Hyperactivation of active cGVHD patient B cells to NOTCH ligand and anti-IgM is NOTCH2-dependent. (A-B) Anti-N2 mAb (N2) or ragweed isotype control mAb (Iso) were diluted in medium and added to 96-well plates containing OP9-DL1 feeder cell monolayers to achieve a final concentration of 1 μg/mL. B cells purified from viably frozen PBMCs of active cGVHD patients (n = 6) were then added to the plates. Following an initial culture period of 30 minutes, agonistic anti-IgM Ab was 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 Ki-67 expression (A), and relative cell size by forward scatter (FSC) (B). P values were determined using a paired Student t test (*P < .05; **P < .01). (C) NOTCH-BCR stimulation with or without specific NOTCH2 blockade with anti-N2, as described in panels A and B. The cells were cultured for 24 hours, the B cells purified away from the feeder cells using magnetic microbeads, and RNA isolated. B-cell RNA was then subjected in NanoString gene-expression profiling to assess differences in NOTCH pathway-regulated genes between groups. Data points represent NanoString nCounter target count number of individual samples for each gene, graphed on a relative scale. Statistical analysis comparing the anti-N2– and isotype control–treated groups was performed using a paired, negative binomial test (*P < .05).