Trajectory and Functional Analysis of PD-1high CD4+CD8+ T Cells in Hepatocellular Carcinoma by Single-Cell Cytometry and Transcriptome Sequencing

Bo Zheng; Dongfang Wang; Xinyao Qiu; Guijuan Luo; Tong Wu; Shuai Yang; Zhixuan Li; Yanjing Zhu; Shan Wang; Rui Wu; Chengjun Sui; Ziqi Gu; Siyun Shen; Seogsong Jeong; Xuan Wu; Jin Gu; Hongyang Wang; Lei Chen

doi:10.1002/advs.202000224

Trajectory and Functional Analysis of PD-1^high CD4⁺CD8⁺ T Cells in Hepatocellular Carcinoma by Single-Cell Cytometry and Transcriptome Sequencing

Adv Sci (Weinh). 2020 May 18;7(13):2000224. doi: 10.1002/advs.202000224. eCollection 2020 Jul.

Authors

Bo Zheng^{1

2}, Dongfang Wang³, Xinyao Qiu⁴, Guijuan Luo^{1

2}, Tong Wu^{1

2}, Shuai Yang⁴, Zhixuan Li^{1

2}, Yanjing Zhu^{1

2}, Shan Wang⁴, Rui Wu⁵, Chengjun Sui^{1

2}, Ziqi Gu^{1

2}, Siyun Shen^{1

2}, Seogsong Jeong⁶, Xuan Wu⁷, Jin Gu³, Hongyang Wang^{1

2

4}, Lei Chen^{1

2

4}

Affiliations

¹ National Center for Liver Cancer Shanghai 200438 China.
² The International Cooperation Laboratory on Signal Transduction Eastern Hepatobiliary Surgery Hospital Second Military Medical University Shanghai 200438 China.
³ MOE Key Laboratory for Bioinformatics BNRIST Bioinformatics Division Department of Automation Tsinghua University Beijing 100084 China.
⁴ Fudan University Shanghai Cancer Center Department of Oncology Shanghai Medical College Fudan University Shanghai 200032 China.
⁵ Department of Biliary Surgery I Eastern Hepatobiliary Surgery Hospital Second Military Medical University Changhai Road 225 Shanghai 200438 China.
⁶ Department of Liver Surgery Renji Hospital School of Medicine Shanghai JiaoTong University Shanghai 200127 China.
⁷ Department of Laboratory Medicine The Tenth People's Hospital of Shanghai Tongji University Shanghai 200072 China.

Abstract

The spatial heterogeneity of immune microenvironment in hepatocellular carcinoma (HCC) remains elusive. Here, a single-cell study involving 17 432 600 immune cells of 39 matched HCC (T), nontumor (N), and leading-edge (L) specimens by mass cytometry is conducted. The tumor-associated CD4/CD8 double-positive T (DPT) cells are found enriched in L regions with synergetic expression of PD-1/HLA-DR/ICOS/CD45RO and exhibit a higher level of IFN-γ, TNF-α, and PD-1 upon stimulation. The enrichment of DPT and PD-1⁺DPT in L regions indicates favorable prognosis. These tumor-associated DPT cells with similar phenotype are also verified in other tumors and HCC animal models. Single-cell RNA-seq further characterizes the molecular features of DPT cells and uncovers 11 clusters with different cytotoxicity, exhaustion, and activation scores. TCR-based trajectory analysis reveals that tumor-associated DPT clusters share separated ancestries with local CD4⁺ or CD8⁺SPT cells rather than CD3⁺PBMC cells. TCR clones with frequency above 10 are mainly found coexisting in DPT and CD8⁺SPT cells. Specifically, PD-1^highDPT cluster (TDPT_10) shares the same ancestry with exhausted CD8⁺SPT cluster (TCD8T_2) and shows higher expression similarity and closer pathological location to PD-1⁺CD8⁺ than PD-1⁺CD4⁺T cells. Together, this study systematically characterizes the unique distribution of PD-1⁺DPTs in HCC and puts forward new insights for the function and origin of tumor-associated DPT cells.

Keywords: double positive T cells; liver cancer; mass cytometry; single‐cell sequencing; tumor microenvironment.