Development of an in vitro immunosuppression assay using natural and induced regulatory T cells for functional assessment of CAR-T cells

• Grant number: 24/17902-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: January 01, 2025
• End date: December 31, 2025
• Field of knowledge: Biological Sciences - Immunology - Applied Immunology
• Principal Investigator: Lucas Eduardo Botelho de Souza
• Grantee: Raissa Raminelli Rezende
• Host Institution: Hemocentro de Ribeirão Preto. Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da USP (HCMRP). Secretaria da Saúde (São Paulo - Estado). Ribeirão Preto , SP, Brazil

Abstract

Adoptive transfer of T cells engineered to express chimeric antigen receptors (CAR) has shown impressive complete remission rates against B-cell malignancies. However, the efficacy of CAR-T cells against solid tumors, which constitute the majority of neoplasms, is still limited. This is because the microenvironment of solid tumors is dense and decorated with immunosuppressive cells and molecules, which hinders the infiltration and cytolytic activity of CAR-T cells. Current widely available models to evaluate the antitumor activity of CAR-T cells are based on immunodeficient animals or cocultures with tumor cells only, which do not recapitulate the obstacles associated with immunosuppression. Therefore, it is necessary to develop testing platforms that include immunosuppressive elements to challenge the performance of CAR-T cells, such as regulatory T cells (Tregs), which are associated with poor prognosis in patients with glioblastoma. Therefore, the aim of this project is to determine the relative amounts of natural or induced Treg cells via ectopic expression of Foxp3 (iTreg) capable of suppressing the antitumor activity of anti-GD2 CAR-T cells against glioblastoma cells in vitro. For this purpose, coculture assays will be performed between CAR-T cells, T98G glioblastoma cells and different proportions of Treg or iTreg cells. The suppression of antitumor activity will be determined by quantifying the bioluminescence emitted by the tumor cells. The direct immunosuppressive effect on CAR-T cells will be evaluated by a proliferation suppression assay via CFSE labeling and by quantifying the release of the immunosuppressive cytokines IL-10 and TGF-beta in the supernatants of the cocultures. We hope that this project will result in the creation of a platform to evaluate strategies that render CAR-T cells resistant to immunosuppression caused by Treg cells and, ultimately, contribute to increasing their therapeutic efficacy against solid tumors.