Dynamic hydrogen peroxide levels reveal a rate-dependent sensitivity in B-cell lymphoma signaling

Although in vivo extracellular microenvironments are dynamic, most in vitro studies are conducted under static conditions. Here, we exposed diffuse large B-cell lymphoma (DLBCL) cells to gradient increases in the concentration of hydrogen peroxide (H₂O₂), thereby capturing some of the dynamics of the tumour microenvironment. Subsequently, we measured the phosphorylation response of B-cell receptor (BCR) signalling proteins CD79a, SYK and PLCγ2 at a high temporal resolution via single-cell phospho-specific flow cytometry. We demonstrated that the cells respond bimodally to static extracellular H₂O₂, where the percentage of cells that respond is mainly determined by the concentration. Computational analysis revealed that the bimodality results from a combination of a steep dose-response relationship and cell-to-cell variability in the response threshold. Dynamic gradient inputs of varying durations indicated that the H₂O₂ concentration is not the only determinant of the signalling response, as cells exposed to more shallow gradients respond at lower H₂O₂ levels. A minimal model of the proximal BCR network qualitatively reproduced the experimental findings and uncovered a rate-dependent sensitivity to H₂O₂, where a lower rate of increase correlates to a higher sensitivity. These findings will bring us closer to understanding how cells process information from their complex and dynamic in vivo environments.