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Evidence for the presentation of major histocompatibility complex class I-restricted Epstein-Barr virus nuclear antigen 1 peptides to CD8+ T lymphocytes.

Voo KS, Fu T, Wang HY, Tellam J, Heslop HE, Brenner MK, Rooney CM, Wang RF - J. Exp. Med. (2004)

Bottom Line: We also demonstrate that new protein synthesis is required for the generation of the HLA-B8 epitope for T cell recognition, suggesting that defective ribosomal products (DRiPs) are the major source of T cell epitopes.Experiments with protease inhibitors indicate that some serine proteases may participate in the degradation of EBNA1 DRiPs before they are further processed by proteasomes.These findings not only provide the first evidence of the presentation of an MHC class I-restricted EBNA1 epitope to CD8+ T cells, but also offer new insight into the molecular mechanisms involved in the processing and presentation of EBNA1.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
The Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is expressed in all EBV-associated tumors, making it an important target for immunotherapy. However, evidence for major histocompatibility complex (MHC) class I-restricted EBNA1 peptides endogenously presented by EBV-transformed B and tumor cells remains elusive. Here we describe for the first time the identification of an endogenously processed human histocompatibility leukocyte antigen (HLA)-B8-restricted EBNA1 peptide that is recognized by CD8+ T cells. T cell recognition could be inhibited by the treatment of target cells with proteasome inhibitors that block the MHC class I antigen processing pathway, but not by an inhibitor (chloroquine) of MHC class II antigen processing. We also demonstrate that new protein synthesis is required for the generation of the HLA-B8 epitope for T cell recognition, suggesting that defective ribosomal products (DRiPs) are the major source of T cell epitopes. Experiments with protease inhibitors indicate that some serine proteases may participate in the degradation of EBNA1 DRiPs before they are further processed by proteasomes. These findings not only provide the first evidence of the presentation of an MHC class I-restricted EBNA1 epitope to CD8+ T cells, but also offer new insight into the molecular mechanisms involved in the processing and presentation of EBNA1.

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Specific inhibition of T cell recognition of EBNA1 by proteasomes inhibitors. (A) Blocking of T cell recognition of EBNA1 by a ZAL proteasome inhibitor. HEK 293 cells cotransfected with EBNA1-GFP were treated with various concentrations of ZAL inhibitor for 10 h. After washing, cells were incubated with T cells overnight for IFN-γ release assays. Various dilutions of DMSO were used as controls. T cell activity in the absence of inhibitor was used at 100% activity. Two CD4+ T cells were used to demonstrate the specificity of ZAL inhibitor. TIL102 and P3-B7 CD4+ T cells able to recognize 102mel and HEK293/DP3/Ii-EBNA1 target cells, respectively, were not inhibited by ZAL. (B) Inhibition of M3-W1-B9 CD8+ T cell recognition of 1359mel target cells stably expressing EBNA1-GFP by lactacystin proteasome inhibitor. The lactacystin inhibitor did not affect recognition of 102mel tumor cells by TIL102 CD4+ cells. (C) Blocking of MHC class II antigen processing by chloroquine. Inhibition of T cell recognition of 102mel cells by TIL102 CD4+ was observed after treatment with chloroquine in a dose-dependent fashion. By contrast, T cell recognition of LCL 111 and HEK293 transfected with HLA-B8 and EBNA1-GFP cDNAs was not significantly affected after the treatment of chloroquine.
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fig8: Specific inhibition of T cell recognition of EBNA1 by proteasomes inhibitors. (A) Blocking of T cell recognition of EBNA1 by a ZAL proteasome inhibitor. HEK 293 cells cotransfected with EBNA1-GFP were treated with various concentrations of ZAL inhibitor for 10 h. After washing, cells were incubated with T cells overnight for IFN-γ release assays. Various dilutions of DMSO were used as controls. T cell activity in the absence of inhibitor was used at 100% activity. Two CD4+ T cells were used to demonstrate the specificity of ZAL inhibitor. TIL102 and P3-B7 CD4+ T cells able to recognize 102mel and HEK293/DP3/Ii-EBNA1 target cells, respectively, were not inhibited by ZAL. (B) Inhibition of M3-W1-B9 CD8+ T cell recognition of 1359mel target cells stably expressing EBNA1-GFP by lactacystin proteasome inhibitor. The lactacystin inhibitor did not affect recognition of 102mel tumor cells by TIL102 CD4+ cells. (C) Blocking of MHC class II antigen processing by chloroquine. Inhibition of T cell recognition of 102mel cells by TIL102 CD4+ was observed after treatment with chloroquine in a dose-dependent fashion. By contrast, T cell recognition of LCL 111 and HEK293 transfected with HLA-B8 and EBNA1-GFP cDNAs was not significantly affected after the treatment of chloroquine.

Mentions: Next, we sought to elucidate the mechanism(s) by which HLA-B8–restricted peptides are generated. One possibility is that HLA-B8 peptides are generated through the conventional MHC class I pathway. If so, specific inhibitors of proteasomes should inhibit the degradation of proteins and thus the presentation of peptides by MHC class I molecules for T cell recognition (22). Alternatively, MHC class I molecules bind and present peptides in MHC class II compartments, one of which is transported to the plasma membrane for T cell recognition (23). Because the EBNA1-P518–526 peptide overlaps with HLA-DR1– and HLA-DP3–restricted peptides that are shown to be endogenously processed and presented to CD4+ T cells (10, 11), MHC class I molecules might acquire and present a peptide generated through the MHC class II pathway. To test this possibility, we transfected HEK293 cells with EBNA1-GFP and HLA-B8 cDNAs, and then treated them with different concentrations of ZAL and lactacystin, specific inhibitors of proteasomes (22). When antigen-specific CD8+ T cells were then cocultured with target cells to evaluate T cell responses, we found that T cell reactivity of HEK293 transfected with HLA-B8 plus EBNA1-GFP cDNAs by the M3W1-B9 CD8+ T cells decreased with increasing concentrations of proteasome inhibitor ZAL, but not in the presence of control DMSO (Fig. 8 A). Similarly, T cell reactivity against LCL 111 cells was inhibited with increasing concentration of ZAL inhibitor. To exclude the potential nonspecific effect of ZAL on T cell recognition, we tested the effects of ZAL inhibitor on melanoma-reactive TIL102-CD4+ T cells, which recognize an MHC class II–restricted epitope on the cell surface of 102mel tumor cells, and of P3-B7 CD4+ T cells, which recognize HLA-DP3–expressing HEK293 cells transfected with Ii-EBNA1 as target cells. The ZAL inhibitor did not have any inhibitory effect on recognition of target cells by TIL102 CD4+ T cells or P3-B7 T CD4+ cells (Fig. 8 A). We also tested the effects of lactacystin, another specific proteasome inhibitor, on T cell recognition. As shown in Fig. 8 B, recognition of 1359mel/EBNA1-GFP cells by M3-W1-B9 CD8+ T cell was decreased with increasing concentrations of lactacystin, whereas no inhibitory effect was observed with TIL102 CD4+ T cell recognition of 102mel target cells. These results suggest that the inhibitory effect of ZAL and lactacystin is specific for the presentation of HLA-B8–restricted epitope to CD8+ T cells, but not for MHC class II antigen processing and presentation.


Evidence for the presentation of major histocompatibility complex class I-restricted Epstein-Barr virus nuclear antigen 1 peptides to CD8+ T lymphocytes.

Voo KS, Fu T, Wang HY, Tellam J, Heslop HE, Brenner MK, Rooney CM, Wang RF - J. Exp. Med. (2004)

Specific inhibition of T cell recognition of EBNA1 by proteasomes inhibitors. (A) Blocking of T cell recognition of EBNA1 by a ZAL proteasome inhibitor. HEK 293 cells cotransfected with EBNA1-GFP were treated with various concentrations of ZAL inhibitor for 10 h. After washing, cells were incubated with T cells overnight for IFN-γ release assays. Various dilutions of DMSO were used as controls. T cell activity in the absence of inhibitor was used at 100% activity. Two CD4+ T cells were used to demonstrate the specificity of ZAL inhibitor. TIL102 and P3-B7 CD4+ T cells able to recognize 102mel and HEK293/DP3/Ii-EBNA1 target cells, respectively, were not inhibited by ZAL. (B) Inhibition of M3-W1-B9 CD8+ T cell recognition of 1359mel target cells stably expressing EBNA1-GFP by lactacystin proteasome inhibitor. The lactacystin inhibitor did not affect recognition of 102mel tumor cells by TIL102 CD4+ cells. (C) Blocking of MHC class II antigen processing by chloroquine. Inhibition of T cell recognition of 102mel cells by TIL102 CD4+ was observed after treatment with chloroquine in a dose-dependent fashion. By contrast, T cell recognition of LCL 111 and HEK293 transfected with HLA-B8 and EBNA1-GFP cDNAs was not significantly affected after the treatment of chloroquine.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211826&req=5

fig8: Specific inhibition of T cell recognition of EBNA1 by proteasomes inhibitors. (A) Blocking of T cell recognition of EBNA1 by a ZAL proteasome inhibitor. HEK 293 cells cotransfected with EBNA1-GFP were treated with various concentrations of ZAL inhibitor for 10 h. After washing, cells were incubated with T cells overnight for IFN-γ release assays. Various dilutions of DMSO were used as controls. T cell activity in the absence of inhibitor was used at 100% activity. Two CD4+ T cells were used to demonstrate the specificity of ZAL inhibitor. TIL102 and P3-B7 CD4+ T cells able to recognize 102mel and HEK293/DP3/Ii-EBNA1 target cells, respectively, were not inhibited by ZAL. (B) Inhibition of M3-W1-B9 CD8+ T cell recognition of 1359mel target cells stably expressing EBNA1-GFP by lactacystin proteasome inhibitor. The lactacystin inhibitor did not affect recognition of 102mel tumor cells by TIL102 CD4+ cells. (C) Blocking of MHC class II antigen processing by chloroquine. Inhibition of T cell recognition of 102mel cells by TIL102 CD4+ was observed after treatment with chloroquine in a dose-dependent fashion. By contrast, T cell recognition of LCL 111 and HEK293 transfected with HLA-B8 and EBNA1-GFP cDNAs was not significantly affected after the treatment of chloroquine.
Mentions: Next, we sought to elucidate the mechanism(s) by which HLA-B8–restricted peptides are generated. One possibility is that HLA-B8 peptides are generated through the conventional MHC class I pathway. If so, specific inhibitors of proteasomes should inhibit the degradation of proteins and thus the presentation of peptides by MHC class I molecules for T cell recognition (22). Alternatively, MHC class I molecules bind and present peptides in MHC class II compartments, one of which is transported to the plasma membrane for T cell recognition (23). Because the EBNA1-P518–526 peptide overlaps with HLA-DR1– and HLA-DP3–restricted peptides that are shown to be endogenously processed and presented to CD4+ T cells (10, 11), MHC class I molecules might acquire and present a peptide generated through the MHC class II pathway. To test this possibility, we transfected HEK293 cells with EBNA1-GFP and HLA-B8 cDNAs, and then treated them with different concentrations of ZAL and lactacystin, specific inhibitors of proteasomes (22). When antigen-specific CD8+ T cells were then cocultured with target cells to evaluate T cell responses, we found that T cell reactivity of HEK293 transfected with HLA-B8 plus EBNA1-GFP cDNAs by the M3W1-B9 CD8+ T cells decreased with increasing concentrations of proteasome inhibitor ZAL, but not in the presence of control DMSO (Fig. 8 A). Similarly, T cell reactivity against LCL 111 cells was inhibited with increasing concentration of ZAL inhibitor. To exclude the potential nonspecific effect of ZAL on T cell recognition, we tested the effects of ZAL inhibitor on melanoma-reactive TIL102-CD4+ T cells, which recognize an MHC class II–restricted epitope on the cell surface of 102mel tumor cells, and of P3-B7 CD4+ T cells, which recognize HLA-DP3–expressing HEK293 cells transfected with Ii-EBNA1 as target cells. The ZAL inhibitor did not have any inhibitory effect on recognition of target cells by TIL102 CD4+ T cells or P3-B7 T CD4+ cells (Fig. 8 A). We also tested the effects of lactacystin, another specific proteasome inhibitor, on T cell recognition. As shown in Fig. 8 B, recognition of 1359mel/EBNA1-GFP cells by M3-W1-B9 CD8+ T cell was decreased with increasing concentrations of lactacystin, whereas no inhibitory effect was observed with TIL102 CD4+ T cell recognition of 102mel target cells. These results suggest that the inhibitory effect of ZAL and lactacystin is specific for the presentation of HLA-B8–restricted epitope to CD8+ T cells, but not for MHC class II antigen processing and presentation.

Bottom Line: We also demonstrate that new protein synthesis is required for the generation of the HLA-B8 epitope for T cell recognition, suggesting that defective ribosomal products (DRiPs) are the major source of T cell epitopes.Experiments with protease inhibitors indicate that some serine proteases may participate in the degradation of EBNA1 DRiPs before they are further processed by proteasomes.These findings not only provide the first evidence of the presentation of an MHC class I-restricted EBNA1 epitope to CD8+ T cells, but also offer new insight into the molecular mechanisms involved in the processing and presentation of EBNA1.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
The Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is expressed in all EBV-associated tumors, making it an important target for immunotherapy. However, evidence for major histocompatibility complex (MHC) class I-restricted EBNA1 peptides endogenously presented by EBV-transformed B and tumor cells remains elusive. Here we describe for the first time the identification of an endogenously processed human histocompatibility leukocyte antigen (HLA)-B8-restricted EBNA1 peptide that is recognized by CD8+ T cells. T cell recognition could be inhibited by the treatment of target cells with proteasome inhibitors that block the MHC class I antigen processing pathway, but not by an inhibitor (chloroquine) of MHC class II antigen processing. We also demonstrate that new protein synthesis is required for the generation of the HLA-B8 epitope for T cell recognition, suggesting that defective ribosomal products (DRiPs) are the major source of T cell epitopes. Experiments with protease inhibitors indicate that some serine proteases may participate in the degradation of EBNA1 DRiPs before they are further processed by proteasomes. These findings not only provide the first evidence of the presentation of an MHC class I-restricted EBNA1 epitope to CD8+ T cells, but also offer new insight into the molecular mechanisms involved in the processing and presentation of EBNA1.

Show MeSH
Related in: MedlinePlus