T cell self-reactivity during thymic development dictates the timing of positive selection

Functional tuning of mature T cells based on their degree of self-reactivity is established during positive selection in the thymus, although how positive selection differs for thymocytes with relatively low versus high self-reactivity is unclear. In addition, preselection thymocytes are highly sensitive to low-affinity ligands, but the mechanism underlying their enhanced TCR sensitivity is not fully understood. Here we show that thymocytes with low self-reactivity experience briefer TCR signals and complete positive selection more slowly than those with high self-reactivity. Additionally, we provide evidence that cells with low self-reactivity retain a preselection gene expression signature as they mature, including genes previously implicated in modulating TCR sensitivity and a novel group of ion channel genes. Our results imply that thymocytes with low self-reactivity down-regulate TCR sensitivity more slowly during positive selection, and suggest that modulation of membrane ion channel function may play a role in regulating TCR tuning throughout development. Overall design: RNA-seq was performed on FACS sorted thymocytes from three OT-1 Rag2KO, three F5 Rag1KO, and three TG6 H2b/d mice (biological replicates). Thymocytes were sorted into 3 populations representing 3 distinct stages of CD8 T cell positive selection prior to sequencing: CD4+CD8+CXCR4+CCR7- (C, early positive selection), CD4+CD8+CXCR4-CCR7+ (B, late positive selection) [TG6 only has two biological replicates for this population], and CD4-CD8+CXCR4-CCR7+ (A, Mature CD8SP). This resulted in 26 samples in total.