Figure 4
From: Placenta-derived gp96 as a multivalent prophylactic cancer vaccine
Placenta-derived gp96 binds tumor-associated antigens.
(a) Relative tumor associated-gene expression analysis of placenta and liver tissues in BALB/c mice (n = 5) or rats (n = 5) by real-time-PCR. The mRNA levels in liver tissues were arbitrarily set as 1.0. (b) Female BALB/c mice (n = 5) were immunized with P-gp96, L-gp96, TFA-gp96 or PBS as a control for three times. Splenocytes were isolated and stimulated with the mouse Kd-restricted Her2 epitope E63 (TYLPTNASL) or control peptide HBcAg87-95 (SYVNTNMGL) for background evaluation and analyzed by IFN-γ ELISPOT assay. (c) Bulk peptides associated with P-gp96 or L-gp96 from BALB/c mice (n = 3) were analyzed by MALDI mass spectrometry. Representative spectra are shown. (d) Relative tumor-associated gene expression between human placenta (n = 5) and liver tissue samples (n = 5) by real-time PCR analysis. (e, f) PBMCs from HLA-A*0201-positive healthy donors (n = 5) were stimulated with P-gp96, TFA-gp96, or BSA for 3 days. Then, stimulated PBMCs were either incubated with the HLA-A*0201-restricted Muc1 epitope (STAPPVHNV) or Her2 epitope (ILHNGAYSL) or a control peptide HBcAg18-27 (FLPSDFFPSV) for background evaluation and assayed by IFN-γ ELISPOT (e) or analyzed for cytotoxic activity by FACS using CFSE-labeled MCF-7 cells as target cells (f). Error bars indicate s.d. * P < 0.05 and ** P < 0.01 (Two-tailed t-test or analysis of variance (ANOVA) and post-hoc comparison using Tukey's test).
