Cells respond to their environments and self-organise into multicellular assemblies with dedicated functions. The migratory and homing response of cells to soluble ligands can be studied by using different techniques, but for real time studies of complex multicellular self-organisation, novel and simpler systems are required. We fabricated a flexible open access microsystem and tested the design by studying cell recruitment from an immune cell reservoir towards an infectious compartment. The two compartments were connected by a network of bifurcated microchannels allowing diffusion of signalling molecules and migration of cells. Bacterial filters were incorporated in the design to prevent bacteria and activated cells from entering the network, permitting migration only from the recruitment reservoir. The fabricated microsystem allows real-time continuous monitoring of cellular decision-making based on biologically produced gradients of cytokines and chemokines. It is a valuable tool for studying cellular migration and self-organisation in relation to infections, autoimmunity, cancer, stem cell homing, and tissue and wound repair.
Exocytosis of cytotoxic granules (CG) by lymphocytes is required for the elimination of infected and malignant cells. Impairments in this process underly a group of diseases with dramatic hyperferritinemic inflammation termed hemophagocytic lymphohistiocytosis (HLH). Although genetic and functional studies of HLH have identified proteins controlling distinct steps of CG exocytosis, the molecular mechanisms that spatiotemporally coordinate CG release remain unclear. We studied a patient exhibiting characteristic clinical features of HLH associated with markedly impaired cytotoxic T lymphocyte (CTL) and natural killer (NK) cell exocytosis functions, who was bearing biallelic deleterious mutations in the gene encoding the small GTPase RhoG. Experimental ablation of RHOG in a model cell line and primary CTLs from healthy individuals uncovered a hitherto unappreciated role of RhoG in retaining CGs in the vicinity of the plasma membrane (PM), a fundamental prerequisite for CG exocytotic release. We discovered that RhoG engages in a protein-protein interaction with Munc13-4, an exocytosis protein essential for CG fusion with the PM. We show that this interaction is critical for docking of Munc13-4-positive CGs to the PM and subsequent membrane fusion and release of CG content. Thus, our study illuminates RhoG as a novel essential regulator of human lymphocyte cytotoxicity, and provides the molecular pathomechanism behind the identified here and previously unreported genetically-determined form of HLH.
Natural killer (NK) cells recognize virally infected cells and tumors. NK cell function depends on balanced signaling from activating receptors, recognizing products from tumors or viruses, and inhibitory receptors (such as KIR/Ly49), which recognize major histocompatibility complex class I (MHC-I) molecules. KIR/Ly49 signaling preserves tolerance to self but also conveys reactivity toward MHC-I–low target cells in a process known as NK cell education. Here, we found that NK cell tolerance and education were determined by the subcellular localization of the tyrosine phosphatase SHP-1. In mice lacking MHC-I molecules, uneducated, self-tolerant Ly49A + NK cells showed accumulation of SHP-1 in the activating immune synapse, where it colocalized with F-actin and the signaling adaptor protein SLP-76. Education of Ly49A + NK cells by the MHC-I molecule H2D d led to reduced synaptic accumulation of SHP-1, accompanied by augmented signaling from activating receptors. Education was also linked to reduced transcription of Ptpn6 , which encodes SHP-1. Moreover, synaptic SHP-1 accumulation was reduced in NK cells carrying the H2D d -educated receptor Ly49G2 but not in those carrying the noneducating receptor Ly49I. Colocalization of Ly49A and SHP-1 outside of the synapse was more frequent in educated compared with uneducated NK cells, suggesting a role for Ly49A in preventing synaptic SHP-1 accumulation in NK cell education. Thus, distinct patterning of SHP-1 in the activating NK cell synapse may determine NK cell tolerance.
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