Background Hepatocellular carcinoma (HCC) is the leading cause of death in patients with cirrhosis, with increasing incidence annually. Curative treatments such as surgical resection, local ablation, or liver transplantation are options for early-stage patients. However, most patients are diagnosed at intermediate to advanced stages, missing the window for surgery. Immune checkpoint therapy has proven effective for advanced liver cancer, yet only a subset of patients respond. Predicting response to this therapy often involves assessing PD-L1 expression in liver tumor tissues, but this method lacks significant clinical results. Other biomarkers like tumor mutation burden, mismatch repair deficiency, immune microenvironment, and RNA characteristics have been proposed but none have proven valuable for routine clinical use. Identifying an economical, rapid, and accurate biomarker for assessing immune checkpoint therapy effectiveness is crucial for clinicians.

Methods We enrolled treatment-naive patients with advanced unresectable HCC and administered PD-1 monoclonal antibody injections. Peripheral blood samples were collected before treatment and 1–3 weeks after for single-cell sequencing and flow cytometry analysis. Liver samples were also collected before and after treatment for sequencing and histological staining. In a murine orthotopic liver cancer model, we purified CD8+HLADR+CD38+ cells from the liver and spleen to identify their function.

Results Single-cell sequencing and flow cytometry indicated a significant increase in HLADR+CD38+CD8+ T cells in the blood of PR patients after PD-1 treatment, characterized by high expression of CXCR3, KI67, and GZMB. This increase was also observed in tumor resection tissues post-treatment. In animal experiments, CD8+HLADR+CD38+ T cells from treated mice exhibited cytotoxic capabilities when co-cultured with HEPA1-6 cells, exerting effects through GZMB.

Conclusions Treatment with PD-1 monoclonal antibody in advanced unresectable HCC patients significantly increased the proportion of HLADR+CD38+CD8+ T cells, characterized by high CXCR3, KI67, and GZMB expression. These cells also increased in tumor tissues post-treatment. In murine models, these cells demonstrated cytotoxicity through GZMB. HLADR+CD38+CD8+ T cells may serve as a valuable biomarker for predicting and evaluating the response to PD-1 inhibitor therapy in HCC, providing a potential target for enhancing antitumor immune responses.

Ethics Approval The experimental protocol was established according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of West China Hospital, Sichuan University(2023-288). Written informed consent was obtained from individual participants or their guardian.