intact (Belperron et al., 2005, 2007). MZ B cells are retained within the MZ by integrin adhesion and are released into circulation by administration of anti­ integrin mAbs (Fig. S2, A and B; Lu and Cyster, 2002). Consistent with the splenec­ tomy data, depletion of MZ B cells led to a large reduction in the NP­ specific IgM response to NP­ Ficoll ± poly(I:C) at 4 d after immunization as compared with controls (Fig. 3 D). Although a similar reduction in NP­ specific IgM was not ob­ served at day 7 after immunization, this time point coincided with the return of MZ B cells to the spleen after integrin treatment and MZ B cell depletion (Fig. S2). In contrast, mice depleted of MZ B cells generated equivalent poly(I:C)­ elicited NP­specific IgG2c responses at all time points evalu ­ ated (Fig. 3 D). Although these results do not formally eliminate a role for MZ B cells in the poly(I:C)­induced IgG2c response, they illustrate that MZ B cells do not make a significant contribution to this response. It remained possible that MZ B cells relocated outside the spleen but were still responsive to poly(I:C) and able to gen­ erate NP­specific IgG2c from this extrasplenic site. How ­ ever, this was excluded as the frequency of splenic NP­specific IgG2c ASCs generated upon immunization with NP­Ficoll + poly(I:C) was the same regardless of MZ B cell depletion (Fig. 3 E). These findings strongly suggest that FO B cells are the predominant source of the poly(I:C)­ induced IgG2c antibody response to the TI­2 antigen NP­Ficoll. Poly(I:C) promotes FO B cells to participate in the IgG2c class-switched TI-2 antibody response Our findings indicated that FO B cells contributed signifi­ cantly to the poly(I:C)­induced NP­Ficoll response, although the capacity of lymph node B cells to contribute to the TI­ 2 antibody response has been considered minimal (Ron and Sprent, 1985; Goud et al., 1988; Liu et al., 1988). To directly examine if FO B cells mounted poly(I:C)­ induced IgG2c antibody responses to NP­Ficoll in vivo, we adoptively trans ­ ferred lymph node cells from IghB1­8/B1­8 (Igha) mice (Sonoda et al., 1997) into recipients and immediately assessed their ability to respond to NP­ Ficoll ± poly(I:C). The B cells within the lymph node were phenotypically homogenous as CD21int CD23high FO B cells and maintained this pheno­ type after adoptive transfer (Fig. 3 F). The B1­ 8 IgH chain con­ fers NP­specificity when paired with an Ig  light chain (Jack et al., 1977; Reth et al., 1978; Bothwell et al., 1981), effec­ tively increasing the NP­specific cells to 5% of the B cell population (unpublished data). Before immunization, the adoptively transferred FO B cells comprised 2.5% of the total splenic B cell compartment, and thus 0.1% of the B cells in the recipient were IgMa (B1­8) and NP specific (unpublished data). After immunization with NP­ Ficoll alone, adoptively transferred FO B cells generated a relatively weak and de­ layed NP­specific IgG2a response (Fig. 3 F). As predicted, 1490 Figure 4. The TI-2 IgG2c response induced by poly(I:C) is not mediated by B cell–intrinsic TLR3 signaling. (A) NP- specific IgG2c responses in wild type, Tlr3/, Mda5/, and Tlr3/ Mda5/ mice mea- sured from sera collected preimmunization (day 0) and at 7 d after NP-Ficoll + poly(I:C) immunization. Symbols represent individual mice, and horizontal bars indicate geometric mean. Shown is one representative of two independent experiments performed, with four to five mice per group. *, P < 0.05. (B) NP-specific IgG2c/a antibody responses in mixed bone marrow chimeras 7 d after immunization with NP-Ficoll + poly(I:C). A mixture of bone marrow cells from B6.C20 (Igha) and Tlr3/ (Ighb; experimental chime- ras; filled) or B6.C20 and wild-type C57BL/6 (Ighb; control chimeras; open) was adoptively transferred to lethally irradiated B6.C20 hosts. 8 wk after reconstitution, chimeras were im- munized and NP-specific antibody measured in preimmune and day-7 sera. Reconstitution frequencies of IgMa- and IgMb-expressing B cells were measured as described in the Materials and methods and found to be equivalent between both experimental and control chimeras. Symbols represent indi- vidual chimeric mice, and horizontal bars indicate geometric mean. Data were combined from two independent experiments with five to six mice per group. n.d., not detected. IFN- drives FO B cell TI-2 responses | Swanson et al.