FIGURE 6.

Effects of immune education on NP-specific B cell populations. C57Bl/6 laboratory mice underwent immune education or treatment with isotype control antibody. Thirty-five days later were treated with intraperitoneal 4-hydroxy-3-nitrophenylacetic acid (NP), 5 μg suspended in PBS (T₀) or subjected to CLP and treated with NP (7 days CLP). Mice were euthanized 7 days later. Spleens were homogenized and B cell populations were analyzed using flow cytometry. Each point represents results in an individual animal, central horizontal line indicates mean, upright and inverted Ts indicate standard deviation, data representative of two independent experiments. Filled circles – T₀ in control mice; Filled square – T₀ in immune educated mice; Open circle – 24 h post-CLP in control mice; Open square – 24 h post-CLP in immune educated mice. Data analyzed using two-way ANOVA with Sidak’s post hoc correction for multiple comparisons. *p < 0.05, significantly different from value in control mice at same time point; ^#p < 0.05, slope of line connecting T₀ mean and mean 24 h post-CLP significantly different than slope of line for control mice. (A) Total splenic NP-specific B cells per spleen. Gating: FSC/SSC, singlets, Live, CD19⁺/B220⁺, CD19⁺/NP⁺; N = 5–9/group. (B) Total NP-specific follicular (Left) and marginal zone (Right) B cells per spleen. Gating: Follicular B cells: FSC/SSC, singlets, Live, CD19⁺/B220⁺, CD19⁺/NP⁺, CD93^–, B220⁺/CD138^–, IgM^lo/CD21/35^lo, Marginal Zone B cells FSC/SSC, singlets, Live, CD19⁺/B220⁺, CD19⁺/NP⁺, CD93^–, B220⁺/CD138^–, IgM^hi/CD21/35^hi; N = 5–6/group.