Simple SummaryLung cancer is the leading cause of cancer-related deaths. Immunotherapy activates the patient’s immune system to identify and kill cancer cells. Moreover, memory immune cells are formed that prevent the recurrence of cancer, leading to durable responses. However, only 20% of patients benefit from immunotherapy because the tumor-derived factors suppress the immune response. Herein, we tested if Withaferin A (a herbal compound) can make immunotherapy more effective in lung cancer patients. We found that Withaferin A induces the production of molecules from lung cancer cells that increase the infiltration of immune cells but are not able to kill cancer cells. Notably, in an immunocompetent mouse model of lung cancer, treatment with a combination of Withaferin A and an immunotherapy regimen showed more effectiveness than immunotherapy alone in activating immune cells and reducing tumor growth. This study presents a novel approach that can be tested clinically to improve lung cancer immunotherapy.AbstractTreatment of late-stage lung cancers remains challenging with a five-year survival rate of 8%. Immune checkpoint blockers (ICBs) revolutionized the treatment of non-small cell lung cancer (NSCLC) by reactivating anti-tumor immunity. Despite achieving durable responses, ICBs are effective in only 20% of patients due to immune resistance. Therefore, synergistic combinatorial approaches that overcome immune resistance are currently under investigation. Herein, we studied the immunomodulatory role of Withaferin A (WFA)—a herbal compound—and its effectiveness in combination with an ICB for the treatment of NSCLC. Our in vitro results show that WFA induces immunogenic cell death (ICD) in NSCLC cell lines and increases expression of the programmed death ligand-1 (PD-L1). The administration of N-acetyl cysteine (NAC), a reactive oxygen species (ROS) scavenger, abrogated WFA-induced ICD and PD-L1 upregulation, suggesting the involvement of ROS in this process. Further, we found that a combination of WFA and α-PD-L1 significantly reduced tumor growth in an immunocompetent tumor model. Our results showed that WFA increases CD-8 T-cells and reduces immunosuppressive cells infiltrating the tumor microenvironment. Administration of NAC partially inhibited the anti-tumor response of the combination regimen. In conclusion, our results demonstrate that WFA sensitizes NSCLC to α-PD-L1 in part via activation of ROS.