Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering

Abstract

Elucidating the rules for receptor triggering in cell-cell and cell-matrix contacts requires precise control of ligand positioning in three dimensions. Here, we use the T cell receptor (TCR) as a model and subject T cells to different geometric arrangements of ligands, using a nanofabricated single-molecule array platform. This is comprised of monovalent TCR ligands anchored to lithographically patterned nanoparticle clusters surrounded by mobile adhesion molecules on a supported lipid bilayer (SLB). The TCR ligand could be co-planar with the SLB (2D), excluding the CD45 transmembrane tyrosine phosphatase, or elevated by 10 nm on solid nanopedestals (3D), allowing closer access of CD45 to engaged TCR. The two configurations resulted in different T cell responses, depending on the lateral spacing between the ligands. These results identify the important contributions of lateral and axial components of ligand positioning and create a more complete foundation for receptor engineering for immunotherapy.

Molecular Scale Spatio-Chemical Control of the Activating-Inhibitory Signal Integration in NK Cells

“…To date, state-of-the-art biomimetic patterns for controlled cellular environment consisted of up to two spatially segregated biochemical functionalities, i.e. one ligand and antifouling agent(32), or two ligands (19). Here, we developed a novel ternary functionalization process for the sitespecific immobilization of three biochemical functionalitiestwo ligands and an antifouling agent, by combining chemical modifications of Si, Au, and TiO2 surfaces with organic silanes, thiols, and phosphonic acids, respectively ( Fig.…”

“…For this reason, the effect of within-membrane segregation different receptors onto the cell function could not be systematically studied.Receptors can be spatially guided within the cell membrane by artificial cell niches based on tunable patterns of extracellular ligands. Various concepts for such niches have evolved in the last two decades thanks to the progressing advances in nanofabrication, and they were applied to study the effect of ligand distribution on different cell functions, such as adhesion(13-15), chemokinesis (16), mechanosensing(17), differentiation(18), and immune activity (19)(20)(21)(22). Functionalized surfaces were also the basis of mimetic cell models, which were used, in conjunction with theoretical modelling, to identify the physical foundation for the recognition of membrane-confined ligands and receptors, which, mostly focused on single protein pairs(23).…”

“…Functionalized surfaces were also the basis of mimetic cell models, which were used, in conjunction with theoretical modelling, to identify the physical foundation for the recognition of membrane-confined ligands and receptors, which, mostly focused on single protein pairs(23). However, all of the state-of-the-art ligand patterns, even those including multiple ligands (19,24), have been limited to spatially control ligands of one type only. Simultaneous control over two or more ligands, on the other hand, has not been realized so far, as it requires new, paradigm shifting fabrication and functionalization approaches with precision and complexity far beyond those exploited for the state-of-the-art ligand patterns.In this paper, we achieved, for the first time to the best of our knowledge, the molecular-scale spatial control of two extracellular ligands, in order to discover how the spatial juxtaposition between two receptors: (i) NKG2Dthe major activating receptor in NK cells, and (ii) KIR2DL1an inhibitory receptor that belongs to the family of killer cell immunoglobulin-like receptors (KIRs)(25, 26), regulate KIR2DL1-mediated inhibition of NK cell cytotoxic activity.…”

“…Functionalized surfaces were also the basis of mimetic cell models, which were used, in conjunction with theoretical modelling, to identify the physical foundation for the recognition of membrane-confined ligands and receptors, which, mostly focused on single protein pairs(23). However, all of the state-of-the-art ligand patterns, even those including multiple ligands (19,24), have been limited to spatially control ligands of one type only. Simultaneous control over two or more ligands, on the other hand, has not been realized so far, as it requires new, paradigm shifting fabrication and functionalization approaches with precision and complexity far beyond those exploited for the state-of-the-art ligand patterns.…”

Electron beam lithography fabrication of SU-8 polymer structures for cell studies

“…For example, Cai et al used nanofabrication based on electron beam lithography (EBL) to define gold structures on a glass surfaces to precisely control position receptor ligands and to subject T cells to defined arrangements of ligands. 6 This is due to the fact that T cell receptor clustering by surface-bound ligands is believed to be a highly effective method to triggering the tyrosine kinase cascade leading to T cell activation. 7 Another type of nanostructured cell culture substrates that has recently attracted a lot of attention, are based on flat and cell compatible supports decorated with vertically aligned nanowires or nanopillars.…”