Extended Data Fig. 6: Acetylcholine modulates the threshold for GC B cell selection; related to Fig. 6.
From: Lymphocyte-derived cholinergic circuits modulate germinal center output and B cell activation
a Left: Representative flow cytometry plots of total tyrosine phosphorylation (p-Tyr) in B cells that were isolated from spleens of WT mice, activated for 48 h in vitro. Cells were then treated (or not) with the indicated AChR inhibitors, with or without 10 mM ACh, and restimulated with 1 μg/mL α-IgM F(ab’)2 (BCR crosslinking) for 90 seconds. Right: Summary statistics showing gMFI of p-Tyr in the left panel, reported relative to unstimulated control cells; N = 8. b Representative flow cytometry plots of c-MYC expression on GC B cells that were isolated from spleens of WT mice and cultured for 24 h with 0.5 mM or 5 mM ACh, and then stimulated for 24 h with serial dilutions of a cocktail containing α-IgM, α-CD40, IL-21 and IL-4. Plots were gated on single cells/viable cells/B220+ B cells/CD95+CD38− GC B cells/activated caspase-3− cells before determining c-MYC expression. c Left: Proportion of activated caspase-3+ GC B cells among the B cells in B after 24 h in culture. Right: EC50 of the fitted dose-response curve in the left panel; N = 6. Data are representative of at least 2 experiments, where data are the mean ± SEM. P values of **< 0.01, ***< 0.001 and ****< 0.0001 were determined for Extended data Fig. 6a by Friedman test with Dunn’s multiple comparison test, and for Extended data Fig. 6c by two-sided Wilcoxon matched pairs test relative to 0 mM control. Exact P values are reported in the Source Data.
