Fig. 3

Effect of MITF on the anti-melanoma immune response. a to c Luciferase imaging of C57BL/6 a, c and athymic b mice after tail vein injection with B16/F10-MITF-KO cells. The experimental conditions were as those described in Fig. 1. d Vaccination assay. Tumors in non-immunized animals subcutaneously injected with irradiated B16/F10 and B16/F10-MITF-KO cells (Groups 1 and 4, respectively). Animals in Group 2 were immunized with ex vivo irradiated B16/F10 cells. After 4 weeks, non-irradiated B16/F10 cells were injected subdermally. Groups 3 and 5 were immunized with ex vivo irradiated B16/F10-MITF-KO cells. After 4 weeks, non-irradiated B16/F10 (Group 3) or non-irradiated B16/F10-MITF-KO (Group 5) cells were injected subdermally. Analysis of primary tumors was carried out 4 weeks after subcutaneous injection. Color scale for Groups 1–3 (Min = 1.00e6; Max = 1.00e7) and for Groups 4–5 (Min = 5.00e4; Max = 5.00e5). e Lungs from mice injected in the tail vein with vehicle (PBS) or indicated cells. f Microscopic view of the lung tissue structure (H&E stain, × 100) (Bars, 600 μm). g Analysis and quantification (histogram) of NKp46-positive cells in mouse lungs performed by flow cytometry of single cell suspensions. *p < 0.005 when compared with vehicle-treated group. **p < 0.005 when compared with B16/F10-treated group. h H&E stain and IHC (with anti-NKp46) of lung tissues. Yellow arrows indicate areas of immune infiltration. The proportion of infiltrated area per total area of lung H&E slide (× 40) was calculated by using ImageJ software (histogram). Quantification was carried out on three different areas of each lung and in 3 animals per group (*p = 0.0026). In panels e to h, lungs were obtained 3 weeks after vehicle/cells injection and n = 3 per group