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

Abstract

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.

Molecular Tuning of Actin Dynamics in Leukocyte Migration as Revealed by Immune-Related Actinopathies

“…Moreover, studies of immune cell motility are further complicated by the diversification of motility strategies adopted by the different immune cell subsets and restricted amount of patient material available. In an effort to address some of those limitations we have recently introduced morphological profiling of leukocytes via high-content cell imaging ( 90 ). Such approach can be applied to restricted sample sizes, can be used in standardizing the comparison of morphological defects in primary leukocytes from patients and has proven to be efficient at discriminating defects in closely related deficiencies such as ARPC1B and WASP deficiencies.…”

“…ARPC1B is an essential subunit of the ARP2/3 complex that is critical for its assembly and maintenance ( 140 ). Upon interaction with a surface coated with ICAM-1 and α-CD3 antibodies to mimic APC encounter, ARPC1B-deficient T cells fail to assemble a circular lamellipodium and instead emit aberrant thin filopodia ( 20 , 21 , 90 ). ARPC1B deficiency in human T cells is also associated with defective migration in response to chemokines ( 20 ), presumably because of a defective lamellipodium.…”

“…Indeed, WASP is not essential for lamellipodium emergence, but rather controls its stability and dynamics, as shown in T cells in the context of IS assembly ( 134 , 135 ). Instead of emitting a radially distributed lamellipodium, WASP-deficient CD8 + T cells display a disrupted lamellipodium that irradiates in different directions ( 87 , 90 ). Alternation of arrest phases at the contact with APC and motility phases to search for new APC is key to the tuning of lymphocyte activation and function.…”

ARPC1B binds WASP to control actin polymerization and curtail tonic signaling in B cells

“…It follows an autosomal recessive inheritance pattern and is clinically marked by leukocytoclastic vasculitis, thrombocytopenia, immune defects, eczema, otitis media, and bloody eosinophilic colitis. ARPC1B deficiency in T cells impairs immune synapse formation and the targeted release of lytic granules, ARP2/3-dependent processes, which helps to explain the reported immune defects ( 3 , 4 ). Similar to WAS, ARPC1B-deficient patients also have increases in circulating CD19 + cells, soluble IgE, sIgA, and autoantibodies, which is suggestive of an intrinsic, autoactivated B cell compartment ( 1 , 5 , 6 ).…”

BioProfiling.jl: profiling biological perturbations with high-content imaging in single cells and heterogeneous populations

“…Depending on the application, the analysis of HCS experiments may involve a variety of tasks. For instance, one might perform a classification task to infer the mechanism of action of candidate drugs ( Ando et al , 2017 ; Ljosa et al , 2013 ; Pawlowski et al , 2016 ), compare cellular phenotypes in various conditions ( German et al , 2021 ; Gustafsdottir et al , 2013 ; Rohban et al , 2017 ) or describe interactions between cellular perturbations ( Billmann et al , 2016 ; Breinig et al , 2015 ; Caldera et al , 2019 ; Fischer et al , 2015 ; Heigwer et al , 2018 ). All these cases involve numerous experimental and analytical steps.…”