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.

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.