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CMV pp65 and IE-1 T cell epitopes recognized by healthy subjects.

Slezak SL, Bettinotti M, Selleri S, Adams S, Marincola FM, Stroncek DF - J Transl Med (2007)

Bottom Line: Several new IE-1 HLA class I epitopes were identified, including 4 restricted to HLA-C antigens.One region of IE-1 spanning amino acids 300 to 327 was rich in class I epitopes.The HLA class I restrictions of IE-1 peptides were more promiscuous than those of pp65 peptides.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Transfusion Medicine, Warren G Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland, USA. sslezak@cc.nih.gov

ABSTRACT

Background: Adoptive immune and vaccine therapies have been used to prevent cytomegalovirus (CMV) disease in recipients of hematopoietic progenitor cell transplants, but the nature of T cell responses to CMV have not been completely characterized.

Methods: Peptide pools and individual peptides derived from the immune-dominant CMV proteins pp65 and IE-1 and antigen-specific, cytokine flow cytometry were used to characterize the prevalence and frequency of CD4+ and CD8+ memory T cells in 20 healthy CMV-seropositive subjects.

Results: CD8+ T cell responses to pp65 were detected in 35% of subjects and to IE-1 in 40% of subjects. CD4+ T cell responses to pp65 were detected in 50% of subjects, but none were detected to IE-1. Several new IE-1 HLA class I epitopes were identified, including 4 restricted to HLA-C antigens. One region of IE-1 spanning amino acids 300 to 327 was rich in class I epitopes. The HLA class I restrictions of IE-1 peptides were more promiscuous than those of pp65 peptides.

Conclusion: Since naturally occurring CD4+ and CD8+ T cell responses to pp65 were detectable in many subjects, but only CD8+ T cell responses to IE-1 were detected, pp65 may be better than IE-1 for use in vaccine and adoptive immune therapies.

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Identification of CMV pp65 epitopes using 15-mer and nanomer peptides. PBMCs were stimulated with a library of 138 15-mer peptides overlapping by 11 residues and covering the entire pp65 protein. IFN-γ flow cytometry was used to detect T cell responses. To identify specific epitopes, PBMCs were stimulated with subpools containing 10 overlapping 15-mer peptides. PBMCs from donors reactive with CD4+ T cells were tested further with individual 15-mers. The results of analysis of CD4+ T cell responses in donor 14 to pp65 peptides are shown.
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Figure 1: Identification of CMV pp65 epitopes using 15-mer and nanomer peptides. PBMCs were stimulated with a library of 138 15-mer peptides overlapping by 11 residues and covering the entire pp65 protein. IFN-γ flow cytometry was used to detect T cell responses. To identify specific epitopes, PBMCs were stimulated with subpools containing 10 overlapping 15-mer peptides. PBMCs from donors reactive with CD4+ T cells were tested further with individual 15-mers. The results of analysis of CD4+ T cell responses in donor 14 to pp65 peptides are shown.

Mentions: In this study, 20 CMV-seropositive and 5 CMV-seronegative healthy donors were evaluated. To determine the CD8+ and CD4+ T cell responses to CMV in the 25 donors, peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with a cocktail of 15-mer peptides overlapping by 11 residues and covering the pp65 and IE-1 proteins. Antigen-specific cytokine flow cytometry was used to detect specific T cell responses[15,16]. To define immunodominant epitopes, PBMCs were also stimulated with subpools of pp65 and IE-1, each subpool containing 10 overlapping 15-mer peptides. PBMCs reactive to a subpool were then tested further against the individual peptides contained within the subpool to determine the reactive 15-mer(s). In order to further characterize epitopes presented to CD8+ T cells by HLA class I antigens, PBMCs from donors with reactive CD8+ cells were tested again with nanomer peptides spanning the reactive 15-mer peptide and overlapping at 8 amino acids (Figures 1 and 2). These studies were approved by a National Institutes of Health Institutional Review Board on the use of Human Subjects in Research.


CMV pp65 and IE-1 T cell epitopes recognized by healthy subjects.

Slezak SL, Bettinotti M, Selleri S, Adams S, Marincola FM, Stroncek DF - J Transl Med (2007)

Identification of CMV pp65 epitopes using 15-mer and nanomer peptides. PBMCs were stimulated with a library of 138 15-mer peptides overlapping by 11 residues and covering the entire pp65 protein. IFN-γ flow cytometry was used to detect T cell responses. To identify specific epitopes, PBMCs were stimulated with subpools containing 10 overlapping 15-mer peptides. PBMCs from donors reactive with CD4+ T cells were tested further with individual 15-mers. The results of analysis of CD4+ T cell responses in donor 14 to pp65 peptides are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851947&req=5

Figure 1: Identification of CMV pp65 epitopes using 15-mer and nanomer peptides. PBMCs were stimulated with a library of 138 15-mer peptides overlapping by 11 residues and covering the entire pp65 protein. IFN-γ flow cytometry was used to detect T cell responses. To identify specific epitopes, PBMCs were stimulated with subpools containing 10 overlapping 15-mer peptides. PBMCs from donors reactive with CD4+ T cells were tested further with individual 15-mers. The results of analysis of CD4+ T cell responses in donor 14 to pp65 peptides are shown.
Mentions: In this study, 20 CMV-seropositive and 5 CMV-seronegative healthy donors were evaluated. To determine the CD8+ and CD4+ T cell responses to CMV in the 25 donors, peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with a cocktail of 15-mer peptides overlapping by 11 residues and covering the pp65 and IE-1 proteins. Antigen-specific cytokine flow cytometry was used to detect specific T cell responses[15,16]. To define immunodominant epitopes, PBMCs were also stimulated with subpools of pp65 and IE-1, each subpool containing 10 overlapping 15-mer peptides. PBMCs reactive to a subpool were then tested further against the individual peptides contained within the subpool to determine the reactive 15-mer(s). In order to further characterize epitopes presented to CD8+ T cells by HLA class I antigens, PBMCs from donors with reactive CD8+ cells were tested again with nanomer peptides spanning the reactive 15-mer peptide and overlapping at 8 amino acids (Figures 1 and 2). These studies were approved by a National Institutes of Health Institutional Review Board on the use of Human Subjects in Research.

Bottom Line: Several new IE-1 HLA class I epitopes were identified, including 4 restricted to HLA-C antigens.One region of IE-1 spanning amino acids 300 to 327 was rich in class I epitopes.The HLA class I restrictions of IE-1 peptides were more promiscuous than those of pp65 peptides.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Transfusion Medicine, Warren G Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland, USA. sslezak@cc.nih.gov

ABSTRACT

Background: Adoptive immune and vaccine therapies have been used to prevent cytomegalovirus (CMV) disease in recipients of hematopoietic progenitor cell transplants, but the nature of T cell responses to CMV have not been completely characterized.

Methods: Peptide pools and individual peptides derived from the immune-dominant CMV proteins pp65 and IE-1 and antigen-specific, cytokine flow cytometry were used to characterize the prevalence and frequency of CD4+ and CD8+ memory T cells in 20 healthy CMV-seropositive subjects.

Results: CD8+ T cell responses to pp65 were detected in 35% of subjects and to IE-1 in 40% of subjects. CD4+ T cell responses to pp65 were detected in 50% of subjects, but none were detected to IE-1. Several new IE-1 HLA class I epitopes were identified, including 4 restricted to HLA-C antigens. One region of IE-1 spanning amino acids 300 to 327 was rich in class I epitopes. The HLA class I restrictions of IE-1 peptides were more promiscuous than those of pp65 peptides.

Conclusion: Since naturally occurring CD4+ and CD8+ T cell responses to pp65 were detectable in many subjects, but only CD8+ T cell responses to IE-1 were detected, pp65 may be better than IE-1 for use in vaccine and adoptive immune therapies.

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