A r t i c l e Figure 3. Poly(I:C) elicits recruitment of FO B cells to participate in the enhanced IgG2c TI-2 antibody response. (A) Fre- quency of splenic NP-specific IgM and IgG2c ASCs as measured by ELISPOT 7 d after immu- nization with either NP-Ficoll alone or NP- Ficoll + poly(I:C). Each symbol represents an individual mouse and horizontal bars indicate either mean (IgM) or geometric mean (IgG2c). Data was combined from four independent experiments with three to five mice per group per experiment. *, P < 0.0001. (B) ELISPOT of NP-specific IgG2c ASCs in lymph nodes from C57BL/6 mice at 7 d after immunization with either NP-Ficoll alone or NP-Ficoll + poly(I:C). Lymph nodes were pooled from three immu- nized mice. Data was from two representative samples shown for each immunization condi- tion. Data was from one experiment of two independent experiments performed. (C) Wild- type splenectomized or sham-operated mice were immunized with NP-Ficoll alone or NP- Ficoll + poly(I:C) and serum NP-specific IgM and IgG2c antibody measured at days 0, 4, and 7 after immunization. Data are expressed as the geometric mean + SEM. Results were combined from two independent experiments with four to six mice per group per experi- ment. *, P < 0.05. (D) C57BL/6 mice were treated with 100 µg each of anti-4 + anti- L antibodies or isotype control antibodies. 7 d after antibody injection, mice were immu- nized with either NP-Ficoll alone or NP-Ficoll + poly(I:C) and serum NP-specific IgM and IgG2c antibody responses measured at the indicated times. Data are expressed as the geometric mean + SEM. Results are from one experiment with four to five mice per group. *, P < 0.05. (E) Frequency of NP-specific IgG2c splenic ASCs measured from antibody-treated wild-type mice. Symbols are the same as those described in D and represent the fre- quency measured from individual animals. Horizontal bars indicate geometric mean. Results are from one experiment with four mice per group. (F) Representative flow cyto- metric analysis of CD21 and CD23 expression in spleen and lymph node of BL6-IghB1-8/B1-8 (Igha) mice. Total lymph node cells (5 × 107) were transferred to C57BL/6 (Ighb) mice and immunized 1 d later. The presence and pheno- type of transferred Igha+ lymph node B cells within a splenic FO B cell gate (CD21int CD23high) and MZ B cell gate (CD21high CD23low) in one representative recipient 1 d after transfer is shown. Recipi- ent mice were immunized with either NP-Ficoll alone or NP-Ficoll + poly(I:C) and NP-specific IgG2a antibodies measured in sera at the indicated time points. Antibody concentrations are shown as relative titers and data are expressed as the geometric mean ± SEM. Results shown from one representative experiment of two independent experiments performed, with four mice per group. n.d., not detected. *, P ≤ 0.0003. et al., 1988; Vinuesa et al., 2003). Collectively, our findings suggest that in the presence of poly(I:C), a B cell population distinct from MZ B cells is the predominant source of NP­ specific IgG2c. To provide further support for this notion, we used a second model in which the MZ B cell population was selec­ tively depleted by in vivo treatment with anti­ integrin (4/L) antibodies (Lu and Cyster, 2002) while FO B cells were left JEM VOL. 207, July 5, 2010 1489