Introduction

T cells play a vital role in immunology by fighting invading pathogens, destroying tumor cells and orchestrating vaccine response. T cell antigen specificity, a factor that is vital to T cell function and for the development of T cell-based immunotherapy, has traditionally been difficult to study due to technical factors. However, the advent of multi-omics technology in recent years has made it possible to profile antigen specificities from single cells in a relatively convenient fashion.

In this technical report, Ma, K.Y et.al describe a high-dimensional tetramer-associated T cell antigen receptor (TCR) sequencing method (TetTCR-SeqHD), that enabled them to simultaneously profile cognate antigen specificities, TCR sequences, targeted gene expression and surface-protein expression from tens of thousands of single-cells. Using human polyclonal CD8+ cells with known antigen specificity and TCR sequences, the authors state that they could demonstrate 98% precision for detecting the correct antigen specificity. The authors also claim that TetTCR-SeqHD is a powerful approach for T cell response profiling in humans and mice.

Results

The authors used the TetTCR-SeqHD technology to detect accurate antigen-specificity of  CD8+ T cells. In this technology, a DNA barcode for a pMHC tetramer is synthesized with a 3’poly(A) tail. Then a fluorophore-labeled streptavidin conjugate with an oligonucleotide sequence complementary to the 5’ end of the tetramer DNA barcode is used to anneal to each unique tetramer DNA barcode to yield barcoded streptavidin. The barcoded streptavidin is then used to form tetramers with peptide-loaded MHCs. Human T cell samples are stained with a panel of DNA-barcoded MHC tetramers, a unique DNA barcoded anti-CD50 antibody as a SampleTag and a panel of 59 DNA barcoded antibodies. The stained cells are then loaded into the BD Rhapsody™ Single Cell Analysis System single-cell analysis platform for high -throughput and high-dimensional molecular profiling of cognate specificity, TCR sequences, targeted gene expression and surface protein level.

Using a panel of Type 1 diabetes (T1D)-related and foreign antigens, the authors report that foreign pathogen-specific T cells exhibited infection-dependent states. They identified three peptides that have elevated antigen-specific CD8+ T cell frequencies in patients with T1D compared with healthy controls. The authors also reported a TCR that cross-reacts with a T1D-related peptide and a peptide derived from the microbiome.

Conclusion

The authors conclude that combined with in-vitro transcription and translation for rapid generation of peptides and pMHCs, TetTCR-SeqHD enables the screening of hundreds of antigens on primary T cells. Due to the advantages of multidimensional profiling of single-cells, TetTCR-SeqHD also enables the identification of phenotypic differences of antigen-specific T cells, distinguish disease status, screen antigens with high throughput and identify TCRs with therapeutic potential.

Read the nature article entitled, “High-throughput and high-dimensional single-cell analysis of antigen-specific CD8+ T cells”