Induction and characterization of CTLs against autologous MM cells. (A) Immature DCs were induced from adherent cells in BMMCs of patients with MM (Pt 1 [λ-type] and 2 [IgA, κ]) by culturing with GM-CSF (1000 U/mL), IL-4 (10 ng/mL), and 10% AB serum, followed by coculture for 12 to 18 hours at a ratio of 5:1 with autologous MM cells incubated for 4 hours after irradiation (30 Gy) and then cultured for 3 days in the presence of TNF-α (20 ng/mL) to induce maturation. MNCs from the same MM patients were then cocultured with mature DCs at a ratio of 10:1 to 20:1 in AIM-V medium and stimulated with the apoptotic body–pulsed DCs weekly. Seven days after the second stimulation, cytotoxicity of the CTL against autologous MM cells (▪), RPMI 8226 cells (□), or K562 cells (▦) was examined using a lactate dehydrogenase assay at indicated effector-to-target (E/T) ratios. Significantly greater specific lysis of autologous MM cells was observed than of RPMI 8226 cells or K562 cells (*, P = .02). Spontaneous release of target cells was less than 20%. Results shown are mean ± SD of triplicate wells. (B) Cell surface phenotype of patient-derived CTLs was analyzed by flow cytometry using specific Abs against CD3, CD8, CD4, and CD56 (filled histogram). Percentages positive cells are calculated relative to an isotype-matched control Ab (open histogram). (C) HLA restriction of CTLs derived from patients with MM (Pt 3, nonsecretory; Pt 4, κ-type MM) was examined using target-blocking LDH assay. CTLs stimulated with apoptotic MM body–pulsed DCs were cocultured with autologous MM cells after incubation with neutralizing antibody against HLA class I (□), HLA class II (▨), control antibody (▦), or without antibody (▪). Cytotoxicity against autologous MM cells was significantly inhibited by anti–HLAclass IAbs (*, P = .02). Spontaneous release of target cells was less than 20%. Results shown are mean ± SD of triplicate wells from 2 representative patient-derived CTLs. (D) Cytotoxicity of CTLs induced by stimulation with DCs cocultured with apoptotic primary MM bodies of HLA-A2–positive patient against autologous MM cells (▪), U266 cells (▴), K562 cells (▵), or autologous PBMCs (□) was assessed with LDH assay at indicated effector-to-target (E/T) ratios for 4 hours at 37°C. CTLs induced by stimulations with DCs cocultured with primary MM bodies showed significantly greater cytotoxicity against autologous MM cells than against U226 cells, K562 cells, or autologous PBMCs. Spontaneous release of target cells was less than 20%. Results shown are mean ± SD of triplicate wells.