Abstract

The detection and neutralization of infected cells and tumors by cytotoxic lymphocytes is a vital immune defense mechanism. The immunological synapse orchestrates the target recognition process and the subsequent cytotoxic activity. Here, we present an integrated experimental and computational strategy to systematically characterize the morphological properties of the immunological synapse of human cytotoxic lymphocytes. Our approach combines high-content imaging with an unbiased, data-driven identification of high-resolution morphological profiles. Such profiling discriminates with high accuracy immunological synapse perturbations induced by an array of actin drugs in both model cell lines and primary lymphocytes. It reveals inter-individual heterogeneity in lymphocyte morphological traits. Furthermore, it uncovers immunological synapse alterations in functionally defective CD8+ T cells from immunodeficient patients carrying ARPC1B mutations. Our study thus provides a foundation for the application of morphological profiling as a powerful and scalable approach to monitor lymphocyte activation status in experimental and disease settings.