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Probing TCR Specificity Using Artificial In Vivo Diversification of CDR3 Regions
Orlando B Giorgetti1, Annette Haas-Assenbaum1, Thomas Boehm1,2,3
1Department of Developmental Immunology, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.
Abstract:
The T-cell receptor sequences expressed on cells recognizing a specific peptide in the context of a given MHC molecule can be explored for common features that might explain their antigen specificity. However, despite the development of numerous experimental and bioinformatic strategies, the specificity problem remains unresolved. To address the need for additional experimental paradigms, we report here on an in vivo experimental strategy designed to artificially diversify a transgenic TCR by CRISPR/Cas9-mediated mutagenesis of Tcra and Tcrb chain genes. In this system, an initially monoclonal repertoire of known specificity is converted into an oligoclonal pool of TCRs of altered antigen reactivity. Tracking the fate of individual clonotypes during the intrathymic differentiation process illuminates the strong selective pressures that shape the repertoire of naïve T cells. Sequence analyses of the artificially diversified repertoires identify key amino acid residues in the CDR3 regions required for antigen recognition, indicating that artificial diversification of well-characterized TCR transgene sequences helps to reduce the complexities of learning the rules of antigen recognition.
Insights
This study introduces a novel in vivo method to diversify T-cell receptor (TCR) sequences using CRISPR/Cas9. This approach helps identify key amino acid residues crucial for T-cell receptor antigen recognition.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Understanding T-cell receptor (TCR) specificity is crucial for immunology.
- Current experimental and bioinformatic strategies have not fully resolved the TCR antigen specificity problem.
Purpose of the Study:
- To develop a new in vivo experimental strategy to artificially diversify a transgenic TCR.
- To identify key amino acid residues in TCRs essential for antigen recognition.
Main Methods:
- Utilized CRISPR/Cas9-mediated mutagenesis of T-cell receptor alpha (Tcra) and T-cell receptor beta (Tcrb) chain genes in vivo.
- Converted an initially monoclonal TCR repertoire into an oligoclonal pool with altered antigen reactivity.
- Tracked individual T-cell clonotypes during intrathymic differentiation and analyzed sequences of diversified repertoires.
Main Results:
- Demonstrated that artificial diversification of transgenic TCRs can alter antigen reactivity.
- Identified specific amino acid residues within the CDR3 regions critical for TCR antigen recognition.
- Highlighted the significant selective pressures influencing the repertoire of naive T cells during intrathymic differentiation.
Conclusions:
- Artificial diversification of well-characterized TCR transgene sequences aids in deciphering the rules of antigen recognition.
- This in vivo strategy provides a powerful tool for studying TCR specificity and repertoire development.
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