Single Cell TCR Immune Profiling

Introduction

Single-cell TCR immune profiling is a transformative approach for understanding immune responses, particularly in antigen recognition and T cell activation. Biotinylated MHC (Major Histocompatibility Complex) monomers serve as powerful tools for identifying antigen-specific T cell receptor (TCR) interactions at the single-cell level.

Here, we describe how biotinylated MHC monomers are used across various platforms to profile TCR specificities, focusing on their applications in high-throughput single-cell technologies, such as 10x Genomics’ Chromium system, BioLegend’s TotalSeq™-C system, and BD Rhapsody™ Single-Cell Analysis System.

 

Note: Some applications involving barcoded DNA oligonucleotides may be subject to third-party intellectual property rights. Users are responsible for ensuring freedom to operate for their intended use.

Why Biotinylated Empty MHC Monomers?

Biotinylated Empty MHC monomers provide distinct advantages for immune profiling:

  • Specificity: MHC molecules present specific peptide antigens to TCRs, enabling precise identification of antigen-specific T cells.
  • Flexibility: These monomers can be loaded with various peptides, allowing researchers to explore diverse immune responses.
  • Versatility: They are compatible with streptavidin-based detection systems, making them adaptable to various platforms.

Overview of Single Cell TCR Immune Profiling

Single-cell TCR immune profiling involves isolating individual T cells and determining their antigen specificity through TCR sequencing paired with MHC tetramer-binding assays. This approach provides insights into:

  • T cell clonal expansion
  • TCR diversity and antigen specificity
  • Immune responses in infections, cancer, and autoimmune conditions

Workflow for Single-Cell TCR Profiling with DNA-Barcoded MHC Tetramers

Here is an overview of using biotinylated MHC monomers in single-cell TCR profiling:

  1. MHC Tetramer Preparation and Peptide Loading
    • Load Empty MHC monomers with the peptide of interest to present the specific peptide-MHC (pMHC) complex to TCRs on T cells.
    • Tetramerize by binding biotinylated MHC molecules to streptavidin conjugated with a fluorochrome and DNA barcode.
  2. Single-Cell Staining and Isolation
    • Label Antigen-Specific T Cells: Incubate the cell sample with DNA-barcoded MHC tetramers to bind TCRs that recognize the specific peptide.
    • Isolate Labeled T Cells: Use FACS to isolate tetramer-positive or CD8-positive T cells, or other methods like magnetic bead isolation.
    • Isolate Single T Cells: Capture individual T cells in droplets using platforms like the 10x Genomics Chromium system, BD Rhapsody™ system, or other single-cell technologies.
  3. Single-Cell Sequencing of TCRs
    • Capture antigen-specific T cells in individual droplets, generate cDNA libraries, and perform next-generation sequencing (NGS) to determine the full-length TCR sequences. Match sequences to specific antigen-binding patterns identified by DNA-barcoded MHC tetramers.
  4. Data Analysis
    • Use specialized software (e.g., Cell Ranger from 10x Genomics) for clonotype identification, antigen-specific TCR mapping, and analysis of clonal expansion and diversity.

Platforms for Single-Cell TCR Profiling

Several high-throughput platforms integrate DNA-barcoded MHC tetramers in single-cell TCR profiling workflows:

  1. 10x Genomics Chromium Single Cell Immune Profiling
    • Overview: This droplet-based platform isolates individual T cells, sequencing paired TCR α and β chains while profiling protein and gene expression.
    • Application: Biotinylated MHC monomers with streptavidin-fluorophore conjugates identify antigen-specific TCRs, capturing full-length TCR sequences.
  2. BioLegend TotalSeq™-C System
    • Overview: TotalSeq™-C uses DNA-barcoded antibodies and reagents, including streptavidin conjugates, for high-dimensional protein and antigen detection paired with TCR sequencing.
    • Application: Biotinylated MHC monomers can be conjugated to streptavidin-DNA barcodes, tracking antigen-specific TCRs through the TotalSeq™-C system.
  3. BD Rhapsody™ Single-Cell Analysis System
    • Overview: The BD Rhapsody™ system enables high-throughput single-cell analysis, with options for targeted mRNA and protein profiling.
    • Application: This system supports flexible integration with biotinylated MHC tetramers, allowing for precise TCR profiling and antigen-specific T cell analysis.

Conclusion

DNA-barcoded Empty Loadable MHC tetramers are vital tools for single-cell TCR immune profiling, offering flexibility and specificity for identifying antigen-specific T cells. Platforms like the 10x Genomics Chromium system, BioLegend TotalSeq™-C, and BD Rhapsody™ enable high-throughput profiling of TCR diversity and specificity, advancing our understanding of immune responses in cancer, infectious diseases, and autoimmune conditions, and driving discovery in immunotherapy.