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. Author manuscript; available in PMC: 2020 Nov 28.

Published in final edited form as: Cancer Lett. 2019 Sep 3;465:24–35. doi: 10.1016/j.canlet.2019.08.018

Figure 5. — 1×10⁶ WT, or Grn^−/−B16-F10 cells were injected subcutaneously in the right flank to C57BL/6 mice.18 days later, primary tumors were dissected. Single cell suspensions were obtained from these tissues and stained for (A) CD3⁻ NK1.1⁺ cells, (B) CD11b⁺ F4/80⁺ macrophages, (C) CD11c⁺ MHC II⁺ dendritic cells, (D) CD4⁺ T cells, (E) CD8⁺ T cells. The percentage of each type cells were calculated based on total CD45⁺ cells. (F) MHC I and PD-L1 expression on the B16-F10/Grn^−/− tumors were analyzed by FCM. Data were presented as mean ± SD (* P < 0.05, two-tailed Student’s t test). n=3 mice per group. (G) Immunohistochemical staining for NK cells in ctrl and B16-F10/Grn^−/− tumor section by using anti-NK1.1 antibody (Scale bar, 100 μm). (H) Live cells were gated and the gate including CD45⁺ cells were analyzed for NK1.1, CD69, IFN-γ, and GzmB. Quantification of active NK cells (NK1.1⁺CD69⁺, NK1.1⁺IFN-γ⁺, NK1.1⁺GzmB⁺) infiltrating to the tumor was reported as a percentage of total NK cells. Data were presented as mean ± SEM (* P < 0.05, *** P < 0.001, two-tailed Student’s t test). n=3 mice per group.

Figure 5. — 1×10⁶ WT, or Grn^−/−B16-F10 cells were injected subcutaneously in the right flank to C57BL/6 mice.18 days later, primary tumors were dissected. Single cell suspensions were obtained from these tissues and stained for (A) CD3⁻ NK1.1⁺ cells, (B) CD11b⁺ F4/80⁺ macrophages, (C) CD11c⁺ MHC II⁺ dendritic cells, (D) CD4⁺ T cells, (E) CD8⁺ T cells. The percentage of each type cells were calculated based on total CD45⁺ cells. (F) MHC I and PD-L1 expression on the B16-F10/Grn^−/− tumors were analyzed by FCM. Data were presented as mean ± SD (* P < 0.05, two-tailed Student’s t test). n=3 mice per group. (G) Immunohistochemical staining for NK cells in ctrl and B16-F10/Grn^−/− tumor section by using anti-NK1.1 antibody (Scale bar, 100 μm). (H) Live cells were gated and the gate including CD45⁺ cells were analyzed for NK1.1, CD69, IFN-γ, and GzmB. Quantification of active NK cells (NK1.1⁺CD69⁺, NK1.1⁺IFN-γ⁺, NK1.1⁺GzmB⁺) infiltrating to the tumor was reported as a percentage of total NK cells. Data were presented as mean ± SEM (* P < 0.05, *** P < 0.001, two-tailed Student’s t test). n=3 mice per group.