Rapid identification of tumor-reactive T-cell receptors by RNA preamplification-based single-cell sequencing
Yipeng Ma1, Fenglan Liu1, Bin Li1
1Department of Research and Development, Shenzhen Institute for Innovation and Translational Medicine, Shenzhen International Biological Valley-Life Science Industrial Park, Dapeng New District, Shenzhen, China.
Abstract:
T-cell receptor (TCR)-transduced T (TCR-T) cell therapy has shown promising efficacy in the clinical treatment of malignant cancers. However, the populations covered by reported TCRs are still limited. Tumor infiltrating lymphocytes (TILs) are natural reservoirs of tumor-reactive T cells and TCRs. Approaches are required for the fast and cost-effective identification of tumor-reactive TCRs from TILs. The widely employed TCR identification approaches by the clonal expansion of TILs involve a TCR singularization process for the direct pairing of TCR Vα and the Vβ chain. However, the clonal expansion of T cells is well known to require extensive time and effort due to the involvement of T cell cultures. Several single-cell multiplexing PCR methods followed by Sanger sequencing have been developed, representing a cost-effective and fast approach for single-cell TCR identification. In this study, an RNA-based preamplification step was included in the single-cell TCR sequencing, which can reduce the multiplexing PCR amplification to one round. Moreover, the cDNA product of RNA preamplification is derived from the whole genome mRNA, instead of TCR mRNA only by multiplexing primers-based DNA preamplification, which is valuable for many other analyses (e.g., phenotypic analysis) of the tumor-reactive T cells that can be correlated with the identified TCRs. The feasibility for both single α chain and dual α chain TILs of this approach highlights its potential value as a rapid and cost-effective sequencing strategy for the development of TCR-T therapies for solid cancers.
Insights
This study introduces a rapid, cost-effective method for identifying tumor-reactive T-cell receptors (TCRs) from tumor-infiltrating lymphocytes (TILs). This advance accelerates the development of TCR-T cell therapies for solid cancers.
Area of Science:
- Immunology
- Oncology
- Molecular Biology
Background:
- T-cell receptor (TCR)-transduced T (TCR-T) cell therapy shows promise for cancer treatment, but the range of identified TCRs is limited.
- Tumor-infiltrating lymphocytes (TILs) are crucial sources of tumor-reactive T cells and TCRs, necessitating efficient identification methods.
- Current TCR identification methods, like clonal expansion, are time-consuming and labor-intensive.
Purpose of the Study:
- To develop a fast and cost-effective strategy for identifying tumor-reactive TCRs from TILs.
- To improve upon existing single-cell TCR sequencing methods by incorporating an RNA-based preamplification step.
- To enable broader downstream analyses of tumor-reactive T cells alongside TCR identification.
Main Methods:
- Incorporation of an RNA-based preamplification step into single-cell TCR sequencing.
- Reduction of multiplexing PCR amplification to a single round.
- Utilizing whole-genome mRNA for cDNA synthesis, enabling comprehensive T-cell analysis.
Main Results:
- The developed method significantly reduces the time and cost associated with TCR identification.
- RNA preamplification allows for a single round of multiplexing PCR, streamlining the process.
- cDNA derived from whole-genome mRNA supports additional analyses, such as phenotypic characterization, correlated with identified TCRs.
- The approach demonstrated feasibility for both single alpha chain and dual alpha chain TILs.
Conclusions:
- This RNA-based single-cell TCR sequencing strategy offers a rapid and cost-effective solution for identifying tumor-reactive TCRs.
- The method's ability to facilitate correlated analyses enhances its value for TCR-T cell therapy development.
- The approach holds significant potential for advancing TCR-T cell therapies targeting solid cancers.
