An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Abstract

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells ('APCs') referred to as 'immunological synapses'. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of 'semi-professional APCs'. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic 'planar cellular APC model' for improved imaging and interrogation of fundamental antigenic signaling processes will be described. Video LinkThe video component of this article can be found at

Protein Kinase C δ Regulates the Depletion of Actin at the Immunological Synapse Required for Polarized Exosome Secretion by T Cells

“…As a drawback, only those APC-T cell interfaces that were perpendicular to the plane of focus along the Z-axis could be properly analyzed (45). Since other experimental models that may facilitate image capture and analyses do not mimic the complex, irregular surface of an APC or a target cell, and may raise to non-physiological interactions in the IS (60–63), we consider the experimental system used here appropriate to address some biological events occurring at the IS. In addition, with the use of the PKCδ C1-based U.DAG2 sensor, we have shown that upon synaptic activation, DAG species are produced that may recruit PKCδ at endomembranes including MVB.…”

Regulation of Immune Functions by Non-Neuronal Acetylcholine (ACh) via Muscarinic and Nicotinic ACh Receptors

“…That finding and the detection of ACh in the plasma and blood of various animal species, including humans, along with the development of highly sensitive radioimmunoassays [21], led to investigations into the origin of ACh in the blood and its physiological function (see also the reviews [15,22,23]). VECs constitutively express major histocompatibility complex (MHC) I and II and a range of costimulatory molecules, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), under the regulation of inflammatory cues (see a review by Carman and Martinelli [24]). VECs thus play an essential locally tuned role in the regulation of T cell migration and information exchange.…”