CD40L induces mTORC signaling and arrests FO B-cell maturation in vitro. (A) Levels of (p)-S6 by intracellular flow cytometry in transitional (T) and FO B-cell subsets, sorted and cultured for 24 hours in vitro with CD40L treatment (+CD40L) compared with without (untx) as indicated. Histograms are representative of 3 independent experiments. Quantified data, from 3 independent experiments, are means (± SD) of all data. ∗∗P < .01 by unpaired Student t test. (B) Differentiation by extracellular flow cytometry of T3a and T3b B cells, sorted and cultured for 24 hours in vitro with, compared with, without CD40L treatment or AICAR treatment to inhibit mTORC signaling as a control. Histograms are representative of 3 independent experiments, here showing differences in T3a B-cell levels of MitoTracker (MTG) and CD73 at 24 hours in culture by treatment condition as indicated. Differentiation to the FO B-cell stage was scored by the acquisition of the CD73⁺MTG⁻ phenotype. Quantified data, from 2 independent experiments, are means (± SD) of all data. ∗∗P < .01; ∗∗∗∗P < .0001 by unpaired Student t test. AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; untx, untreated.