APRIL induces PD-L1 expression on human MM cell line cells mainly via paracrine mechanism. (A) CD14⁺ monocytes were stimulated with M-CSF and/or RANKL for 14 days. APRIL expression in these cells was examined by real-time qRT-PCR; 18S was used to normalize APRIL expression. (B) Transwell experiments were performed in which MM cells were placed in the upper chamber and medium alone, or OCs were placed in the lower chambers. After 4 days, mRNA was collected from MM cells and subjected to real-time qRT-PCR for APRIL normalized to 18S. Fold increases compared with controls were shown. (C) MM cell lines were cultured with APRIL (200 ng/mL) for 7 days and PD-L1 expression was examined by flow cytometry. (D) MM cell lines were treated with human recombinant APRIL and/or anti-APRIL mAb (200 ng/mL) for 4 hours, and PD-L1 expression was examined by real-time qRT-PCR. (E) Indicated MM cell lines were stimulated with APRIL and PD-L1 expression was examined by real-time qRT-PCR. (F) MM1R and JJN3 cells were cultured with IFN-γ (500 IU/mL, 24 hours), IL-6 (10 ng/mL, 48 hours), and APRIL (200 ng/mL, 7 days). PD-L1 and pMEK1/2 expression was assessed by immunoblotting of cell lysates. *P < .05; **P < .001; by unpaired 2-sided Student t test.