Figure 1.

The IgE response is broadly suppressed in vivo under multiple adjuvant conditions. (A) Representative flow cytometry of GC B cells and PCs from the draining LNs from a mouse immunized with NP-CGG in alum adjuvant (left) versus splenocytes cultured for 4 d after in vitro activation with anti-CD40 and IL-4 (right). (B) Representative flow cytometry to show the gating strategy used to identify NP-specific PCs and enumerate the relative frequencies of PCs expressing each isotype from the draining LNs of a mouse immunized with NP-CGG in alum adjuvant. (C) Cell number and frequency of IgE⁺, IgG1⁺, or IgG2c⁺ GC B cells and NP-specific PCs from the draining LNs of mice immunized with NP-CGG with the indicated adjuvants or no adjuvant (Ctrl). WT mice (B6/J from our colony [A] or purchased NCI B6 CD45.1 congenics [B and C]) were analyzed 7 d after subcutaneous immunization. Cells cultured in A were from a B6/J mouse bred in our colony. GC B cells were gated as B220^hi CD138⁻ PNA^hi CD38^lo IgD⁻ cells, and PCs were gated as B220^lo CD138⁺ CD38^lo IgD⁻ cells, as shown previously (Yang et al., 2012). NP-specific PCs were identified by total (intracellular and surface) staining with NP-APC. Isotype-specific cells were identified by total (IgG1 and IgG2c) or intracellular (IgE) staining (see Yang et al., 2012 and Materials and methods). IgM⁺ cells were excluded from the analysis in B and C by total IgM staining. Dots represent individual mice. Xs represent cell counts that were below the limit of detection. Bars represent the mean (frequency plots) or geometric mean (cell number plots). n.s., not significant; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA with Dunnett’s post-test comparing each group to the leftmost control group). Data in A and B are representative of >10 and 5 experiments, respectively. Similar IgE and IgG1 responses as in C were observed in a separate experiment when mice were immunized with NP-KLH in alum, CFA, and SAS (data not shown). PNA, peanut agglutinin.