High-throughput sequencing technology for T-cell receptor specificity

TetTCR-seq is a method for high-throughput pairing of T cell receptor (TCR) sequences with their specific antigens using DNA-barcoded pMHC tetramers, enabling rapid, cost-effective immune profiling, disease diagnosis, and therapeutic development.

Background

The identification of antigen-specific T-cell receptors (TCRs) is crucial for understanding and treating immune-related diseases, such as viral infections, autoimmune disorders, and cancers. Traditional methods, such as using fluorescently labeled peptide-major histocompatibility complex (pMHC) tetramers, are limited by spectral overlap, restricting the number of antigens that can be studied simultaneously. Although time-of-flight mass cytometry (CyTOF) can analyze more antigens, it is destructive and prevents linking antigen binding to specific TCR sequences. DNA-barcoded pMHC dextramer technology allows for bulk analysis of antigen-binding T cells but fails to provide single-cell resolution, losing critical information on individual TCR-antigen interactions. Additionally, the high cost and lengthy process of chemically synthesizing peptides hinder the rapid generation of pMHC libraries tailored to specific pathogens or neoantigens.

These limitations demand the development of a high-throughput, cost-effective method to link TCR sequences with their cognate antigens at the single-cell level, enabling comprehensive analysis of T-cell specificity and cross-reactivity in various disease contexts.

Technology description

This invention introduces a method called TetTCR-seq for high-throughput pairing of T-cell receptor (TCR) sequences with their cognate peptide-major histocompatibility complex (pMHC) antigens at the single-cell level. The process involves creating a library of DNA-barcoded pMHC tetramers using in vitro transcription and translation (IVTT) to generate peptides, which are then loaded onto MHC monomers and linked to a multimer backbone. These tetramers are used to stain T cells, which are subsequently sorted and analyzed. The DNA barcodes attached to the tetramers allow for the identification of the specific peptides bound to each T cell, and the TCR sequences are determined through sequencing.

This method enables the rapid and cost-effective generation of pMHC libraries, facilitates the identification of TCR cross-reactivity, and provides detailed information on T-cell antigen specificity, developmental status, and activation status. The approach significantly reduces the time (from weeks to 2-3 days) and cost associated with traditional peptide synthesis and allows for the scalable analysis of T cells, making it applicable for immune profiling, disease diagnosis, and therapeutic development.

Benefits

Rapid and cost-effective generation of pMHC libraries
Facilitates identification of TCR cross-reactivity
Provides detailed information on T-cell antigen specificity, developmental status, and activation status
Enables scalable analysis of T cells
High-throughput pairing of TCR sequences with pMHC antigens at the single-cell level
Allows for the identification of TCRs specific to neoantigens without cross-reactivity to wild-type antigens
Applicable for immune profiling, disease diagnosis, and therapeutic development
Compatible with single-cell transcriptomics and proteomics
Can be integrated into high-throughput platforms for broader applications in immunology and personalized medicine