loss of S1P1 responsiveness would also be expected to direct TI­2 antigen­activated B cells to the T–B cell border where associated TD antigens could be presented to antigen­specific T cells. Protective humoral immunity to pathogens is contributed by distinct B cell subsets with unique activation requirements and response patterns. Studies from dissected model patho­ gens, such as S. pneumoniae and influenza, have taught us that multifaceted protective humoral immune responses are com­ prised by a collection of antibodies derived from distinct B cell subsets (Baumgarth et al., 1999, 2008; Snapper, 2006). Features that dictate which B cell subset responds have been characterized, including the intrinsic responsiveness of each B cell subset and the biochemical properties of the antigens. Yet, it is still not understood how innate signals encountered during these infections dictate which B cell subset participates in each facet of the antibody response. The TI­2 response to NP­Ficoll has been characterized by the domination of IgM and IgG3 isotypes derived from MZ B cells (Otipoby et al., 1996; Guinamard et al., 2000; Samardzic et al., 2002; Shih et al., 2002). We demonstrate in this paper that in the pres­ ence of type I IFN innate signals, FO B cells are rapidly pro­ moted to participate in a switched TI­2 antibody response through B cell–intrinsic IFN­ / signaling that leads to antigen­specific IgG2c antibody production. Whether type I IFN recruits additional FO B cells to respond to a TI­2 anti ­ gen or promotes enhanced clonal expansion of responding antigen­specific FO B cells remains to be established. Rapid low­ affinity extrafollicular innate­ like antibody responses are crucial to providing early acquired immunity against pathogens. This study provides the first direct evi­ dence for an innate­like switched FO B cell antibody re ­ sponse to a TI­2 antigen. The participation by FO B cells to this switched TI­2 response was unexpected given the long­ standing model that FO B cells are a slower responding B cell subset and predominately produce higher affinity post­ germinal center antibodies during TD responses (Martin and Kearney, 2002; McHeyzer­Williams, 2003). Economically, one rationale behind the enhanced contribution of FO B cells to providing the IFN­ /–induced IgG2c response, as opposed to the subset which produces the TI­2 IgM re ­ sponse, is that this division of labor allows for IgG2c induc­ tion but not at the expense of the crucial initial IgM response. Furthermore, this division of labor illustrates the importance of both arms of the anti­pathogen humoral response: a rapid IgM response to provide antibody capable of neutralizing and controlling the infection and a robust and highly efficient IgG2c response effective at specialized FcR­mediated clear ­ ance (Nimmerjahn and Ravetch, 2005, 2008). These findings propose a novel role for FO B cells in responding to innate signals in vivo to participate in innate­like antibody responses. Future studies will be required to address whether a discreet subset of innate FO B cells exist that solely have the capacity to respond in this manner or whether this phenomenon is a general characteristic of all FO B cells recruited in response to particular innate signals. JEM VOL. 207, July 5, 2010 A r t i c l e MATERIALS AND METHODS Mice. C57BL/6 mice (The Jackson Laboratory) were used directly or bred and maintained within the Biological Resource Center at National Jewish Health (NJH; Denver, CO). C57BL/6­ IghB1­8/B1­8 ;Igha mice (Sonoda et al., 1997; gift from K. Rajewsky, Harvard University, Boston, MA) and B6.C20 mice (C57BL/6 mice congenic for Igha; gift from L. Herzenberg, Stanford University, Palo Alto, CA) were both bred and maintained within the Bio­ logical Resource Center at NJH. Ifnar1/ mice (Müller et al., 1994) on the C57BL/6 genetic background were provided by P. Marrack (NJH, Denver, CO). MDA5/ (Gitlin et al., 2006), Tlr3/, and Mda5/, Tlr3/ mice on C57BL/6 genetic backgrounds were maintained at Washington University (St. Louis, MO). Tlr3/ mice (Alexopoulou et al., 2001) on the C57BL/6 background were provided by R. Flavell (Yale University, New Haven, CT) and also bred and housed within the NJH Biological Resource Center. Animals were used between 8 and 12 wk of age, unless otherwise specified. All experiments were performed in accordance with NJH and Washington University Institutional Animal Care and Use Committees. Flow cytometric analyses and antibodies. Spleen and lymph node cells were harvested and stained according to standard protocols. Fluorescently labeled mAbs against the following mouse surface antigens were used for staining and either purchased or isolated and conjugated in our laboratory: B220 (RA3­6B2; BD and eBioscience), IgM (R33­24.12; hybridoma), IgMb (AF6­78; hybridoma), CD21 (7G6; hybridoma), CD19 (1D3; hybrid ­ oma and BD), CD1d (1B1; eBioscience), CD69 (H1.2F3; eBioscience), CD86 (GL1; eBioscience), CD23 (B3B4; BD), TCR­  (H57­597; BD), IgHB1­8 (anti­idiotype; Ac146; hybridoma; Reth et al., 1979), IgM a (DS­1; BD), Ig (JC5­1; SouthernBiotech), IgD (11­26c; BD), CD11a (2D7; BD), IFNR1 (MAR1­5A3; BioLegend), and Mouse IgG1,  isotype control (MOPC­21; BD). For detection of NP­specific B cells by flow cytometry, cells were stained with 4­Hydroxy­3­nitrophenylacetyl hapten conjugated to Phycoerythrin (NP40–PE; Biosearch Technologies) after standard flow cytometric staining protocol. The secondary detection reagent for detecting biotin­conjugated antibodies by flow cytometry was streptavidin­PE (eBio ­ science). Flow cytometric analyses were performed by acquiring data on either a FACSCalibur (BD) or a BD LSRII (BD) and with FlowJo v8.8.6 software (Tree Star, Inc.). Immunizations. Mice were immunized i.p. with 5 µg NP (4­Hydroxy­3­ nitrophenylacetic) hapten conjugated to NP–Ficoll (Amino­Ethyl­Carboxy­ Methyl­FICOLL) at NP 32–, NP41–, or NP50–Ficoll valencies (Biosearch Technologies). Unless otherwise specified, the following doses of adjuvants or cytokines were included with NP­Ficoll in immunizations as detailed: 100 µg poly(I:C) (polyinosine­ polycytidylic acid; InvivoGen), 100 µg Pam3CSK4 (N­ Palmitoyl­ S­ [2,3­ bis(palmitoyloxy)­ (2, RS)­ propyl]­ [R]­ Cys­[ S]­Ser­[ S]­Lys4; InvivoGen), 100 µg 3M­012 (33080; TLR7 agonist; 3M Pharmaceuticals; gift from R. Kedl, NJH), or 8 × 105 U recombinant mouse IFN­ 11 (Genbank accession no. AY225954; gift from R. Kedl, NJH). Antigens, adjuvants, and cytokines were either purchased as endo­ toxin free (≤0.125 EU/mg) or tested by an endpoint chromogenic Limulus Amebocyte Lysate (LAL) assay (Cambrex) and found to be at levels ≤0.1 EU per mouse for immunization. NP-specific ELISAs. To measure NP­specific serum antibody responses, 96­well flat­bottom MaxiSorp MicroWell plates (Thermo Fisher Scientific) were coated overnight with 2 µg/ml (4­ Hydroxy­ 3­ iodo­ 5­ nitrophenylacetyl) NIP15–BSA (Biosearch Technologies) diluted in PBS at 4°C. Plates were washed once (PBS, 0.1% Tween 20; Thermo Fisher Scientific), blocked (PBS, 1% BSA, and 0.05% NaN3) for a minimum of 2 h at 37°C, and washed once again. For capture of antigen­specific antibodies, sera were initially di ­ luted 1:20 and subsequent threefold serial dilutions were made into blocking buffer and incubated overnight at 4°C. Plates were washed three times be­ fore incubation with an alkaline phosphatase (AP)–conjugated goat anti– mouse isotype­specific detection antibody (SouthernBiotech) for 1 h at 37°C. After three washes, plates were developed by the addition of alkaline 1495