Targeting Mouse Glioblastoma cells with gp100/Pmel-Specific Cytotoxic T lymphocytes to evaluate the role of the tumor redox state on the CTL efficacy.

Glioblastoma (GBM), the most aggressive primary brain tumor, consists of a subset of glioma cells with self-renewal abilities that can recapitulate the entire tumor even after surgical resection or chemoradiotherapy. Patients diagnosed with this difficult-to-treat disease have only 14.6 months of median survival due to its poor prognosis since it can evade immune responses and resist treatments. Immunotherapy, especially the use of chimeric antigen receptor (CAR) T cells, shows promise, but it doesn't work very well in the hostile tumor microenvironment of GBM. A critical component of this environment is oxidative stress, which can impact immune cell function and lead to decreased immune cell activity. Due to its rather hypoxic and inflamed nature, the GBM tumor microenvironment is hostile to the T cells. Excessive ROS compromises CTL activity. The host laboratory has shown that the tumor’s redox status influences the efficacy of the CTL in vitro. These results need to be validated in vivo. Therefore, my project consisted of building an in vivo model to test the impact of the target cells redox status on the CTL. This model consisted of murine glioma cells SB28 that I transfected to express melanoma tumor antigen gp100/Pmel in order to render the SB28 sensitive to pmel CTL isolated from transgenic mice expressing T cell receptor specific for the gp100/pmel antigen. As effector cells I crossed the pmel mice with CD30 knockout animals to generate pmel/CD30 knockout mice. This model will be used to evaluate the ability of pmel CTL to eliminate SB28pmel and oxidized SB28pmel where thioredoxin reductase 2 and catalase expression has been disrupted, respectively, by CRISPR/CAS9 and shRNA technology. This in vivo model will allow a better understanding of the ROS impact on the adaptive immune response. This is essential for the development of more potent immunotherapies and therapeutic interventions against GBM

Glioblastoma (GBM), the most aggressive primary brain tumor, consists of a subset of glioma cells with self-renewal abilities that can recapitulate the entire tumor even after surgical resection or chemoradiotherapy. Patients diagnosed with this difficult-to-treat disease have only 14.6 months of median survival due to its poor prognosis since it can evade immune responses and resist treatments. Immunotherapy, especially the use of chimeric antigen receptor (CAR) T cells, shows promise, but it doesn't work very well in the hostile tumor microenvironment of GBM. A critical component of this environment is oxidative stress, which can impact immune cell function and lead to decreased immune cell activity. Due to its rather hypoxic and inflamed nature, the GBM tumor microenvironment is hostile to the T cells. Excessive ROS compromises CTL activity. The host laboratory has shown that the tumor’s redox status influences the efficacy of the CTL in vitro. These results need to be validated in vivo. Therefore, my project consisted of building an in vivo model to test the impact of the target cells redox status on the CTL. This model consisted of murine glioma cells SB28 that I transfected to express melanoma tumor antigen gp100/Pmel in order to render the SB28 sensitive to pmel CTL isolated from transgenic mice expressing T cell receptor specific for the gp100/pmel antigen. As effector cells I crossed the pmel mice with CD30 knock out animal to generate pmel/CD30 knockout mice. This model will be used to evaluate the ability of pmel CTL to eliminate SB28pmel and oxidized SB28pmel where thioredoxin reductase 2 and catalase expression have been disrupted, respectively, by CRISPR/CAS9 and shRNA technology. This in vivo model will allow a better understanding of the ROS impact on the adaptive immune response. This is essential for the development of more potent immunotherapies and therapeutic interventions against GBM