Pik3cd GOF B cells show failed induction of anergy. (A) Splenocytes from BM chimeras were assessed for expression of IgM on transitional, follicular, and MZ SW_HEL cells. Representative histogram plots are shown. (B) Graphs show IgM MFI expressed relative to IgM expression on WT CD45.2⁺ polyclonal follicular cells. Each point represents an individual mouse; bars show means (n = 8–9). N.D., not done, as insufficient numbers of MZ B cells were generated to be confidently analyzed. (C and D) Splenocytes from chimeric mice were either unstimulated or stimulated in vitro with HEL for 18 h; expression of CD86 was then determined by flow cytometry. Histograms show representative staining of CD86 on HEL-stimulated SW_HEL cells (C), and the graph shows CD86 MFI of unstimulated or HEL-stimulated cells (D). Each point represents an individual mouse; bars show means ± SEM (n = 5 mice per group, combined from two experiments). Significant differences were determined using two-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. See also Fig. S4.