OX40L and IL-2 combination strategy for gastric cancer immunotherapy.

Background: Gastric cancer (GC) remains a major global health challenge with persistently low remission rates, largely because most patients are diagnosed at advanced stages. Although chemotherapy, targeted therapy, and immune checkpoint inhibitors have achieved progress, their benefits remain limited due to the highly immunosuppressive tumor microenvironment (TME), characterized by inadequate T-cell activation and impaired costimulatory signaling. Recent genomic and single-cell studies highlight the need for strategies that can more effectively enhance antitumor immunity. Given these challenges, this study aimed to evaluate whether dual stimulation with interleukin-2 (IL-2) and OX40 ligand (OX40L) could synergistically enhance T-cell activation in GC. Specifically, we analyzed OX40/OX40L expression patterns in GC tissues and tumor-infiltrating lymphocytes (TILs), assessed the immunostimulatory effects of IL-2 and OX40L in PBMCs and TILs, and tested a recombinant adenoviral vector co-expressing IL-2 and OX40L to determine its ability to potentiate T-cell-mediated cytotoxicity against primary GC tumor cells. Methods: Based on preliminary analyses of The Cancer Genome Atlas (TCGA) database and single-cell RNA sequencing (scRNA-seq) data, we identified the costimulatory molecules IL-2 and OX40L as potential therapeutic targets. We collected tumor tissues and paired peripheral blood mononuclear cells (PBMCs) from 70 untreated GC patients, and used bioinformatics tools to analyze T cell subpopulations and OX40/OX40L expression in the TME. The immunostimulatory effects of IL-2 and OX40L, used alone or in combination, were evaluated in vitro on PBMCs and TILs. A recombinant adenoviral vector co-expressing IL-2 and OX40L was also constructed to assess its pro-apoptotic effect in primary GC tumor cell cultures. Results: The combination of IL-2 and OX40L significantly enhanced T cell activation in both PBMCs and TILs, and increased the expression of antitumor effector molecules. The IL-2/OX40L adenoviral vector effectively activated TILs, which subsequently induced prominent apoptotic responses in primary GC tumor cells. Conclusions: Our study demonstrates that a costimulatory immunotherapy strategy combining IL-2 and OX40L significantly enhances antitumor responses in PBMCs and TILs from GC patients, showing strong therapeutic potential as a complementary approach for the treatment of GC.