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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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Identification of peptide-specific T cells by intracellular cytokine staining for IFN-γ. (A) Schematic representation of the three CMV-derived glycoproteins studied. The identification of a CD4-restricted gB-derived epitope is shown as an example in (B). PBMCs were stimulated overnight, then stained with Live/Dead fixable violet stain and anti-CD19 (dump channel) as well as anti-CD4 and anti-CD8. After fixation (4% paraformaldehyde) and permeabilization with 0.5% saponin, activated T cells were identified by staining with anti–IFN-γ. DMSO and staphylococcal enterotoxin B (SEB)–stimulated cells served as negative or positive controls, respectively. Shown are the gating strategy (top row) and results of the first round of screening (middle row). The individual peptide epitope was then identified in a subsequent screening (bottom row). Numbers indicate the frequency of IFN-γ–producing cells within the total CD4+ T cell population. FSC-A, forward scatter area; FSC-H, forward scatter height; SSC-A, side scatter area.
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fig01: Identification of peptide-specific T cells by intracellular cytokine staining for IFN-γ. (A) Schematic representation of the three CMV-derived glycoproteins studied. The identification of a CD4-restricted gB-derived epitope is shown as an example in (B). PBMCs were stimulated overnight, then stained with Live/Dead fixable violet stain and anti-CD19 (dump channel) as well as anti-CD4 and anti-CD8. After fixation (4% paraformaldehyde) and permeabilization with 0.5% saponin, activated T cells were identified by staining with anti–IFN-γ. DMSO and staphylococcal enterotoxin B (SEB)–stimulated cells served as negative or positive controls, respectively. Shown are the gating strategy (top row) and results of the first round of screening (middle row). The individual peptide epitope was then identified in a subsequent screening (bottom row). Numbers indicate the frequency of IFN-γ–producing cells within the total CD4+ T cell population. FSC-A, forward scatter area; FSC-H, forward scatter height; SSC-A, side scatter area.

Mentions: Following overnight stimulation, activated T cells were identified by flow cytometric detection of intracellular IFN-γ expression. For this, cells were washed with 1× PBS and stained with Live/Dead fixable violet stain (Invitrogen) for 15 min at room temperature (RT) followed by one wash in 1× PBS and one in staining buffer (1× PBS plus 0.5% BSA and 2 mM EDTA). T cells were then identified by staining with anti–CD4-PE (BD Biosciences) and anti–CD8-PECy5 (Beckman Coulter), and B cells were excluded by staining with anti-CD19 Pacific Blue (eBioscience; dump channel). Cells were incubated for 15 min at 4°C before washing off excess Ab with staining buffer. Fixing was carried out with 4% paraformaldehyde (in PBS; Sigma-Aldrich) for 15 min at RT before permeabilizing with 0.5% saponin (in PBS; Sigma-Aldrich) for 5 min. Intracellular IFN-γ was then stained with an anti–IFN-γ FITC Ab (BD Biosciences) followed by a final wash in staining buffer. Acquisition was carried out on an LSR II flow cytometer and FACSDiva software (BD Biosciences) collecting 300,000 live lymphocytes, and data were analyzed using FlowJo software version 7.6.5 (Tree Star). For the analysis, doublets were excluded based on forward scatter height versus forward scatter area, followed by exclusion of dead cells and CD19+ cells. The lymphocyte population was then gated on a forward scatter area versus side scatter area scatter plot (see Fig. 1B), and the proportion of IFN-γ–producing cells was determined within the CD4 or CD8 T cell subset. A cytokine response was defined as positive when the frequency of cytokine-producing cells was at least 2-fold increased above background frequency detected in the corresponding DMSO-stimulated sample.


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)

Identification of peptide-specific T cells by intracellular cytokine staining for IFN-γ. (A) Schematic representation of the three CMV-derived glycoproteins studied. The identification of a CD4-restricted gB-derived epitope is shown as an example in (B). PBMCs were stimulated overnight, then stained with Live/Dead fixable violet stain and anti-CD19 (dump channel) as well as anti-CD4 and anti-CD8. After fixation (4% paraformaldehyde) and permeabilization with 0.5% saponin, activated T cells were identified by staining with anti–IFN-γ. DMSO and staphylococcal enterotoxin B (SEB)–stimulated cells served as negative or positive controls, respectively. Shown are the gating strategy (top row) and results of the first round of screening (middle row). The individual peptide epitope was then identified in a subsequent screening (bottom row). Numbers indicate the frequency of IFN-γ–producing cells within the total CD4+ T cell population. FSC-A, forward scatter area; FSC-H, forward scatter height; SSC-A, side scatter area.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4592104&req=5

fig01: Identification of peptide-specific T cells by intracellular cytokine staining for IFN-γ. (A) Schematic representation of the three CMV-derived glycoproteins studied. The identification of a CD4-restricted gB-derived epitope is shown as an example in (B). PBMCs were stimulated overnight, then stained with Live/Dead fixable violet stain and anti-CD19 (dump channel) as well as anti-CD4 and anti-CD8. After fixation (4% paraformaldehyde) and permeabilization with 0.5% saponin, activated T cells were identified by staining with anti–IFN-γ. DMSO and staphylococcal enterotoxin B (SEB)–stimulated cells served as negative or positive controls, respectively. Shown are the gating strategy (top row) and results of the first round of screening (middle row). The individual peptide epitope was then identified in a subsequent screening (bottom row). Numbers indicate the frequency of IFN-γ–producing cells within the total CD4+ T cell population. FSC-A, forward scatter area; FSC-H, forward scatter height; SSC-A, side scatter area.
Mentions: Following overnight stimulation, activated T cells were identified by flow cytometric detection of intracellular IFN-γ expression. For this, cells were washed with 1× PBS and stained with Live/Dead fixable violet stain (Invitrogen) for 15 min at room temperature (RT) followed by one wash in 1× PBS and one in staining buffer (1× PBS plus 0.5% BSA and 2 mM EDTA). T cells were then identified by staining with anti–CD4-PE (BD Biosciences) and anti–CD8-PECy5 (Beckman Coulter), and B cells were excluded by staining with anti-CD19 Pacific Blue (eBioscience; dump channel). Cells were incubated for 15 min at 4°C before washing off excess Ab with staining buffer. Fixing was carried out with 4% paraformaldehyde (in PBS; Sigma-Aldrich) for 15 min at RT before permeabilizing with 0.5% saponin (in PBS; Sigma-Aldrich) for 5 min. Intracellular IFN-γ was then stained with an anti–IFN-γ FITC Ab (BD Biosciences) followed by a final wash in staining buffer. Acquisition was carried out on an LSR II flow cytometer and FACSDiva software (BD Biosciences) collecting 300,000 live lymphocytes, and data were analyzed using FlowJo software version 7.6.5 (Tree Star). For the analysis, doublets were excluded based on forward scatter height versus forward scatter area, followed by exclusion of dead cells and CD19+ cells. The lymphocyte population was then gated on a forward scatter area versus side scatter area scatter plot (see Fig. 1B), and the proportion of IFN-γ–producing cells was determined within the CD4 or CD8 T cell subset. A cytokine response was defined as positive when the frequency of cytokine-producing cells was at least 2-fold increased above background frequency detected in the corresponding DMSO-stimulated sample.

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