Extended Data Fig. 5: AChR expression is coordinated during B cell activation; related to Fig. 5.
From: Lymphocyte-derived cholinergic circuits modulate germinal center output and B cell activation
a Representative flow cytometry plots showing the gating strategy (top), and expression (bottom left panels), of the M2 and M4 mAChRs on the indicated B cell populations from WT mice at day 10 post-SRBC immunization. Plots were gated on single cells/viable cells/B220+ B cells, with non-GC B cells then subdivided into activated cells (CD86+CD95+) and naïve cells (CD86−CD95−). Bottom right panels: Summary statistics for M2 and M4 gMFI; N = 11. b Representative flow cytometry plots of protein expression of the indicated AChRs on GC B cells from the WT mice in Fig. 5d at days 5 and 10 post-SRBC immunization. c Summary statistics showing gMFI of expression of CD138 on the activated B cells in Fig. 5e. Statistics are reported relative to unstimulated cells; N = 6. d Summary statistics showing levels of secreted IgM in culture supernatants of the activated B cells in Fig. 5e. Statistics are reported relative to unstimulated cells; N = 6. e qPCR determination of the relative induction of Aicda in the B cells cultured and treated as in Fig. 5e; N = 4. f Flow cytometric determination of Ca2+ mobilization in WT splenic B cells that were activated in vitro for 48 h with 1 μg/mL α-IgM and loaded with Indo-I. Cells were then stimulated with ACh (10 mM) as indicated; N = 13. g Left: Representative flow cytometry plot showing p-CREB expression on GC B cells isolated from WT mice at day 6 post-SRBC immunization. Plot was gated on single cells/viable cells/B220+ B cells. Right: Summary statistics of p-CREB expression on GC B cells from mice of the indicated genotypes at day 6 post-SRBC immunization; N = 10. Data are the mean ± SEM. P values of *<0.05, **<0.01, ***<0.001, and ****<0.0001 were determined for Extended Data Fig. 5a by Friedman test with Dunn’s multiple comparison test; for Extended Data Fig. 5c-e by RM-ANOVA with Dunnett’s multiple comparison test reported relative to Unstim.; and for Extended Data Fig.5g by two-sided Mann-Whitney test, relative to Chat flox/flox WT control. Exact P values are reported in the Source Data.
