ABSTRACT: CAR-T cell therapy has transformed the treatment of hematological malignancies; however, its broad application is limited due to toxicities and high cost. As an alternative, NK cells offer advantages, including allogeneic use and intrinsic antitumoral activity. Nevertheless, efficacy can be compromised by the immunosuppressive microenvironment, especially through the action of Treg cells. To overcome this obstacle, we proposed the coexpression of GITRL in CAR19-NK cells, aiming to enhance cytotoxicity against CD19⁺ cells and modulate the tumor microenvironment. CAR19-NK-92 and CAR19-GITRL-NK-92 cells, as well as peripheral blood-derived NK cells (PB-NK), were generated. These cells were evaluated regarding their expansion, expression stability, immunophenotype, metabolism, cytotoxicity, cytokine secretion, degranulation, and effects on Tregs. In vivo assay in a murine leukemia model was performed to evaluate tumor burden and survival. CAR19-GITRL-NK-92 cells exhibited greater expansion capacity, a more active metabolic profile (glycolysis and ATP production), in addition to increased expression of NKp44 and NKp46. Both CAR cells exhibited cytotoxicity nearly 40% higher than that of NK-92 cells, accompanied by increased cytokine secretion (TNF-α and IFN-γ) and degranulation. Although the presence of GITRL did not directly enhance tumor cell lysis in vitro, its expression was upregulated following CAR activation by target cells. RNA-Seq analyses revealed activation of pro-effector pathways (KIT, IRF5) and downregulation of inhibitory signals (IL1RN). For the in vivo assay, CAR19-GITRL-NK-92 cells exhibited enhanced tumor control and prolonged survival. In primary NK cells, coexpression of GITRL increased in vitro cytotoxicity and conferred partial resistance to Treg-mediated suppression, with a 43% reduction in growth, compared to 79% observed in CAR19-PB-NK cells. In conclusion, GITRL coexpression in CAR19-NK cells led to a more active metabolism, enhanced activation of pro-cytotoxic pathways, and superior tumor control in a murine model, as well as partial resistance to Treg-mediated immunosuppression in primary cells. Future studies, particularly in vivo, will be essential to validate and extend these findings. These results emphasize the translational potential of CAR19-GITRL-NK cells as an innovative strategy for the treatment of B-cell leukemias and lymphomas.
