FIGURE 4.

Targeting Ag to Clec9A enhances early B cell activation, which occurs independent of T cell help. To examine B cell activation, 1 × 10⁶ OB1^GFP+ cells were adoptively transferred into B6 or Clec9a^−/− mice 1 d before vaccination with 0.5 μg of αClec9A-OVA_FGD. (A) Activation of OB1 cells in the spleen (GFP⁺CD19⁺CD3⁻PI⁻) was assessed 24 h postimmunization based on the expression levels of CXCR5, CCR7, CD86, and MHC II. (B) geometric mean fluorescence intensity (gMFI) values plotted are normalized against cells from naive mice. The activation phenotype of endogenous B cells (GFP⁻CD19⁺CD3⁻PI⁻) is shown in Supplemental Fig. 2A and was assessed as in (A). (C) Early Clec9A-mediated B cell activation is independent of T cells. A total of 1 × 10⁶ CTV⁺ OB1 cells were adoptively transferred into B6 or MHC II^−/− mice 1 d before vaccination with 2 μg of αClec9A–OVA_FGD. Activation of OB1 cells (CTV⁺CD19⁺CD3⁻PI⁻) in the spleen was assessed based on the expression levels of CXCR5 and CCR7. gMFI values plotted are normalized against cells from naive mice. (D) Rapid activation of B cells is a general feature of Clec9A-targeted vaccination. Targeting the B cell hapten NP to Clec9A also enhances early activation to NP-specific B cells. A total of 1 × 10⁶ GFP⁺ or CTV-labeled B1-8^hi cells were adoptively transferred into B6 or Clec9a^−/− mice 1 d before vaccination with 0.1 μg of αClec9A-NP. Activation of B1-8^hi cells (GFP⁺/CTV⁺ IgLκ⁻ NP-binding CD19⁺CD3⁻PI⁻). Similar experiments were performed using alternative αClec9A mAbs labeled with NP (Supplemental Fig. 2B). Each symbol represents a mouse, and horizontal lines indicate the mean. (B–D) Pooled data from two experiments with n = 6–8 mice (B), two experiments with n = 2–5 mice (C), or three independent experiments with n = 8–12 mice (D). ns = p ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA, followed by Tukey test (B–D).