Probing the Minimum Geometric Requirements for T-Cell Stimulation

Abstract

The immune recognition process involves an elaborate arrangement of adhesion, costimulatory and signaling molecules organized into a stereotypic geometric structure known as the immunological synapse (IS). We have developed a versatile engineered platform to probe the minimum geometric requirements (in terms of spacing and stoichiometry) for T-cell stimulation. Arrays of metallic nanodots, ∼ 2-10 nm in size, to which a UCHT1 Fab antibody was bound, were created by nanolithography. These served as individual T-cell receptor (TCR) binding sites. The adhesion molecule ICAM-1 was either statically bound to a PEG-silane brush surrounding the nanodots, or it was allowed to move freely within the arrays in a supported lipid bilayer. T-cells were plated on arrays comprising individual TCR binding sites, small clusters and extended hexagonal close packed arrays, with spacings ranging from 40 nm and below to 1 μm. TCR signaling strength was monitored by measuring phosphorylated tyrosine (pY) intensity. T-cell adhesion was assessed by the number of cells bound to the arrays, as well as by the area of spread cells.Systematic variation of the spacing and cluster size of TCR binding sites in the arrays enabled determination of the minimum conditions that support T-cell signaling and the formation of the IS. TCR signaling increased with decreasing spacing below a threshold spacing ∼ 60 nm. For clusters with these spacings the formation of the stereotypic “bullseye” geometry that characterizes the immune synapse became evident, with ICAM-1 excluded to the periphery. In terms of stoichiometry, at least 4 TCR-binding sites (within ∼ 60 nm) were required for T-cell adhesion and spreading. Further, in the absence of ICAM-1 (or at low concentrations), a threshold density of agonist sites was found, above which the TCR apparently plays a dual role of immune activity and adhesion.