DNA origami demonstrate the unique stimulatory power of single pMHCs as T cell antigens

Abstract

T cells detect with their T cell antigen receptors (TCRs) the presence of rare agonist peptide/MHC complexes (pMHCs) on the surface of antigen-presenting cells (APCs). How extracellular ligand binding triggers intracellular signaling is poorly understood, yet spatial antigen arrangement on the APC surface has been suggested to be a critical factor. To examine this, we engineered a biomimetic interface based on laterally mobile functionalized DNA origami platforms, which allow for nanoscale control over ligand distances without interfering with the cell-intrinsic dynamics of receptor clustering. When targeting TCRs via stably binding monovalent antibody fragments, we found the minimum signaling unit promoting efficient T cell activation to consist of two antibody-ligated TCRs within a distance of 20 nm. In contrast, transiently engaging antigenic pMHCs stimulated T cells robustly as well-isolated entities. These results identify pairs of antibody-bound TCRs as minimal receptor entities for effective TCR triggering yet validate the exceptional stimulatory potency of single isolated pMHC molecules.

Strategies for the site-specific decoration of DNA origami nanostructures with functionally intact proteins

“…For the quantitative comparison of different functionalization strategies, we employed a recently introduced platform 6 based on rectangular DNA origami tiles anchored to a fluid-phase supported lipid bilayer (SLB) via cholesterol-modified oligonucleotides 25 . DNA origami constructs were assembled from a 65x54 nm DNA origami tile 1 featuring a centrally located and elongated staple strand to create a target for quantitative functionalization with proteins (SI Fig.…”

“…S1). As protein we selected for this study a monovalent single-chain antibody fragment of the variable domain derived from the TCRβreactive monoclonal antibody H57-597 (H57-scFV) 26 , which was shown to induce T-cell activation when displayed on SLBs 6,27 . We further equipped the H57-scFV C-terminally with either a biotin ligase recognition sequence or an unpaired cysteine for the site-specific attachment to DNA origami structures, that would not interfere with TCR binding (Fig.…”

“…Given that exact placement of proteins within DNA origami structures is a primary reason for their use in the first place, this obvious lack of experimental control diminishes the usefulness of tetravalent streptavidin in many biological and biophysical applications. In fact, we have previously observed that the presence of only 0.5% DNA origami structures carrying two H57-scFVs at a distance of 10 nm instead of a single H57-scFV dominates T-cell activation behavior 6 with obvious consequences for the kind of conclusions that can be drawn from such experiments.…”

“…By using short oligonucleotides ("staples") to guide the folding of a long single stranded DNA scaffold, it is not only possible to program the shape of a DNA origami structure but also to arrange functional elements with nanometer resolution and precision 1 . Proteindecorated DNA origami structures have thus far been interfaced with cells as soluble agents 2,3 , attached to a solid substrate 4,5 or anchored to a fluid supported lipid bilayer (SLB) 6,7 ; biological questions addressed range from studying cellular signaling and adhesion processes [2][3][4][5][6]8,9 , to creating synthetic multienzyme cascades 10,11 to more and more elaborate robotic DNA machines 12 .…”

“…While this may be acceptable for some applications, many mechanistic studies, e.g. those focusing on receptor-ligand interactions 2,3,6 , depend on the functionalization with not more than one protein at a specific site. We have recently circumvented this potential shortcoming of streptavidin by using divalent SAv (dSAv) 24 as a connector to strictly avoid double or triple occupancies at a single modification site 6 .…”