Morphological profiling of human T and NK lymphocytes by high-content cell imaging

The immunological synapse is a complex structure that decodes stimulatory signals into adapted lymphocyte responses. It is a unique window to monitor lymphocyte activity because of development of systematic quantitative approaches. Here we demonstrate the applicability of high-content imaging to human T and natural killer (NK) cells and develop a pipeline for unbiased analysis of high-definition morphological profiles. Our approach reveals how distinct facets of actin cytoskeleton remodeling shape immunological synapse architecture and affect lytic granule positioning. Morphological profiling of CD8+ T cells from immunodeficient individuals allows discrimination of the roles of the ARP2/3 subunit ARPC1B and the ARP2/3 activator Wiskott-Aldrich syndrome protein (WASP) in immunological synapse assembly. Single-cell analysis further identifies uncoupling of lytic granules and F-actin radial distribution in ARPC1B-deficient lymphocytes. Our study provides a foundation for development of morphological profiling as a scalable approach to monitor primary lymphocyte responsiveness and to identify complex aspects of lymphocyte micro-architecture. [Display omitted] •Cytotoxic lymphocytes can be profiled in depth using high-content imaging•Morphological changes are identified with dedicated analytical tools•Distinct immunological synapse alterations in ARPC1B- and WASP-deficient T cells•3D distribution of lytic granules is impaired in ARPC1B-deficient CD8+ T cells German et al. provide a workflow to study activation of cytotoxic T and NK lymphocytes by high-content confocal microscopy. Images are analyzed to characterize morphological variation in immunological synapses induced by actin compounds in primary cells as well as in CD8+ T cells derived from ARPC1B- and WASP-deficient individuals.