Identification of unconventional Dual TCR clonotypes and their capacity to evade thymic selection 4707

Description Greater than 15% of human and mouse αβ T cells express 2 functional T cell receptors (dual TCR cells) due to allelic inclusion during thymopoiesis. Co-expression of 2 TCR clonotypes decreases the stringency of thymic selection, enabling untolerated, atypical T cell receptors to emerge. We hypothesize that these TCRs enable the dual TCR repertoire’s increased autoreactivity, alloreactivity, and tumor neoantigen reactivity. However, identifying atypical TCRs has been challenging due to limitations in dual TCR identification and sequencing. Using our novel dual TCRα transgenic reporter mouse (B6.TCRα-GFP/RFP) we isolated single (GFP+/RFP+) and dual (GFP+RFP+) CD4+ & CD8+ T cells, and developed a pipeline to identify dual TCR-associated clonotypes. From single-cell TCR sequences of the dual TCR repertoire, we distinguished conventional TCRs from the unique TCRs we hypothesize harbor atypical reactivity. We find that dual TCR cells that contain atypical TCRs are paired with conventional TCRs by necessity and that atypical dual TCR chains are associated with known autoreactive TCRs. Via independent retrogenic cloning of dual TCRs into RAG deficient mice, we are examining that in the absence of a conventional receptor, the atypical receptor will fail to emigrate from the thymus. These findings confirm our hypothesis that dual TCR expression enables thymic emergence of atypical receptors and validate our pipeline as a method in identifying atypical dual T cell receptors in bulk.