The disialyl gangliosides GD2/GD3 have been implicated in the enhancement of malignancy in a number of human and animal cancers and as a tumor antigen target for immunotherapy. Earlier we reported on a GD3-based vaccine, which protects canine melanoma patients when provided as an adjunct to standard of care (1). Despite a significant increase in the median survival time, the mechanism by which vaccine works remains unclear; therefore, we vaccinated C57BL/6 mice with the GD3-vaccine formulation and appropriate controls and evaluated changes in the immune cells. Methods: C57BL/6 mice were vaccinated weekly for 4 weeks followed by a week of rest. Mice were then euthanized to collect blood, liver, and spleen for evaluation of NKT-cells. Liver lymphocytes were collected by making mono-cellular suspension of liver obtained after enzymatic digestion. Collected lymphocytes were stained for CD3, CD4, TCRβ, NK1.1, CD49b and dead cells. In these experiments untreated and α-galactosylceramide (α-GalCer) treated mice were used as controls. Results: Mice treated with the subcutaneous injections of the GD3 vaccine showed an increase in NKT-cells (NK1.1+CD49b+CD3+CD4+TCRb+) in the liver but failed to show an increase in the spleen and blood when compared with the untreated mice. The mice treated with α-GalCer also showed an accumulation of NKT-cells in the liver; however, the increase in the livers obtained from vaccine-treated mice was 5-10-fold higher compared to the α-GalCer treated mice. Discussion: NKT-cells are the Swiss army knife of the immune system which are capable of producing different cytokines and chemokines to regulate the overall immune system. After their discovery two decades ago, activation of NKT-cells have shown to have a crucial protective role in various infectious, and non-infectious diseases. These cells form a bridge between the innate and adaptive immune cells. The activation and maintenance of these cells are dependent upon the presentation of lipid molecules on CD1 receptors by dendritic cells. Since the target antigen in the vaccine is a lipid (GD3) it is very likely it would be presented on CD1 receptors for the activation of NKT-cells. Due to the lack of validated and suitable canine CD1 receptor reagents, we were unable to define the mechanism behind the hypothetical protection found in the canine GD3 based vaccine (1). In the murine model NKT-cells are well characterized, and provided us with an opportunity to understand the possible selective activation of NKT-cells by the GD3 based vaccine. Our data found an increase in NKT-cells in the liver for mice vaccinated with GD3 and α-GalCer, but no discernible differences were found in blood and spleen between vaccinated mice and normal controls. Further investigation using the B16 melanoma cell line in C57BL/6 mice vaccinated with the GD3 vaccine and suitable controls may identify the NKT response in the tumor microenvironment. Conclusions: While the GD3-based vaccine and α-GalCer both increased NKT-cells in the murine liver, GD3 increased NKT counts 5-10 fold over α-GalCer. In addition, monitoring changes in NKT numbers in the peripheral blood may not be of benefit due to low cell counts. Acknowledgment: The study is funded by a grant from the American Kennel Club Health Foundation and The UF CVM Foundation. Reference: 1. Milner RJ. The immune response to disialoganglioside GD3 vaccination in dogs with spontaneous occurring melanoma: A large animal translational model. Poster # 123628_1 presented at: Proceeding American Association for Cancer Research, Tumor Immunology: Basic and Clinical Advances; 2010 Dec 30; Miami Beach, Florida.

Citation Format: Rowan J. Milner, Bikash Sahay, Matthew Cascio, Marc Salute. A protective GD3-based vaccine increases NKT-cells in a C57BL/6 murine model [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A204.