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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

Role of LCL-secreted IL-10 in CD8+ T cell recognition of WT and ΔLMP2A LCLs.(A) WT and ΔLMP2A LCLs were washed and incubated at 0.5x106 cells/ml for 18 hours, and released IL-10 was measured by ELISA. Each dot represents the mean of quadruplicates for a single independently established cell line. Data shown are representative for three independent experiments. WT and ΔLMP2A lines established from 5 different donors were analyzed in parallel. The horizontal line indicates the median; the Mann-Whitney U test was applied. (B) mRNA levels for human IL-10 and BCRF1 (viral IL-10) were measured by quantitative RT-PCR in WT and ΔLMP2A LCLs. Expression relative to the housekeeping gene GUSB is displayed. Each dot represents an independently established LCL. LCLs were from 4 different donors, WT and ΔLMP2A LCLs from each donor were analyzed in parallel. Data are representative of two independent experiments. The horizontal line indicates the median; the Mann-Whitney U test was applied. (C-F) Reactivity of EBV-specific CD8+ T cell clones against WT or ΔLMP2A LCLs was determined after antibody blocking of the IL-10 receptor on the T cells (C, E) or in the presence of a neutralizing IL-10-specific antibody (D, F). Conditions with no antibody (No Ab) or a matched isotype control antibody (iso) were tested in parallel. After 16 hours, IFN-γ release into the supernatant was quantified by ELISA. Mean and range of duplicates are shown. (C, D) Exemplary experiments. (E, F) Analyses of the relative change in recognition due to antibody blocking, with isotype controls set to 100%. A synopsis of experiments with three different T cell clones with overall mean and SEM is shown. Statistical analysis was performed with the Mann-Whitney U test.
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ppat.1004906.g004: Role of LCL-secreted IL-10 in CD8+ T cell recognition of WT and ΔLMP2A LCLs.(A) WT and ΔLMP2A LCLs were washed and incubated at 0.5x106 cells/ml for 18 hours, and released IL-10 was measured by ELISA. Each dot represents the mean of quadruplicates for a single independently established cell line. Data shown are representative for three independent experiments. WT and ΔLMP2A lines established from 5 different donors were analyzed in parallel. The horizontal line indicates the median; the Mann-Whitney U test was applied. (B) mRNA levels for human IL-10 and BCRF1 (viral IL-10) were measured by quantitative RT-PCR in WT and ΔLMP2A LCLs. Expression relative to the housekeeping gene GUSB is displayed. Each dot represents an independently established LCL. LCLs were from 4 different donors, WT and ΔLMP2A LCLs from each donor were analyzed in parallel. Data are representative of two independent experiments. The horizontal line indicates the median; the Mann-Whitney U test was applied. (C-F) Reactivity of EBV-specific CD8+ T cell clones against WT or ΔLMP2A LCLs was determined after antibody blocking of the IL-10 receptor on the T cells (C, E) or in the presence of a neutralizing IL-10-specific antibody (D, F). Conditions with no antibody (No Ab) or a matched isotype control antibody (iso) were tested in parallel. After 16 hours, IFN-γ release into the supernatant was quantified by ELISA. Mean and range of duplicates are shown. (C, D) Exemplary experiments. (E, F) Analyses of the relative change in recognition due to antibody blocking, with isotype controls set to 100%. A synopsis of experiments with three different T cell clones with overall mean and SEM is shown. Statistical analysis was performed with the Mann-Whitney U test.

Mentions: It was recently shown that LMP2A increases IL-10 production in infected B cells [38]. The possibility of a similar effect in our system was intriguing, because cellular IL-10 and its viral homolog reduce the antiviral activity of different types of immune effector cells [41–43]. In accordance with Incrocci and colleagues [38], we found that WT LCLs released higher amounts of IL-10 than LCLs lacking LMP2A (Fig 4A). These levels of secreted IL-10 were not mirrored by transcription levels for human IL-10 (Fig 4B), which suggested an effect of LMP2A on post-transcriptional regulation of IL-10 [44]. In contrast to cellular IL-10, transcription of viral IL-10 was very low in each type of LCL (Fig 4B), in accordance with its description as a lytic-cycle gene [45]. To determine whether differences in IL-10 release could directly influence T cell reactivity to LCLs, we used specific antibodies to block IL-10 receptor on CD8+ T cells (Fig 4C and 4E), or to neutralize IL-10 in the supernatant (Fig 4D and 4F). In each case, recognition of WT or ΔLMP2A LCLs was not altered. Thus, modulation of IL-10 secretion by LMP2A did not directly affect the ability of CD8+ T cells to recognize infected B cells. This experiment did not rule out indirect effects of secreted IL-10, which may act back on the LCLs over time in culture and modulate their immunogenicity.


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)

Role of LCL-secreted IL-10 in CD8+ T cell recognition of WT and ΔLMP2A LCLs.(A) WT and ΔLMP2A LCLs were washed and incubated at 0.5x106 cells/ml for 18 hours, and released IL-10 was measured by ELISA. Each dot represents the mean of quadruplicates for a single independently established cell line. Data shown are representative for three independent experiments. WT and ΔLMP2A lines established from 5 different donors were analyzed in parallel. The horizontal line indicates the median; the Mann-Whitney U test was applied. (B) mRNA levels for human IL-10 and BCRF1 (viral IL-10) were measured by quantitative RT-PCR in WT and ΔLMP2A LCLs. Expression relative to the housekeeping gene GUSB is displayed. Each dot represents an independently established LCL. LCLs were from 4 different donors, WT and ΔLMP2A LCLs from each donor were analyzed in parallel. Data are representative of two independent experiments. The horizontal line indicates the median; the Mann-Whitney U test was applied. (C-F) Reactivity of EBV-specific CD8+ T cell clones against WT or ΔLMP2A LCLs was determined after antibody blocking of the IL-10 receptor on the T cells (C, E) or in the presence of a neutralizing IL-10-specific antibody (D, F). Conditions with no antibody (No Ab) or a matched isotype control antibody (iso) were tested in parallel. After 16 hours, IFN-γ release into the supernatant was quantified by ELISA. Mean and range of duplicates are shown. (C, D) Exemplary experiments. (E, F) Analyses of the relative change in recognition due to antibody blocking, with isotype controls set to 100%. A synopsis of experiments with three different T cell clones with overall mean and SEM is shown. Statistical analysis was performed with the Mann-Whitney U test.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004906.g004: Role of LCL-secreted IL-10 in CD8+ T cell recognition of WT and ΔLMP2A LCLs.(A) WT and ΔLMP2A LCLs were washed and incubated at 0.5x106 cells/ml for 18 hours, and released IL-10 was measured by ELISA. Each dot represents the mean of quadruplicates for a single independently established cell line. Data shown are representative for three independent experiments. WT and ΔLMP2A lines established from 5 different donors were analyzed in parallel. The horizontal line indicates the median; the Mann-Whitney U test was applied. (B) mRNA levels for human IL-10 and BCRF1 (viral IL-10) were measured by quantitative RT-PCR in WT and ΔLMP2A LCLs. Expression relative to the housekeeping gene GUSB is displayed. Each dot represents an independently established LCL. LCLs were from 4 different donors, WT and ΔLMP2A LCLs from each donor were analyzed in parallel. Data are representative of two independent experiments. The horizontal line indicates the median; the Mann-Whitney U test was applied. (C-F) Reactivity of EBV-specific CD8+ T cell clones against WT or ΔLMP2A LCLs was determined after antibody blocking of the IL-10 receptor on the T cells (C, E) or in the presence of a neutralizing IL-10-specific antibody (D, F). Conditions with no antibody (No Ab) or a matched isotype control antibody (iso) were tested in parallel. After 16 hours, IFN-γ release into the supernatant was quantified by ELISA. Mean and range of duplicates are shown. (C, D) Exemplary experiments. (E, F) Analyses of the relative change in recognition due to antibody blocking, with isotype controls set to 100%. A synopsis of experiments with three different T cell clones with overall mean and SEM is shown. Statistical analysis was performed with the Mann-Whitney U test.
Mentions: It was recently shown that LMP2A increases IL-10 production in infected B cells [38]. The possibility of a similar effect in our system was intriguing, because cellular IL-10 and its viral homolog reduce the antiviral activity of different types of immune effector cells [41–43]. In accordance with Incrocci and colleagues [38], we found that WT LCLs released higher amounts of IL-10 than LCLs lacking LMP2A (Fig 4A). These levels of secreted IL-10 were not mirrored by transcription levels for human IL-10 (Fig 4B), which suggested an effect of LMP2A on post-transcriptional regulation of IL-10 [44]. In contrast to cellular IL-10, transcription of viral IL-10 was very low in each type of LCL (Fig 4B), in accordance with its description as a lytic-cycle gene [45]. To determine whether differences in IL-10 release could directly influence T cell reactivity to LCLs, we used specific antibodies to block IL-10 receptor on CD8+ T cells (Fig 4C and 4E), or to neutralize IL-10 in the supernatant (Fig 4D and 4F). In each case, recognition of WT or ΔLMP2A LCLs was not altered. Thus, modulation of IL-10 secretion by LMP2A did not directly affect the ability of CD8+ T cells to recognize infected B cells. This experiment did not rule out indirect effects of secreted IL-10, which may act back on the LCLs over time in culture and modulate their immunogenicity.

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