A Membrane-Activated, Universal T-Cell Receptor Agonist

Abstract

transmembrane signaling in response to extracellular stimuli. Inherent PM organizational principles often include, but are not limited to, ordered proteo-lipid nanodomains and cortical actin cytoskeleton. Such structural features underpin the steady-state environment of signaling components to provide an optimal platform for signal initiation. Therefore, it is necessary to evaluate the 'resting state' organization of signaling components and its modulation after stimulation for complete understanding of the PM-localized stage of the cell surface signaling. In this study, we used RBL mast cells as a general paradigm of ligand induced transmembrane signaling. Here, extracellular triggering of immunoglobin E complexed to its receptor, FcεRI, (IgE-FcεRI) by multivalent antigen leads to receptor clustering and subsequent phosphorylation by Lyn tyrosine kinase to initiate signaling. We first examined lateral environment 'sensed' in the resting state by IgE-FcεRI and Lyn by monitoring their respective lateral diffusion modes in RBL cells with multiplexed Imaging Fluorescence Correlation Spectroscopy (Imaging FCS) and associated spot variation FCS (svFCS). The diffusion of both receptor and kinase deviate from free diffusion due to interaction with different confinement sources. Furthermore, we identified the simultaneous presence of lipid-dependent and actin-dependent confinement sources for the diffusion of Lyn kinase, whereas diffusion of IgE-FcεRI was weakly dependent on cortical actin organization. Importantly, our Imaging FCS results for a dozen different membrane proteins indicate that 70% of the plasma membrane exhibits the diffusion properties of an ordered environment. We further elucidate the association of Lyn kinase and stimulated IgE-FcεRI receptor with their lipiddriven ordered environments, which effectively enhances their spatial proximity that is essential for optimal phosphorylation of antigen-activated receptors. The interaction of T cells with antigen presenting cells (APC) plays a central role in the adaptive immune system. However, the molecular mechanisms by which binding of the T cell receptor (TCR) to its ligand triggers a signal within the cell are still debated. Here, we use DNA origami decorated with TCR ligands anchored to a planar glass-supported lipid bilayer to assess the effects of local ligand density, arrangement and mobility on T cell activation. Our experimental setup allows for controlling the nanoscale arrangement of TCR ligands on the DNA origami scaffold as well as the re-organization of ligand and TCR during T cell activation. Further, the spatial distribution of ligands can be tuned independently of ligand concentration. TCRb-reactive single-chain antibody fragment (scFv) and peptide-loaded major histocompatibility complex II (pMHC) were used as ligands and placed on the DNA origami at engineered capture sites in different layouts and densities. Functionalized DNA origami platforms were characterized using single molecule fluorescence microscopy and high-speed atomic force micr...