Coexpression of GITRL confers resistance to Treg cell-mediated immunosuppression to anti-CD19 CAR-NK cells

Abstract

Introduction Regulatory T cells play a pivotal role in shaping immune interactions, limiting antitumor immunity, and impairing the activity of engineered effector cells. Overcoming regulatory T (Treg) cell–mediated immunosuppression is critical to unlock the full therapeutic potential of chimeric antigen receptor (CAR)-cell therapies. Here, we develop “armored” anti-CD19 CAR-NK cells coexpressing GITR ligand (GITRL) (CAR19-GITRL-NK) to both target malignant B cells and locally counteract Treg-mediated suppression. GITR is highly expressed on Tregs, and engagement by its ligand GITRL can modulate their function, providing a rational strategy to reprogram the tumor microenvironment. Methods We generated anti-CD19 NK-92 and primary peripheral blood NK (PB-NK) cells with or without GITRL coexpression using lentiviral vectors, and evaluated their functionality and therapeutic potential. CAR19-GITRL-NK-92 cells were analyzed regarding their expansion, metabolism, cytotoxicity, cytokine secretion, degranulation, and in vivo antitumor potential. The cytotoxic activity of CAR19-GITRL PB-NK cells was examined, and their growth was measured in the presence of T CD4⁺ cells transduced with FoxP3 expression vector (iTreg-FoxP3) used as a model for Treg cells. Results CAR19-GITRL-NK-92 cells displayed superior expansion and enhanced metabolic fitness. Functionally, both CAR constructs increased the cytotoxicity of NK-92 and cytokine secretion against CD19⁺ Nalm-6 and Namalwa cells in vitro. RNA-Seq revealed 54 upregulated and 34 downregulated genes in CAR19-GITRL-NK-92 versus CAR19-NK-92 cells, with enrichment of pathways linked to immune activation, proliferation, chemotaxis, and cytotoxicity, including increased expression of NCR2, CD2, CMKLR1, IRF5, KIT, and PTPN3. In vivo, CAR19-GITRL-NK-92 cells achieved superior tumor control and the longest overall survival. In primary NK cells, CAR19-GITRL enhanced killing of CD19⁺ targets, and GITRL expression attenuated iTreg-FoxP3 mediated inhibition in CAR-NK cells, indicating a resistance mechanism to Treg suppression. Discussion Collectively, these data position CAR19-GITRL-NK cells as a potent and innovative strategy that couples direct tumor targeting with active undermining of Treg-driven immunosuppression for the treatment of B-cell leukemias and lymphomas.