High-Throughput Discovery of MHC Class I- and II-Restricted T Cell Epitopes Using Synthetic Cellular Circuits

Infectious Diseases Rheumatology and Immunology Medicine Basic Medical Sciences Cell Biology Oncology Life Sciences Immunology
high-throughput discovery MHC class I MHC class II T cell epitopes synthetic cellular circuits antigen discovery

High-Throughput Discovery of MHC Class I and II Restricted T Cell Epitopes Using a Novel Synthetic Cellular Circuit

Research Background and Issues

In recent years, antigen discovery technologies have made remarkable progress, especially in the application of human T-cell receptors (TCRs) in the recognition of major histocompatibility complex (MHC) class I restriction. However, the research and technology concerning MHC class II restriction and mouse TCR activity are relatively lagging. This limitation hinders the treatment and research of various immune-related diseases such as cancer, infectious diseases, and autoimmune diseases. This paper aims to report a novel TCR antigen discovery method, namely TCR mapping of Antigenic Peptides (TCR-MAP), to fill this research gap.

Source of the Paper

This article was co-authored by scholars such as Ayano C. Kohlgruber, Mohammad H. Dezfulian, Brandon M. Sie, Charlotte I. Wang, Tomasz Kula, Uri Laserson, H. Benjamin Larman, and Stephen J. Elledge. The participating research units include Brigham and Women’s Hospital, Harvard Medical School, Boston Children’s Hospital, Massachusetts General Hospital, Icahn School of Medicine at Mount Sinai, Johns Hopkins School of Medicine, among others. The paper was accepted on November 5, 2023, published on April 16, 2024, and appeared in “Nature Biotechnology.”

Research Methods and Process

Overview of the Method

TCR-MAP is a method that leverages synthetic TCR stimulation circuits to activate immune cell extracellular tags. Specifically, through the use of synthetic circuits in immortalized Jurkat T cells, tags are activated in Sortase-mediated engineered antigen-presenting cells (APCs). This allows tagged live APCs to be directly purified and decoded via sequencing. This method can query MHC class I and II restricted TCR autologous or viral responses with extremely high throughput and sensitivity.

Detailed Process

  1. Engineering Jurkat Cells: First, Jurkat cells were genetically modified to express synthetic circuits, including mCD40L-Srta. This construct is expressed after TCR activation and catalyzes the tagging of cognate APCs.

  2. Modification of Target APCs: By transduction, target APCs express processed peptide fragments and target MHC molecules. When these peptide fragments are recognized by TCRs, tags are transferred to the APC surface.

  3. Experimental Verification:

    • Initially, trials using peptides derived from human cytomegalovirus (CMV) demonstrated that the method could identify specific peptides and tag them.
    • Further verification included experiments with cancer/testis antigen 1b (CTAG1B/NY-ESO-1), showing the method’s high specificity and sensitivity in detecting cancer peptide fragments.
    • Similarly, validation was conducted on a mouse model using the known OT-I TCR-SIINFEKL epitope, indicating that the system is also applicable to mice.

Algorithms and Data Analysis

The study also employed complex computational models and data analysis algorithms, including flow cytometry and bioinformatics analysis, to precisely determine antigen tagging responses. Additionally, custom-developed software was used for data processing, ensuring result accuracy and reproducibility.

Main Results

  1. Effective Capture of TCR-MHC Interactions: TCR-MAP can effectively capture the specificity interactions between MHC class I and II molecules and TCR through cell surface tagging methods, with verified sensitivity and specificity.

  2. High-Throughput Detection of Autoreactive and Viral Responses: The system can sensitively and in high throughput detect autologous or viral responses, suitable for antigen discovery in cancer, infectious diseases, and autoimmune diseases.

  3. Mapping of Diverse Response Libraries: In experiments containing multiple protein libraries from humans, viruses, and mice, TCR-MAP demonstrated efficient antigen mapping capabilities, especially in complex antigen environments.

  4. Sensitive Detection and Future Expansion: Experimental results proved TCR-MAP’s sensitivity in handling both low-affinity and high-affinity antigens, providing potential for future applications in diverse TCR clonotype libraries. Additionally, the system also showed potential in capturing non-peptide antigens such as lipids or metabolites.

Conclusion and Significance

Scientific Value

The TCR-MAP method developed in this study provides the scientific community with an efficient, flexible, and sensitive new method for high-throughput analysis of TCR-specific antigens. It overcomes many limitations of traditional methods, including dependence on primary T cells and the requirement for target cell lysis.

Application Value

The broad application potential of TCR-MAP includes antigen discovery and therapy for cancer, infectious diseases, and autoimmune diseases, particularly significant in autoimmune disease and tumor treatment.

Research Highlights

  • Flexibility and Specificity: The method is broadly applicable, including models such as humans and mice, suitable for both MHC class I and II restricted TCRs.
  • High Throughput and Sensitivity: By achieving efficient antigen discovery and tagging, the sensitivity and specificity of the detection are improved.
  • Cross-Species Usability: The study proved that TCR-MAP could be effectively applied across multiple species, facilitating both basic and applied research.

TCR-MAP provides an innovative, flexible, and efficient antigen discovery method that is sure to bring significant breakthroughs in immunology research, clinical applications, and other related fields.