Analysis of the MHC-II monoclonal antibody HB12-18. (A and B) Anti-MHC-II (M5/114, 25 μg/g body weight) or control IgG were injected i.p. into WT mice on days 0, 4, 7, 10, 13, and 16 after B16F10 inoculation on day 0 into the flank of mice (n = 5 per group). (A) Tumor growth curve. (B) Frequencies of myeloid DC (CD11b+CD11c+) in the peripheral blood on day 15. (C) Splenocytes from C57BL/6J, H2-Aacit/cit, or BALB/cJ mice were incubated with different concentrations of HB12-18 (n = 3 per group). MHC-II expression (HB12-18 reactivity) on CD19+ B cells was determined by flow cytometry. MFI, mean fluorescence intensity. (D) WT (CD45.1) mice were injected i.v. with CellTrace Violet (CTV)-labeled OT-I CD8 T cells (CD45.2) and CellTrace Far Red (CTF)-labeled OT-II CD4 T cells (CD45.2), and 1 day later, recipients were mock injected (left panel), or injected i.p. with OVA+IgG (middle panel) or OVA+HB12-18 (200 μg/mouse, right panel) (n = 3 per group). Representative flow cytometry plots of CTV-positive OT-I CD8 T cells (CD45.2) and CTF-positive OT-II CD4 T cells (CD45.2) in the spleens of WT recipients (CD45.1) 4 days after immunization. Data points represent individual mice (B). Data are representative of two independent experiments (A–D). Error bars indicate SD (B and C) or SEM (A). P values were determined by Student’s t test (B) or two-way ANOVA with post-hoc Tukey test (A); no difference between treatments was found in A. ****P < 0.0001.