“Do not eat me’’: LILRB4 signaling as a novel macrophage immunotherapy in lung cancer 2254327

Abstract Introduction Lung cancer is the leading cause of cancer-related deaths. Although current T cell-targeting immunotherapies have improved patient survival, the efficacy is still low. Today, macrophage-mediated immune escape via “do not eat me” signaling is brought to light. The leukocyte immunoglobulin-like receptors (LILRs) are new targets for cancer immunotherapy aimed at tumor-associated macrophages (TAMs). Methods TAMs from lung cancer patients were FACS-sorted and processed for RNA sequencing. Opal multiplex staining on patient microarrays was used to investigate the cellular expression of LILRB4 in the tumor microenvironment. Peripheral blood mononuclear cells (PBMCs) were differentiated into macrophages and co-cultured with cancer cells to obtain in vitro TAMs. qPCRs and Western blotting were performed to evaluate the expression levels of LILRs on TAMs. The functional effect of LILRB4 knockdown TAMs (KD) on cancer cells was studied by flow cytometry. RNA-seq was conducted to elucidate how LILRB4 regulates phagocytosis in TAMs. Further LILRB4 blockage was applied on the ex vivo model of human precision-cut lung slices (PCLS) and in in vivo lung cancer models. Results A comprehensive analysis of the expression profiles of LILRs revealed that among LILRs, LILRB4 is highly expressed in TAMs. In vitro, TAMs-specific blockage of LILRB4 had no effect on cancer cell proliferation and apoptosis, but significantly increased their phagocytosis. RNA sequencing identified differential expressed genes in LILRB4 KD TAMs, which indicate that silencing of LILRB4 inhibits cell cycle progression of TAMs. This result was confirmed by flow cytometry and correlated with reduced phagocytic activity in the G2/M phase. In addition, blocking LILRB4 ex vivo and in vivo reduced tumor growth and differentially affected the immune cell composition. Conclusion Taken together, our data suggest that LILRB4 may play a role in macrophage-specific immune evasion in lung cancer and is a promising target for lung cancer immunotherapy. Funding Source Institute for Lung Health Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)