Overcoming Radio-Immunotherapy Treatment Resistance through ILT4 Blockade and Reversal of HFRT Induced CXCL1-CXCR2 Axis Activation and Tumor-Associated Macrophage Immunosuppression

Abstract

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. Immunoglobulin-like transcript (ILT) 4 is a potential immune checkpoint molecule, highly expressed in various tumor cells, but its role in radiotherapy is still unknown. This study confirmed the role and molecular mechanism of ILT4 in suppressing radiotherapy immunosuppressive microenvironment formation and promoting tumor radiotherapy resistance. We propose a new therapeutic strategy that block ILT4 to enhance the efficacy of radiotherapy, and cooperate with radiotherapy to reverse immunotherapy resistance. Using multiplex immunohistochemistry, we analyzed ILT4 expression, tumor-associated macrophage (TAM) /T cell phenotype and quantity in tumor patient treated with SBRT. Using mice subcutaneous tumor models, Single-cell RNA sequencing and multiplex flowcytometry, we assessed the role of ILT4 inhibition and hyper-fractionated radiotherapy (HFRT) on preventing tumor growth and immune escape. The molecular signaling and cytokines regulated by ILT4 under HFRT were analyzed by transcriptome sequencing and further verified by molecular experiments. By establishing cancer cell/TAM co-culture system in vitro, using CXCL1 protein or CXCR2 inhibitor and macrophage/CD8+ T cell deletion antibody in vivo, we identified the downstream pathway and cytokine of ILT4 to enhancing HFRT -induced TAM immune response. In the tumor specimens of NSCLC patients treated with SBRT, we found that high ILT4 expression predicted poor progression-free survival and more M2-TAM recruitment. Among the C57BL/6 mice model, ILT4 inhibition in cancer cells reduced HFRT mediated M2-TAMs accumulation, and to sustain activation and proliferation of CD8+ T cells, and eventually suppressed tumor progression. Mechanistically, RT promoted ILT4 expression, which subsequently induced NF-κB pathway activation and CXCL1 secretion to enhance M2-TAMs migration in vitro. Using CXCL1 protein or CXCR2 inhibitor administration, inferring that ILT4 promotes TAMs migration via NF-κB-CXCL1-CXCR2 axis. Consistently, depletion of TAMs blocked the T cell function impairment and radiotherapy resistance induced by ILT4 in vivo. Importantly, targeting ILT4 potentiated the effect of radiotherapy, overcomes radio-immunotherapy treatment resistance. ILT4 mediates HFRT-induced M2-like TAMs recruitment and subsequently T cell response impairment by regulating NF-κB-CXCL1-CXCR2 axis. ILT4 is an attractive drug target for enhancing radiotherapy and overcomes radio-immunotherapy treatment resistance.