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

Fluorochrome-conjugated anti-mouse monoclonal antibodies (Abs) specific for CD8α, CD274, CD279, CTLA-4, CD86, tumor necrosis factor (TNF), interferon gamma (IFNγ), interleukin-2 (IL-2), CD4, FoxP3, NKp46, CD3, and interleukin-10 (IL-10) were purchased from eBiosciences (San Diego, CA) and diluted 1:200 prior to use. Anti-CD8 depletion antibodies were purified from the mouse 2.43 hybridoma cell line purchased from ATCC (Manassas, VA). Bioluminescent GL261-Luc 2 cells were derived and grown as previously described [6].

GL261-Luc2 cells were harvested by trypsinization, washed and resuspended at a concentration of 1–2x10⁷ cells/ml in DMEM without FCS and implanted into ten week old B6 (Cg)-Tyr ^c-23 /J (albino C57BL/6) mice (The Jackson Laboratory, Bar Harbor, ME) at an average weight of 19–20 g as previously described [5, 6, 13]. Briefly, animals were anesthetized by an intraperitoneal injection of ketamine (10 mg/kg) and xylazine (80 mg/kg), placed in a stereotactic apparatus and an incision was made over the cranial midline. A burrhole was made 0.1 mm posterior to the bregma and 2.3 mm to the right of the midline. A needle was inserted to a depth of 3 mm and withdrawn 0.4 mm to a depth of 2.6 mm. Two μl of GL261-luc2 cells (10⁷ cells/ml) were infused over the course of 3 min. The burrhole was closed with bonewax and the incision was sutured.

Following implantation surgery, animals were fed standard rodent chow for 3 days. Animals were then randomized to remain on standard diet (SD) or changed to a KD (KetoCal®; Nutricia North America, Gaithersburg, MD). The KD was obtained directly from the manufacturer and is a nutritionally complete diet providing a 4:1 ratio of fats to carbohydrates plus protein (72 % fat, 15 % protein, and 3 % carbohydrate). The KD was prepared by mixing KetoCal® with water (2:1) and fed to the animals each day (ad libitum). Bioluminescence was analyzed to quantify tumor burden as described [6]. Serum β-hydroxybutyrate (βHB) and glucose levels were measured using a Precision Xtra® blood monitoring system (Abbott Laboratories, Abbott Park, IL). Animals were weighed every 3–5 days and euthanized upon occurrence of visible symptoms of impending death such as hunched posture, reduced mobility and weight loss [5, 14]. Measurements of animal body weight, blood βHB, and glucose can be found in (Additional file 1: Figure S1).

Supernatant from 2.43 hybridoma cells was precipitated in saturated ammonium sulfate to 45 % (v/v) overnight at 4 °C and dialyzed against PBS for 24 h. The concentration of dialyzed antibody was determined by UV spectroscopy, and 0.3 mg of purified antibody was administered via intraperitoneal injection twice before tumor inoculation (day −5 and −3), and continued every three days after inoculation until euthanasia. CD8 T cell depletion was confirmed by flow cytometry analysis of peripheral blood mononuclear cells, as previously described [15]. Confirmation of CD8 depletion can be found in (Additional file 2: Figure S2).

When mice became symptomatic they were anesthetized with 80 mg/kg ketamine, 10 mg/kg xylazine followed by cardiac perfusion with ice-cold RPMI media just prior to euthanization. Tumor tissue and non-tumor contralateral brain were collected in RPMI media and run through a 70 μm filter. Tumor-infiltrating cells were isolated from tumor tissue by centrifugation over a 30/70 % Percoll gradient (Sigma-Aldrich, St. Louis, MO) before antibody staining and analysis of cell populations on an LSRFortessa flow cytometer (BD Biosciences, San Jose, CA). Flow cytometry data were analyzed with FlowJo8.8 (Tree Star Inc., Ashland, OR) and graphs were generated using Prism 5 software (GraphPad Software, La Jolla, CA). Gating strategies and isotype controls can be found in the Additional file 3: Figure S3 and Additional file 4: Figure S4 section.

Lymphocytes were cultured alone or stimulated with GL261-Luc2 cells at a density of 10⁶ cells per well (6-well plate). GolgiStop (BD Biosciences) was added at 1 h to inhibit export of cytokines and after a further 5 h of incubation, cells were stained for extracellular proteins. Permeabilization and intracellular staining for cytokines was done according to manufacturer’s instructions using the Cytofix/Cytoperm kit (BD Biosciences). Gating strategies and isotype controls can be found in the Additional file 5: Figure S5 and Additional file 6: Figure S6 section.

Lymphocytes were isolated from tumor tissue, and cultured alone or with GL261-Luc2 cells at varying effector to target cell ratios. Lactate dehydrogenase (LDH) ELISA was performed using CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI). Absorbance was recorded at 490 nm.

Six to 8-week-old female C57BL/6 mice were obtained from The Jackson Laboratory. All experiments were conducted under Arizona State University IACUC approval and followed all relevant federal guidelines and institutional policies. The Armstrong and clone 13 strains of Lymphocytic Choriomeningitis Virus (LCMV) were grown as previously described [16]. Mice were infected with 2 x 10⁵ PFU of LCMV (Armstrong) injected intraperitoneally or 2 x 10⁶ PFU of LCMV (clone 13) injected intravenously.

Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, San Diego, CA). All values are represented as the mean ± SD and significance was determined using both the Student’s t test and the Mann Whitney non-parametric test. P < 0.05 was considered statistically significant. For the Kaplan Meier survival data the log-rank (Mantel-Cox) test was used to assess statistical significance.

Tumor bearing animals maintained on the ketogenic diet (KD) had a greater median survival when compared to animals fed a standard diet (SD) (Fig. 1a). In order to effectively evaluate the importance of tumor-reactive CD8+ T cells in slowing tumor progression, CD8+ T cells were depleted from immune competent albino C57BL/6 mice bearing tumors. There was a significant decrease in survival of mice depleted of CD8+ T cells prior to tumor cell inoculation in comparison to wild type mice maintained on SD (Fig. 1b). In order to determine the importance of CD8+ T cells in the anti-tumor effects of the KD, CD8+ T cell depleted animals were treated with the KD and survival was measured. Although the KD significantly improved survival in immune intact mice when compared to those maintained on SD, that difference in survival is lost when CD8+ T cells are depleted and mice are treated with the KD in comparison to immune intact mice fed SD (Fig. 1c). Furthermore, the KD significantly increased survival in immune intact mice when compared to CD8 depleted mice fed KD (Fig. 1d). Analysis of bioluminescence data also shows slower tumor growth in animals treated with KD when compared to SD in both immune competent and CD8 depleted mice (Fig. 1e).

To evaluate the effects of the KD on immune cell infiltration into the tumor, amounts of tumor-infiltrating CD8+, CD4+, CD4 + FoxP3+, and NKp46 + CD3- cells were tested. There was no significant difference in the percentage of tumor-infiltrating CD8+ T cells between mice fed the KD and the SD (Fig. 2a). However, mice fed the KD had a significant increase in the percentage of CD4+ T cells infiltrating the tumor in comparison to SD (Fig. 2b). This increase in the percentage of CD4+ T cells was not due to an increase in the percentage of FoxP3 + CD4+ T regulatory (Treg) cells (Fig. 2c), and therefore the ratio of CD4+ T cells to Treg cells is significantly increased in tumors from mice fed a KD (Fig. 2e). In comparison, the CD8+ T cell to Treg cell ratio remained unchanged when comparing the two treatment groups (Fig. 2d). Lastly, there was no difference in the percentage of tumor-infiltrating NK cells in tumors from mice fed a KD compared to SD (Fig. 2f). Similar results were found when looking at the total number of infiltrating cells (data not shown). Therefore, the KD enhances CD4+ T cell presence at the tumor site, and this increase is not associated with an increase in the T regulatory cell subset.

Tumor cell expression of immune inhibitory checkpoint proteins is a major mechanism by which tumors limit the efficacy of immune responses in vivo. To examine the influence of the KD on immune inhibitory checkpoints we evaluated changes of immune inhibitory receptor expression on CD8+ tumor-infiltrating lymphocytes (TILs), and changes in expression of inhibitory ligands on the tumor cells. Mice fed the KD had significantly reduced expression of two inhibitory ligands, PD-1 (Fig. 3a) and CTLA-4 on CD8+ TILs (Fig. 3b). Additionally, mice fed the KD had reduced expression of CD86 (Fig. 3c) and PD-L1 (Fig. 3d) on the tumor cells. This suggests that the KD may alter tumor-mediated T cell suppression by reducing the number of cells that are susceptible to inhibition through the PD-1 and CTLA-4 inhibitory pathways.

To evaluate the influence of the KD on tumor-reactive immune cells at the tumor site, immune cell function from TILs removed from the tumor site at time of necropsy was tested. Intracellular cytokine staining following stimulation with tumor cells showed that when compared to SD, the KD significantly increases the ability of tumor-reactive CD8+ T cells to produce interferon gamma (IFNγ), tumor necrosis factor (TNF), and interleukin 2 (IL-2) when stimulated with GL261-Luc2 cells (Fig. 4a). Additionally, the KD significantly increased cytotoxic capabilities of tumor-reactive T cells from mice when compared to SD (Fig. 4b). The function of T regulatory cells was also assessed by intracellular cytokine staining for interleukin 10 (IL-10). Although we did not find a difference in the number of tumor-infiltrating Tregs, those found in the tumors from animals fed the KD produced significantly less IL-10 in response to GL261-Luc2 cells when compared to animals maintained on SD (Fig. 4c). Lastly, we studied natural killer (NK) cell function and found that tumor-infiltrating NK cells from mice fed the KD produce significantly more IFNγ and TNF in response to GL261-Luc2 cells than the cells isolated from SD fed animals (Fig. 4d). Whether through direct interaction with immune cells, or through alleviation of tumor immune suppression in the microenvironment, the KD significantly enhances tumor-reactive immune function.

To determine if the KD specifically enhances TIL function in the tumor microenvironment or alters global immune status, the effects of the KD on immune responses to two strains of Lymphocytic Choriomeningitis Virus (LCMV) was examined. Non-tumor bearing mice were infected with either LCMV Armstrong or Clone 13, and CD8 T cell function was accessed at Day 6 and 30. There was no significant difference in cytokine production by CD8+ T cells responding to either LCMV dominant epitopes, GP33 or NP396 (Fig. 5a) at either time point or with either infection regardless of diet. Additionally, there was no significant difference in the percentage of PD-1 + CD8+ T cells between KD and SD fed mice (Fig. 5b). Although the KD did not alter CD8 function against acute and chronic viral infections, it did alter immune mediated killing at the tumor site suggesting alleviation of immune suppression is specific to the tumor microenvironment.

This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of St. Joseph’s Hospital and Medical Center (protocol number 334 (A3510-01)). All surgery was performed under ketamine/xylazine anesthesia, and every effort was made to minimize suffering.

Not applicable.

The datasets supporting the conclusions of this article are included within the article and its supplementary files.