A Bayesian framework reveals heterogeneous and stochastic decision-making in NK cell cytotoxicity

Natural killer (NK) cells display striking variability in their cytotoxic responses, but whether this variation reflects random events, stable differences between cells, or effects of previous encounters remains unclear. We developed a Bayesian framework that uses single-cell interaction histories to disentangle these sources of variability. The framework models target encounters and killing decisions to quantify population heterogeneity, determine the sample sizes needed to distinguish competing mechanisms, and separate stable cell-to-cell differences from history-dependent behaviour. Synthetic data established when these mechanisms can be reliably distinguished in practice. We tested the framework using time-lapse imaging of NK cells exposed to rituximab or CC-96673, an antibody co-targeting CD20 and CD47. Despite similar overall killing, the framework identified distinct underlying responses: rituximab mainly increased mean killing rate, whereas CC-96673 reduced cell-to-cell variation. Event-count analysis supported continuous population heterogeneity, while ordered interaction histories revealed that both stable differences in killing propensity and previous encounters shape cytotoxic decisions. To make the framework directly usable, we introduce BARRACUDA, an open-source web platform and Python package implementing these analyses, including donor-aware extensions, for single-cell cytotoxicity datasets.