IL4/STAT6 pathway is inactivated in P2/CD23^–cells and inhibits differentiation of P2/CD23⁺cells. (A) IL4R and STAT6 mRNA expression levels as assessed by RNA-seq in the different cell populations (n = 3 paired samples). (B) STAT6 and IL-4R protein expression as quantified by western blotting in NBCs, D4 cells, and different cell subsets on D6. The western blot is representative of 3 independent experiments. (C) The strength of pSTAT6 signaling (assessed by flow cytometry) in IL4 nonstimulated or stimulated (stim) D4 cells (at 10 or 30 minutes) and in different cell subsets on D6. The data are representative of 3 independent experiments. (D) Top left, experiment scheme: after a short stimulation with cocktail 2 (IL-2 + IL-10 + IL-4), P2/CD23⁺ cells were sorted and cultured for 40 hours either with cocktail 2 (+IL-4) or only with IL-2 and IL-10 (without IL-4). Top right, strength of pSTAT6 signaling (assessed by flow cytometry) in both cell populations showing an activation of STAT6 only in IL-4–treated cells. Bottom left, number of CASP3-negative cells, showing an effect of IL-4 on cell survival (left). Treatment with the pan-caspase inhibitor QVD-OPH relieves this effect (right). Bottom right, number of plasmablasts (assessed by flow cytometry) obtained after 40 hours of culture without (left) and with (right) QVD-OPH showing a significant increase of generated plasmablasts in the condition without IL-4. FACS, fluorescence-activated cell sorting.