Extended Data Fig. 3: B cells lacking PI3Kγ activate normally and are capable of forming germinal centers and normal IgA responses.
From: PI3Kγ in B cells promotes antibody responses and generation of antibody-secreting cells
(a) Littermate control or conditional Pik3cg KO mice were immunized and boosted with NP-OVA precipitated in alum. Antigen-specific IgG2c (day 28) was measured via NP-BSA ELISA, and arbitrary units were defined using pooled immunized sera as a standard. (b) Immunofluorescence imaging of spleens from indicated mice 28 days after SRBC immunization. Data are representative of two independent repeats (n = 6 for each genotype). (c-h) Murine naïve B cells were stimulated using TI stimulation with TLR agonists (CpG or LPS) or TD stimulation with anti-CD40, anti-IgM F(ab’)2 and assessed for proliferation using cell trace violet (gating on live cells after 4 days) and activation by staining for CD69 or CD86 (gating on live cells after 2 days). Data (n = 3 for each condition) are representative of two independent experiments. (i-n) Quantification of B cell subsets from Peyer’s patches of mice assessed by flow cytometry. Data are representative of three independent experiments (n = 13 for control and 14 for KO). (o) Quantification of fecal IgA in control versus B cell-specific PI3Kγ KO mice (n = 6 per group). (p) Quantification of surface IgG-expressing memory and GC B cells, normalized to control animals, in control versus B cell-specific PI3Kγ KO mice (n = 3 for control and 5 for KO). (q-r) In-vitro generation of GC B cells using 40LB feeder cells, as in Nojima et al. 2011. Data are from two independent experiments (n = 5 for each condition). Data are presented as a bar graph (median ± SD) or box-and-whisker plots showing median, min, and max. Each dot represents different biological samples and statistical analysis was performed using non-parametric two-tailed unpaired T-tests, except for panels q-r which uses non-parametric paired T-test.
