High ratio of resident to exhausted CD4+ T cells predicts favorable prognosis and potentially better immunotherapeutic efficacy in hepatocellular carcinoma

Background: Tumor-infiltrating lymphocytes (TILs) are significantly implicated in regulating the tumor immune microenvironment (TIME) and immunotherapeutic response. However, little is known about the impact of the resident and exhausted status of TILs in hepatocellular carcinoma (HCC). Methods: Single-cell RNA sequencing data was applied to discover resident and exhausted signatures of TILs. Survival outcomes, biological function, immune infiltration, genomic variation, immunotherapeutic efficacy, and sorafenib response were further explored the clinical significance and molecular association of TILs in HCC. Moreover, a candidate gene with predictive capability for the dismal subtype was identified through univariate Cox regression analysis, survival analysis, and BEST website. Results: Single-cell analysis revealed that CD8+ T, CD4+ T, and NK cells were strongly associated with resident and exhausted patterns. Specific resident and exhausted signatures for each subpopulation were extracted in HCC. Further multivariate Cox analysis revealed that the ratio of resident to exhausted CD4+ T cells in TIME was an independent prognosis factor. After incorporating tumor purity with the ratio of resident to exhausted CD4+ T cells, we stratified HCC patients into three subtypes and found that (i) CD4 residency_highexhaustion_low subtype was endowed with favorable prognosis, immune activation, and sensitivity to immunotherapy; (ii) CD4 exhaustion_highresidency_low subtype was characterized by genome instability and sensitivity to sorafenib; (iii) Immune-desert subtype was associated with malignant-related pathways and poor prognosis. Furthermore, spindle assembly abnormal protein 6 homolog (SASS6) was identified as a key gene, which accurately predicted the immune-desert subtype. Prognostic analysis and in vitro experiments further demonstrated that SASS6 was closely associated with tumor prognosis, proliferation, and migration. Conclusions: The ratio of resident to exhausted CD4+ T cells could serve as a candidate biomarker for evaluating prognosis and potential response to immunotherapy in HCC and SASS6 was a novel biomarker and candidate therapeutic target for prognostic assessment of HCC.