Effect of myeloma cells on RANKL secretion by activated T lymphocytes.

Aliquots of conditioned media were assessed for sRANKL by ELISA. Activated CD3⁺ T lymphocytes (A) or CD4⁺/CD8⁺ subsets (B) were cocultured with HMCL RPMI-8266 or U266 placed in a transwell insert. Autologous CD3⁺ T cells were cocultured with fresh MM cells (panel C). (C indicates activated CD3⁺ T cells.) Graphs represent the mean levels ± SE of 3 repeated experiments. *P < .05. Secretion of RANKL into conditioned medium was checked by immunoprecipitation with RANK-Fc followed by gel electrophoresis. RANKL protein was detected either by gel staining with silver stain plus in conditioned medium of activated T cells cocultured with HMCL (D) or by immunoblot analysis using anti-RANKL mAb (E). Data are representative of 3 independent experiments. C indicates activated CD3⁺ lymphocytes).