Fig 2: 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

Fig 3: gMoxi-mediated tumour regression and NK cell promotion depend on adrenergic signalling. Lewis lung cancer (LLC) cells were inoculated on the upper back of C57BL6 mice on day 0, and gMoxi was performed at the acupoint Zusanli (ST36) with three moxa cones every other day from day 1. Propranolol (Prop) in drinking water was given 1 wk before LLC inoculation. Epinephrine (Epi) was daily administered by IP injection from day 1. Mice were killed on day 21, and tumours and spleens were isolated and analysed by flow cytometry. A, Tumour growth curve; B, tumour photograph; C, tumour weight; D, splenocytes were co-cultured with YAC1 cells at 10:1, and tumour lysis was detected by apoptosis analysis. E, the proportion of CD3-NKp46+ NK cells in spleens; F, the proportion of CD3+CD4+ T cells in spleens; G, the proportion of CD3+CD8+ T cells in spleens; H, the proportion of CD3-NKp46+ NK cells in tumours; I, the proportion of CD3+CD4+ T cells in tumours; J, the proportion of CD3+CD8+ T cells in tumours; K, splenocytes were incubated in the present or absent Prop or Epi with or without PK136 antibody for 24 h and then co-cultured with YAC1 cells at 5:1 for 2.5 h, and tumour lysis was detected by apoptosis analysis. Independent experiments were repeated twice. *P < .05; **P < .01; ***P < .001; NS, non-statistically significant

Fig 4: YPF did not influence the population of immunity cells in spleen. Mononuclear cells were isolated from the spleen of orthotopic LLC-bearing C57BL/6 mice treated with normal saline or YPF at Day 14 after inoculation and then analyzed by flow cytometry after stained with CD3, CD4, CD8, NKp46, Foxp3, CD11b, F4/80, and Gr-1. (A) Percentages of CD3+ T, CD4+ T, and CD8+ T cells in spleen. (B) Percentage of NK cells in spleen. (C) Percentage of regulatory T cells (Treg) in spleen. (D) Percentage of macrophage in spleen. (E) Percentage of myeloid-derived suppressor cells (MDSCs) in spleen. NS, non-significant.

Fig 5: DHA feeding improved IFN-? production and degranulation by NK cells in the spleen at day 7 postinfection. A Flow cytometric analysis of the ratio and the geometric mean fluorescence intensity (gMFI) of IFN-?+ splenic NK cells (CD3-CD19-NK1.1+NKp46+) from control versus DHA-fed mice following stimulation with PMA and ionomycin in the presence of GolgiPlug and GolgiStop for 6 h. B, C Flow cytometric analysis depicting the frequencies and gMFI of perforin (B) and granzyme B (GZMB) (C) by splenic NK cells (CD3-CD19-NK1.1+NKp46+) from control versus DHA-fed mice at day 7 postinfection. D Purified splenic NK cells from both DHA-fed and control mice at day 7 after MCMV infection were cocultured with CTV-labeled YAC-1 (left) or MCMV-infected 3T3 cells (right) for 6 h at the E:T ratio of 16:1. Representative flow cytometry plots of CTV+ singlets are shown on the left. The percentages of NK cell-specific lysis are shown on the right, which were calculated by the following formula: [(% CTV + 7-AAD+ cell specific lysis - %CTV + 7-AAD+ cell spontaneous lysis)/ (100 - % CTV + 7-AAD+cell spontaneous lysis)] × 100. For each experiment, n = 9 pooled from 3 independent experiments (A–C); n = 5 pooled from 2 independent experiments (D). Each symbol represents an individual mouse, and the blue dots and red square represent control and DHA-enriched diet-fed mice, respectively. All experiments were replicated 3 times. Error bars represent interquartile ranges; *p < 0.05; **p < 0.01