Purpose: T-cell inhibitory receptors and their ligands, and their roles in the tumor immune response have been extensively studied recently. Cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed cell death 1 (PD-1) or T cell immunoglobulin and mucin domain 3 (TIM-3) have been identified on the CD4 and CD8 T-cell surface, and it has been shown that the pathways of CTLA-4 and its ligand CD80 or CD86 (B7-2), PD-1 and its ligand PD-L1 or PD-L2 (B7-DC), and TIM-3 and its ligand Galectin-9 axes contributed to effector T-cell dysfunction in the tumor immune microenvironment. Those T-cell inhibitory receptors and their ligands also play important roles in the immune response in lung adenocarcinoma. In this study, we investigated inhibitory roles of TIM-3/Galectin-9 pathway in lung adenocarcinoma.
Experimental Design: TILs or PBMCs were obtained from lung cancer patients. Expression of inhibitory molecules on those cells was analyzed by flow cytometry using FACS. PD-L1 and Galectin-9 expression in lung cancer was analyzed using tissue microarray by immunohistochemistry (IHC). To investigate soluble Galectin-9 released from the tumor cells, an EGFR-mutated tumor cell line, PC-9 and control EGFR wild type cell line, OU-LC-SK were treated with Afatinib at a concentration of 10 nM for 3 days. Then, the amount of Galectin-9 in culture supernatant was measured by ELISA. To investigate T-cell apoptosis induction by Galectin-9, established XAGE1-specifiic CD8 cloned T-cells were incubated with Galectin-9 protein for 8 hrs.
Results: First, we examined expression of PD-1, TIM-3, BTLA and LAG-3 on CD4 and CD8 T-cells obtained from PBMCs and TILs in 11 lung cancer patients. Augmented expression in TILs compared to PBMCs was observed on PD-1 and TIM-3, but not on BTLA or LAG-3 in CD4 and CD8 T-cells. Next, we analyzed expression of the T-cell inhibitory receptor ligands PD-L1 and Galectin-9 in lung cancer by IHC. With adenocarcinoma, the frequencies of high PD-L1 and Galectin-9 expression in the cell membrane or cytoplasm were 49% and 31%, respectively. On the other hand, with squamous cell carcinoma, the frequencies were 32% and 16%, respectively. Furthermore, correlated expression of PD-L1, Galectin-9 and CD3 (T-cell infiltration) was observed at the periphery of the tumor nest. Those findings suggest the relevance of the PD-1/PD-L1 and TIM-3/Galectin-9 pathways in the tumor microenvironment in lung cancer. To investigate soluble Galectin-9 released from the tumor cells, EGFR-mutated and -wild type tumor cell lines were treated with EGFR-TKI. Galectin-9 was detected in the medium of EGFR-mutated lung cancer cell lines following treatment with Afatinib. Moreover, apoptosis was induced in TIM-3-positive CD8 T-cell clones following interaction with Galectin-9 protein and this was inhibited by the addition of anti-Galectin-9 or an anti-TIM-3 antibody. The findings suggested that a significant amount of Galectin-9 could be released and induced T-cell apoptosis in tumor microenvironment.
Conclusions: The results suggested the relevance of the PD-1/PD-L1 and TIM-3/Galectin-9 pathways in the tumor microenvironment in lung cancer and that release of soluble Galectin-9 from tumor cells could negatively regulate T-cell function.
Citation Format: Yoshihiro Ohue, Koji Kurose, Yumi Nishio, Midori Isobe, Mikio Oka, Eiichi Nakayama. Role of TIM-3/Galectin-9 pathway in lung cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A101.
- ©2016 American Association for Cancer Research.