Natural killer T cell activation increases iNOS⁺CD206^- M1 macrophage and controls the growth of solid tumor

NKT cells play an important role in anti-tumor immunity. Alpha-galactosylceramide (α-GalCer), a synthetic glycolipid is presented to natural killer T (NKT) cells by most antigen-presenting cells through CD1d molecules leading to activation of NKT cells. However, the precise mechanisms of how α-GalCer-activated NKT regulate the polarization of the macrophages and effector T cells in the solid tumor are not studied adequately.

We induced solid tumor in C57BL/6 mice by subcutaneous injection of B16F10 cell line (1 X 10⁶ cells) and monitored the tumor growth. Animals were given an intraperitoneal injection of α-GalCer (2 μg/injection) in 200 μl PBS on day + 1, + 5, + 10, + 15, and + 20 (with respect to tumor cell injection). Immune cells were characterized using flow cytometry and immunofluorescence staining. NK cells, Gr1⁺ cells, and F4/80⁺ macrophages in the mice were depleted by intravenous injection of cell-specific antibodies. Statistical analysis was performed using Student's t-test or one-way ANOVA.

Our results showed that intratumoral NKT cells have a lower frequency of CD69, CD25, CD122, and IFN-γR expression; produced less inflammatory cytokines such as IFN-γ, TNF-α, and GM-CSF; higher frequency CD62L⁺ NKT cells; and also showed reduced proliferation as compared to the splenic NKT cells. Mice treated with α-GalCer showed a significantly increased frequency of IFN-γ-producing NKT cells, CD8⁺ T cells, and effector Th1 cells. Depletion of NK cells in α-GalCer-treated mice showed a lower frequency of IFN-γ-producing CD4⁺ and CD8⁺ T cells in the tumor and prevented the α-GalCer-induced tumor growth. NKT cell activation with α-GalCer treatment significantly increased the iNOS⁺CD206^- M1-macrophages and reduced the iNOS^-CD206⁺ M2-macrophages in the spleen and tumor, and depletion of F4/80⁺ macrophages prevented the α-GalCer-induced reduction in the tumor growth.

We showed that activation of NKT cell with α-GalCer modulates the frequency of M1-macrophages and effector Th1 cells in the secondary lymphoid tissues and tumor microenvironment and inhibit tumor growth. The finding suggests that activation of NKT cells with α-GalCer may provide an effective anti-cancer outcome.