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Updated: Dec 21, 2025
T and B Cell Receptor Immune Repertoire Analysis using Next-generation Sequencing
Published on: January 12, 2021
Massively parallel interrogation and mining of natively paired human TCRαβ repertoires
Matthew J Spindler1, Ayla L Nelson1, Ellen K Wagner1
1GigaMune, Inc., South San Francisco, CA, USA.
Abstract:
T cells engineered to express antigen-specific T cell receptors (TCRs) are potent therapies for viral infections and cancer. However, efficient identification of clinical candidate TCRs is complicated by the size and complexity of T cell repertoires and the challenges of working with primary T cells. Here we present a high-throughput method to identify TCRs with high functional avidity from diverse human T cell repertoires. The approach used massively parallel microfluidics to generate libraries of natively paired, full-length TCRαβ clones, from millions of primary T cells, which were then expressed in Jurkat cells. The TCRαβ-Jurkat libraries enabled repeated screening and panning for antigen-reactive TCRs using peptide major histocompatibility complex binding and cellular activation. We captured more than 2.9 million natively paired TCRαβ clonotypes from six healthy human donors and identified rare (<0.001% frequency) viral-antigen-reactive TCRs. We also mined a tumor-infiltrating lymphocyte sample from a patient with melanoma and identified several tumor-specific TCRs, which, after expression in primary T cells, led to tumor cell killing.
Insights
We developed a high-throughput method to find T cell receptors (TCRs) with high functional avidity. This approach efficiently identifies rare viral-antigen and tumor-specific TCRs from millions of cells for potential therapies.
Area of Science:
- Immunology
- Molecular Biology
- Biotechnology
Background:
- T cell receptors (TCRs) engineered to express specific antigens are promising therapies for viral infections and cancer.
- Identifying clinical-grade TCRs is challenging due to T cell repertoire complexity and difficulties working with primary T cells.
Purpose of the Study:
- To develop a high-throughput method for identifying T cell receptors (TCRs) with high functional avidity from diverse human T cell repertoires.
- To enable efficient screening and panning for antigen-reactive TCRs.
Main Methods:
- Utilized massively parallel microfluidics to generate libraries of natively paired, full-length TCRαβ clones from millions of primary T cells.
- Expressed TCRαβ clones in Jurkat cells to create libraries for screening.
- Employed peptide-major histocompatibility complex binding and cellular activation assays for screening and panning.
Main Results:
- Captured over 2.9 million natively paired TCRαβ clonotypes from six healthy human donors.
- Identified rare viral-antigen-reactive TCRs (<0.001% frequency).
- Discovered tumor-specific TCRs from a melanoma patient's tumor-infiltrating lymphocytes, which mediated tumor cell killing upon expression in primary T cells.
Conclusions:
- The developed high-throughput method enables efficient identification of clinically relevant TCRs with high functional avidity.
- This approach facilitates the discovery of rare antigen-specific TCRs for potential therapeutic applications in viral infections and cancer.
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