Figure 3

Vaccination with scFv^NLDC-145-HER2 induced HER2-specific cellular immune response. BALB/c mice were vaccinated with HER2, scFv^NLDC-145-neu or scFv^NLDC-145-HER2. Control animals received PBS or pcDNA3.1. Spleens and peripheral blood were harvested from the vaccinated animals after two vaccinations. A splenocytes isolated from the vaccinated animals were cultured in the presence of 10 μg/mL recombinant HER2 or TRP2 protein for 4 d with the addition of [³H] thymidine in the last 16 h. T-cell proliferation was determined by [³H] thymidine incorporation (left panel). Right panel, the supernatant recovered from the assay in left was tested for cytokine production by ELISA. B intracellular staining for IFN-γ and TNF-α in splenocytes from the vaccinated animals stimulated with 10 μg/mL recombinant HER2 or TRP2 protein for 24 h and brefeldin A added during the final 4 h of incubation. One representative dot plots from scFv^NLDC-145-HER2-vaccinated animal. C percentage of CD4⁺TNF-α⁺, CD4⁺IFN-γ⁺, CD8⁺ TNF-α⁺ and CD8⁺ IFN-γ⁺ cells in total CD4⁺ and CD 8⁺ T cells from each group are shown. The data are mean percentages ± SE. D splenocytes were cocultured with D2F2/E2 cells for 5 d. The resultant splenocytes (E) were cocultured for 4 h with the ⁵¹Cr-labeled target cells (T) (left panel). Right panel, the restimulated splenocytes from scFv^NLDC-145-HER2 vaccinated mice were also cocultured for 4 h with the ⁵¹Cr-labeled D2F2 or EL4/E2 (different gene background control) target cells. Percentages of target cells killing by the splenocytes from the vaccinated mice are shown. Data represent the means of triplicate cultures and are representative of two independent experiments. Bars, SE. *, P < 0.01, **, P < 0.001, scFv^NLDC-145-HER2 compared with other groups.