CD4+ T-cell–mediated killing of myeloma is mediated by bone marrow macrophages. (A) Flow cytometry quantitation of F4/80⁺CD64⁺ macrophages in femoral bone marrow isolated from mice on day 5 after IV challenge with 1 × 10⁶ MOPC.BM cells. Mice were treated with an anti-asialo-GM1 (a-AGM1) or isotype control (Is ctr.) mAb. Gating strategy is shown on the left. The histogram shows means ± SD for 6 mice per treatment group; **P < .01. (B) In vitro coculture of MOPC.BM cells with ex vivo–isolated F4/80⁺CD64⁺ macrophages from the experiment described in panel A; effector/target ratio, 10:1. Results are plotted as tumor cell growth of a percentage of tumor cells cultured alone, calculated on the basis of 18-hour [³H]-thymidine release (n = 6-12 per treatment group, mean ± SD; ***P < .001). (C) Flow cytometry, with the nitric oxide–reactive fluorescent dye DAF-FM diacetate. Dot plots show representative results for staining of cells isolated from femoral bone marrow of TCR-Tg and wild-type (WT) BALB/c mice on day 7 after tumor challenge with 1 × 10⁶ MOPC315.BM. Histograms show staining for costimulatory receptors CD80/86, asialo-GM1, and MHC -II on the DAF-FM^High population found in TCR-Tg mice. (D) Representative flow cytometry staining using a pId315:I-Ed peptide/MHC-II–specific single-chain variable fragment (TCRm). Bone marrow cells were isolated from TCR-Tg and wild-type (WT) mice on day 5 after IV challenge with 1 × 10⁶ MOPC.BM cells, with a rabbit anti-asialo-GM1 antibody, followed by magnetic bead separation. Nonchallenged TCR-Tg mouse bone marrow was used as a control. Dorsal whole-body bioluminescence staining (E) and survival (F) for TCR-Tg and WT BALB/c mice challenged with 1 × 10⁶ MOPC315.BM. Mice received daily injections of 200 μg depleting mAb against Ly6G/C (RB6-8C5) or a relevant isotype control mAb, starting 4 days before tumor challenge (n = 6 to 8 per treatment group). Bioluminescence data are shown as means ± SD of total flux.