¹Department of Urology, Nanjing Tongren Hospital, School of Medicine, Southeast University, Nanjing 210000, Jiangsu Province, CHINA DOI : 10.9775/kvfd.2026.36599 This study aimed to investigate whether sequential intravesical instillation of epirubicin (EPI) followed by interleukin-15 (IL-15)/interleukin-15 receptor alpha (IL-15Rα) could integrate cytotoxic and immune-stimulatory effects for improved antitumor efficacy. EPI-induced immunogenic cell death (ICD) was assessed in MB49 cells by measuring calreticulin (CRT) exposure and adenosine triphosphate (ATP) release. Cytotoxic activity of splenocytes against MB49 cells was evaluated by co-culture apoptosis assays and IFN-γ quantification. In vivo efficacy was examined in C57BL/6 mice bearing orthotopic bladder tumors. Tumor volume survival, and immune infiltration were analyzed, and CD8+ T cell depletion was performed to assess the contribution of these cells to therapeutic efficacy. EPI treatment increased surface CRT and ATP release. In co-culture assays, EPI pretreatment enhanced splenocyte-mediated MB49 cell apoptosis and IFN-γ secretion, and these effects were further augmented by IL-15/IL-15Rα. In vivo, sequential therapy significantly reduced tumor volume, prolonged survival, and promoted a favorable immune microenvironment, characterized by increased CD8+ T and NK1.1+ cell infiltration, reduced Treg population, an elevated CD8/Treg ratio, and increased IFN-γ level. Importantly, CD8+ T cell depletion attenuated these therapeutic benefits. Sequential intravesical administration of EPI and IL-15/IL-15Rα enhanced antitumor efficacy in an orthotopic bladder cancer model by promoting ICD-associated changes and cytotoxic immune responses. These findings provide preclinical evidence supporting further investigation of EPI-based chemo-immunotherapy for bladder cancer, including BCG-unresponsive or high-risk non-muscle-invasive bladder cancer (NMIBC). Keywords : Chemo-immunotherapy, EPI, IL-15/IL-15Rα, NMIBC