26The detection and neutralization of infected cells and tumors by cytotoxic lymphocytes is a 27 vital immune defense mechanism. The immunological synapse orchestrates the target 28 recognition process and the subsequent cytotoxic activity. Here, we present an integrated 29 experimental and computational strategy to systematically characterize the morphological 30properties of the immunological synapse of human cytotoxic lymphocytes. Our approach 31 combines high-content imaging with an unbiased, data-driven identification of high-32 resolution morphological profiles. Such profiling discriminates with high accuracy 33 immunological synapse perturbations induced by an array of actin drugs in both model cell 34 lines and primary lymphocytes. It reveals inter-individual heterogeneity in lymphocyte 35 morphological traits. Furthermore, it uncovers immunological synapse alterations in 36 functionally defective CD8 + T cells from immunodeficient patients carrying ARPC1B 37 mutations. Our study thus provides a foundation for the application of morphological 38 profiling as a powerful and scalable approach to monitor lymphocyte activation status in 39 experimental and disease settings. 40 41 42The immunological synapse (IS) is a complex cellular structure that sets lymphocyte 43 activation and function during encounter with antigen-presenting cells and target cells. The 44 canonical IS is characterized by a symmetrical architecture consisting of concentric rings of 45 F-actin and integrins, while the antigen receptors occupy a central position 1,2 . The 46 lymphocyte spreading associated with IS assembly, as well as the molecular organization 47 defining IS architecture, rely on actin cytoskeleton dynamics. In cytotoxic lymphocytes, 48 including CD8+ T cells and NK cells, the IS is particularly important because it sustains the 49 polarized delivery of cytolytic molecules such as perforin and granzymes towards target 50 cells 3 . Indeed, activation of the integrin LFA-1 via an inside-out signaling from the T-cell 51 receptor (TCR) in T cells, and several stimulatory receptors in NK cells 4-7 , leads to the 52 formation of a tight adhesive ring allowing confinement of the degranulation process. 53Additional layers of control of lytic granule delivery at the IS are their polarization via the 54 orientation of the microtubule organizing center 8,9 and their restricted passage through 55 pervasive actin cytoskeleton clearances 10 . Given the key events occurring at the IS, this 56 structure is a window of choice to monitor lymphocyte activation and function. Indeed, the 57 positioning and dynamic behavior of multiple receptors and signaling molecules have been 58 characterized within the IS 11 , and alterations of the its architecture have been reported in 59 multiple disease settings 12,13 . However, the various microscopy approaches employed so far 60 to characterize spatial organization of the IS have remained low throughput and have been 61 restricted to the analysis of a limited number of morphological features. A more ...
