NP­specific IgM b and IgG2c antibody responses (Fig. 5 C). As predicted from immunization of IFNR1/ mice (Fig. 5 A), Ifnar1/ (Ighb) B cells also produced an NP­ specific IgMb antibody response (Fig. 5 C). In contrast, Ifnar1/ (Ighb) B cells did not generate any significant NP­specific IgG2c antibody (Fig. 5 C). Moreover, wild­ type (B6.C20­ Igha) B cells in competition with IgMb­expressing B cells in both sets of chimeras made comparable NP­ specific IgMa and IgG2a antibody responses (Fig. 5 C), further eliminating a role for non–B cell hematopoietic populations and indicating a B cell–autonomous defect. This impaired ability of Ifnar1/ B cells to class switch to IgG2c was not a result of abnormal B cell development, as Ifnar1/ mice have normal composi­ tions of mature splenic B cell compartments (Le Bon et al., 2006; Green et al., 2009). These data show that Ifnar1/ B cells contribute normally to TI­ 2 IgM responses but that in the presence of type I IFN– inducing PAMPs such as poly(I:C), they are unable to generate antigen­ specific IgG2c TI­ 2 antibody response. This establishes an in vivo role for type I IFN receptor signaling by FO B cells in directly promoting the production of the antiviral IgG2c iso­ type and, thus, enhancing the B cell antibody response. IFN- acts selectively on BCR-activated FO B cells to increase CD69 expression FO and MZ B cells express similar levels of the type I IFN receptor (Fig. 6 A), excluding differential receptor expression as an origin for the dominant FO B cell response to type I IFN­ promoting adjuvants. Thus, we predicted that type I IFN differentially influenced FO B cells relative to MZ B cells. Type I IFN can directly activate B cells in vitro (Morikawa et al., 1987; Braun et al., 2002; Coro et al., 2006; Kamphuis et al., 2006; Shiow et al., 2006) and in vivo (Chang et al., 2007; Coro et al., 2006; Shiow et al., 2006). Thus, we assessed the ability of IFN­  to activate either FO or MZ B cells and found that both populations displayed similar sensi­ tivity and kinetics to recombinant IFN as measured by induc­ tion of surface expression of CD69 and CD86 activation antigens (Fig. 6 B and not depicted). We next questioned whether FO and MZ B cells might differ in their response to IFN­  stimulation in the context of BCR­mediated activation similar to the situation during an antibody response. Concomitant treatment of FO B cells with anti­IgM and suboptimal levels of IFN­  led to CD69 expression to levels greater than those detected with anti­ IgM stimulation alone (Fig. 6 C). In contrast, anti­ IgM treatment of purified MZ B cells failed to increase CD69 expression to any appreciable level and, importantly, this expression was not enhanced by concomitant IFN­  treat­ ment (Fig. 6 C). The inability of anti­IgM to induce CD69 expression on MZ B cells was not true of all activation markers as anti­IgM ± IFN­  increased CD86 expression similar to FO B cells (Fig. 6 C). Furthermore, MZ B cells exhibited increased sensitivity to LPS activation as mea­ sured by CD86 expression (Fig. 6 D) as previously shown (Oliver et al., 1999). JEM VOL. 207, July 5, 2010 A r t i c l e Overall, these data demonstrate that IFN­  selectively acts on BCR­ stimulated FO, but not MZ, B cells to elevate CD69 surface expression. Type I IFN­ mediated surface expression of CD69 is inversely correlated with S1P1 chemo­ attractant receptor responsiveness (Shiow et al., 2006), suggesting that selective CD69 expression by antigen­ experienced FO B cells could alter the trafficking of these cells during a response. DISCUSSION Study of B cell antibody response to hapten model antigens over the last several decades has provided considerable insight into how humoral immunity deals with different types of antigens. In particular, the reductionist nature of these model antigens facilitated our understanding of how different B cell subpopulations respond to distinct antigens. However, it is now evident that extrapolating from these studies is limited with respect to modeling humoral immunity to physiological pathogens. Pathogens such as viruses and bacteria not only present TI and TD antigens to B cells but also display a vari­ ety of PAMPs capable of alerting the innate immune system. This study addresses how these PAMPs influence humoral immunity to TI­2 antigens. The most dramatic changes elicited by poly(I:C) to the TI­2 antibody response were measured in the acceleration of the kinetics and overall increase in the magnitude of the antigen­specific IgG2c response. Heightened production of IgG2c is significant, as this isotype is the most efficient IgG subclass for anti­pathogen FcR­mediated effector functions (Nimmerjahn and Ravetch, 2005) and has long been recog­ nized as the predominant isotype elicited by viral infections (Coutelier et al., 1987). Thus, these findings support the no­ tion that PAMPs polarize antibody responses to those iso­ types best suited to deal with particular pathogens (Janeway, 1989). We further show that during the TI­2 antibody re ­ sponse, poly(I:C) promotes a persistence of elevated levels of antigen­specific IgG2c serum antibody, which are similar to those previously shown for TD antibody responses (Le Bon et al., 2001). The durability of this enhanced response has implications for vaccine design given the importance of neu­ tralizing antibody concentration in providing efficacious antiviral protective immunity (Bachmann and Zinkernagel, 1997; Bachmann et al., 1997; Pulendran, 2009). Generally, pathogen­specific antibody responses divert from characterized hapten­specific responses, as they are typ ­ ically faster and elicit IgG isotypes earlier than anti­hapten responses (Bachmann and Zinkernagel, 1997). We show that including a type I IFN­inducing PRR agonist with the NP­ Ficoll TI­2 antigen results in a rapid anti­hapten IgG2c re ­ sponse. This class­switched anti­hapten response more closely reflects previously characterized antiviral and antimicrobial antibody responses with regard to kinetics, isotype specificity, and extrafollicular antibody production, illustrating the ap­ plicability of these studies to model pathogens. For instance, among the many arms of the influenza antibody response, 1493