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Latent Membrane Protein LMP2A Impairs Recognition of EBV-Infected Cells by CD8+ T Cells.

Rancan C, Schirrmann L, Hüls C, Zeidler R, Moosmann A - PLoS Pathog. (2015)

Bottom Line: We found that CD8+ T cell clones showed higher reactivity against LMP2A-deficient LCLs compared to LCLs infected with complete EBV.We identified several potential mediators of this immunomodulatory effect.Our results demonstrate that LMP2A reduces the reactivity of CD8+ T cells against EBV-infected cells, and we identify several relevant mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Clinical Cooperation Group Immunooncology, Department of Medicine III, Klinikum der Universität München, and Research Unit Gene Vectors, Helmholtz Zentrum München, Munich, Germany; German Center for Infection Research (DZIF), Munich, Germany.

ABSTRACT
The common pathogen Epstein-Barr virus (EBV) transforms normal human B cells and can cause cancer. Latent membrane protein 2A (LMP2A) of EBV supports activation and proliferation of infected B cells and is expressed in many types of EBV-associated cancer. It is not clear how latent EBV infection and cancer escape elimination by host immunity, and it is unknown whether LMP2A can influence the interaction of EBV-infected cells with the immune system. We infected primary B cells with EBV deleted for LMP2A, and established lymphoblastoid cell lines (LCLs). We found that CD8+ T cell clones showed higher reactivity against LMP2A-deficient LCLs compared to LCLs infected with complete EBV. We identified several potential mediators of this immunomodulatory effect. In the absence of LMP2A, expression of some EBV latent antigens was elevated, and cell surface expression of MHC class I was marginally increased. LMP2A-deficient LCLs produced lower amounts of IL-10, although this did not directly affect CD8+ T cell recognition. Deletion of LMP2A led to several changes in the cell surface immunophenotype of LCLs. Specifically, the agonistic NKG2D ligands MICA and ULBP4 were increased. Blocking experiments showed that NKG2D activation contributed to LCL recognition by CD8+ T cell clones. Our results demonstrate that LMP2A reduces the reactivity of CD8+ T cells against EBV-infected cells, and we identify several relevant mechanisms.

No MeSH data available.


Related in: MedlinePlus

Effect of NKG2D blocking on CD8+ T cell recognition of LCLs with or without LMP2A.(A) Expression of NKG2D on CD8+ T cell clones specific for a variety of EBV or HCMV epitopes. Specificity is indicated by the first three amino acids of the peptide. Each dot represents a single analysis of one CD8+ T cell clone. (B–E) CD8+ T cell clones were co-incubated with WT or ΔLMP2A LCLs in the presence of an anti-NKG2D antibody (α-NKG2D), a matched isotype control (iso), or no antibody (No Ab). T cells had been pre-treated with the antibodies for 1 hour before incubation with LCLs. After 16 hours, IFN-γ in the supernatant was evaluated by ELISA. (B) A representative experiment for a CD8+ T cell clone specific for the FLY epitope from EBV latent antigen LMP2. “pep”, WT LCL exogenously loaded with corresponding peptide as a positive control; “∅t”, T cells alone. Mean and range of duplicates are shown. (C) Summary of experiments with EBV-specific CD8+ T cell clones. Epitopes from the latent proteins LMP2 (FLY and CLG), EBNA1 (HPV) and EBNA3A (RPP) were tested. Each pair of values linked by a line represents the same LCL, treated with α-NKG2D or isotype control antibody, and here the Wilcoxon signed-rank test was applied. A total of six LCL donors were analyzed. WT and ΔLMP2A LCLs from the same donor were always analyzed in parallel. For comparison of WT and ΔLMP2A LCLs, the Mann-Whitney U test was used. (D) Relative change of recognition upon NKG2D blocking. Isotype controls were set to 100% recognition. Statistical analysis was performed as in C. (E) WT and ΔLMP2A LCLs were loaded with peptides CRV and VLE from the HCMV protein IE-1 at 10–8 M. Antibody blocking and analysis of specific T cell recognition were performed as above.
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ppat.1004906.g007: Effect of NKG2D blocking on CD8+ T cell recognition of LCLs with or without LMP2A.(A) Expression of NKG2D on CD8+ T cell clones specific for a variety of EBV or HCMV epitopes. Specificity is indicated by the first three amino acids of the peptide. Each dot represents a single analysis of one CD8+ T cell clone. (B–E) CD8+ T cell clones were co-incubated with WT or ΔLMP2A LCLs in the presence of an anti-NKG2D antibody (α-NKG2D), a matched isotype control (iso), or no antibody (No Ab). T cells had been pre-treated with the antibodies for 1 hour before incubation with LCLs. After 16 hours, IFN-γ in the supernatant was evaluated by ELISA. (B) A representative experiment for a CD8+ T cell clone specific for the FLY epitope from EBV latent antigen LMP2. “pep”, WT LCL exogenously loaded with corresponding peptide as a positive control; “∅t”, T cells alone. Mean and range of duplicates are shown. (C) Summary of experiments with EBV-specific CD8+ T cell clones. Epitopes from the latent proteins LMP2 (FLY and CLG), EBNA1 (HPV) and EBNA3A (RPP) were tested. Each pair of values linked by a line represents the same LCL, treated with α-NKG2D or isotype control antibody, and here the Wilcoxon signed-rank test was applied. A total of six LCL donors were analyzed. WT and ΔLMP2A LCLs from the same donor were always analyzed in parallel. For comparison of WT and ΔLMP2A LCLs, the Mann-Whitney U test was used. (D) Relative change of recognition upon NKG2D blocking. Isotype controls were set to 100% recognition. Statistical analysis was performed as in C. (E) WT and ΔLMP2A LCLs were loaded with peptides CRV and VLE from the HCMV protein IE-1 at 10–8 M. Antibody blocking and analysis of specific T cell recognition were performed as above.

Mentions: We tested the functional relevance of differential NKG2D ligand expression for CD8+ T cell recognition. An analysis of NKG2D levels on several CD8+ T cell clones showed that all were positive for NKG2D (Fig 7A). Differences in NKG2D expression levels were not correlated with antigen specificity. When we blocked NKG2D on EBV-specific CD8+ T cells with a specific antibody, IFN-γ release after contact with LCLs was reduced (Fig 7B–7D). A reduction in the reactivity of CD8+ T cells to both WT and mutant LCLs was observed after blocking, but reduction was even slightly stronger for ΔLMP2A LCLs than for WT LCLs (Fig 7C and 7D). Likewise, blocking NKG2D on HCMV-specific CD8+ T cell clones led to reduced recognition of peptide-loaded LCLs (Fig 7E). Thus, NKG2D ligands on LCLs contribute to their recognition by CD8+ T cells irrespective of antigen specificity. LMP2A reduces CD8+ T cell recognition of EBV-infected B cells by reducing the expression of NKG2D ligands.


Latent Membrane Protein LMP2A Impairs Recognition of EBV-Infected Cells by CD8+ T Cells.

Rancan C, Schirrmann L, Hüls C, Zeidler R, Moosmann A - PLoS Pathog. (2015)

Effect of NKG2D blocking on CD8+ T cell recognition of LCLs with or without LMP2A.(A) Expression of NKG2D on CD8+ T cell clones specific for a variety of EBV or HCMV epitopes. Specificity is indicated by the first three amino acids of the peptide. Each dot represents a single analysis of one CD8+ T cell clone. (B–E) CD8+ T cell clones were co-incubated with WT or ΔLMP2A LCLs in the presence of an anti-NKG2D antibody (α-NKG2D), a matched isotype control (iso), or no antibody (No Ab). T cells had been pre-treated with the antibodies for 1 hour before incubation with LCLs. After 16 hours, IFN-γ in the supernatant was evaluated by ELISA. (B) A representative experiment for a CD8+ T cell clone specific for the FLY epitope from EBV latent antigen LMP2. “pep”, WT LCL exogenously loaded with corresponding peptide as a positive control; “∅t”, T cells alone. Mean and range of duplicates are shown. (C) Summary of experiments with EBV-specific CD8+ T cell clones. Epitopes from the latent proteins LMP2 (FLY and CLG), EBNA1 (HPV) and EBNA3A (RPP) were tested. Each pair of values linked by a line represents the same LCL, treated with α-NKG2D or isotype control antibody, and here the Wilcoxon signed-rank test was applied. A total of six LCL donors were analyzed. WT and ΔLMP2A LCLs from the same donor were always analyzed in parallel. For comparison of WT and ΔLMP2A LCLs, the Mann-Whitney U test was used. (D) Relative change of recognition upon NKG2D blocking. Isotype controls were set to 100% recognition. Statistical analysis was performed as in C. (E) WT and ΔLMP2A LCLs were loaded with peptides CRV and VLE from the HCMV protein IE-1 at 10–8 M. Antibody blocking and analysis of specific T cell recognition were performed as above.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4465838&req=5

ppat.1004906.g007: Effect of NKG2D blocking on CD8+ T cell recognition of LCLs with or without LMP2A.(A) Expression of NKG2D on CD8+ T cell clones specific for a variety of EBV or HCMV epitopes. Specificity is indicated by the first three amino acids of the peptide. Each dot represents a single analysis of one CD8+ T cell clone. (B–E) CD8+ T cell clones were co-incubated with WT or ΔLMP2A LCLs in the presence of an anti-NKG2D antibody (α-NKG2D), a matched isotype control (iso), or no antibody (No Ab). T cells had been pre-treated with the antibodies for 1 hour before incubation with LCLs. After 16 hours, IFN-γ in the supernatant was evaluated by ELISA. (B) A representative experiment for a CD8+ T cell clone specific for the FLY epitope from EBV latent antigen LMP2. “pep”, WT LCL exogenously loaded with corresponding peptide as a positive control; “∅t”, T cells alone. Mean and range of duplicates are shown. (C) Summary of experiments with EBV-specific CD8+ T cell clones. Epitopes from the latent proteins LMP2 (FLY and CLG), EBNA1 (HPV) and EBNA3A (RPP) were tested. Each pair of values linked by a line represents the same LCL, treated with α-NKG2D or isotype control antibody, and here the Wilcoxon signed-rank test was applied. A total of six LCL donors were analyzed. WT and ΔLMP2A LCLs from the same donor were always analyzed in parallel. For comparison of WT and ΔLMP2A LCLs, the Mann-Whitney U test was used. (D) Relative change of recognition upon NKG2D blocking. Isotype controls were set to 100% recognition. Statistical analysis was performed as in C. (E) WT and ΔLMP2A LCLs were loaded with peptides CRV and VLE from the HCMV protein IE-1 at 10–8 M. Antibody blocking and analysis of specific T cell recognition were performed as above.
Mentions: We tested the functional relevance of differential NKG2D ligand expression for CD8+ T cell recognition. An analysis of NKG2D levels on several CD8+ T cell clones showed that all were positive for NKG2D (Fig 7A). Differences in NKG2D expression levels were not correlated with antigen specificity. When we blocked NKG2D on EBV-specific CD8+ T cells with a specific antibody, IFN-γ release after contact with LCLs was reduced (Fig 7B–7D). A reduction in the reactivity of CD8+ T cells to both WT and mutant LCLs was observed after blocking, but reduction was even slightly stronger for ΔLMP2A LCLs than for WT LCLs (Fig 7C and 7D). Likewise, blocking NKG2D on HCMV-specific CD8+ T cell clones led to reduced recognition of peptide-loaded LCLs (Fig 7E). Thus, NKG2D ligands on LCLs contribute to their recognition by CD8+ T cells irrespective of antigen specificity. LMP2A reduces CD8+ T cell recognition of EBV-infected B cells by reducing the expression of NKG2D ligands.

Bottom Line: We found that CD8+ T cell clones showed higher reactivity against LMP2A-deficient LCLs compared to LCLs infected with complete EBV.We identified several potential mediators of this immunomodulatory effect.Our results demonstrate that LMP2A reduces the reactivity of CD8+ T cells against EBV-infected cells, and we identify several relevant mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Clinical Cooperation Group Immunooncology, Department of Medicine III, Klinikum der Universität München, and Research Unit Gene Vectors, Helmholtz Zentrum München, Munich, Germany; German Center for Infection Research (DZIF), Munich, Germany.

ABSTRACT
The common pathogen Epstein-Barr virus (EBV) transforms normal human B cells and can cause cancer. Latent membrane protein 2A (LMP2A) of EBV supports activation and proliferation of infected B cells and is expressed in many types of EBV-associated cancer. It is not clear how latent EBV infection and cancer escape elimination by host immunity, and it is unknown whether LMP2A can influence the interaction of EBV-infected cells with the immune system. We infected primary B cells with EBV deleted for LMP2A, and established lymphoblastoid cell lines (LCLs). We found that CD8+ T cell clones showed higher reactivity against LMP2A-deficient LCLs compared to LCLs infected with complete EBV. We identified several potential mediators of this immunomodulatory effect. In the absence of LMP2A, expression of some EBV latent antigens was elevated, and cell surface expression of MHC class I was marginally increased. LMP2A-deficient LCLs produced lower amounts of IL-10, although this did not directly affect CD8+ T cell recognition. Deletion of LMP2A led to several changes in the cell surface immunophenotype of LCLs. Specifically, the agonistic NKG2D ligands MICA and ULBP4 were increased. Blocking experiments showed that NKG2D activation contributed to LCL recognition by CD8+ T cell clones. Our results demonstrate that LMP2A reduces the reactivity of CD8+ T cells against EBV-infected cells, and we identify several relevant mechanisms.

No MeSH data available.


Related in: MedlinePlus