On June 15^th, 2017, CELL published ‘Landscape of Infiltrating T Cells in Liver Cancer Revealed by Single-cell Sequencing’ from the laboratories of Zemin Zhang, Jirun Peng and Wenjun Ouyang. In this study, the immune landscape in the liver cancer microenvironment was depicted at the single cell level for the first time.

Cancer immunotherapies have dramatically altered the oncological treatment landscape over the past decades. While such therapies as checkpoint blockade can lead to remarkable clinical responses, their efficacies are not uniform among cancer patients or cancer types. Several factors such as mutational loads, tumor infiltrating lymphocytes (TILs) levels, and the expression of drug targets, are only partially correlated with patient responses for anti-CTLA4 or anti-PD1 therapies. Due to high degree of heterogeneity and complexity of the tumor microenvironment, single cell analysis of infiltrating lymphocytes is required to characterize many fundamental properties of such diverse class of immune cells, which hold keys to many unanswered questions in cancer immunotherapies.

Hepatocellular carcinomas (HCC), a major form of liver cancer, is one of the leading causes of cancer-related death worldwide and chronic HBV infection is the major cause of HCC in China. There are limited treatment options and there is a lack of clinical success in immunotherapies. Although HCC tumors harbor a significant level of infiltrating lymphocytes, those TILs are by inference incapable of killing tumor cells. Deeply understanding the tumor-immune microenvironment to explore novel cancer immunotherapy and to find new targets is of great significance for the diagnosis and treatment of hepatocellular carcinoma.

The research team applied deep single-cell RNA sequencing analysis to >5000 single T cells isolated from the peripheral blood, tumors and adjacent normal tissues of 6 HCC patients to comprehensively study their complete TCR sequences and transcriptomes. Using sophisticated bioinformatics methods, they performed a series of in-depth analyses of infiltrating T cells, including their composition, classification, lineage, functional states and TCR sequences. They discovered that tumor infiltrating T cells exhibit remarkably distinct composition, functions, and development status from those from blood and normal tissues. Although the clinical efficacy of various checkpoint inhibitors hinges at least in part upon their ability to unleash the CD8+ cytotoxic T cells in the tumor microenvironment, the cytotoxic activity of these T cells could be rendered ineffective primarily by the suppression of Tregs or by reaching a T cell dysfunction state called exhaustion, defined by poor effector function, sustained expression of inhibitory receptors and a unique transcriptional state. Signature genes for exhausted CD8+ T cells and tumor-specific Tregs, such as Layilin (LAYN), were examined in detail, and they found that overexpressing LAYN in primary CD8+ T cells resulted in inhibition of IFN-γ production, suggesting a regulatory function of LAYN. Furthermore, the research team observed a high degree of tumor-specific clonal T cells in tumor microenvironment but most of those cells were exhausted, which explained the tumor escape from immune surveillance. Finally, combined expression and TCR-based analyses revealed connectivity and potential developmental path of different subsets. Among the exhausted T cell subset, a unique subpopulation CD8+FOXP3+ regulatory-like cells were identified from the exhausted CD8+ T cells, hinting the possibility such cells could actually gain the suppressive function.

This is the first time that the tumor infiltrating lymphocytes were comprehensively analyzed at the single-cells resolution. The large compendium of T cell transcriptome data provides a key resource for understanding immune-cancer cell interactions and developing novel immunotherapy strategies, thereby promoting the clinical application of existing immunotherapy regimens and help to identify effective targets for the treatment of liver cancer.

Dr. Chunhong Zheng, Liangtao Zheng, Dr. Jae-Kwang Yoo, Huahu Guo, Yuanyuan Zhang and Xinyi Guo are the co-first authors of this paper. Prof. Wenjun Ouyang at AMGEN, Prof. Jirun Peng at Beijing Shijitan Hospital of Capital Medical University and Prof. Zemin Zhang at BIOPIC of Peking University are the co-correspondence authors. This work was supported by Beijing Advanced Innovation Center for Genomics at Peking University, Key Technologies R&D Program, NSF China and Peking-Tsinghua Center for Life Sciences. Dr. Chunhong Zheng was supported by the Postdoctoral Foundation of Center for Life Sciences at Peking University-Tsinghua University.

Landscape of Infiltrating T Cells in Liver Cancer Revealed by Single-Cell Sequencing

Chunhong Zheng, Liangtao Zheng, Jae-Kwang Yoo, Huahu Guo, Yuanyuan Zhang, Xinyi Guo, Boxi Kang, Ruozhen Hu, Julie Y. Huang,Qiming Zhang, Zhouzerui Liu, Minghui Dong, Xueda Hu, Wenjun Ouyang, Jirun Peng, Zemin Zhang. Cell 169(7), 1342–1356( 2017). DOI: http://dx.doi.org/10.1016/j.cell.2017.05.035