Affinity measurement and sequencing of T cell receptor

The technology enables the measurement of T cell receptor (TCR) affinity and sequencing of antigen-specific T cells using a micropipette adhesion assay and single-cell paired TCRα/TCRβ sequencing. The method isolates antigen-specific T cells, measures TCR affinity, and determines TCR sequences for applications in immunotherapy and monitoring immune responses.

Background

The accurate determination of T cell receptor (TCR) affinity and sequence is crucial for understanding immune responses and developing effective immunotherapies for viral infections and cancers and for monitoring patients undergoing immunotherapy.

Traditional methods, such as surface plasmon resonance (SPR), are labor-intensive and not suitable for analyzing large, polyclonal TCR repertoires, especially from primary human samples. These methods also often require high frequencies or counts of antigen-specific cytotoxic T lymphocytes (CTLs), which are typically not obtainable from a single blood draw due to their low frequency in humans. Additionally, current techniques fail to easily link TCR biophysical binding parameters to their sequences, which is essential for comprehensive T cell profiling. Consequently, there is a need for a method that can profile TCR affinity and sequence from a small number of T cells efficiently and accurately.

Technology description

The technology for determining the affinity and sequence of T cell receptors on antigen-specific T cells involves a sophisticated process utilizing a micropipette adhesion assay combined with single-cell paired TCRα/TCRβ sequencing. Initially, antigen-specific T cells are isolated from a sample using peptide-major histocompatibility complex (pMHC)-tagged streptamers. These streptamers bind to the TCRs on the T cells, allowing for their isolation via flow cytometry. Subsequently, the streptamers are dissociated from the T cells using a biotin-containing buffer, freeing the TCRs for further analysis.

The TCR affinity is measured by bringing T cells into contact with red blood cells coated with pMHC and recording the frequency of adhesion events, which is then used to calculate the two-dimensional (2D) TCR affinity. Following affinity measurement, the T cells are individually transferred into lysis buffer for reverse transcription and PCR amplification of the TCRα and TCRβ chains. The amplified TCR sequences undergo next-generation sequencing, enabling the determination of the TCR sequences. This method is applicable for identifying high-affinity TCRs for adoptive transfer in treating viral infections or cancers and monitoring the TCR repertoire in patients undergoing immunotherapy.

This technology is differentiated by its ability to simultaneously measure TCR affinity and sequence directly from primary T cells, which is a significant advancement over traditional methods. The use of micropipette adhesion assays allows for precise measurement of 2D TCR-pMHC binding affinities, which correlates better with T cell function compared to conventional three-dimensional (3D) affinity measurements. Additionally, the integration of single-cell paired TCRα/TCRβ sequencing provides detailed insights into the TCR repertoire at the single-cell level.

This dual capability enables rapid selection of high-affinity TCRs for adoptive immunotherapy, offering a more efficient and reliable approach for treating diseases like cancer and persistent viral infections. Furthermore, the method's sensitivity allows for the analysis of rare antigen-specific T cell populations, making it a powerful tool for both basic research and clinical applications in immune monitoring and therapy optimization.

Benefits

Directly measures TCR affinity using a micropipette adhesion assay
Determines TCR sequences through single-cell paired TCRα/TCRβ sequencing
Requires only a small number of antigen-specific T cells for analysis
Provides rapid and accurate TCR affinity and sequence information
Predicts functional capacity of T cells based on TCR affinity
Allows for the selection of optimal T cells for adoptive immunotherapies
Enables tracking of T cell clonality and repertoire diversity
Identifies high-affinity TCRs for adoptive transfer in viral infection and cancer treatment
Monitors TCR repertoire in patients undergoing immunotherapy
Offers a method to assess immune response quality in health and disease