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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

CD8+ T cell recognition of heterologous peptides on LCLs with or without LMP2A.WT and ΔLMP2A LCLs were loaded with peptides CRV and VLE from HCMV IE-1 or peptide NLV from HCMV pp65 at the indicated concentrations, and were coincubated for 16 hours with cognate antigen-specific CD8+ T cell clones. IFN-γ release by CD8+ T cells into supernatant was quantified by ELISA. (A) Exemplary dose-response curves. Dotted lines indicate the peptide concentration that induced half-maximal IFN-γ release. (B) Paired analysis of results by donor. Each pair of values represents LCLs from one donor tested in one experiment with one peptide. Three HCMV peptides and LCLs from 3 donors were included in this analysis. The Wilcoxon signed-rank test was applied. (C) Synoptic analysis of the relative change in recognition in the absence of LMP2A, setting recognition of WT LCLs to 100% for each pair of values shown in B. The Mann-Whitney U test was applied.
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ppat.1004906.g003: CD8+ T cell recognition of heterologous peptides on LCLs with or without LMP2A.WT and ΔLMP2A LCLs were loaded with peptides CRV and VLE from HCMV IE-1 or peptide NLV from HCMV pp65 at the indicated concentrations, and were coincubated for 16 hours with cognate antigen-specific CD8+ T cell clones. IFN-γ release by CD8+ T cells into supernatant was quantified by ELISA. (A) Exemplary dose-response curves. Dotted lines indicate the peptide concentration that induced half-maximal IFN-γ release. (B) Paired analysis of results by donor. Each pair of values represents LCLs from one donor tested in one experiment with one peptide. Three HCMV peptides and LCLs from 3 donors were included in this analysis. The Wilcoxon signed-rank test was applied. (C) Synoptic analysis of the relative change in recognition in the absence of LMP2A, setting recognition of WT LCLs to 100% for each pair of values shown in B. The Mann-Whitney U test was applied.

Mentions: Next, we investigated whether LMP2A modulated the reactivity of CD8+ T cells to EBV-infected B cells by mechanisms other than altering the availability of EBV antigens. We loaded WT and ΔLMP2A LCLs exogenously with peptides CRV and VLE, derived from the human cytomegalovirus (HCMV) protein IE-1, and we analyzed LCL recognition by HCMV-specific CD8+ T cell clones (Fig 3). Peptide-loaded ΔLMP2A LCLs were more strongly recognized by these CD8+ T cells than peptide-loaded WT LCLs, resulting in higher IFN-γ release. We also investigated direct killing by cytotoxic CD8+ T cells, but did not observe differences in killing of WT and ΔLMP2A LCLs loaded with HCMV peptides (S2 Fig). The reasons for differential regulation of IFN-γ secretion and direct cytotoxicity in this setting remain to be elucidated.


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)

CD8+ T cell recognition of heterologous peptides on LCLs with or without LMP2A.WT and ΔLMP2A LCLs were loaded with peptides CRV and VLE from HCMV IE-1 or peptide NLV from HCMV pp65 at the indicated concentrations, and were coincubated for 16 hours with cognate antigen-specific CD8+ T cell clones. IFN-γ release by CD8+ T cells into supernatant was quantified by ELISA. (A) Exemplary dose-response curves. Dotted lines indicate the peptide concentration that induced half-maximal IFN-γ release. (B) Paired analysis of results by donor. Each pair of values represents LCLs from one donor tested in one experiment with one peptide. Three HCMV peptides and LCLs from 3 donors were included in this analysis. The Wilcoxon signed-rank test was applied. (C) Synoptic analysis of the relative change in recognition in the absence of LMP2A, setting recognition of WT LCLs to 100% for each pair of values shown in B. The Mann-Whitney U test was applied.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004906.g003: CD8+ T cell recognition of heterologous peptides on LCLs with or without LMP2A.WT and ΔLMP2A LCLs were loaded with peptides CRV and VLE from HCMV IE-1 or peptide NLV from HCMV pp65 at the indicated concentrations, and were coincubated for 16 hours with cognate antigen-specific CD8+ T cell clones. IFN-γ release by CD8+ T cells into supernatant was quantified by ELISA. (A) Exemplary dose-response curves. Dotted lines indicate the peptide concentration that induced half-maximal IFN-γ release. (B) Paired analysis of results by donor. Each pair of values represents LCLs from one donor tested in one experiment with one peptide. Three HCMV peptides and LCLs from 3 donors were included in this analysis. The Wilcoxon signed-rank test was applied. (C) Synoptic analysis of the relative change in recognition in the absence of LMP2A, setting recognition of WT LCLs to 100% for each pair of values shown in B. The Mann-Whitney U test was applied.
Mentions: Next, we investigated whether LMP2A modulated the reactivity of CD8+ T cells to EBV-infected B cells by mechanisms other than altering the availability of EBV antigens. We loaded WT and ΔLMP2A LCLs exogenously with peptides CRV and VLE, derived from the human cytomegalovirus (HCMV) protein IE-1, and we analyzed LCL recognition by HCMV-specific CD8+ T cell clones (Fig 3). Peptide-loaded ΔLMP2A LCLs were more strongly recognized by these CD8+ T cells than peptide-loaded WT LCLs, resulting in higher IFN-γ release. We also investigated direct killing by cytotoxic CD8+ T cells, but did not observe differences in killing of WT and ΔLMP2A LCLs loaded with HCMV peptides (S2 Fig). The reasons for differential regulation of IFN-γ secretion and direct cytotoxicity in this setting remain to be elucidated.

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