Abstract Introduction: Immune checkpoint inhibitors (ICI) targeting the PD-1/PD-L1 axis in Non-Small Cell Lung Cancer (NSCLC) have led to improved clinical benefit. However not all patients respond to treatment due to immunosuppressive barriers in the tumor microenvironment. Radiation Therapy (RT) in combination with ICI is a promising regimen however the underlying mechanisms are poorly understood. Our work identified combining a low dose Stereotactic Body Radiation Therapy (SBRT) in combination with ICI led to reduced tumor growth and improved survival in mouse models of NSCLC. The immunomodulatory role of RT was dependent on lung resident Scgb1a1+ club cells. In the absence of club cells, the improved survival benefit and cytotoxic T cell activity was lost. (Ban et al, Nature Cancer, 2021). These findings led to the hypothesis that radiation-induced club cell factors may synergize with ICI to improve therapeutic efficacy. Methods and Results: To expand upon radiation intrinsic mechanisms that can promote tumor immunity through activation of club cells, we decided to utilize C22 cells, an immortalized murine club cell line. Upon Irradiation of C22 cells with 3x 4Gy RT, the same dose that improves the efficacy of ICI in-vivo, we found that irradiated cells had increased G2M cell cycle arrest and had slightly increased apoptosis. We performed RNA sequencing analysis of C22 cells treated with RT (0gy or 4gy x3). Gene set enrichment analysis revealed upregulation of hallmark pathways such as Inflammatory response, Interferon alpha response and Tnfa signaling via Nf-kb pathway. RT-qPCR analysis confirmed a significant increase in mRNA expression of Isg15, Stat1, Oasl2, Il6, Ptgs2 and Ifnb1, key factors in Type 1 Interferon response pathway. Utilizing IFNb-YFP reporter mice, we have shown that irradiating the lungs at 3x-4GyRT leads to significantly increased expression of IFNb in club cells as compared to non-radiated controls. Moreover, pharmacologically blocking IFN signaling by treatment with anti-IFNAR1 antibody abrogates the therapeutic efficacy of RT in our mouse model of NSCLC. Conclusions: Irradiation can cause DNA damage in cells which can trigger the activation of the innate cGAS-STING pathway leading to the production of type 1 IFNs. Type 1 IFNs are potent cytokines that can enhance antigen presentation, induce Dendritic cell maturation, increase immune cell recruitment and cytotoxic capacity of T cells. By further understanding radiation induced inflammatory response role of club cells, our studies can guide future clinical trials of ICI to attenuate immunosuppressive barriers in NSCLC. Citation Format: Aakanksha R. Kapoor, Arshdeep Singh, Bharati Swami, Yongfeng He, Vivek Mittal. Novel insights into the immunoregulatory role of radiation activated club cells in non-small cell lung cancer abstract. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C043.
Kapoor et al. (Wed,) studied this question.