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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 lysis of HLA-B8–matched EBV-transformed LCLs by M3-W1-B9 CD8+ T cells. (A) Recognition of HLA-B8–matched LCLs by M3-W1-B9 CD8+ T cells. LCLs were cocultured with M3-W1-B9 CD8+ T cells at an E/T ratio of 1:1. Mismatched LCLs were used as negative controls. (B) Specific lysis of HLA-B8–matched LCL 111 cells by CD8+ T cells at different E/T ratios. LCL 1 was used as a negative control. LCL cells were labeled with 51chromium. Cytolysis by CD8+ T cells was determined in a 16-h chromium release assay. (C) Cold target inhibition of recognition of LCL 111 cells by M3-W1-B9 CD8+ T cells. Lysis of LCLs by M3-W1-B9 CD8+ T cells was specifically inhibited when EBNA1-P518–526–pulsed cold LCL 111 targets were used. Lysis was tested with an effector to hot target ratio of 40:1. Cold LCL 111 target cells were pulsed with EBNA1-P518–526 or EBNA1-P572–584 peptide at a concentration of 1 μM and were mixed with hot targets at a ratio of 4:1. All experiments were repeated twice with similar results.
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fig5: Specific lysis of HLA-B8–matched EBV-transformed LCLs by M3-W1-B9 CD8+ T cells. (A) Recognition of HLA-B8–matched LCLs by M3-W1-B9 CD8+ T cells. LCLs were cocultured with M3-W1-B9 CD8+ T cells at an E/T ratio of 1:1. Mismatched LCLs were used as negative controls. (B) Specific lysis of HLA-B8–matched LCL 111 cells by CD8+ T cells at different E/T ratios. LCL 1 was used as a negative control. LCL cells were labeled with 51chromium. Cytolysis by CD8+ T cells was determined in a 16-h chromium release assay. (C) Cold target inhibition of recognition of LCL 111 cells by M3-W1-B9 CD8+ T cells. Lysis of LCLs by M3-W1-B9 CD8+ T cells was specifically inhibited when EBNA1-P518–526–pulsed cold LCL 111 targets were used. Lysis was tested with an effector to hot target ratio of 40:1. Cold LCL 111 target cells were pulsed with EBNA1-P518–526 or EBNA1-P572–584 peptide at a concentration of 1 μM and were mixed with hot targets at a ratio of 4:1. All experiments were repeated twice with similar results.

Mentions: Although peptide-specific CD8+ T cells against putative tumor antigens or peptides can often be generated from human PBMCs, they show no reactivity with tumor cells. Possible explanations include the low affinity of the T cells for the MHC–peptide complexes or the failure of presentation of naturally processed peptides on the surface of tumor cells (21). Indeed, EBNA1 peptide-specific CD8+ T cells generated from human PBMCs after in vitro stimulation failed to recognize autologous EBV+ LCLs (5). T cell reactivity was only found when autologous EBV+ LCL cells were pulsed with exogenous protein or peptides (5). To test whether the CD8+ T cells generated in this study were capable of recognizing naturally processed peptides on EBV-infected cells, we used several EBV B cell lines as target cells. As shown in Fig. 5 A, a cytokine release assay demonstrated that M3W1-B9 CD8+ T cells strongly recognized HLA-B8+ LCL 8, 111, and 1088 cells, but did not respond to HLA-mismatched LCL 1 and 2 cells. To further test whether M3W1-B9 CD8+ T cells can lyse tumor target cells, we performed chromium release assays. CD8+ T cells specifically lysed LCL 111 cells but not the HLA-mismatched LCL1 cells (Fig. 5 B). Cold target inhibition experiments showed that the specific killing of chromium-labeled (hot) LCL 111 target cells by the M3W1-B9 CD8+ T cells could be inhibited by the 9-mer EBNA1-P518–526 peptide–pulsed unlabeled (cold) LCL 111 cells, but not by control peptide–pulsed cold LCL 111 cells (Fig. 5 C). Taken together, these results indicate that the 9-mer EBNA1-P518–526 peptide can specifically block the recognition and lysis of EBV+ LCL 111 cells by the M3W1-B9 CD8+ T cells, implying that a similar or identical EBNA1 peptide is endogenously processed and presented to T cells by HLA-B8 molecules on the surface of EBV+ LCL cells by a mechanism that overrides the inhibitory effect of the GAr domain on processing and presentation of EBNA1.


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 lysis of HLA-B8–matched EBV-transformed LCLs by M3-W1-B9 CD8+ T cells. (A) Recognition of HLA-B8–matched LCLs by M3-W1-B9 CD8+ T cells. LCLs were cocultured with M3-W1-B9 CD8+ T cells at an E/T ratio of 1:1. Mismatched LCLs were used as negative controls. (B) Specific lysis of HLA-B8–matched LCL 111 cells by CD8+ T cells at different E/T ratios. LCL 1 was used as a negative control. LCL cells were labeled with 51chromium. Cytolysis by CD8+ T cells was determined in a 16-h chromium release assay. (C) Cold target inhibition of recognition of LCL 111 cells by M3-W1-B9 CD8+ T cells. Lysis of LCLs by M3-W1-B9 CD8+ T cells was specifically inhibited when EBNA1-P518–526–pulsed cold LCL 111 targets were used. Lysis was tested with an effector to hot target ratio of 40:1. Cold LCL 111 target cells were pulsed with EBNA1-P518–526 or EBNA1-P572–584 peptide at a concentration of 1 μM and were mixed with hot targets at a ratio of 4:1. All experiments were repeated twice with similar results.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211826&req=5

fig5: Specific lysis of HLA-B8–matched EBV-transformed LCLs by M3-W1-B9 CD8+ T cells. (A) Recognition of HLA-B8–matched LCLs by M3-W1-B9 CD8+ T cells. LCLs were cocultured with M3-W1-B9 CD8+ T cells at an E/T ratio of 1:1. Mismatched LCLs were used as negative controls. (B) Specific lysis of HLA-B8–matched LCL 111 cells by CD8+ T cells at different E/T ratios. LCL 1 was used as a negative control. LCL cells were labeled with 51chromium. Cytolysis by CD8+ T cells was determined in a 16-h chromium release assay. (C) Cold target inhibition of recognition of LCL 111 cells by M3-W1-B9 CD8+ T cells. Lysis of LCLs by M3-W1-B9 CD8+ T cells was specifically inhibited when EBNA1-P518–526–pulsed cold LCL 111 targets were used. Lysis was tested with an effector to hot target ratio of 40:1. Cold LCL 111 target cells were pulsed with EBNA1-P518–526 or EBNA1-P572–584 peptide at a concentration of 1 μM and were mixed with hot targets at a ratio of 4:1. All experiments were repeated twice with similar results.
Mentions: Although peptide-specific CD8+ T cells against putative tumor antigens or peptides can often be generated from human PBMCs, they show no reactivity with tumor cells. Possible explanations include the low affinity of the T cells for the MHC–peptide complexes or the failure of presentation of naturally processed peptides on the surface of tumor cells (21). Indeed, EBNA1 peptide-specific CD8+ T cells generated from human PBMCs after in vitro stimulation failed to recognize autologous EBV+ LCLs (5). T cell reactivity was only found when autologous EBV+ LCL cells were pulsed with exogenous protein or peptides (5). To test whether the CD8+ T cells generated in this study were capable of recognizing naturally processed peptides on EBV-infected cells, we used several EBV B cell lines as target cells. As shown in Fig. 5 A, a cytokine release assay demonstrated that M3W1-B9 CD8+ T cells strongly recognized HLA-B8+ LCL 8, 111, and 1088 cells, but did not respond to HLA-mismatched LCL 1 and 2 cells. To further test whether M3W1-B9 CD8+ T cells can lyse tumor target cells, we performed chromium release assays. CD8+ T cells specifically lysed LCL 111 cells but not the HLA-mismatched LCL1 cells (Fig. 5 B). Cold target inhibition experiments showed that the specific killing of chromium-labeled (hot) LCL 111 target cells by the M3W1-B9 CD8+ T cells could be inhibited by the 9-mer EBNA1-P518–526 peptide–pulsed unlabeled (cold) LCL 111 cells, but not by control peptide–pulsed cold LCL 111 cells (Fig. 5 C). Taken together, these results indicate that the 9-mer EBNA1-P518–526 peptide can specifically block the recognition and lysis of EBV+ LCL 111 cells by the M3W1-B9 CD8+ T cells, implying that a similar or identical EBNA1 peptide is endogenously processed and presented to T cells by HLA-B8 molecules on the surface of EBV+ LCL cells by a mechanism that overrides the inhibitory effect of the GAr domain on processing and presentation of EBNA1.

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