant recognition of three different wild-type B*3501+ LCLs at levels that though below those seen for the RT-LCL (dl7a) transformant, were clearly well above the background shown by HLA-mismatched LCL controls. We went on to compare the efficiency of dl7 and wild-type LCL recognition by representative EBNA1-specific clones in titration assays, varying the numbers of either effector or target cells added. Fig. 2 B shows data from such an experiment assaying two HPV-specific clones, c32 and c33, against the RT (dl7a) and RT (B95) LCL pair. Assaying different numbers of effectors against a standard number of targets (Fig. 2 B, left), levels of wild-type LCL recognition by the HPV-specific clones were consistently 40–70% of those seen against the dl7 transformant. A similar pattern was observed when assaying a standard number of HPV-specific effectors against different numbers of targets (Fig. 2 B, right). Interestingly, when we repeated these experiments using ELISPOT assays (21) as a read out, the different levels of response were apparent not in the numbers of T cells that responded but in the intensity of the IFN-γ produced per responding cell (not depicted). Fig. 2 B also shows the results obtained in the same titration assays with a CD8+ T cell clone (c98) specific for the YPL epitope. Like other YPL-specific effectors tested, this clone did not discriminate between the two types of target LCLs.

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