Background 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. Methods We induced solid tumor in C57BL/6 mice by subcutaneous injection of B16F10 cell line (1 X 10 6 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. Results 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. Conclusions 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. Electronic supplementary material The online version of this article (10.1186/s40425-019-0697-7) contains supplementary material, which is available to authorized users.

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BackgroundType I NK T cells, also known as iNKT cells, can recognize self or microbial lipids presented through CD1d molecules on antigen-presenting cells. Activation of NKT cells induces inflammatory cytokines and help in mounting anti-tumor immunity. How does stimulation of iNKT cells in vivo alter the tumor microenvironment is not clearly understood.MethodsC57BL/6 mice were given a subcutaneous injection of B16F10 melanoma cell line (1 X 106 cells). Mice were given intraperitoneal injection of alpha-galactosylceramide (a-GalCer, a ligand for iNKT cells; 2 microgram/injection) on day +1, +5, +10, +15 and +20. NK cells, Gr1+ cells and F4/80+ macrophages in mice were depleted using cell-specific antibodies. The growth of tumors was monitored, and immune cells were characterized using flow cytometry and immunofluorescence staining. Student’s t-test and one-way ANOVA were used for statistical analysis.ResultsOur results showed that intratumoral NK T cells had significantly low expression of CD25, CD69, CD122, and IFN-gamma receptor molecules and produced lower inflammatory cytokines (IFN-gamma, TNF-alpha, and GM-CSF) as compared to splenic NK T cells. The soluble factor produced by B16F10 cells reduces the expression of these cytokines and cytokine receptors in vitro on the NK T cells purified from the spleen. Treatment of tumor-bearing mice with a-GalCer significantly increased the IFN-gamma-producing NK T cells, CD8+ T cells, and effector Th1 cells in secondary lymphoid organs, and tumors, also significantly reduced the tumor growth. Furthermore, a-GalCer treatment significantly increased the iNOS+CD206- M1-macrophages and reduced the iNOS-CD206+ M2-macrophages in the spleen and tumor. The depletion of F4/80+ macrophages prevented the a-GalCer-induced reduction of tumor growth.ConclusionsOur results showed that tumor produced soluble factors alter the phenotype of NK T cells. Activation of NKT cells with a-GalCer promotes the M1-macrophages, and effector CD8+ T cells, Th1 cells in the secondary lymphoid organs and tumor microenvironment. This finding suggests that activation of NKT cells may provide an effective anti-tumor response.AcknowledgementsThis work was supported by the Science and Engineering Research Board grant (EMR/2016/007108) and SwarnJayanti Fellowship (DST/SJF/LSA-01/2017–18) from the Department Science and Technology (DST), Government of India.Ethics ApprovalAll the procedures performed in the experiments involving mice were in accordance with the ethical standards of (NCCS) Institutional Ethics Committee of Animals Usage (Approval ID: EAF/B-166/2011 and EAF/B-256/2016).