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 (HO), 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 HO, 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 HO concentration is not the only determinant of the signalling response, as cells exposed to more shallow gradients respond at lower HO levels. A minimal model of the proximal BCR network qualitatively reproduced the experimental findings and uncovered a rate-dependent sensitivity to HO, 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.