Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and the third leading cause of cancer-related death (1). HCC is the most common primary liver cancer, accounting for approximately 70%–80% of all primary liver cancers (2). HCC usually has a poor prognosis because it is usually multifocal at the time of discovery, prone to metastasis and a high recurrence rate after treatment (3, 4).

Chronic hepatitis B virus (HBV) infection remains a serious global public health problem, affecting 350 million people worldwide. Patients with chronic hepatitis B (CHB) are at increased risk for HBV-related cirrhosis, liver failure, and hepatocellular carcinoma (5). Although the exact mechanisms that determine the outcome of the disease remain unclear, increasing evidence suggests that the prognosis of HBV infection depends on the interaction between the virus and the host immune system. More than 80% of HCC patients in China are caused by chronic hepatitis B virus infection (6–8). Therefore, HBV infection is a key cause of HCC, and HBV-associated hepatocellular carcinoma (HBV-HCC) has always been the focus and hotspot of research. The risk of HCC development is much greater in patients with HBV-associated cirrhosis (HBV-LC) (9–11).

Studies have shown that immune system dysregulation plays an important role in the development of HCC (12, 13), and the coordinated immune response of tumor-associated immune cells, such as cytotoxic T cells, CD4⁺ T cells, Treg cells, natural killer (NK) cells has a significant impact on the development of HCC (14, 15). In particular, cytotoxic T cells play an important role in the clearance of infected or malignant hepatocytes (16). Meanwhile, recent studies have shown that B cells exhibit distinct roles in carcinogenesis and tumor progression (17–19).

Most immune checkpoint molecules include programmed cell death protein-1 (PD-1), T cell immunoglobulin domain and mucin domain-3 (TIM-3), lymphocyte-activation gene 3 (LAG-3) and T cell immune receptor with Ig and ITIM domains (TIGIT) have shown immunosuppressive activity, which can suppress T cell responses and mediate T cell exhaustion, cancer cells can evade immune surveillance by upregulating ligands for inhibitory checkpoint molecules (20–23). A new type of tumor immunotherapy, immune checkpoint inhibitors (ICIs) treat tumors by specifically blocking antibodies to block the abnormal interaction between tumor cells and T lymphocytes, thereby releasing immunosuppression and enhancing immune responses (24, 25). Immune checkpoint inhibitors are currently used as a treatment in a variety of tumors and have achieved good therapeutic effects (16, 26, 27).

A comprehensive characterization of the circulating immune landscape of HBV-HCC is still lacking, and understanding the expression of immune checkpoint molecules in different immune cells is crucial for tailoring novel immunotherapies against HBV-HCC. In addition, dynamic observation of the changes in the number of circulating lymphocytes may help to monitor the immune status of patients with high risk for HCC and HCC after treatment. Therefore, in this study, we analyzed the frequency and phenotype of various immune cells in the periphery of HBV-HCC, including T cells, B cells, NK cells, NKT cells, γδ T cells and their subsets, and the expression of immune checkpoints including PD-1, TIGIT, TIM3, and LAG3 in circulating immune cells in HBV-HCC patients. We further analyzed the relationship between immune cells and clinical parameters in patients with HBV- HCC.

T cell exhaustion is a state of T cell dysfunction caused by persistent antigenic stimulation during chronic infection and cancer, leading to tumor immune escape by hindering tumor clearance (20, 34). We found that αβ T cells in the peripheral circulation of patients with HBV-HCC were significantly decreased. And the increased expression of inhibitory receptors was a key feature of this phenomenon, consistent with the increased expression of TIGIT on CD4⁺ T cells of HBV-HCC patients in our study, this provides a potential rationale for developing therapeutic modalities targeting TIGIT to prevent HCC progression and promote survival. Moreover, our results showed the correlation between TIGIT⁺CD4⁺ T cells and clinical inflammatory indicators, suggesting that TIGIT⁺CD4⁺ T cells may be used as an indicator to predict the occurrence and development of HBV-HCC. At the same time, we found that compared with HBV-LC patients, the expression of TIM3 on peripheral blood CD4⁺ T cells and TIGIT on CD8⁺ T cells in HBV-HCC patients increased, which was consistent with previous research results (35). This suggests that targeted immunotherapy based on TIGIT on the surface of CD8⁺ T cells may be an effective treatment for HCC. Substantial evidence suggests that dysfunction of the host antiviral immune response is the main cause of persistent HBV replication, and we found that TIM3 expression on CD4⁺ T cells was up-regulated in HCC patients with high HBV DNA load, suggesting that TIM3 may serve as a potential therapeutic target for HCC patients with high viral burden. Naïve CD8⁺ T cells are elevated in the viral replication group, which appeared to be associated with continued exposure to high levels of viral antigens. A similar explanation could be provided by a mouse model of previous chronic polyomavirus infection, in which a sustained recruitment of naïve cells was demonstrated in the face of sustained antigenic stimulation (36).

The αβ T cells compartment can be divided into distinct subpopulations based on their ability to maintain long-term immune memory, and the naïve CD4⁺ T cell compartment has long been thought to consist of a homogeneous population of cells that have not experienced antigens (37). In this study, we observed a lower proportion of naïve CD4⁺ T cells in HBV-HCC patients and HBV-LC compared with healthy controls, which may be related to HBV infection and liver disease status, and persistent antigenic stimulation leads to differentiation of CD4⁺ T cells, which may be related to the occurrence and development of tumors. We found that among CD8⁺ T cell subsets, Compared with healthy controls and HBV-LC patients, TEMRA was significantly increased in PBMCs of HBV-HCC patients, which is consistent with previous findings (38), which may be related to the decreased recruitment of TEMRA to tumor sites. The co-expression of human leukocyte antigen DR (HLA-DR) and CD38 is related to the activation of CD8⁺ T cells, and the circulating activated CD8⁺ T cells (CD38⁺HLADR⁺) in patients with HBV-HCC are significantly higher than those in healthy controls, indicating that HBV-HCC patients have an effective adaptive immune response. CD4⁺ helper T (Th) cells are key modulators of tumor immunity. Based on cytokine production and immune function, cells are divided into distinct subpopulations: Th1 cells, Th2 cells, and Th17 cells, etc (39). Th1 cells produce characteristic cytokines interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, which mainly mediate antitumor immunity and are associated with favorable prognosis in HCC patients. Meanwhile, Th2 cells secrete IL-4 and IL-10, which promote tumor growth or metastasis through immunosuppression (40, 41). Our findings showed an increase in Th2 cells in the peripheral circulation of HBV-HCC patients. Similarly, the study by Foerster et al. showed an increased frequency of Th2 cells in HCC tissues compared to normal tissues (42), suggesting that developing novel strategy to alleviate Th2 cell infiltration may be helpful for the treatment of HBV-HCC.

Normal B cell development and survival can promote an efficient immune response to pathogen clearance, whereas abnormal B cell differentiation and activation can lead to disruption of B-cell homeostasis (43). Recent evidence suggests that B cells exhibit dual biological effects in the development and progression of several cancers (44). Studies have shown that MZ B cells may be able to mimic the antigen-presenting activity of traditional dendritic cells (cDCs) and present antigens to CD4⁺ T cells to modulate their immune responses (45). In the present study, we found an increase in naïve B cells and a significant decrease in the frequency of MZ B cells, class-switched B cells and plasma cells in HBV-HCC patients, consistent with previous reports (43, 46), which reflects immune deficiency in HBV-HCC patients. And the results were contrary in advanced HBV-HCC. At present, the role of B lymphocytes in the development of HBV-HCC is still controversial and needs further study.

NK and γδ T cells provide the first line of defense against virus-infected cells and tumors, and their function is regulated by a balance between inhibitory and activating receptors. NK cell function is regulated by HLA class I-specific inhibitory receptors (KIR and CD94) and many non-HLA-specific receptors including activation markers NKp30, NKp44, NKp46 and NKG2D (47). Previous studies have shown that the expression of NKp46 is positively correlated with cytotoxicity (48, 49) and plays an important role in eliminating virus-infected cells by recognizing viral proteins (50, 51). Our study showed that the frequency of NK cells in peripheral blood of HBV-HCC patients is higher than that of HBV-LC patients, especially CD56^dim NK cells, which is different from previous research results (52), which may be related to the small sample size in this study. At the same time, the NKp46 of NK cells in the peripheral blood of HBV-HCC patients was significantly up-regulated, which was inconsistent with the reported decrease in the expression of NKp46 in peripheral NK cells of HCC (47), which may be related to chronic HBV infection in the HCC patients (53, 54). Two main subsets of γδ T cells in human peripheral blood, Vδ1 and Vγ9Vδ2, we found that the expression of PD-1 on Vδ1 cells was increased in HBV-HCC patients, and we found that TIM3⁺ γδ T in peripheral blood of patients with advanced HBV-HCC was higher than that in early stage, suggesting that the expression of TIM3 on γδ T cells may be related to disease progression. Virus replication leads to the increased expression of NKG2D on Vδ2 T cells, which may be related to the enhanced cell activation and killing ability caused by continuous virus stimulation.

To determine whether peripheral circulating lymphocytes are associated with diagnosis of HBV-HCC, we plotted ROC curve for the frequency of lymphocytes which was different between healthy controls and HBV-HCC patients, the results demonstrated that the combination of CD3⁺ T cell and CD8⁺HLADR⁺CD38⁺ T cell may be a potential diagnostic indicator for HBV-HCC. Immunotherapy is a very promising approach to treat HCC patients, such as PD-1 inhibitors, chimeric antigen receptor T-cell therapy, and adoptive immunotherapy based on NK or γδ T cells, especially for patients who are not candidates for surgery, interventional or patients with recurrence. Monitoring immune status is crucial for personalized immunomodulatory therapy to achieve better therapeutic effects. Peripheral blood immune cell detection is non-invasive, convenient, reproducible, and relatively easy to apply in clinical practice. In view of the different responses of HCC patients to treatment, especially HCC patients with HBV infection, it is necessary to find new immunotherapy targets, and at the same time, we can formulate targeted treatment strategies according to the immune damage of each patient. At present, on the basis of the existing traditional treatment methods for HCC, it is still a difficult problem to determine the optimal regimen and timing of combined immunotherapy. There are still some limitations in our study. Firstly, in this study, we carried out a detailed analysis of the peripheral immune characteristics of HBV-HCC patients, but lacked the exploration of the liver immune microenvironment in HBV-HCC patients, a recent study showed that immune cell subsets have an immunosuppressive gradient in the tumor microenvironment of circulating blood, non-tumor tissues and liver cancer tissues (55). Further studies are needed to elucidate the interactions and mechanisms between tumors and the immune system. Secondly, due to the limited sample size, some immunophenotypic changes were slightly different from previous findings and further large sample size study is warranted to validate these findings. In addition, there were fewer female HBV-HCC patients in our study, which may lead to bias in the results. We need to continue to expand the sample size to reduce gender bias between groups, which will help to discover the characteristics of HBV-HCC circulating immunity in the population and explore whether there are differences in circulating immunity between genders in HBV-HCC. Thirdly, HCC has many causes, including hepatitis B virus, hepatitis C virus, alcohol, etc. In this study, we only described peripheral immunity in patients with HBV-HCC. We need to compare the peripheral immune characteristics of patients with HCC with different causes and to achieve the purpose of individualized treatment for HCC of different etiologies in future. Finally, Single-cell RNA sequencing (scRNA-seq) is a new technique for transcriptome analysis of a large number of single cells. scRNA-seq can provide a deeper understanding of the biological behavior of cells in the complex tumor microenvironment (TME), by analyzing single cell populations (56). Single-cell based transcriptomic analysis has been increasingly used to study the composition of tumor cells and immune cells in normal and disease tissues to better reflect the changes in tumor microenvironment (57). scRNA-seq can provide a comprehensive understanding of the pathogenesis of HCC and is conducive to individualized treatment of HCC. Multiple studies analyzed the scRNA-seq data from healthy controls, tumor tissues and adjacent tissues, and the results showed that the difference of proportion of various immune cells, including CD8⁺ T cells, NK cells, macrophages, B cells and other cells (58–61). The changes of infiltrated immune cells may be related to the immunosuppressive microenvironment in HCC and correlated with HCC progression (62). Previous studies mostly conducted scRNA-seq analysis on HCC tumor tissues and focused on the heterogeneity of tumor microenvironment, while few studies on scRNA-seq of HCC peripheral blood single cells (63). The scRNA-seq analysis of peripheral blood immune cells combined with the immune characteristics of tumor microenvironment would be more beneficial to explore the mechanism of development and the treatment strategy of HCC. Therefore, it is necessary to conduct further research on the scRNA-seq analysis of HBV-HCC peripheral blood immune cells.

Our study systematically describes the immune landscape of peripheral circulation in HBV-HCC patients and showed that circulating lymphocytes and their subsets in HBV-HCC patients exhibited features of immune exhaustion, especially in HCC patients with persistent viral replication and in patients with advanced HBV-HCC, including decreased frequency of αβ T cells and increased expression of inhibitory receptors including TIGIT and TIM3 on CD4⁺ T cells and γδ T cells, as well as changes in the B cells immune compartment, these changes may be used as potential predictors for evaluating the disease outcome based on the viral replication and disease progression.

Meanwhile, our research suggests that the combination of CD3⁺ T cell and CD8⁺HLADR⁺CD38⁺ T cell may be a potential diagnostic indicator for HBV-HCC. Finally, this study will also provide new evidence for development of novel immunotherapeutic strategies in HBV-HCC patients.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by The Third Affiliated Hospital of Sun Yat-sen University. The patients/participants provided their written informed consent to participate in this study.Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

The authors have contributed to the manuscript by proposing the concept, planning the study and revising the manuscript (BW, ZX, YX, ZNY), executing the experiment, analyzing experimental data and writing the manuscript (RNS), collecting patient samples and clinical data (RNS, JWL, XYL, YDY, BL), following up patients and collecting information (RNS, TBL, SX, AYD). All authors contributed to the article and approved the submitted version.