NOTCH2 expression is maintained on active cGVHD B cells in the presence of NOTCH ligand and BCR stimulation. (A-B) B cells were purified from viably frozen PBMCs of HCT patients with active cGVHD (Active, n = 4) or no cGVHD (No, n = 4) and plated in medium onto OP9-DL1 feeder cell monolayers. In some wells, the γ-secretase inhibitor DAPT was added for a final concentration of 10 μM, with DMSO alone used as the vehicle control in parallel. Following an initial culture period of 30 minutes, agonistic anti-IgM Ab was then added to the appropriate wells at a concentration of 0.625 μg/mL. The cells were cultured for 72 hours, harvested, and flow cytometry analysis performed to assess NOTCH2 surface expression on CD19⁺ B cells. (A) Representative histogram overlay showing relative NOTCH2 expression between patient groups and activation conditions (as indicated). (B) MFI values for all patients assessed under conditions of anti-IgM stimulation, either without or with DAPT. (C) B cells were purified from viably frozen PBMCs from healthy donors and then plated onto monolayers of parental OP9 cells or OP9-DL1 cells, with various concentrations of anti-IgM Ab (micrograms per milliliter, indicated by numbers in the OP9 histogram overlay). Following a culture period of 72 hours, the cells were harvested and analysis of NOTCH2 expression on CD19⁺ cells was performed by flow cytometry. In panel B, P values were determined using a nonpaired Student t test for Active vs No comparison, and a paired Student t test for Active (no DAPT) vs Active (DAPT) comparison. *P < .05.