The role and mechanisms of interleukin-9 in facilitating anticancer effect of immune checkpoint blockade in lung cancer animal model

Abstract

Lung cancer remains the major reason for cancer-related deaths. Immune checkpoint blockade (ICB), particularly with the use of programmed cell death protein 1 (PD-1)/programmed cell death protein ligand 1 (PD-L1) antibodies, has gained major advancement in cancer therapy throughout the recent decade, nonetheless only a minority of patients may benefit. Therefore, novel immunotherapeutic targets and combination strategies are urgently needed to II expand the clinical benefit to wider patient population. Interleukin-9 (IL-9) is known to be important in modifying tumor biology and has pleiotropic effects in different cancers. In lung cancer, the function of IL-9 is not fully elucidated. The purpose of this study was to better understand the function(s) of IL-9 in lung cancer and the underlying mechanisms. Lewis lung carcinoma (LLC) and CMT167 murine lung cancer cell lines were utilized in this study. IL-9 receptor (IL-9R) was only expressed in CMT167 cells, not LLC cells, but neither of their viability was altered by IL-9 in vitro. However, IL-9 led to tumor suppression and facilitated intratumoral T lymphocyte immunity in IL-9R-positive CMT167 model, not in IL-9R-lacking LLC model. Upon IL-9R knockdown, IL-9 lost its efficacy to induce anticancer immunity and tumor inhibition in CMT167 model. In CMT167 tumors, although IL-9 elevated both tumor-infiltrating CD4+ and CD8+ T lymphocytes, it was the cytotoxic T cell subset that mediated the tumor suppression by IL-9. To understand the mechanisms of IL-9-enhanced CD8+ T cell responses in CMT167 tumors, T cell activation markers, recruitment chemokines, dendritic cells (DCs) frequencies, and tumor cell-surface major histocompatibility complex (MHC)-I expression were investigated. Increased DCs and upregulated MHC-I were observed in CMT167 tumors upon IL-9 therapy, both of which were III absent in IL-9R knockdown CMT167 model. In vitro, IL-9 increased MHC-I expression in CMT167 and human lung adenocarcinoma A549 cells consistently. The signal transducer and activator of transcription 1 (STAT1) and protein kinase B (Akt) pathways in CMT167 cells were not affected, while phosphorylation of extracellular signal-regulated kinase (ERK) was enhanced by IL-9. The upregulation of MHC-I by IL-9 in CMT167 cells was abrogated by ERK inhibition. Previously, others have reported a positive correlation of IL-9-producing T helper type 9 (Th9) cells with the therapeutic efficacy of PD-1 blockade in cancers, demonstrating that IL-9 and PD-1/PD-L1 inhibition might have a potential synergism. Indeed, besides the enhanced anti-tumor T cell immunity, IL-9 also increased the PD-1 expression on CD8+ T lymphocytes and PD-L1 expression on CMT167 cells. Combined treatment with IL-9 and PD-1 blockade further increased intratumoral cytotoxic T lymphocytes and synergistically inhibited the growth of CMT167 tumors. In summary, IL-9/IL-9R interaction in CMT167 tumors could promote tumoral MHC-I presentation and increase DCs frequencies, leading to tumor growth suppression by enhanced cytotoxic T lymphocyte immunity. Intratumoral IL-9R expression might potentially be investigated as a selection biomarker for lung cancer subsets susceptible to IL-9 therapy. The findings from this study lend support IV for future clinical evaluation of IL-9 as an immunomodulatory agent in combination with PD-1 blockade in lung cancer.