The Planar Lipid Bilayer System Serves as a Reductionist Approach for Studying NK Cell Immunological Synapses and Their Functions

Abstract

Summary The immunological synapse (IS) is the junction between an immune cell (e.g. a T or NK cell) and another cell (e.g. an antigen-presenting cell (APC), or a tumor cell). The formation of the IS is crucial for cell-mediated immunity, and as such, an understanding of both the composition of the IS and the factors that drive its formation are essential for understanding how and when NK cells eliminate susceptible target cells. The supported lipid bilayer (SLB) system is a highly effective tool for directly studying the IS. SLBs confer three main advantages: 1) they allow for synapse formation on a level horizontal surface, allowing for direct visualization of the IS under high resolution imaging systems, 2) they mimic the surface of a target cell by providing a fluid mosaic into which surface proteins can be embedded while permitting free motion in two dimensions, which is important for studying the dynamics of synapse formation, and 3) they allow investigators to determine the exact composition of the bilayer, thus in turn allowing them to answer very specific questions about the IS. It is our hope that this chapter will furnish readers with an awareness of the applications of the SLB system for studying the IS in NK cells, and also of a basic knowledge of how to use this system for themselves.

Imaging Polarized Secretory Traffic at the Immune Synapse in Living T Lymphocytes

“…Unfortunately, this technique can be somewhat reductionist since it is not possible to guarantee that all the molecular interactions occurring in a cell–cell synapse will occur also upon interaction with the bilayer ( 5 ). In fact, supported lipid bilayers do not mimic the complex surface of an APC or target cell, giving rise to non-physiological interactions in the IS ( 51 ). In addition, capture in a defined Z in the very initial moments of the interaction with the coverslip will exclude intracellular structures (i.e., secretory vesicles) located beyond a certain Z distance from the coverslip (>200–300 nm).…”

Understanding T cell signaling using membrane reconstitution

“…We recently used a similar system to reveal that PD‐1 strongly co‐localizes with CD28, but partially segregates from TCR in CD8 + cytotoxic T cells . Finally, SLB has been used to study the immunological synapse of various innate immune cells …”

Chimeric antigen receptor (CAR)-modified natural killer cell-based immunotherapy and immunological synapse formation in cancer and HIV

“…In addition to the cell-cell conjugation system, the structure, function, and signaling cascades at the IS have been further confirmed by imaging T cell interactions with a glass-supported, planar lipid bilayer, which contains the MHC-peptide complex and other costimulatory molecules (Tozeren et al, 1992 ; Grakoui et al, 1999 ; Lee et al, 2002 , 2003 ; Mossman et al, 2005 ). The general consensus in the field of immunology is that a glass-supported, planar lipid bilayer system can mimic target cells for the study of the IS at high resolution (Dustin et al, 2007 ; Choudhuri et al, 2014 ; Zheng et al, 2015a ; Bertolet and Liu, 2016 ). Previous studies have demonstrated that IS quality determines the efficacy of T cell-mediated cytotoxicity (Grakoui et al, 1999 ; Jenkins et al, 2009 ; Dustin and Long, 2010 ).…”