Figure 2. Poly(I:C) promotes a long-lived, extrafollicular, and low-affinity IgG2c antibody response to NP-Ficoll. (A) C57BL/6 mice were immunized with either NP-Ficoll alone or with poly(I:C) and serum NP-specific IgG2c antibodies measured before and at the indicated days after immunization. Data are expressed as the geometric mean ± SEM. Results shown are from one representative experiment of two indepen- dent experiments performed. *, P ≤ 0.002. (B) Histological spleen sections from C57BL/6 mice 7 d after immunization with NP-Ficoll or NP-Ficoll + poly(I:C). Top panels show sections stained for Ig, IgM, and IgG2c. B cell follicles within the white pulp were revealed by IgM staining to provide orientation for the general lymphoid architecture. Top panels are 10× original magnification with enlarged areas within white outlined boxes represented in middle and bottom panels. Bottom panels depict only green Ig+ ASCs and white arrows identify IgG2c ASCs. Bars, 50 µm. Data are representative of two independent experiments. (C) Relative affinity of NP-specific IgG2c antibodies was determined as the binding ratio of NP4/ NP16 and was measured by ELISA. Sera samples were analyzed from mice at 7, 14, and 31 d after immunization with NP-Ficoll or NP-Ficoll + poly(I:C). Results were combined from two independent experiments with four to eight mice per immunization group. Data are expressed as the 1488 concomitant decline in the frequency of splenic NP­specific IgM ASCs. Furthermore, no significant differences were mea­ sured in the frequency of either NP­specific IgG2c or IgM ASCs in the bone marrow of mice regardless of poly(I:C) immunization (unpublished data). Given the accelerated kinetics of the IgG2c response in­ duced by poly(I:C), we initially considered that MZ B cells destined to produce IgM were subverted to class switch to IgG2c during this TI­2 response. However, antigen­specific IgG2c ASCs were also present in the lymph nodes of mice immunized with NP­Ficoll + poly(I:C) and were virtually absent in mice immunized with antigen alone (Fig. 3 B). As MZ B cells do not normally recirculate in rodents (Gray et al., 1982; Kumararatne et al., 1982), the presence of lymph node NP­specific IgG2c ASCs indicated that a non­MZ popula ­ tion contributed at least partially to the poly(I:C)­induced IgG2c response. Although the capacity of lymph node B cells to respond to TI­2 antigens has been shown (Ron and Sprent, 1985; Goud et al., 1988; Liu et al., 1988; Vinuesa et al., 2003), this response is limited with respect to other B cell subsets (Martin et al., 2001; Balázs et al., 2002). Thus, our data sug­ gest that innate signals augment the TI­2 antibody response in part via enhanced production of antigen­specific IgG2c by lymph node B cells. MZ B cells are not required for the IgG2c antibody responses to NP-Ficoll + poly(I:C) B1 and MZ B cells are considered the major contributors to the TI­2 antibody response (Fagarasan and Honjo, 2000; Martin et al., 2001). However, with regards to NP­Ficoll in particular, MZ B cells have been shown to be predominantly responsible for the TI­2 antibody response (Otipoby et al., 1996; Guinamard et al., 2000; Samardzic et al., 2002; Shih et al., 2002). Because MZ B cells are restricted to the spleen in rodents, we addressed whether MZ B cells contributed to the poly(I:C)­induced IgG2c response by splenectomy and immunization with NP­Ficoll ± poly(I:C). As previously re ­ ported (Amlot et al., 1985), splenectomized mice mounted significantly reduced NP­specific IgM responses at days 4 and 7 after immunization with NP­Ficoll compared with sham­ operated animals and irrespective of poly(I:C) (Fig. 3 C). Likewise, at day 7 the antigen­specific IgG2c response to NP­Ficoll alone was diminished approximately sevenfold in splenectomized mice (Fig. 3 C). However, in the presence of poly(I:C) there was only a twofold reduction in the NP­ specific IgG2c response measured in asplenic mice as compared with controls (Fig. 3 C). These data confirm MZ B cells are largely responsible for the NP­Ficoll IgM response and further show this is true in the presence of poly(I:C). Conversely, the small IgM and IgG2c antibody responses generated in asplenic mice highlight a minor role for FO B cells in the NP­Ficoll TI­2 response (Ron and Sprent, 1985; Goud et al., 1988; Liu arithmetic mean ± SEM. The NP-specific IgG2c mAb S43-10 was used as a positive control for a high-affinity binding antibody. IFN- drives FO B cell TI-2 responses | Swanson et al.