CD28 activation protects against induced cell death. (A) Serum starvation. The indicated myeloma cells were cultured in media (RPMI 1640) without serum (SS), or medium without serum plus soluble anti-CD28 mAb (SS+CD28). After 48 hours, viability was determined by annexin V/PI staining. Data are the aggregate mean ± SD of 3 independent experiments. P = .07 for MM.1S serum starvation versus SS + anti-CD28; P = .12 for 8226S. (B) Dex-mediated cell death. The indicated myeloma cell lines were cultured in 0.1% FBS with or without soluble anti-CD28 mAb and 100 μM dex. After 72 hours, viability was determined by Annexin V/PI staining. Data are the aggregate mean ± SD of 3 independent experiments. In comparing the survival in dex versus dex plus anti-CD28: P = .02 for MM.1S, P = .03 for 8226S, and P = .002 for U266. (C) Primary myeloma cells. Primary myeloma cells were purified from 3 different patient samples (bone marrow aspirates; recurring MM) by CD138 immunomagnetic selection (Miltenyi Biotech). The samples were more than 90% CD138⁺ and were all CD28⁺. Input cells (2 × 10⁵) were cultured in 10% FCS (PS 1; left panel) or no serum (PS 1-3; right panel) with or without anti-CD28 mAb (9.3; 1 μg/mL) for 24 hours. Total viable cell numbers were then enumerated in trypan blue and expressed as the percentage of the starting input number of cells.