Altered IRF4 and IRF8 gene expression in B cells from active cGVHD patients, with normalization by ATRA. B cells from HCT patients with active cGVHD vs no cGVHD (n = 6), or from healthy donors (n = 6) were cultured in medium alone, or with low-dose (0.625 μg/mL) or high-dose (5 μg/mL) concentrations of anti-IgM for 24 hours. The B cells were then harvested, total RNA isolated, and real-time qPCR analysis performed to assess the abundance of IRF4 and IRF8 transcripts. The relative expression level shown for each gene is normalized to the median value for healthy donors with no anti-IgM stimulation, represented as a value of 1. (A) IRF4 and (B) IRF8 mRNA levels in each of the 3 sample groups with and without BCR stimulation. (C) Normalized IRF4 and IRF8 mRNA expressed as the ratio of IRF4 to that of IRF8 transcripts (IRF4-to-IRF8). P values were determined using a nonpaired Student t test (*P < .05; **P < .01). (D) B cells from HCT patients with active cGVHD (n = 6) were cultured with ATRA (0.1 μM) or vehicle alone, followed 30 minutes later by the addition of 0.625 μg/mL anti-IgM. Following a 24-hour culture period, the B cells were then harvested, total RNA isolated, and real-time qPCR analysis performed to determine relative IRF4 and IRF8 expression, and the IRF4-to-IRF8 ratio, as described for panels B and C. (E) B cells from HCT patients with active cGVHD (n = 4-6) were plated onto OP9-DL1 monolayers in the presence of 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 24 hours, harvested, and intracellular staining with flow cytometry analysis performed to assess IRF4 and IRF8 levels. For each protein, data represent the ratio of ATRA-treated B cells over vehicle-treated B cells, with the dashed line included for reference to a value of 1.0.