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Updated: Jul 15, 2025
Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs
Published on: September 10, 2017
Barcoding intracellular reverse transcription enables high-throughput phenotype-coupled T cell receptor analyses
Sahana Jayaraman1, Janelle M Montagne2, Thomas R Nirschl3
1Institute for Cell Engineering, Division of Immunology, Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
We developed INCERTS, a new method for linking T-cell receptor (TCR) sequences with cellular phenotypes. This approach efficiently processes millions of cells, enabling discovery of antigen-specific TCRs for cancer vaccine development.
Area of Science:
- Immunology
- Molecular Biology
- Genomics
Background:
- Characterizing adaptive immune responses requires linking cellular phenotypes with T-cell receptor (TCR) and B-cell receptor (BCR) sequences.
- Current methods for this analysis suffer from low sample throughput and high costs, limiting their widespread application.
Purpose of the Study:
- To introduce INtraCEllular Reverse Transcription with Sorting and sequencing (INCERTS), a novel assay for high-throughput, cost-effective association of TCR/BCR sequences with cellular phenotypes.
- To demonstrate INCERTS' capability in identifying antigen-specific TCRs from cancer patients vaccinated with a novel mutant KRAS peptide.
Main Methods:
- INCERTS combines molecular indexing of receptor repertoires within intact cells with fluorescence-activated cell sorting (FACS).
- Millions of cells from pooled human peripheral blood mononuclear cell (PBMC) samples were processed.
- Following ex vivo stimulation, 28 barcoded samples were pooled and sorted into peptide-reactive and non-reactive CD4+ and CD8+ populations using FACS.
- Complementary patient-matched single-cell RNA sequencing (scRNA-seq) data were integrated.
Main Results:
- INCERTS enabled efficient processing of millions of cells while maintaining robust association between TCR sequences and cellular phenotypes.
- The method successfully identified antigen-specific TCRs in cancer patients immunized with a mutant KRAS peptide vaccine.
- Full-length, paired TCR alpha and beta chain sequences were retrieved for therapeutic validation.
Conclusions:
- INCERTS offers a powerful and efficient approach for high-throughput analysis of adaptive immune responses.
- This method facilitates the discovery of antigen-specific TCRs, crucial for developing targeted immunotherapies and vaccines.
- The successful application in a cancer vaccine context highlights INCERTS' potential for advancing translational immunology research.
