Editor’s Note
T lymphocytes play a crucial role in human adaptive immunity by recognizing antigens major histocompatibility complex (MHC) molecules through specific T-cell receptors (TCRs) on their surface. 95% of T cells have TCRs composed of α and β peptide chains connected by disulfide bonds, forming an α/β heterodimer. Each chain consists of constant and variable domains. TCR diversity arises from the rearrangement of the variable (V), diversity (D), and joining (J) gene segments of the peptide chains, along with random nucleotide additions or deletions.
Hepatocellular carcinoma ( HCC) ranks fourth globally in cancer-related deaths. The immune microenvironment of HCC includes various T lymphocyte subgroups with different functions. Currently, the TCR diversity and clonality characteristics at different stages of HCC are not well-understood. Dr. Yijin Wang’s team’s latest research published in Cancer Science aims to explore T-cell immune characteristics from different tissue sources at various stages of HCC. This research may help clarify HCC progression mechanisms and develop cell therapy methods targeting specific neoantigens[1].
- Research Overview
- Methods：
The study prospectively included 10 patients each with early (BCLC_A), intermediate (BCLC_B), and advanced (BCLC_C) stage HCC, diagnosed through imaging and pathology. Liver tumor tissues, adjacent non-tumor tissues, and peripheral blood mononuclear cells (PBMCs) were collected from 30 HCC patients. All samples underwent TCR-β sequencing using the ImmuHub technology platform. The V, D, J, and C segments in each sample were matched against the IMGT database, identifying clone CDR3 nucleotide and amino acid sequences, and counting and calculating the frequency of each clone. Early-stage HCC patients were further divided into recurrence and non-recurrence groups, and the heterogeneity of CDR3 sequences between these groups was analyzed.
Figure 1. Experimental design process
- Results:
1. Analysis of CDR3 Sequence Diversity and Clonality
The CDR3 sequence in TCRs is crucial for TCR diversity. The proportion of the most frequent 100 CDR3s (TOP100 CDR3) is often used to evaluate TCR diversity. Figure 2 in the article provides a quantitative analysis of TCR CDR3 sequences in various samples from HCC patients at different stages of the Barcelona Clinic Liver Cancer (BCLC) classification. In tumor tissues, the frequency of TOP 100 CDR3 in early, intermediate, and advanced-stage patients was 52.81%, 35.11%, and 41.04% respectively, showing statistically significant differences between BCLC_A and BCLC_B stages (P=0.042). In PBMC samples, the proportions in early, intermediate, and advanced stages were 32.53%, 25.87%, and 46.53%, suggesting the lowest TCR diversity in late-stage HCC (P=0.015).The study also calculated the cumulative frequency of CDR3 in each sample and divided the clone distribution into 14 groups from highest to lowest frequency (Fig. 2D-L). For tumor tissues, the frequency of TOP 50 CDR3 in BCLC_A, BCLC_B, BCLC_C stage patients was 46.67%, 27.87%, and 34.27% respectively (Fig. 2D-F). In PBMC samples, these frequencies were 29.23%, 23.10%, and 43.59% respectively (Fig. 2J-L), indicating a significant increase in clonality of TCR in PBMCs in late-stage HCC.
Considering the differences in Clone Fraction, CDR3 sequences were categorized into High (Clone Fraction ≥0.1%), Mid (0.01% < Clone Fraction < 0.1%), and Low (Clone Fraction ≤0.01%) groups. In PBMCs, compared to early and intermediate stages, late-stage HCC patients had the lowest frequency of low-frequency CDR3s and the highest frequency of high-frequency CDR3s, indicating reduced TCR diversity and increased clonality in T cells from late-stage HCC peripheral blood (Fig. 2N).
Figure 2. Quantitative analysis of TCR CDR3 sequences in tumor, paracancer and PBMC samples in BCLC-A, BCLC-B and BCLC-C stage HCC patients
This section of the article details the analysis of the diversity and clonality of CDR3 sequences in T-cell receptors (TCRs) in the context of liver cancer (hepatocellular carcinoma, HCC) research.
2. Analysis of TCR Diversity and Clonality in Recurrent Patients
To clarify the relationship between TCR and prognosis, the study analyzed the frequency of V(D)J gene segment usage, CDR3 sequence distribution, and amino acid composition in 6 recurrent and 3 non-recurrent patients at the BCLC_A stage.The TCR clonality in tumor tissues of recurrent patients was lower compared to those in non-recurrent patients (Fig. 3A).Diversity indices such as Shannon, D75, and Singleton were analyzed. These analyses confirmed that the TCR diversity in PBMC samples was higher than in tumor samples, regardless of recurrence status (Fig. 3B-D). The study also found that the usage frequency of hydrophobic amino acids was lower in the PBMC (P=0.02) and tumor samples (P=0.686) of the recurrent group compared to the non-recurrent group. This finding is consistent with previous reports that hydrophobic amino acids can activate autoreactive T cells (Fig. 3G-H).
These analyses provide insights into the differences in TCR characteristics between recurrent and non-recurrent HCC patients and highlight the complex interplay between TCR diversity, clonality, and cancer recurrence.
Figure 3. Analysis of TCR characteristics in recurrent and non-recurrent patients
- Research Conclusions
The study illuminated the TCR characteristics in HCC tumor tissues, adjacent non-tumor tissues, and peripheral blood mononuclear cells (PBMCs) across different Barcelona Clinic Liver Cancer (BCLC) stages. It involved both longitudinal comparisons across different stages and lateral comparisons across different samples, revealing the TCR characteristics at various stages of HCC. Notably, TCR clonality significantly increased and diversity decreased in late-stage (BCLC_C) PBMCs compared to early and intermediate stages. This suggests that PBMCs may better depict the TCR characteristics of HCC patients at different tumor stages than tumor tissues. The study also established a relationship between TCR characteristics and patient prognosis, with higher TCR clonality correlating with lower tumor recurrence risk and better prognosis. TCR clonality in early-stage tissues could be an important predictor of postoperative recurrence in patients.
- Researchers’ Insights
The analysis of TCR characteristics in HCC at different stages is crucial for understanding the mechanisms of tumor immune escape and developing more effective immunotherapies. After delineating the TCR characteristics across different BCLC stages, further exploration of the causal relationship between TCR characteristics and HCC progression is needed. Additionally, with the advancement of targeted therapies and the need for precision medicine, developing TCR-T therapy strategies through tumor-specific TCR detection and T-cell modification in patients represents a future direction.
About the Corresponding Author
Dr. Yijin Wang is an Associate Professor, Researcher, and Doctoral Supervisor at the Southern University of Science and Technology. Specializing in the immunoregulatory mechanisms of infectious and metabolic liver diseases, Wang has published over 30 papers as the first or corresponding author in prestigious journals, including Science Advances and Lancet Respiratory Medicine. Selected among the top 2% of global scientists in 2022, Wang has received numerous awards and leads significant projects in hepatology research.
About the First Author
Rui Li, a Ph.D. student at the Southern University of Science and Technology, focuses on the mechanisms of mitochondrial quality control in the development and metastasis of hepatocellular carcinoma.
Reference:
Li R, Wang J, Li X, et al. T-cell receptor sequencing reveals hepatocellular carcinoma immune characteristics according to Barcelona Clinic liver cancer stages within liver tissue and peripheral blood. Cancer Sci. 2023 Nov 14. doi: 10.1111/cas.16013.
TAG: review; Hepatocellular Carcinoma;
