The adoptive transfer of T lymphocytes expressing chimeric antigen receptors (CAR) has resulted in impressive complete remission rates in B-cell malignancies. However, the therapeutic efficacy of CAR-T cells is still low or non-existent against solid tumors, which make up the vast majority of neoplasms. One of the main reasons for this failure is the presence of ligands and immunosuppressive cells in the tumor microenvironment. The complete activation of T cells requires the engagement of costimulatory molecules whose expression is temporally segregated and whose nature of biochemical signals are complementary. This is the case of CD28, expressed constitutively, and GITR, expressed right after the initial activation of T cells. Therefore, our hypothesis is that the combined expression of a CAR containing the costimulatory domain CD28 and the GITR ligand (GITRL) will potentiate the antineoplastic action of CAR-T cells. In addition to providing complementary costimulatory signals for effector CAR-T cells, GITRL can suppress the action of regulatory T cells in the tumor microenvironment, thus possibly reducing local immunosuppression. Then, the aim of this study is to evaluate the therapeutic efficiency of anti-GD2 CAR-T cells coexpressing GITRL in a preclinical model of glioblastoma multiforme. In this project, we used as a model a CAR against the ganglioside GD2, which is highly immunogenic and expressed in tumors of neuroectodermal origin, such as glioblastoma. To test our hypothesis, we generated two lentiviral vectors to express CAR anti-GD2 and another one coexpressing GITRL as a costimulatory domain. After that, we produced lentiviral particles to transduced human T lymphocytes and we detected CAR expression in the T cells transduced with both vectors. To verify the functional activity of these CAR-T cells, we performed a coculture assay against T98G cell line, which express high levels of GD2, and HCT-166 cell, that dont express. These cell lines were previously modified to express luciferase to enable the analysis through bioluminescence imaging. We tested two ratios of effector:target cells (E:T) and we observed that both CAR.GD2 and CAR.GD2-GITRL T cells demonstrated powerful capacity of lysis only against cells that express GD2. Then, we interrogated whether these CAR-T cells could keep their lysis capacity after additional rechallenges. So, another two rounds of coculture were performed and the cells showed persistence of cytotoxicity. In addition, tumor cell killing was associated with secretion of IFNy as evaluated by ELISA quantification of coculture supernatants. These data encouraged us to compare the antineoplastic activity and persistence of CAR-T cells anti-GD2 in an orthotopic pre-clinical model of glioblastoma multiforme, which is under way. We expect that the potential results of this project will foster the development of a new advanced cellular immunotherapy strategy for the treatment of solid GD2+ neoplasms and will pave the way to increase the antineoplastic efficiency of CAR-T cells against other malignancies.