ATRA suppresses NOTCH2-BCR hyperresponsiveness of B cells from active cGVHD patients. (A-D) B cells from HCT patients with active cGVHD (n = 6) were plated in medium on monolayers of OP9-DL1 feeder cells and exposed to ATRA (0.1 μM) or vehicle alone. Following an initial culture period of 30 minutes, agonistic anti-IgM Ab was then added to the appropriate wells at a final concentration of 0.625 μg/mL. The cells were cultured for 72 hours, harvested, and flow cytometry analysis performed to assess B-cell Ki-67 expression (A), FSC as a measure of relative cell size (B), cell-surface NOTCH2 expression (C), and number of live cells (D). (E-F) B cells from HCT patients with active cGVHD (n = 4) were plated in medium on monolayers of OP9-DL1 feeder cells and exposed to ATRA (0.1 μM), vehicle alone, anti-N2 mAb, or isotype control mAb. Following an initial culture period of 30 minutes, agonistic anti-IgM Ab was then added to the appropriate wells at a final concentration of 0.625 μg/mL. The cells were cultured for 72 hours, harvested, and flow cytometry analysis performed to assess B-cell surface proteins (E) and intracellular ERK1/2 protein phosphorylation (F). (E) Panel i shows representative dot plots from 1 patient for the treatments indicated; panel ii shows the frequency of CD38⁺IgD⁻ B cells from all 4 patients under the indicated culture conditions. (F) Panel i shows representative histograms from 1 patient for the treatments indicated; panel ii shows phospho-ERK MFI levels in B cells from all 4 patients under the indicated culture conditions. P values were determined using a paired Student t test. n.s., not significant.