Abstract
T cell receptor beta chain (TCRβ) diversity (Dβ) gene segments are highly conserved across evolution, with trout Dβ1 sequence identical to human and mouse Dβ1. A key conserved feature is enrichment for glycine in all three Dβ reading frames (RFs). Previously, we found that replacement of mouse Dβ1 with a typical immunoglobulin DH sequence, which unlike Dβ is enriched for tyrosine, leads to an increase in the use of tyrosine in TCRβ complementarity determining region 3 (CDR-B3) after thymic selection, altering T cell numbers, CDR-B3 diversity, and T cell function. To test whether the incorporation of charged amino acids into the Dβ sequence in place of glycine would also influence T cell biology, we targeted the TCRβ locus with a novel glycine-deficient DβDKRQ allele that replaces Dβ1 coding sequence with charged amino acids in all three reading frames. Developing T cells using DβDKRQ expressed TCR CDR-B3s depleted of tyrosine and glycine and enriched for germline-encoded lysine, arginine, and glutamine. Total thymocytes declined in number during the process of β selection that occurs during the transition from the DN3bc to DN4 stage. Conventional thymocyte and T cell numbers remained reduced at all subsequent thymic stages and in the spleen. By contrast, regulatory T cell numbers were increased in Peyer’s patches and the large intestine. In terms of functional consequences, T cell reactivity to an ovalbumin immunodominant epitope was reduced. These findings buttress the view that natural selection of Dβ sequence is used to shape the pre-immune TCRβ repertoire, affecting both conventional and regulatory T cell development and influencing epitope recognition.
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Acknowledgements
We thank Pratibha Kapoor and Yingxin Zhuang for assistance with the creation of the targeting constructs and the gene targeting. We thank Robert Schelonka for assistance in planning the studies and Barry Sleckman for contributing the original DB2KO construct and for reviewing and discussing the results of our studies.
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This work was supported, in part, by AI090902 (HWS) and AI163555 (HWS).
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ML took the lead role in performing the FACS analysis of T cell subsets, the sequencing of the thymocyte and T cell subsets, sequence analysis, performing the ovalbumin challenge, preparing of the figures, and writing initial version of the manuscript. MK participated in interpreting and analyzing the data, and in revising and editing the manuscript. Both ML and MK contributed equally to the manuscript. PDB participated in the planning of the experiments, in interpreting the data and in editing the manuscript. HWS developed the concept of the project, directed the planning and execution of the studies, reviewed the data, and directed the writing of the manuscript.
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Levinson, M., Khass, M., Burrows, P.D. et al. Germline-enforced enrichment for charged amino acids in TCR beta chain (TCRβ) complementarity determining region 3 (CDR-B3) alters T cell development, repertoire content, and antigen recognition. Immunogenetics 75, 341–353 (2023). https://doi.org/10.1007/s00251-023-01304-w
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DOI: https://doi.org/10.1007/s00251-023-01304-w