Figure 5.

Grp78-treated CD11c⁺ cells kept regulatory function ex vivo. NIT lysate-pulsed cells (5 × 10⁶) were injected into 6- to 8-week-old BALB/c mice every 7 days for three times. Mice were sacrificed 1 week after final injection, and PLNs, blood, and spleen were dissected. Lymphocyte subsets from PLNs (A) and blood (B) were analyzed by flow cytometry for percent values. Cells were gated for CD3⁺. Percentage of CD4⁺ in CD3⁺ cells (left), percentage of CD25⁺FoxP3⁺ in CD4⁺ cells (median), and representative flow cytometric dot plots (right) were depicted. (C) Splenocytes were analyzed by flow cytometry for percent and absolute values. Frequencies and representative flow cytometric dot plots of Treg (CD4⁺CD25⁺FoxP3⁺), Th1 (CD4⁺IFNγ⁺), Th2 (CD4⁺IL-4⁺), and Th17 (CD4⁺IL-17⁺) cells were also indicated. (D,E) Splenocytes were reprimed with mitomycin-treated NITs for 5 days (D) or 3 days (E). Then, cell proliferation was assayed by CFSE dilution (D) or ³H-TdR incorporation (E). (F) Splenocytes were reprimed by mitomycin-treated NITs for 7 days and then subjected to CFSE staining. After coculture with NITs for 12 h, cell apoptosis in CFSE⁺CD3⁺ T cells was assayed. All results were representative of three independent experiments. Error bars are presented in SEM. *P < 0.05, **P < 0.01.