Arrays of protein nanodots with dot-size tuned independently of spacing (e.g., ∼100 to 600 nm diameter for 900 nm spacing) are fabricated. The mechanism of size control is demonstrated, by numerical simulations, to arise from shadow effects during deposition of a sacrificial metal mask. We functionalize the nanodots with antibodies and embed them in a polymer-cushion or in lipid-bilayers or transfer them to soft elastomers. Their ability to influence cell architecture and local membrane organization is demonstrated in T-lymphocytes, using reflection interference contrast and total internal reflection fluorescence microscopy.

We created APC-mimetic synthetic substrates to study the impact of ligand clustering on T cell activation and spreading. The substrates exhibit antibodies directed against the TCR-complex in the form of a patterned array of sub micrometric dots surrounded by a fluid supported lipid bilayer (SLB) which may itself be functionalized with another bio-molecule. We show that for T cell adhesion mediated by T cell receptor (TCR) alone, in the patterned, but not in the corresponding homogeneous controls, the TCR, ZAP-70 and actin are present in the form of clusters or patches that co-localize with the ligand-dots. However, global cell scale parameters like cell area and actin distribution are only weakly impacted by ligand clustering. In presence of ICAM-1 - the ligand of the T cell integrin LFA-1 - on the SLB, the TCR is still clustered due to the patterning of its ligands, but now global parameters are also impacted. The actin organization changes to a peripheral ring, resembling the classical actin distribution seen on homogeneous substrates, the patterned membrane topography disappears and the membrane is flat, whereas the cell area increases significantly. These observations taken together point to a possible pivotal role for LFA-1 in amplifying the effect of TCR-clustering. No such effect is evident for co-engagement of CD28, affected via its ligand B7.2. Unlike on ICAM-1, on B7.2 cell spreading and actin organization are similar for homogeneous and patterned substrates. However, TCR and ZAP-70 clusters are still formed in the patterned case. These results indicate complementary role for LFA-1 and CD28 in the regulation and putative coupling of TCR micro-clusters to actin. The engineered substrates presented here clearly have the potential to act as platform for fundamental research in immune cell biology, as well as translational analyses in immunotherapy, for example to screen molecules for their role in T cell adhesion/activation.