A r t i c l e Figure 1. Poly(I:C) enhances the anti- body response to a model TI-2 antigen. (A) C57BL/6 mice were immunized with 5 µg NP-Ficoll alone or 5 µg NP-Ficoll + 100 µg poly(I:C) and serum NP-specific IgM, IgG3, and IgG2c antibodies were measured in preimmune (day 0 [d0]), day 4, and day 7 after immuniza- tion sera. Symbols represent individual mice, and horizontal bars indicate geometric mean. Data were combined from two independent experiments with five mice per group per experiment. *, P ≤ 0.005. (B) Total serum NP- specific IgG2c, IgG3, IgG2b, IgG1, and IgM were measured 7 d after immunization with NP- Ficoll ± poly(I:C). Data represent geometric means calculated from data combined from two independent experiments, with five mice per group per experiment. Geometric means from NP-specific IgM, IgG3, and IgG2c responses were the same as those represented in A. nonspecific antibody production by polyclonally activated B cells (Fig. S1, B and C). Poly(I:C) alone did not induce either NP­ specific IgG2c or total antigen nonspecific IgG2c. These results were confirmed by measuring the total IgG2c levels after immunization with NP­ Ficoll ± poly(I:C) (Fig. S1 C). Although poly(I:C) was found to increase total IgG2c serum levels on day 7, this increase was fully accounted for by the increase in NP­ specific IgG2c at the same time point (Fig. 1 A). Together, these results negate a role for polyclonal IgG2c antibody production and verify that poly(I:C) enhances the TI­ 2 antibody response by an antigen­ specific mechanism. It is of note that IgG2c (IgG2a in Igha expressing mouse strains) is not only the predominant Ig isotype generated in antiviral antibody responses (Coutelier et al., 1987) but is also considered a highly effective antipathogen Ig isotype (Nimmerjahn and Ravetch, 2005, 2008). Thus, we restricted our subsequent focus to understanding the mechanism by which poly(I:C) altered the production of this IgG2c response. Poly(I:C) augments the long-lived IgG2c TI-2 antibody response NP­ Ficoll elicits relatively long­ lived serum NP­ specific antibody responses in the absence of adjuvants (García de Vinuesa et al., 1999; Obukhanych and Nussenzweig, 2006). We examined the adjuvant efficacy of poly(I:C) on this response and found that poly(I:C) significantly enhanced NP­ specific IgG2c antibody levels at all time points evaluated (Fig. 2 A). However, although the levels of long­ lived antigen­ specific antibody were considerably increased, this did not result from an increased longevity of the antigen­ specific IgG2c antibody response as the rate of decline of NP­ specific IgG2c antibodies was similar regardless of poly(I:C) (Fig. 2 A). Poly(I:C) facilitates an extrafollicular and low-affinity IgG2c antibody response to NP-Ficoll TI­ 2 antigens typically elicit a rapid extrafollicular antibody response with limited isotype class switching and without JEM VOL. 207, July 5, 2010 somatic hypermutation (MacLennan et al., 2003). Given the rapid nature of the IgG2c antibody response induced by poly(I:C) to NP­ Ficoll, we tested whether this would like­ wise be a low­ affinity extrafollicular response. The primary antibody response to NP­ Ficoll is dominated by Ig+ NP­ specific antibodies (Tesch et al., 1984), thus we used Ig expression as a surrogate for NP­ specific ASCs (Claassen et al., 1986; Jacob et al., 1991; García de Vinuesa et al., 1999; Shih et al., 2002). NP­ Ficoll alone elicited Ig+ IgM–expressing ASCs localized to extrafollicular foci in the bridging channel of the red pulp (Fig. 2 B) as previously re­ ported (Claassen et al., 1986; García de Vinuesa et al., 1999; Shih et al., 2002). NP­ Ficoll + poly(I:C) induced not only extrafollicular Ig+ IgM–expressing ASCs but also Ig+ IgG2c– expressing ASCs within extrafollicular foci. Additionally, Ig+ plasma cells that lacked IgM or IgG2c expression (Fig. 2 B) were also observed with poly(I:C) and are likely to be NP­ specific IgG3­ producing ASCs, as this isotype comprised a significant amount of the response (Fig. 1 B). The relative affinity of poly(I:C)­ induced NP­ specific IgG2c antibodies was also measured and found to be com­ prised of relatively low­ affinity antibodies, which is similar to that produced in response to NP­ Ficoll alone (Fig. 2 C). In addition, this relative affinity did not increase with time indi­ cating the absence of affinity maturation (Fig. 2 C). Thus, we show that poly(I:C) promotes a rapid IgG2c class­ switched TI­ 2 antibody response characterized by both extrafollicular antibody secretion and low­ affinity antibodies. Both splenic and lymph node B cells participate in the poly(I:C)- elicited IgG2c antibody response to NP-Ficoll Measuring the frequency of splenic NP­ specific IgG2c ASCs after immunization with poly(I:C) revealed a relative increase in the frequency of splenic NP­ specific B cells that differ­ entiated into IgG2c ASCs (Fig. 3 A). In accordance with anti­ body levels in serum (Fig. 1 A), poly(I:C) also promoted a 1487