Figure 6 (See previous page).

Dab2^KD DC vaccine was more effective than WT DC vaccine in the induction of antitumor immunity in tumor immunotherapy. (A) CD8⁺ T cells proliferation in vivo by WT or Dab2^KD BMDCs was assessed by tetramer assay as described in materials and methods. Naive C57BL/6 mice were transferred i.v with OT-1 T cells together with OVA-pulsed Dab2^KD or WT mDCs, and then boosted with OVA_257–264 peptide on day 7. Three day later, splenocytes from the mice were stained with PE-labeled H-2K^b/OVA_257–264 tetramer and FITC-anti-CD8 antibodies, and subsequently analyzed by flow cytometry. Shown is representative FACS data (upper) and the number of tetramer(Tet)⁺CD8⁺ T cells is represented as mean± SD of six samples from two mice (lower). *p < 0.05, Student t-test. (B) The splenocytes prepared for tetramer assay in (A) were activated with phorbol myristate acetate (PMA)/ionomycin (40 ng/mL each, Sigma–Aldrich) for 4 h. Cells were then examined by flow cytometry after surface and intracellular staining with FITC-anti-CD8 antibody and PerCP/Cy-anti-IFNγ antibody, respectively. Shown is representative FACS data (upper), and the number of IFNγ+CD8+ T cells is represented as mean ± SD of six samples from two mice (lower). ***p < 0.001, Student t-test. (C) OT-1 mice were immunized twice at a 1-week interval with 1 × 10⁶ OVA peptide (OVA_257–264)-pulsed Dab2^KD or WT mBMDCs (C57BL/6). Cells from the spleen and DLN of vaccinated mice were harvested and cultured for 5–7 d in the presence of OVA_257–264 peptide. The level of IFNγ in the culture supernatants on day 2 of culture was assessed by ELISA, and the data are shown as mean ± SD of six simples from two independent experiments. *p < 0.05, Student t-test. (D) CTL activity in the DLNs of vaccinated OT-1 mice was assessed by flow cytometry using CFSE-labeled E.G7 and EL4 as target cells. Quantitative CTL activities are shown as mean ± SD (n = 3). *p < 0.05, Student t-test. (E) C57BL/6 mice were inoculated s.c. with E.G7 and EL4 tumor cells (5 × 10⁵) in the right flank and immunized twice on day 3 and day 10 with 1 × 10⁶ Dab2^KD (si-Dab2) or WT (si-con) mDCs (from C57BL/6 BM cells) that were pulsed with OVA peptide. Tumor growth was monitored and represented as mean ± SD of four mice from each of two experiments (bottom). *p < 0.05, Student t-test.