Research Report: Dominance and Persistence of T-Cell Clones in Tumor-Infiltrating Lymphocytes during Expansion in Breast Cancer

Background

Tumor-infiltrating lymphocytes (TILs) are lymphocytes that naturally infiltrate the tumor microenvironment and exhibit reactivity to the tumor. This subject is one of the most actively researched areas in tumor therapy. For instance, immune checkpoint inhibitors enhance the antitumor reactivity of TILs by inhibiting the tumor’s immune evasion mechanisms. Although immune checkpoint inhibitors have improved the functionality of TILs, the therapeutic approach of directly transferring effectively expanded T cells in vitro, known as Adoptive Cell Therapy (ACT), has been a research hotspot in tumor therapy since the 1980s, especially in melanoma research, and was approved in 2024 as the first T-cell therapy for solid tumors.

Research Aim

Although ACT-TIL has shown great promise in cancer treatment, especially in melanoma research, the clonal dynamics and clinical significance of TILs in breast cancer still require further investigation. This study aims to analyze the clonal dynamics of TILs at different expansion stages in breast cancer patients by tracking TCR (T-cell receptor) lineage changes and exploring the relationship with in vitro reactivity, with the goal of optimizing ACT-TIL treatment strategies.

Source

This paper was authored by Baknoon Ham et al., from NeogenTC Corp. (Seoul, South Korea), University of Ulsan College of Medicine (Seoul, South Korea), and other institutions. It was exclusively authorized by Springer Nature Limited and published in the “British Journal of Cancer” in 2024.

Research Methodology

This study included 19 breast cancer patients who underwent surgical resection to explore changes in T-cell clone types during the expansion of tumor-infiltrating lymphocytes (TILs). The specific research process is as follows:

Pathological Materials and Immunohistochemistry

A total of 19 breast cancer patients participated in the study. Tumor samples from all patients underwent paraffin section immunohistochemical staining and were classified based on estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and tumor subtype.

TIL Extraction and Expansion

TILs were extracted from tumor samples and expanded in two stages: a two-week initial expansion (2w TIL) after tumor tissue extraction, followed by a rapid expansion process (REP TIL). In the 2w TIL stage, high doses of interleukin-2 (IL-2) and anti-CD3 agonistic antibodies were administered to promote the survival and expansion of effector T cells. After rapid expansion, the majority of the initial TILs expanded by 1000 to 2000 times.

TCR Lineage Analysis

RNA samples were extracted using Smart-seq2 technology, and expanded T cells underwent sequencing analysis. TCR sequences were annotated using Mixcr software and performed advanced analysis using the Immunarch software package, including clonotype abundance, lineage diversity, lineage overlap, and gene usage estimation.

Data Analysis and Clonotype Identification

Statistical analysis, Shannon diversity index, Gini coefficient, and other parameters were used to evaluate the relationship between TCR lineage and in vitro reactivity. Results showed that an average of 60.0% (TRB) and 64.7% (TRA) of the top 10% of clones in FFPE samples were retained in 2w TIL.

Results Presentation and Analysis

Consensus analysis and in vitro reactivity test results revealed that no significant influencing factors were found in samples with low retention rates, necessitating further research to determine their relationship with ACT-TIL treatment response.

Key Findings

This study primarily discovered the following points: 1. Common high-frequency T-cell clones in initial FFPE samples have a higher probability of maintaining their dominant position during TILs expansion. 2. CD4+ and CD8+ T cells exhibited similar diversity and CDR3 length distribution. 3. The proportion of clonotypes associated with in vitro reactivity did not show significant differences.

Conclusion

This study tracked TILs lineage changes from FFPE to 2w TIL and REP TIL, confirming the high likelihood of high-frequency clonotypes maintaining their dominance during expansion. Additionally, the study indicated that T-cell clones present in both PBMCs and TILs have higher expansion potential, suggesting PBMC as a potential source for ACT-TILs. Despite some limitations, such as RNA quality differences, small sample size, and lack of single-cell analysis, this study provides important evidence for further optimizing ACT-TIL treatment strategies.

Research Significance

By detailed analysis of the clonal dynamics of TILs in breast cancer patients and their relationship with clinical response, this study revealed the patterns of T-cell clonal changes in ACT-TILs, emphasizing the retention of high-frequency clonotypes during TILs expansion. Additionally, the results provide a scientific basis for using PBMCs in T-cell adoptive therapy, demonstrating its potential in cancer immunotherapy.

Research Highlights

1. A detailed quantitative analysis of TILs’ T-cell clonal dynamics at different expansion stages in breast cancer patients for the first time.
2. Confirmation of the priority of high-frequency T-cell clones during expansion, leading to new considerations for optimizing ACT-TIL treatment.
3. Suggestion of the feasibility of PBMCs as a potential source for ACT-TILs, offering a simpler patient treatment scheme.

Future Prospects

Future research could combine single-cell technologies to further explore the functions of specific T-cell clones in tumor response and their relationship with clinical treatment outcomes. Increasing sample size and clinical data will help uncover the broad application potential of ACT-TILs in different cancer types.