Multiparameter stimulation mapping of signaling states in single pediatric immune cells reveals heightened tonic activation during puberty

Cellular stimulation via factors such as cytokines followed by multiparameter single-cell measurements is a powerful approach to interrogate cellular functions. However, transforming such high-dimensional data into biological insights presents unique challenges, particularly given the extensive response heterogeneity among single cells, such as the presence of bimodal responding versus non-responding subpopulations upon stimulation. Here we present an unsupervised high-dimensional approach for analyzing stimulation responses at the single cell level (HDStIM) and apply it to evaluate how pediatric development may shape peripheral immune cell signaling states and responsiveness to stimulations in 42 subjects (age: 2 - 16). We show that in comparison to the conventional approach of assessing one marker at a time by averaging across single cells, HDStIM can effectively learn, in an unsupervised fashion, the multi-parameter signature of responding versus non-responding cells to accurately quantify responses within cell populations. HDStIM reveals that the extent of pre-stimulation/baseline activation of interferon-related and TCR signaling molecules in myeloid and T cells, respectively, increases during puberty. This suggests that puberty is marked by a heightened "tonic" activation state in these cells, perhaps to strengthen defense against pathogens during this period of human development.