Multi-omics insights for deciphering prognosis-related T cell subsets in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the leading causes of tumour-related death. T cells and cytokines play a critical role in tumour progression, but the T cell landscape correlated with HCC prognosis remains undepicted.

The prognostic significance of intra-tumoural immune cells, chemokines and cytokines were analysed using mass cytometry, bulk RNA sequencing and scRNA-seq data with survival information. The signature of CD4⁺CD8⁺ double positive T (DPT) cells was constructed using scRNA-seq and quantified by ssGSEA scores, whose association with the response to atezolizumab plus bevacizumab was evaluated. Cellular cross-talk and spatial patterns were analysed by scRNA-seq and spatial transcriptomics. Flow cytometry, gene knockdown, transwell migration, co-culture assays, qPCR and wound healing assay were performed to further validate the DPT-associated niche.

Higher intra-tumoural levels of DPT cells, CD45RA⁺CD4⁺ conventional T cells, HBEGF and CX3CR1 were associated with unfavourable prognosis in HCC. In contrast, higher infiltration of CD161⁺CD45RA^-CD4⁺ conventional T cells and CD8⁺ T cells correlated with prolonged survival. CD45⁺EpCAM⁺ and CD45⁺α-SMA⁺ cells were more frequent in short-term survivors. DPT infiltration was identified across HCC multi-cohorts and syngeneic mouse models. In patients receiving atezolizumab plus bevacizumab, responders exhibited higher DPT ssGSEA scores than non-responders. Multi-omics analyses indicated cross-talk and spatial association of DPT cells with capillary-associated endothelial cells, supporting a pro-tumour niche. HBEGF was positively correlated with DPT cells and highly expressed in endothelial compartments. Endothelial-derived HBEGF knockdown reduced DPT migration. Moreover, DPT co-culture increased expression of signatures associated with immunosuppressive checkpoints, chemokine signalling, epithelial-mesenchymal transition and stemness in Hep3B cells and promoted their migration.

Our findings depicted the prognostic immune landscape of HCC by identifying distinct T cell populations and molecular interactions. DPT cells emerged as a critical biomarker for poor prognosis, and the endothelial-derived HBEGF-DPT axis could represent a potential therapeutic target.