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The Cellular Localization of Human Cytomegalovirus Glycoprotein Expression Greatly Influences the Frequency and Functional Phenotype of Specific CD4+ T Cell Responses.

Pachnio A, Zuo J, Ryan GB, Begum J, Moss PA - J. Immunol. (2015)

Bottom Line: In this study, we examine and contrast the magnitude and phenotype of the T cell immune response against gB, gH, and gL within healthy donors. gB-specific CD4(+) T cells were found in 95% of donors, and 29 epitopes were defined with gB-specific response sizes ranging from 0.02 to 2.88% of the CD4(+) T cell pool.Glycoproteins were effectively presented following delivery to APCs but only gB-derived epitopes were presented following endogenous synthesis. gB expression was observed exclusively within vesicular structures colocalizing with HLA-DM whereas gH was distributed evenly throughout the cytoplasm.These results reveal that gB is a uniquely immunogenic CMV glycoprotein and this is likely to reflect its unique pattern of endogenous Ag presentation.

View Article: PubMed Central - PubMed

Affiliation: School of Cancer Sciences, College of Medicine and Dentistry, University of Birmingham, Birmingham B15 2TT, United Kingdom; and a.pachnio@bham.ac.uk p.moss@bham.ac.uk.

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Newly identified T cell epitopes are generated during natural infection without the need for de novo synthesis. To determine whether glycoprotein-derived epitopes are generated in the context of viral infection, HLA-matched fibroblasts, pretreated with IFN-γ (A) or autologous PBMCs (B), were infected with purified CMV for 24 h prior to addition of clonal T cells specific for peptides IRS or QLN, respectively. T cell activation was detected by analysis of IFN-γ in supernatants using ELISA. Shown is a representative of two experiments for each epitope studied.
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fig05: Newly identified T cell epitopes are generated during natural infection without the need for de novo synthesis. To determine whether glycoprotein-derived epitopes are generated in the context of viral infection, HLA-matched fibroblasts, pretreated with IFN-γ (A) or autologous PBMCs (B), were infected with purified CMV for 24 h prior to addition of clonal T cells specific for peptides IRS or QLN, respectively. T cell activation was detected by analysis of IFN-γ in supernatants using ELISA. Shown is a representative of two experiments for each epitope studied.

Mentions: Another major incentive for generating T cell clones was to use these to confirm that the individual peptide epitopes were presented during the course of natural viral infection of a target cell. In case of the DR7-restriced epitope IRS, HLA-matched fibroblasts were treated with IFN-γ to induce HLA class II expression and then infected with a purified preparation of CMV (Merlin strain) for 24 h prior to addition of T cell clones. Recognition of the Ag was determined by detection of IFN-γ within the culture supernatant. Peptide-loaded and uninfected fibroblasts served as controls. Results show that T cells were indeed able to recognize virus-infected fibroblasts (Fig. 5A). Furthermore, infection of fibroblasts with a UV-inactivated CMV preparation established that de novo protein synthesis was not necessary, showing that incoming virus particles contained sufficient amounts of gB to sensitize specific T cells. For the gH-derived epitope QLN, no HLA-matched fibroblasts were available and we therefore infected autologous PBMCs with virus, as monocytes are permissive for infection. This confirmed that the peptide epitope QLN was also generated efficiently in the context of a viral infection (Fig. 5B).


The Cellular Localization of Human Cytomegalovirus Glycoprotein Expression Greatly Influences the Frequency and Functional Phenotype of Specific CD4+ T Cell Responses.

Pachnio A, Zuo J, Ryan GB, Begum J, Moss PA - J. Immunol. (2015)

Newly identified T cell epitopes are generated during natural infection without the need for de novo synthesis. To determine whether glycoprotein-derived epitopes are generated in the context of viral infection, HLA-matched fibroblasts, pretreated with IFN-γ (A) or autologous PBMCs (B), were infected with purified CMV for 24 h prior to addition of clonal T cells specific for peptides IRS or QLN, respectively. T cell activation was detected by analysis of IFN-γ in supernatants using ELISA. Shown is a representative of two experiments for each epitope studied.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4592104&req=5

fig05: Newly identified T cell epitopes are generated during natural infection without the need for de novo synthesis. To determine whether glycoprotein-derived epitopes are generated in the context of viral infection, HLA-matched fibroblasts, pretreated with IFN-γ (A) or autologous PBMCs (B), were infected with purified CMV for 24 h prior to addition of clonal T cells specific for peptides IRS or QLN, respectively. T cell activation was detected by analysis of IFN-γ in supernatants using ELISA. Shown is a representative of two experiments for each epitope studied.
Mentions: Another major incentive for generating T cell clones was to use these to confirm that the individual peptide epitopes were presented during the course of natural viral infection of a target cell. In case of the DR7-restriced epitope IRS, HLA-matched fibroblasts were treated with IFN-γ to induce HLA class II expression and then infected with a purified preparation of CMV (Merlin strain) for 24 h prior to addition of T cell clones. Recognition of the Ag was determined by detection of IFN-γ within the culture supernatant. Peptide-loaded and uninfected fibroblasts served as controls. Results show that T cells were indeed able to recognize virus-infected fibroblasts (Fig. 5A). Furthermore, infection of fibroblasts with a UV-inactivated CMV preparation established that de novo protein synthesis was not necessary, showing that incoming virus particles contained sufficient amounts of gB to sensitize specific T cells. For the gH-derived epitope QLN, no HLA-matched fibroblasts were available and we therefore infected autologous PBMCs with virus, as monocytes are permissive for infection. This confirmed that the peptide epitope QLN was also generated efficiently in the context of a viral infection (Fig. 5B).

Bottom Line: In this study, we examine and contrast the magnitude and phenotype of the T cell immune response against gB, gH, and gL within healthy donors. gB-specific CD4(+) T cells were found in 95% of donors, and 29 epitopes were defined with gB-specific response sizes ranging from 0.02 to 2.88% of the CD4(+) T cell pool.Glycoproteins were effectively presented following delivery to APCs but only gB-derived epitopes were presented following endogenous synthesis. gB expression was observed exclusively within vesicular structures colocalizing with HLA-DM whereas gH was distributed evenly throughout the cytoplasm.These results reveal that gB is a uniquely immunogenic CMV glycoprotein and this is likely to reflect its unique pattern of endogenous Ag presentation.

View Article: PubMed Central - PubMed

Affiliation: School of Cancer Sciences, College of Medicine and Dentistry, University of Birmingham, Birmingham B15 2TT, United Kingdom; and a.pachnio@bham.ac.uk p.moss@bham.ac.uk.

Show MeSH
Related in: MedlinePlus