Figure 9

Co-culture performed with macrophages-depleted spleen cells reduces proinflammatory cytokines production. Spleen cells were obtained from mice submitted to the subcutaneous tumor model and treated with BCG (T) after 22 days post-tumor injection. CD11b⁺F4/80⁺ cells (Macrophages—MØ) were depleted from the spleen cells (T) using anti-PE MicroBeads kit described in the methodology. The percentage of depletion (94%) was addressed by flow cytometry. (A) A representative plot and frequency are shown. Total spleen cells (T) or macrophages-depleted spleen cells (T)^−MØ suspension were used for co-culture in vitro with infected MB49 cells (1 MB49 cell: 2 spleen cells). MB49 cells used for co-culture were previously infected with BCG (MB49 + BCG) for 24 h and washed to remove free BCG in the supernatant. (B, D) Co-cultured cells were maintained together for 24 h. MB49 or spleen cells were also infected separately (not in co-culture) and used as controls. (C, E) Spleen cells were also stimulated with LPS (1 µg/ml) for 24 h as controls. TNF-α (B, C) and IL-6 (D, E) cytokines production were measured in the supernatants after 24 h by ELISA. The values are representative of two independent experiments. *Statistically significant compared to spleen cells (T) before macrophage depletion. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.