Effect of myeloma cells on RANKL expression in activated T lymphocytes in cocultures.

Purified CD4⁺, CD8⁺, and CD3⁺ T lymphocytes were activated with coated CD3 plus CD28 mAb. After 4 days mRNA and proteins were tested for RANKL expression by RT-PCR (A) and Western blot analysis (B), respectively. The 24-hour activated CD3⁺ T lymphocytes were cocultured in a transwell system with HMCLs and RANKL mRNA expression was evaluated in T cells 24 hours later (panel C). Instead RANKL protein was assessed after a further 48 hours of cocultures in activated T cells (D) or activated CD4⁺/CD8⁺ subsets (E) and in the presence or absence of anti–IL-6 mAb (F). Similarly, RANKL protein was evaluated in autologous T lymphocytes cocultured with fresh myeloma cells from MM patients (G). The figures are representative of 3 independent experiments; C indicates control (activated CD3⁺lymphocytes). Graphs represent the mean optical density (OD) of RANKL normalized to OD of β₂-microglobulin (panel C) or actin (D-G; named “OD ratio”) of representative experiments.