Enhanced immunity in a mouse model of malignant glioma is mediated by a therapeutic ketogenic diet

Abstract

Background: Glioblastoma multiforme is a highly aggressive brain tumor with a poor prognosis, and advances in treatment have led to only marginal increases in overall survival. We and others have shown previously that the therapeutic ketogenic diet (KD) prolongs survival in mouse models of glioma, explained by both direct tumor growth inhibition and suppression of pro-inflammatory microenvironment conditions. The aim of this study is to assess the effects of the KD on the glioma reactive immune response.

Methods: The GL261-Luc2 intracranial mouse model of glioma was used to investigate the effects of the KD on the tumor-specific immune response. Tumor-infiltrating CD8+ T cells, CD4+ T cells and natural killer (NK) cells were analyzed by flow cytometry. The expression of immune inhibitory receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed death 1 (PD-1) on CD8+ T cells were also analyzed by flow cytometry. Analysis of intracellular cytokine production was used to determine production of IFN, IL-2 and IFN- in tumor-infiltrating CD8+ T and natural killer (NK) cells and IL-10 production by T regulatory cells.

Results: We demonstrate that mice fed the KD had increased tumor-reactive innate and adaptive immune responses, including increased cytokine production and cytolysis via tumor-reactive CD8+ T cells. Additionally, we saw that mice maintained on the KD had increased CD4 infiltration, while T regulatory cell numbers stayed consistent. Lastly, mice fed the KD had a significant reduction in immune inhibitory receptor expression as well as decreased inhibitory ligand expression on glioma cells.

Conclusions: The KD may work in part as an immune adjuvant, boosting tumor-reactive immune responses in the microenvironment by alleviating immune suppression. This evidence suggests that the KD increases tumor-reactive immune responses, and may have implications in combinational treatment approaches.

Keywords: CTLA-4; Glioblastoma; Glioma; Immune inhibitory checkpoints; Immunology; Immunosuppression; Ketogenic diet; Metabolism; Microenvironment; PD-1.

Figures

Fig. 1

Enhanced survival with the ketogenic diet is mediated in part by CD8 T cells. Kaplan-Meier survival curves for ketogenic diet (KD) versus standard diet (SD) (a), SD versus SD + CD8 depletion (b), SD versus KD + CD8 depletion (c), KD versus KD + CD8 depletion (d). Bioluminescent tumor signals plotted as in vivo photon count versus days post-implantation (e). N = 12 for immune competent mice; N = 5 for CD8 depleted mice; Log-rank (Mantel-Cox) test; p-values indicated on graphs

Fig. 2

CD4+ T cell infiltration increases in mice fed the KD, without increases in Treg cell numbers. Flow cytometry analysis was performed to assess the cell types infiltrating tumors from mice fed both SD and KD. CD8 T cells (a), CD4 T cells (b) and CD4 + FoxP3+ T regulatory cells (c) were assessed. The ratio of CD8 T cells to T regulatory cells (d) and CD4 to T regulatory cells (e) were determined. The percent of infiltrating NKp46 + CD3- natural killer cells (f) were also assessed. N = 5; student’s two-tailed t-test; ***p < 0.001; ****p < 0.0001

Fig. 3

The ketogenic diet reduces expression of immune inhibitory receptors and ligands expressed in glioma tumors. Expression of the immune inhibitory receptors, PD-1 (a) and CTLA-4 (b) on infiltrating CD8 T cells isolated from tumors from mice fed each diet were assessed. Expression of the immune inhibitory ligands, CD86 (c) and PD-L1 (d), on GL261-Luc2 tumor tissue was also assessed. N = 5; student’s two tailed t-test; *p < 0.05; **p < 0.01; ****p < 0.0001

Fig. 4

The ketogenic diet significantly enhances tumor-reactive CD8+ T cell and NK cell activity. Tumor-infiltrating lymphocytes (TILs) isolated from gliomas from mice fed KD versus SD were cultured alone (white bar) or in the presence of GL261-Luc2 tumor cells (black bar) to access activity. Analysis of IFNγ, TNF and IL-2 production in tumor-infiltrating CD8+ T cells was performed (a). Cytotoxic capability of CD8+ T cells isolated from tumors was assessed following exposure to GL261-Luc2 cells (b). IL-10-production in CD4 + FoxP3+ T regulatory cells was also assessed in response to stimulation with GL261-Luc2 cells (c). IFNγ and TNF production in the infiltrating NKp46 + CD3- natural killer cells isolated from tumors were assessed (d). N = 5; student’s two-tailed t-test between the antigen-challenged SD and KD groups only; *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 5

The ketogenic diet had no effect on T cell activity in an acute and chronic mouse model of LCMV infection. Splenocytes from non-tumor bearing mice infected with LCMV Armstrong or Clone 13 were isolated at day 6 and 30, and stimulated with GP33 or NP396 antigens. IFNγ + TNF + CD8+ cells in mice fed SD versus KD (a). PD-1 + CD8+ expression in mice fed SD versus KD (b). N = 5 in each group