A r t i c l e poly(I:C) markedly enhanced both the magnitude and kinet­ ics of the NP­ specific IgG2a response produced from FO B cells in vivo (Fig. 3 F) and similar to that elicited by poly(I:C) in wild­ type mice (Fig. 1 A). Similar findings were found when fivefold fewer (107) LN cells were transferred (unpub­ lished data). These findings directly demonstrate that FO B cells are able to generate IgG2c/a antibody responses to a TI­ 2 antigen and, more importantly, show that this response is both accelerated and amplified by poly(I:C). Collectively, these results reveal that FO B cells provide a rapid and robust innate­ like IgG2c antibody response to a TI­ 2 antigen elic­ ited by poly(I:C). Poly(I:C)-induced NP-specific IgG2c does not require TLR3 signaling by B cells As a dsRNA analogue, poly(I:C) is recognized by both TLR3 and MDA5 PRRs in vivo (Alexopoulou et al., 2001; Gitlin et al., 2006; Kato et al., 2006). To assess the in vivo contribu­ tion of these PRRs in the poly(I:C)­ induced IgG2c antibody response, Tlr3/ and Mda5/ single and compound mutants were immunized with NP­ Ficoll + poly(I:C). These results re­ vealed modest but significant reductions in NP­ specific IgG2c in the absence of either TLR3 or MDA5 that were additive in Tlr3/ Mda5/ double mutants (Fig. 4 A). The combined reduction observed in the double­ deficient mice implicates both TLR3 and MDA5 in the recognition of poly(I:C) and sub­ sequent induction of a class­ switched IgG2c B cell response. The requirement for direct TLR signaling by B cells dur­ ing antibody responses has been the subject of recent debate (Pasare and Medzhitov, 2005; Gavin et al., 2006; Barr et al., 2007, 2009; Jegerlehner et al., 2007; Meyer­ Bahlburg et al., 2007). Thus, we next questioned whether B cell–autonomous TLR3 signaling was required for the poly(I:C)­ induced NP­ Ficoll IgG2c response. To address this, we generated mixed bone marrow chimeras whereby wild­ type (B6.C20­ Igha) and either Tlr3/ or Tlr3+/+ (Ighb) B cells were juxtaposed in direct competition with each other in recipient mice, and we measured the capacity of both populations to generate an IgG2a/c antibody response to NP­ Ficoll + poly(I:C) (Fig. 4 B). In these chimeras, TLR3/ (Ighb) B cells mounted equiv­ alent NP­ specific IgG2c responses upon immunization with NP­ Ficoll + poly(I:C) as compared with wild­ type (C57BL/ 6­ Ighb) B cells in control chimeras (Fig. 4 B). These results demonstrate that direct TLR3 signaling by B cells is not re­ quired for poly(I:C)­ induced TI­ 2 IgG2c responses. Further­ more, these findings suggest that downstream secondary innate cytokine signals induced by PRR signaling may suffice in promoting B cell IgG2c antibody responses. JEM VOL. 207, July 5, 2010 Figure 5. Poly(I:C) induction of IgG2c requires type I IFN receptor signaling by B cells. (A) NP-specific IgM and IgG2c responses in wild-type and Ifnar1/ mice measured from sera collected at days 0 and 7 after immuniza- tion with NP-Ficoll + poly(I:C). Symbols represent individual mice, and bars represent geometric means. Data were combined from two indepen- dent experiments with three to five mice per group per experiment. *, P ≤ 0.005. (B) C57BL/6 mice were immunized with 5 µg NP-Ficoll alone or in the presence of 100 µg Pam3CSK4 (TLR1/2), 100 µg poly(I:C) (TLR3), 100 µg 3M-012 (TLR7), or 8 × 105 U rIFN-11. NP-specific IgG2c re- sponses were measured from sera collected preimmune (day 0) and at 7 d after immuniza- tion. Symbols represent individual mice, and horizontal bars indicate geometric mean. Shown is one representative of two independent experi- ments performed, with four mice per group. (C) Mixed bone marrow chimeras were generated as described in Fig. 2 B, except that experimental chimeras were generated from the transfer of Ifnar1/ (Ighb) + B6.C20 (Igha) bone marrow. After reconstitution, chimeric mice were immu- nized with NP-Ficoll + poly(I:C) and NP-specific IgMa, IgMb, IgG2a, and IgG2c serum antibody responses measured preimmune (day 0) and at 7 d after immunization. Symbols represent indi- vidual mice, and horizontal bars indicate geo- metric mean. Representative data are from one experiment shown of two independent experi- ments performed, with four mice per group. n.d., not detected. *, P < 0.05. 1491