Functional single-cell analysis of T-cell activation by supported lipid bilayer-tethered ligands on arrays of nanowells

Abstract

Supported lipid bilayers are an important biomolecular tool for characterizing immunological synapses. Immobilized bilayers presenting tethered ligands on planar substrates have yielded both spatio-temporal and structural insights into how T cell receptors (TCRs) reorganize during the initial formation of synapses upon recognition of peptide antigen bound to major histocompatibility complex (MHC) molecules. The prototypical configuration of these assays, however, limits the extent to which the kinetics and structure of the supramolecular activation clusters of the synapse (that occur in seconds or minutes) can be related to subsequent complex cellular responses, such as cytokine secretion and proliferation, occurring over hours to days. Here we describe a new method that allows correlative measures of both attributes with single-cell resolution by using immobilized lipid bilayers and tethered ligands on the surface of dense arrays of subnanoliter wells. This modification allows each nanowell to function as an artificial antigen-presenting cell (APC), and the synapses formed upon contact can be imaged by fluorescence microscopy. We show that the lipid bilayers remain stable and mobile on the surface of the PDMS, and that modifying the ligands tethered to the bilayer alters the structure of the resulting synapses in expected ways. Finally, we demonstrate that this approach allows the subsequent characterization of secreted cytokines from the activated human T cell clones by microengraving in both antigen- and pan-specific manners. This new technique should allow detailed investigations on how biophysical and structural aspects of the synapse influence the activation of individual T cells and their subsequent complex functional responses.

Spatially and temporally controlled immune cell interactions using microscale tools

“…Although it is more challenging to modulate dynamic environments, wells can be modified with supported lipid bilayers to present adhered ligands as external stimuli (Figure 2c). Love and colleagues used this approach to turn wells into artificial activating substrates, enabling study of both immune synapse structure and functional analysis of cell activation within the same system [44]. These examples highlight the potential of microscale tools to dissect out both cell-intrinsic and cell-extrinsic factors on intercellular interactions in controlled environments.…”

Single-cell analysis tools for drug discovery and development

“…Each of these methods rely on antibodies, so, unlike mass spectrometry proteomics of bulk samples 82, 83 , single cell proteomics methods cannot yet serve as discovery level tools. For the microfluidic platforms, the microengraving technique 35, 84 , single cell barcode chips (SCBCs), and single-cell Westerns 85 (scWesterns) yield the most advanced capabilities. A number of alternative approaches, typically with reduced levels of multiplexing, have been reported, including high throughput microdroplet-base screening approaches, 33, 86-89 and some of these are reviewed elsewhere 90 .…”

Microfluidics-Based Single-Cell Functional Proteomics for Fundamental and Applied Biomedical Applications

“…This collective work over the past decade has refined the notion that the quality of a T-cell immune response is best captured by the functional performance of the T cells, rather than by their quantity (84). Most reported applications of the microengraving platform have also focused on immunology (25,67,(85)(86)(87)(88)(89). These investigations have emphasized types of experiments that are not tractable using the various cytometry methods.…”

“…Tumor antigen-specific CD8 + T cells secrete from <10 1 to >10 5 copies over 12 h (Figure 1). A secretion rate of 10 copies sec −1 was measured from influenza antigen-specific CD4 + T cells (25). GB This is a serine protease (enzyme) present in cytotoxic T lymphocyte and NK cell granules.…”

“…One major area has been in the design of platforms that integrate different assays, such as measurements of cell motility (24) or immune cell activation (25), correlated with functional proteomics assays from those same cells. A second area has been the ability to execute proteomics assays on quantized cell populations (i.e., 1, 2, 3. .…”