Figure 6. CD84 expression is regulated in a MIF-dependent manner in the MM microenvironment.

(A and B) PB CD14⁺ cells derived from healthy volunteers were cultured alone or with the human myeloma cell lines (MM.1S, U266, KMS11) at a ratio of 1:2. After 48 hours, the CD14⁺ cells were analyzed by flow cytometry for CD84 (A) and PD-L1 (B) expression (n = 3). Representative histograms are shown. (C) MIF protein expression was analyzed in the human MM cell lines NCI-H929, MM.1S, U266, RPMI-8226, L363, MM.1R, and Raji cell line (positive control) by Western blot analysis. (D and E) PB CD14⁺ cells from healthy donors (D) or THP1 cells (E) were cultured with or without 200 ng/mL human recombinant MIF (hrMIF). After 48 hours, the cells were analyzed for CD84 and PD-L1 expression by flow cytometry (n = 3). (F and G) BM from C57BL/KaLwRij WT mice (F) or 5TGM1-injected C57BL/KaLwRij mice (G) was grown until an almost confluent adherent cell layer was formed. Thereafter, 100 ng/mL MIF was added. After 48 hours, the cells were harvested and analyzed by flow cytometry (excluding CD45⁺ and CD138⁺ cells) for CD84 expression (n = 3–5). (H and I) MM.1S cells were transfected using MIF or control siRNA (10 μM/mL). After 48 hours, the medium was replaced with fresh culture medium, and cells were grown an additional 72 hours for collection of supernatant. After 72 hours, the supernatants of the cells were transferred to PB CD14⁺ cells derived from healthy donors. After 48 hours, the CD14⁺ cells were harvested and analyzed by flow cytometry for CD84 (H) and PD-L1 (I) expression (n = 5).