CD28 signaling induced by DC or other MM cells prevents myeloma cell death. MM cells (MM.1S) were cultured alone or with DC at a 1:1 ratio ± 2 µM melphalan ± 50 µg/mL of the blocking αCD28 mAb CD28.6 (A) or ± 100 µg/mL CTLA4-Ig (B) or for 72 hours. Cell survival was analyzed by flow cytometry for Annexin V/7AAD staining and DC were gated out using CD11b. U266 (C), RPMI8226 (D), or KMS11 (E) myeloma cells were cultured as in (B) for 72 hours and survival was assessed by flow cytometry. (F) RPMI8226 myeloma cells were infected with lentiviral particles encoding shRNA specific for CD28, GAPDH, or empty vector (pLKO.1) and cultured for 4 days in full serum media. Survival was assessed by Annexin V staining. (G) MM.1S cells were cultured in either 10% serum or 0.2% (low) serum conditions for 5 days ± 100 µg/mL CTLA4-Ig. Fifty percent of the media was changed every other day to prevent nutrient depletion, and new CTLA4-Ig was added with every media change. Cell survival was analyzed by flow cytometry for Annexin V/7AAD staining. Data for panels A and B) are representative of 4 individual experiments; data for panels C-E are representative of 2 individual experiments. *P < .05, **P < .01, ***P < .005. NS, not significant.