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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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HLA restriction and functional avidity of CD4+ T cell clones specific for gB, gH, and gL. Representative examples of HLA restriction assays: clonal T cells were cocultured with either autologous or partially HLA-matched peptide-loaded LCLs, and IFN-γ was detected in the supernatant to analyze T cell activation (A). The functional avidity of CD4+ T cell clones specific for the same epitopes was investigated by preincubation of autologous LCLs with decreasing concentrations of the peptide, followed by measurement of IFN-γ release after incubation with T cells (B). Each experiment was carried out at least twice in duplicates.
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fig04: HLA restriction and functional avidity of CD4+ T cell clones specific for gB, gH, and gL. Representative examples of HLA restriction assays: clonal T cells were cocultured with either autologous or partially HLA-matched peptide-loaded LCLs, and IFN-γ was detected in the supernatant to analyze T cell activation (A). The functional avidity of CD4+ T cell clones specific for the same epitopes was investigated by preincubation of autologous LCLs with decreasing concentrations of the peptide, followed by measurement of IFN-γ release after incubation with T cells (B). Each experiment was carried out at least twice in duplicates.

Mentions: Table I lists the protein, peptide sequence, and HLA restriction of all seven cloned T cell responses. The HLA restriction of each clone was determined with the use of LCLs. Clones were tested for recognition of peptide-loaded autologous LCLs and also allogeneic, peptide-loaded LCLs that were partially matched for specific HLA class II alleles. Three examples from this analysis are shown in Fig. 4A. The HLA restriction of both the gB-derived epitope IRS and gH-derived epitope QLN was defined with T cell clones from donor 9, and gL-derived epitope T cell clones were generated from donor 11. Peptide IRS was presented only by LCLs sharing HLA-DR7, thus identifying this as the restricting allele. The QLN response was analyzed in the same donor using the same panel of partially matched LCLs. T cell recognition was blocked completely by preincubation with an anti–HLA-DR Ab, however none of the allogeneic LCLs induced T cell recognition. As the DR7 restriction of IRS-specific clones from the same individual was clearly identified using this panel of LCLs, we concluded that this epitope is HLA-DR4 restricted. Several subtypes of HLA-DR4 have been observed, and polymorphisms occur in regions of the molecule that play a role in the interaction of the TCR and its Ag (46, 47). This may explain why this particular epitope was only presented by autologous LCLs. HLA-DR4 and -DR7 are both extremely closely linked to DR53, but no such subtype-specific differences have been reported for this HLA allele, supporting our conclusion that the QLN epitope is HLA-DR4 restricted. The final example shows the HLA restriction of the gL-derived epitope QGD, which was determined to be HLA-DP8.


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)

HLA restriction and functional avidity of CD4+ T cell clones specific for gB, gH, and gL. Representative examples of HLA restriction assays: clonal T cells were cocultured with either autologous or partially HLA-matched peptide-loaded LCLs, and IFN-γ was detected in the supernatant to analyze T cell activation (A). The functional avidity of CD4+ T cell clones specific for the same epitopes was investigated by preincubation of autologous LCLs with decreasing concentrations of the peptide, followed by measurement of IFN-γ release after incubation with T cells (B). Each experiment was carried out at least twice in duplicates.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

fig04: HLA restriction and functional avidity of CD4+ T cell clones specific for gB, gH, and gL. Representative examples of HLA restriction assays: clonal T cells were cocultured with either autologous or partially HLA-matched peptide-loaded LCLs, and IFN-γ was detected in the supernatant to analyze T cell activation (A). The functional avidity of CD4+ T cell clones specific for the same epitopes was investigated by preincubation of autologous LCLs with decreasing concentrations of the peptide, followed by measurement of IFN-γ release after incubation with T cells (B). Each experiment was carried out at least twice in duplicates.
Mentions: Table I lists the protein, peptide sequence, and HLA restriction of all seven cloned T cell responses. The HLA restriction of each clone was determined with the use of LCLs. Clones were tested for recognition of peptide-loaded autologous LCLs and also allogeneic, peptide-loaded LCLs that were partially matched for specific HLA class II alleles. Three examples from this analysis are shown in Fig. 4A. The HLA restriction of both the gB-derived epitope IRS and gH-derived epitope QLN was defined with T cell clones from donor 9, and gL-derived epitope T cell clones were generated from donor 11. Peptide IRS was presented only by LCLs sharing HLA-DR7, thus identifying this as the restricting allele. The QLN response was analyzed in the same donor using the same panel of partially matched LCLs. T cell recognition was blocked completely by preincubation with an anti–HLA-DR Ab, however none of the allogeneic LCLs induced T cell recognition. As the DR7 restriction of IRS-specific clones from the same individual was clearly identified using this panel of LCLs, we concluded that this epitope is HLA-DR4 restricted. Several subtypes of HLA-DR4 have been observed, and polymorphisms occur in regions of the molecule that play a role in the interaction of the TCR and its Ag (46, 47). This may explain why this particular epitope was only presented by autologous LCLs. HLA-DR4 and -DR7 are both extremely closely linked to DR53, but no such subtype-specific differences have been reported for this HLA allele, supporting our conclusion that the QLN epitope is HLA-DR4 restricted. The final example shows the HLA restriction of the gL-derived epitope QGD, which was determined to be HLA-DP8.

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