Journal: Cancer immunology, immunotherapy : CII

Article Title: Identification of the Murine H-2D b and Human HLA-A*0201 MHC Class I Restricted HPV6 E7-Specific Cytotoxic T Lymphocyte Epitopes

doi: 10.1007/s00262-016-1793-x

Figure Lengend Snippet: 5–8 week old C57BL/6 mice (5 mice group) were vaccinated with either 2μg of pcDNA3-HPV6b E7 or 2μg of pcDNA3-HPV6b CRT/E7 DNA via intradermal delivery (gene gun) and were boosted twice with the same regimen at 7-day intervals. One week after the last vaccination, splenocytes were stimulated with the indicated HPV6b E7 overlapping peptide (1μg/ml) in the presence of GolgiPlug overnight at 37°C. Splenocytes stimulated with PMA and ionomycin in the presence of GolgiPlug for 3 hours were used as a positive control. The splenocytes were then analyzed by flow cytometry for the presence of HPV6b E7 peptide-specific CD8+ T cells by staining for cell surface CD8 and intracellular IFN-γ. a. Summary of the frequency of HPV6b E7 peptide-specific CD8+ T cells elicited after pcDNA3-HPV6b E7 DNA vaccination. b. Summary of the frequency of HPV6b E7 peptide-specific CD8+ T cells elicited after pcDNA3-HPV6b CRT/E7 DNA vaccination.

Article Snippet: Seven HPV6b E7 overlapping peptides (18–30 amino acids in length and overlapping by 10 amino acids) that span the full length of E7 protein, and HPV6b E7aa21-29, E7aa28-37, E7aa31-40 and E7aa82-90 peptides were synthesized by GenScript (Piscataway, NJ).

Techniques: Positive Control, Flow Cytometry, Staining

Journal: Cancer immunology, immunotherapy : CII

Article Title: Identification of the Murine H-2D b and Human HLA-A*0201 MHC Class I Restricted HPV6 E7-Specific Cytotoxic T Lymphocyte Epitopes

doi: 10.1007/s00262-016-1793-x

Figure Lengend Snippet: a. Representative flow cytometry of IFN-γ secretion by CD8+ T cells after HPV6b E7peptide incubation, as indicated. Splenocytes of pcDNA3-HPV6b CRT/E7 DNA vaccinated C57BL/6 mice (5 mice/group) were stimulated with either HPV6b E7aa21-50, or HPV6b E7aa21-29, E7aa28-37, or E7aa31-40 peptide (1μg/ml) in the presence of GolgiPlug overnight at 37°C. The HPV6b E7 peptide-specific CD8+ T cells were then analyzed by staining for cell surface CD8 and intracellular IFN-γ. b. Determination of MHC-restriction of HPV6b E7 peptide recognized by CD8+ T cells after HPV6b CRT/E7 DNA vaccination. HPV6b E7aa21-29 peptide-specific CD8+ T cells were stimulated with E7aa21-29 peptide pulsed 293 cells, or 293 cells expressing either the H-2Db or H-2Kb molecules in the presence of GolgiPlug overnight at 37°C. The activation of CD8+ T cells was then analyzed by staining for cell surface CD8 and intracellular IFN-γ, and analyzed by flow cytometry. The number in the upper right quadrant represents the percent of HPV6b E7 peptide-specific CD8+ T cells out of the total CD8+ T cells.

Article Snippet: Seven HPV6b E7 overlapping peptides (18–30 amino acids in length and overlapping by 10 amino acids) that span the full length of E7 protein, and HPV6b E7aa21-29, E7aa28-37, E7aa31-40 and E7aa82-90 peptides were synthesized by GenScript (Piscataway, NJ).

Techniques: Flow Cytometry, Incubation, Staining, Expressing, Activation Assay

Journal: Cancer immunology, immunotherapy : CII

Article Title: Identification of the Murine H-2D b and Human HLA-A*0201 MHC Class I Restricted HPV6 E7-Specific Cytotoxic T Lymphocyte Epitopes

doi: 10.1007/s00262-016-1793-x

Figure Lengend Snippet: 5–8 week old HLA-A2 transgenic mice with a C57BL/6 background (HLA-A2/Dd mice, 5 mice/group) were vaccinated with 2μg of pcDNA3-HPV6b CRT/E7 DNA via intradermal delivery (gene gun), and were boosted twice with the same regimen at 7-day intervals. One week after the last vaccination, splenocytes were prepared and stimulated with the indicated HPV6b E7 overlapping peptide (1μg/ml) in the presence of GolgiPlug overnight at 37°C. The HPV6b E7 peptide-specific CD8+ T cells were then analyzed with flow cytometry by staining for cell surface CD8 and intracellular IFN-γ. a. Representative flow cytometry data. b. Summary of the flow cytometry data. The number in the upper right quadrant represents the percent of HPV6b E7 peptide-specific CD8+ T cells out of the total CD8+ T cells isolated from splenocytes.

Article Snippet: Seven HPV6b E7 overlapping peptides (18–30 amino acids in length and overlapping by 10 amino acids) that span the full length of E7 protein, and HPV6b E7aa21-29, E7aa28-37, E7aa31-40 and E7aa82-90 peptides were synthesized by GenScript (Piscataway, NJ).

Techniques: Transgenic Assay, Flow Cytometry, Staining, Isolation

Journal: Cancer immunology, immunotherapy : CII

Article Title: Identification of the Murine H-2D b and Human HLA-A*0201 MHC Class I Restricted HPV6 E7-Specific Cytotoxic T Lymphocyte Epitopes

doi: 10.1007/s00262-016-1793-x

Figure Lengend Snippet: a. 5–8 week old female HLA-A2/Dd mice (3 mice/group) were vaccinated subcutaneously with HPV6b E7aa69-90 peptide formulated with the histidine-rich amphipathic peptide LAH4 and CpG 1826, and boosted twice at 1-week intervals. One week after the last vaccination, splenocytes were isolated and stimulated with HPV6b E7aa82-90 peptide (1 μg/ml) in the presence of GolgiPlug overnight at 37°C. The HPV6b E7 peptide-specific CD8+ T cells were then analyzed by staining for cell surface CD8 and intracellular IFN-γ, followed by flow cytometry analysis. b. Establishment of the HPV6b E7aa82-90 peptide-specific CD8+ T cell line. The above splenocytes were stimulated with HPV6b E7aa82-90 peptide pulsed, irradiated TC-1/HLA-A2/Dd cells to establish the T cell line. The specificity of the T cell line was confirmed by intracellular IFN-γ staining. c. HPV6b E7aa82-90 peptide recognition by CD8+ T cell is HLA-A*0201 restricted. HPV6b E7aa82-90 peptide-specific CD8+ T cells were co-incubated with either HPV6b E7aa82-90 peptide pulsed TC-1 cells, or TC-1/AAD cells at the indicated E:T ratio for 5 hours. The killing of TC-1 cells or TC-1/AAD cells was then measured by active-caspase-3 staining.

Article Snippet: Seven HPV6b E7 overlapping peptides (18–30 amino acids in length and overlapping by 10 amino acids) that span the full length of E7 protein, and HPV6b E7aa21-29, E7aa28-37, E7aa31-40 and E7aa82-90 peptides were synthesized by GenScript (Piscataway, NJ).

Techniques: Isolation, Staining, Flow Cytometry, Irradiation, Incubation

Journal: Cancer immunology, immunotherapy : CII

Article Title: Identification of the Murine H-2D b and Human HLA-A*0201 MHC Class I Restricted HPV6 E7-Specific Cytotoxic T Lymphocyte Epitopes

doi: 10.1007/s00262-016-1793-x

Figure Lengend Snippet: a. 5–8 week old female HLA-A2/Dd mice (3 mice/group) were vaccinated subcutaneously with HPV6b E7aa69-90 peptide formulated with LAH4 and CpG, or with LAH4 and CpG only, boosted twice with the same regimen at 1-week intervals. Splenocytes from naïve C57BL/6 mice were divided into two populations. The first group was labeled with 5 μM CFSE (CFSEhi) and pulsed with 2 μg/ml of E7aa82-90 peptide. The other population was labeled with 0.05 μM CFSE (CFSElo). The two populations were then mixed at the ratio of 1:1. 3×107 of these cells were injected into either HPV6b E7aa69-90 peptide vaccinated or control mice intravenously. 18 hours later, peripheral blood cells were collected for the analysis of specific cytotoxic activity by flow cytometry. Specific cytotoxic activity was expressed as the ratio between CFSElo over CFSEhi populations. b. Summary of specific in vivo cytotoxic activity.

Article Snippet: Seven HPV6b E7 overlapping peptides (18–30 amino acids in length and overlapping by 10 amino acids) that span the full length of E7 protein, and HPV6b E7aa21-29, E7aa28-37, E7aa31-40 and E7aa82-90 peptides were synthesized by GenScript (Piscataway, NJ).

Techniques: Labeling, Injection, Control, Activity Assay, Flow Cytometry, In Vivo