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

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-gamma, TNF-alpha, 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(high)DPT 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.