Type I IFN mediates poly(I:C)-induced antigen-specific IgG2c during the TI-2 antibody response Both TLR3 and MDA5 dsRNA­ sensing receptors are capable of recognizing poly(I:C) to induce a rapid type I IFN response (Alexopoulou et al., 2001; Gitlin et al., 2006; Kato et al., 2006; Longhi et al., 2009). Given the accelerated IgG2c response to poly(I:C), we assessed the contribution of type I IFN to this response by immunizing Ifnar1/ mice. These results showed that the NP­ specific IgG2c response to poly(I:C) immunization was reduced 90% in Ifnar1/ mice compared with wild type (Fig. 5 A) and inversely mirrored by a modest but significant increase in the NP­ specific IgM response over wild­ type controls (Fig. 5 A). These findings demonstrate that type I IFN signaling is necessary for efficient induction of the TI­ 2 IgG2c antibody response promoted by poly(I:C). Type I IFN is an early hallmark of the antiviral innate im­ mune response initiated from the recognition of viral PAMPs. Thus, we predicted that the instruction to class switch to IgG2c during the TI­ 2 antibody response would be a general property of all PAMPs capable of stimulating endogenous type I IFN production. To test this, we immunized mice with a representative group of TLR agonists known to stim­ ulate type I IFN (TLR7 agonist, 3M­ 012; Gorden et al., 2005) or not (TLR1/2 agonist, Pam3CSK4; Longhi et al., 2009) and measured the IgG2c antibody response elicited to NP­ Ficoll. As predicted, mice immunized with a type I 1492 Figure 6. IFN- induces BCR-stimulated FO B cells to up-regulate CD69 to a greater extent than MZ B cells. (A) Histograms represent flow cytometric analysis of IFNR1 surface expression on CD21int CD23high FO B cells and CD21high CD23low MZ B cells isolated from naive wild-type mice. Isotype control antibody staining for both FO (shaded light gray) and MZ (dashed line) B cells is provided. Data are representative from two independent experiments. (B–D) FO and MZ B cells were sorted to ≥94% purity from naive C57BL/6 mice and separately incubated with the indicated stimuli in vitro for 6 or 10 h. After stimulation, surface expression of CD69 (6 h) or CD86 (10 h) was subsequently assessed in both B cell populations by flow cytometry and data depicted as histograms. The following stimuli and doses were used: (B) rIFN- at 1, 10, or 100 U/ml; (C) rIFN- at 1 U/ml and anti-IgM F(ab)2 at 10 µg/ml; (D) LPS at 10 µg/ml. Med, medium. Data are representative from at least two independent experiments. IFN­ inducing TLR7 agonist produced NP­ specific IgG2c antibody responses similar to that induced by poly(I:C) (Fig. 5 B). Conversely, the Pam3CSK4 TLR1/2 agonist did not promote NP­ specific IgG2c above levels seen with antigen alone (Fig. 5 B). These findings indicate that type I IFN­ inducing adjuvants promote class­ switching to IgG2c during the TI­ 2 antibody response. Fur­ thermore, these results are consistent with results showing that type I IFN augments particular iso­ types during the B cell antibody response to soluble protein antigens and viruses (Finkelman et al., 1991; Le Bon et al., 2001; Coro et al., 2006; Heer et al., 2007). We next considered whether we could bypass the re­ quirement for signaling through PRRs altogether by directly providing exogenous type I IFN during immunization. Mice immunized with NP­ Ficoll and a single administration of rIFN­  were found to mount significantly higher levels of NP­ specific IgG2c than NP­ Ficoll alone (Fig. 5 B). Although the magnitude of the IgG2c response elicited by rIFN­  im­ munization was reduced compared with poly(I:C) immuni­ zation, it was nevertheless notable that a single treatment induced a considerable IgG2c antibody response after 1 wk. Type I IFN receptor expression by B cells is required for the IgG2c TI-2 antibody response To address whether expression of the type I IFN receptor was required by B cells for the poly(I:C)­ induced IgG2c TI­ 2 response antibody response, Ifnar1/ B cells were evaluated in mixed bone marrow chimeras in direct competition with wild­ type B cells. Antibody responses by Ifnar1/ (Ighb) B cells or wild­ type B6 (Ighb) B cells were measured as NP­ specific IgMb and IgG2c, whereas competing wild­ type (B6.C20­ Igha) responses in both experimental and control chimeras were detected as NP­ specific IgMa and IgG2a (Fig. 5 C). Upon immunization with NP­ Ficoll + poly(I:C), wild­ type (B6­ Ighb) B cells in control chimeras mounted significant IFN- drives FO B cell TI-2 responses | Swanson et al.