Rapid Generation of Transgenic Mice Expressing T Cell Receptors Specific for SARS-CoV-2 Spike Epitope Using Single-Cell Sequencing and Repertoire Analysis

Core Concepts

A streamlined approach combining single-cell sequencing, TCR repertoire analysis, and transgenic mouse generation enables rapid development of antigen-specific TCR transgenic mouse models.

Abstract

The authors present an optimized approach for generating TCR transgenic mouse models that overcomes the limitations of the classical hybridoma-based method. Key highlights: The classical hybridoma-based approach for selecting TCR clones was unsuccessful in generating a functional SARS-CoV-2 Spike-specific TCR transgenic mouse line (CORSET8). To improve the selection process, the authors combined single-cell RNA sequencing and TCR repertoire analysis using the DALI software tool. This allowed them to identify an optimal TCR clonotype based on characteristics like clonal expansion, cytokine production, and activation phenotype. The selected TCR sequences were then used to generate the CORSET8 transgenic mouse line, which exhibited robust proliferation and activation of transgenic T cells upon immunization with SARS-CoV-2 Spike protein or vaccination. The CORSET8 mice also showed strong proliferation of transgenic T cells in response to SARS-CoV-2 infection in a mouse model expressing the human ACE2 receptor. The authors conclude that their streamlined approach offers an attractive method for future development of TCR transgenic mouse models, as it is more time- and cost-effective compared to the classical hybridoma-based approach.

Stats

The authors report that 95.17% (± 1.61) of CORSET8 cells underwent division in the draining mediastinal lymph node 4 days after adoptive transfer and Spike/CpG immunization. 99.63% (± 0.03%) of CORSET8 cells had undergone division and displayed an activated phenotype in the draining popliteal lymph node 4 days after adoptive transfer and Pfizer BNT162b2 vaccine immunization. 99.28% (± 0.12%) of CORSET8 cells divided and upregulated activation markers in the draining mediastinal lymph node 4 days after adoptive transfer and SARS-CoV-2 infection in hACE2 transgenic mice.

Quotes

"Owing to the browser-based interactive GUI, immunologists having limited coding experience can effectively analyze these complex datasets." "The streamlined method for generating TCR Tg mice, as presented here, offers an attractive approach for future TCR Tg mouse development."

Key Insights Distilled From

TCR transgenic clone selection guided by immune receptor analysis and single cell RNA expression of polyclonal responders

by Debeuf,N., L... at www.biorxiv.org 03-27-2024

https://www.biorxiv.org/content/10.1101/2024.03.27.586931v1

Deeper Inquiries

How could the authors further validate the functionality and utility of the CORSET8 transgenic mouse model, beyond the experiments presented in this study?

To further validate the functionality and utility of the CORSET8 transgenic mouse model, the authors could consider the following experiments: Long-term immune response: Assess the longevity of the antigen-specific T cell response in CORSET8 mice by monitoring T cell persistence and memory formation over an extended period post-immunization or infection. Functional assays: Conduct additional functional assays to evaluate the effector functions of CORSET8 T cells, such as cytokine production, cytotoxicity, and antigen-specific killing capacity. Tissue distribution: Investigate the tissue distribution of CORSET8 T cells to determine their migration patterns and potential for tissue-specific immune responses. Viral clearance: Evaluate the ability of CORSET8 mice to clear viral infections by challenging them with a live virus and monitoring viral load over time. Immunopathology: Assess the potential for immunopathological responses in CORSET8 mice by examining tissue damage and inflammatory markers in response to antigen exposure.

What are the potential limitations or drawbacks of the single-cell sequencing and repertoire analysis approach compared to the classical hybridoma-based method?

Some potential limitations or drawbacks of the single-cell sequencing and repertoire analysis approach compared to the classical hybridoma-based method include: Cost: Single-cell sequencing can be more expensive than traditional hybridoma methods, especially when analyzing a large number of samples or cells. Complexity: The bioinformatics analysis required for single-cell sequencing data can be complex and may require specialized expertise, which could be a barrier for researchers with limited bioinformatics skills. Cell viability: Single-cell sequencing relies on the isolation of viable single cells, which can be challenging and may lead to cell stress or loss during the process. Cell heterogeneity: Single-cell sequencing captures the heterogeneity of the cell population, which can complicate data analysis and interpretation compared to the clonal populations obtained from hybridoma methods. Validation: The functional validation of TCR sequences identified through single-cell sequencing may require additional experiments to confirm antigen specificity and T cell functionality.

How could the insights gained from the development of the CORSET8 model be applied to generate transgenic mouse models targeting other pathogens or antigens of interest?

The insights gained from the development of the CORSET8 model can be applied to generate transgenic mouse models targeting other pathogens or antigens of interest in the following ways: Antigen selection: Utilize similar strategies to identify immunogenic and stable epitopes for other pathogens or antigens of interest, ensuring the selection of functionally relevant TCR clones. Single-cell analysis: Employ single-cell sequencing and repertoire analysis to identify and characterize antigen-specific T cell clones for other pathogens, allowing for a more rationalized selection of TCR sequences. Functional validation: Conduct thorough functional validation assays to confirm the antigen specificity, affinity, and effector functions of the selected TCR clones before generating transgenic mice. Immunophenotyping: Integrate immune receptor profiling with immunophenotyping to assess the functional properties and precursor frequency of individual T cells within a polyclonal response to vaccination or infection for other pathogens. Optimization: Continuously refine and optimize the process of TCR transgenic mouse generation based on the lessons learned from the CORSET8 model, aiming for improved efficiency and success rates in generating functional TCR Tg mice for diverse antigens.