FIG. 6.

Knockdown of DC CTLA-4 enhances the T_H1 response and antitumor immunity. (A) Human DC were treated with CTLA-4 or NT siRNA for 72 h, matured, and cocultured at a ratio of 1:10 with syngeneic T cells with restimulation on days 9 and 24. T cells were sampled throughout the process by incubation in brefeldin A for 5 h and analysis by flow cytometry to determine CD4:CD8 ratio, CD8 activation (CD25 and intracellular IFN-γ), and quantitation of CD4⁺CD25⁺Foxp3⁺ tregs. Data shown are representative of eight independent experiments with eight biologically distinct products. (B) Relative CTLA-4 concentrations of various siRNA-treated mouse DC culture supernatants were characterized by western blot after which 1 × 10⁶ total splenocytes were cultured in these supernatants with supplemental IL-2 added on days 5, 7, and 9. The data indicated that the proliferation of CD8⁺CD25⁺ cells was dependent upon low levels of CTLA-4-supernatant content as well as proportional to the concentration of CTLA-4 in the supernatant. (C, D) Mouse BMDC were differentiated from mouse bone marrow cultured with GM-CSF and IL-4 for 6 days, treated with CTLA-4 or NT siRNA for 72 h, loaded with B16 mRNA, matured, and injected into the ipsilateral footpad of recipient C57BL/6 mice in which palpable B16 tumors had been preestablished 3 days prior. Mice were given booster vaccinations on day 14, and tumors were measured routinely for >3 weeks. Cohorts consisted of five mice each. *P < 0.05. (E) DC were polarized during in vitro maturation toward either T_H1 or T_H2, and culture supernatants were analyzed for the presence of DC-secreted CTLA-4 by western blot after 24 h. T_H1 = polarized with 1 ng/mL IL-12. T_H2 = polarized with 10 ng/mL (1×) or 100 ng/mL (10×) SEB. BMDC, bone marrow-derived dendritic cells; IM, immature DC.