Zhen Sun ^1,2 Aotian Xu ^2* Zhaojun Wu ² Xiaohao Lan ^2* Ganchen Gao ^2* Bin Guo ^2* Zhongjie Yu ² Lin Shao ^2* Hao Wu ^2* Min Lv ^2* Yongjie Wang ^3* Yi Zhao ^2* Bin Wang ^1,2,4*

• ¹ Department of Pathogenic Biology, College of Basic Medicine, Qingdao University, Qingdao, China
• ² Qingdao Sino-Cell Biomedicine Co., Ltd., Qingdao, China
• ³ Department of Thoracic Surgery, The Affiliated Hospital of Qingdao University, Qingdao, China
• ⁴ Department of special medicine, School of basic medicine, Qingdao University, Qingdao, China

The adoptive cell transfer of tumor-infiltrating lymphocytes (TILs) has proven clinically beneficial in patients with non-small cell lung cancer refractory to checkpoint blockade immunotherapy, which has prompted interest in TIL-adoptive cell transfer. The transgenic expression of IL15 can promote the expansion, survival, and function of T cells ex vivo and in vivo and enhance their anti-tumor activity. The effect of expressing mIL15 regulated by hypoxia in the tumor microenvironment on the expansion, survival, and stem-like properties of TILs has not been explored.Methods: Using TILs expanded from the tumor tissues of lung cancer patients, TILs with or without mIL15 expression (TIL-mIL15 or UN-TIL) were generated by lentiviral transduction. To reflect the advantages of mTIL15, the cells were divided into groups with IL2 (TIL-mIL15+IL2) or without IL2 (TIL-mIL15-IL2). Results: Compared to UN-TIL cells, mIL15 expression had a similar capacity for promoting TIL proliferation and maintaining cell viability. Our experimental findings indicate that, compared to UN-TIL and TIL-mIL15+IL2 cells, the expression of mIL15 in TIL-mIL15-IL2 cells promoted the formation of stem-like TILs (CD8 + CD39 -CD69 -) and led to significant decreases in the proportion and absolute number of terminally differentiated TILs (CD8 + CD39 + CD69 + ). RNA-Seq data revealed that in TIL-mIL15-IL2 cells, the expression of genes related to T cell differentiation and effector function, including PRDM1, ID2, EOMES, IFNG, GZMB, and TNF, were significantly decreased, whereas the expression of the memory stem-like T cell marker TCF7 was significantly increased. Furthermore, compared to UN-TIL and TIL-mIL15+IL2 cells, TIL-mIL15-IL2 cells showed significantly lower expression levels of inhibitory receptors LAG3, TIGIT, and TIM3, which was consistent with the RNA-Seq results. Discussion: This study demonstrates the superior ex vivo 删除[孙振]: -删除[孙振]: -删除[孙振]: -删除[孙振]:2 persistence of TIL-mIL15-IL2 cells, which may serve as a novel treatment strategy for lung cancer patients.