Functional tuning of 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 murine 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 associate membrane ion channel expression with thymocyte self-reactivity and progress through positive selection.

Calcium is a universal second messenger present in all eukaryotic cells. The mobilization and storage of Ca²⁺ ions drives a number of signaling-related processes, stress–responses, or metabolic changes, all of which are relevant for the development of immune cells and their adaption to pathogens. Here, we introduce the Förster resonance energy transfer (FRET)-reporter mouse YellowCaB expressing the genetically encoded calcium indicator TN-XXL in B lymphocytes. Calcium-induced conformation change of TN-XXL results in FRET-donor quenching measurable by two-photon fluorescence lifetime imaging. For the first time, using our novel numerical analysis, we extract absolute cytoplasmic calcium concentrations in activated B cells during affinity maturation in vivo. We show that calcium in activated B cells is highly dynamic and that activation introduces a persistent calcium heterogeneity to the lineage. A characterization of absolute calcium concentrations present at any time within the cytosol is therefore of great value for the understanding of long-lived beneficial immune responses and detrimental autoimmunity.