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Combined megaplex TCR isolation and SMART-based real-time quantitation methods for quantitating antigen-specific T
George Du1, Liyou Qiu, Ling Shen
1Department of Microbiology and Immunology, Center for Primate Biomedical Research, University of Illinois College of Medicine, 835 S. Wocoltt, MC790, Chicago, IL 60612, USA.
Insights
Quantifying antigen-specific T cell clones is challenging. This study introduces a novel method combining megaplex TCR isolation and SMART amplification for accurate T cell clone quantitation from limited samples.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Quantifying antigen-specific T cell clones is crucial for understanding immune responses in disease.
- Current methods face challenges in sensitivity and scalability, especially with limited specimen amounts.
Purpose of the Study:
- To develop and validate a novel assay for accurate quantitation of numerous antigen-specific T cell clones.
- To enable T cell clone analysis using limited biological specimens.
Main Methods:
- Combined megaplex T cell receptor (TCR) isolation to cover a broad TCR repertoire (24 Vbeta, 13 Jbeta segments).
- Purification of antigen-specific T cells (e.g., PPD-specific IFNgamma-producing T cells) via flow cytometry sorting.
- SMART (Signal Amplification Reaction) based real-time quantitative PCR for proportional enrichment and sensitive detection of TCR VDJ clonotypic sequences.
Main Results:
- The combined method accurately quantitates numerous antigen-specific T cell clones from limited samples (e.g., 2x10^6 PBMCs).
- SMART amplification maintained relative TCR gene expression levels compared to unamplified cDNA.
- The assay achieved a detection limit of 10^-5 to 10^-6 antigen-specific T cells, with specific primers discriminating clones differing by >=2 bases in DJ regions.
Conclusions:
- This novel assay system effectively quantitates large numbers of antigen-specific T cell clones.
- The method is valuable for studying T cell responses in infectious diseases, autoimmune conditions, and cancer.
- It offers a sensitive and scalable approach for T cell repertoire analysis.
Abstract:
Despite recent advances in measuring cellular immune responses, the quantitation of antigen-specific T cell clones in infections or diseases remains challenging. Here, we employed combined megaplex TCR isolation and SMART-based real-time quantitation methods to quantitate numerous antigen-specific T cell clones using limited amounts of specimens. The megaplex TCR isolation covered the repertoire comprised of recombinants from 24 Vbeta families and 13 Jbeta segments, and allowed us to isolate TCR VDJ clonotypic sequences from one or many PPD-specific IFNgamma-producing T cells that were purified by flow cytometry sorting. The SMART amplification technique was then validated for its capacity to proportionally enrich cellular TCR mRNA/cDNA for real-time quantitation of large numbers of T cell clones. SMART amplified cDNA was shown to maintain relative expression levels of TCR genes when compared to unamplified cDNA. While the SMART-based real-time quantitative PCR conferred a detection limit of 10(-5) to 10(-6) antigen-specific T cells, the clonotypic primers specifically amplified and quantitated the target clone TCR but discriminated other clones that differed by >or=2 bases in the DJ regions. Furthermore, the combined megaplex TCR isolation and SMART-based real-time quantiation methods allowed us to quantitate large numbers of PPD-specific IFNgamma-producing T cell clones using as few as 2 x 10(6) PBMC collected weekly after mycobacterial infection. This assay system may be useful for studies of antigen-specific T cell clones in tumors, autoimmune and infectious diseases.
