2926 - ILT4, Immunosuppression, and Resistance to Radioimmunotherapy (iRT) in Non-Small Cell Lung Cancer (NSCLC) through Recruiting SHP2 to Induce Mitochondrial Oxidative Stress-Mediated Tumor-Associated Mac

M. Yuan^1,2, X. Chen³, J. Yu⁴, and D. Chen¹; ¹Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China, ²Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China, ³Department of Radiation Oncology, Qilu Hospital of Shandong University, Jinan, Shandong, China, ⁴Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China

Purpose/Objective(s): Immunotherapy combined with radiotherapy (iRT) has unlimited potential, but up to 60% of cancer patients do not benefit from it. Enhancing the anti-tumor immune stimulatory effect triggered by radiotherapy is the key to overcome iRT resistance. The senescence of tumor-associated macrophages (TAMs), which play a crucial role in regulating the tumor microenvironment (TME), and its mechanisms and role in RT remain unclear. This study demonstrates for the first time that RT can induce TAM senescence, thereby fostering an immunosuppressive TME and mediating resistance to iRT; with Immunoglobulin-like transcript 4 (ILT4) being a key regulator. We propose that targeting ILT4 to eliminate senescent TAMs and optimize the TME may reverse resistance to iRT. Materials/

Methods: Using mIF and CyTOF technology to analyze ILT4 expression, the phenotype of senescent macrophages/T cells and clinical efficacy in NSCLC patients before and after iRT. Using full/conditional knockout mice in situ/subcutaneous tumor models, single-cell sequencing, and mFCM, we assessed the immune suppressive and tumor-promoting roles of senescent TAMs dependent on ILT4, as well as using senescent cell and macrophage-specific eliminators. Transcriptomic sequencing of human TAMs treated with ILT4 monoclonal antibodies or Pirb-/- mouse bone marrow cells, along with co-IP, GST-pull down, and further experiments using SHP2/mitochondrial oxidative stress inhibitors, confirmed the specific mechanism by which ILT4 regulates RT-induced TAM senescence. By establishing huHSC-NCG myeloid cell humanized mouse models resistant to ICB treatment, the study assessed the effectiveness of blocking TAMsPirb in reversing resistance to iRT.
Results: Analysis of clinical samples showed a significant increase in TAM senescence and ILT4 expression after RT, associated with decreased CD8+ T cell infiltration and poor iRT treatment outcomes. Mouse models and primary immune cell in vitro further confirmed that RT-induced TAM senescence and secretion of SASP mediated CD8+ T cell dysfunction and RT resistance; RT promoted ILT4 upregulation, and blocking ILT4 significantly reversed this process, enhancing the effectiveness of RT. Effective counteraction of ILT4-induced CD8+ T cell functional suppression and resistance to RT was achieved by eliminating macrophages or senescent cells. Using SHP2 inhibitors and mitochondrial oxidative stress inhibitors, we unveiled that ILT4 promotes TAM senescence and immunosuppression by recruiting SHP2 to activate the mitochondrial oxidative stress pathway. Most critically, blocking ILT4 not only enhanced the efficacy of RT but also synergistically overcame primary and secondary resistance to ICBs.
Conclusion: This study reveals for the first time RT-induced TAM senescence and its role in NSCLC immunosuppression and resistance to iRT, confirming a novel mechanism by which ILT4 promotes TAM senescence through recruiting SHP2 to activate the mitochondrial oxidative stress pathway.