Spatial and Chemical Surface Guidance of NK Cell Cytotoxic Activity

Abstract

Studying how different signaling pathways spatially integrate in cells requires selective manipulation and control of different transmembrane ligand-receptor pairs at the same time. This work explores a novel method for precisely arranging two arbitrarily chosen ligands on a micron-scale two-dimensional pattern. The approach is based on lithographic patterning of Au and TiO films, followed by their selective functionalization with Ni-nitrilotriacetic acid-histidine and biotin-avidin chemistries, respectively. The selectivity of chemical and biological functionalizations is demonstrated by X-ray photoelectron spectroscopy and immunofluorescence imaging, respectively. This approach is applied to produce the first type of bifunctional surfaces with controllably positioned ligands for activating the receptors of natural killer (NK) immune cells. NK cells were used as a model system to demonstrate the potency of the surface in guiding site-selective cell attachment and activation. Upon applying the suitable ligand or ligand combination, the surfaces guided the appropriate single- or bifunctional attachment and activation. These encouraging results demonstrate the effectiveness of the system as an experimental platform aimed at the comprehensive understanding of the immunological synapse. The great simplicity, modularity, and specificity of this approach make it applicable for a myriad of combinations of other biomolecules and applications, turning it into the "Swiss knife" of biointerfaces.

Advanced Materials and Devices for the Regulation and Study of NK Cells

“…We found that flat surfaces stimulated enhanced spreading of NK cells (Figure 10b–d). This finding mirrored our previously published results [50]. Concurrently, nanowires, including those functionalized with MICA, could not stimulate substantial cell spreading.…”

“…Whereas such functionalization is possible via various conjugation approaches, it becomes more complicated when the desired pattern is supposed to mimic the molecular diversity of the immune synapse, and thus contain two or more controllably clustered antigens. Recently, we reported an orthogonal approach for site-selective functionalization of a photolithographic pattern with two ligands for NK cell receptors [50]. The pattern consisted of Au disks surrounded by an oxidized Ti film using negative-tone photolithography and etching.…”

Mechanical Regulation of the Cytotoxic Activity of Natural Killer Cells

“…The cytotoxic activity of NK cells is tightly regulated by the microclustering of their activating receptors such as CD16 (45) and NKG2D (46), as well as of their inhibitory receptors such as KIR2DL1 (47). This microclustering can be regulated in vitro by various micro-/nano-platforms with controlled spatial distribution of activating and inhibitory ligands (48)(49)(50)(51). On the other hand, it was natural to ask whether surface stiffness controls the cytotoxic activity of NK cells by modulating the clustering of target receptors of the used activating ligands, or via a signaling pathway independent of receptor clustering.…”

Probing the nanoscale organisation and multivalency of cell surface receptors: DNA origami nanoarrays for cellular studies with single-molecule control

“…Importantly integrin clustering distributes the forces between the receptor-ligand complexes, increasing the maximum force per bond and enabling further integrin adhesion assembly 4-6, 9, 11 . Additionally, it is known that integrins work synergistically with other membrane receptors to modulate cell behaviour 5, 12-14 . Different strategies have been developed to fabricate biomimetic substrates for the investigation of ECM geometries in cell spreading and focal adhesion formation 6,[15][16][17][18][19][20] . Achieving nanoscale control of adhesion receptors, with single molecule resolution, is essential for such investigations; micellar diblock copolymer self-assembly 21 and nanopatterning approaches 4,17,18,22 have been the most notable strategies to meet this requirement.…”

“…However, a recent study with peptidomimetic ligands indicated that major differences exist in the clustering behaviour between αvβ3 and α5β1 integrins 23 and information about other integrin subtypes or from different cellular systems is lacking. Most importantly, these strategies have shown only partial application for multivalent investigations with single-molecule control 16,19 . In this regard, we recently presented a strategy for the fabrication of biomimetic nanoarrays, based on the use of DNA origami that permit the multivalent investigation of ligand-receptor molecule interactions (simultaneous binding of multiple different ligands) in cancer cell spreading with nanoscale spatial resolution and single-molecule control 20 .…”