DNA Origami as a Nanoscale Platform for T-Cell Activation

Abstract

In the human immune system, the recognition of an antigen by the T-cell receptor (TCR) takes place within the contact area between the T-cell and the antigen-presenting cell (APC). It is thought that the nanoscale spatial distribution of proteins within this contact zone plays an essential role in the initiation of an immune response. Despite extensive studies, the molecular details of this process, in particular the structural requirements for TCR triggering and how nanoscale events are translated into T-cell activation, are still poorly understood. 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 and arrangement on T-cell activation. Thus, our experimental setup allows for the precise nanoscale arrangement of TCR ligands on the DNA origami scaffold, while at the same time permitting the re-organization of ligand and TCR during T-cell activation. We used either fluorescently labeled CD3 antibody or recombinant TCRβ-reactive single chain antibody fragment (scFv) as stimulatory ligands that were placed on the DNA origami at one to 20 engineered capture sites in different layouts and densities. The actual number of ligands per origami was determined using several single molecule fluorescence microcopy methods and atomic force microscopy. The activation of T-cells interfaced with the APC-mimicking surfaces was measured using a calcium-sensitive fluorescent dye and the effects of local ligand density, nanoscale ligand arrangement as well as the nature of the ligand were assessed. Further, the rearrangement of TCR and ligands in the process of T-cell activation was monitored by single molecule microscopy.