Microfluidic platform for characterizing TCR-pMHC interactions

Abstract

The physical characteristics of the T cell receptor (TCR)-peptide-major histocompatibility complex (pMHC) interaction are known to play a central role in determining T cell function in the initial stages of the adaptive immune response. State-of-the-art assays can probe the kinetics of this interaction with single-molecular-bond resolution, but this precision typically comes at the cost of low throughput, since the complexity of these measurements largely precludes "scaling up." Here, we explore the feasibility of detecting specific TCR-pMHC interactions by flowing T cells past immobilized pMHC and measuring the reduction in cell speed due to the mechanical force of the receptor-ligand interaction. To test this new fluidic measurement modality, we fabricated a microfluidic device in which pMHC-coated beads are immobilized in hydrodynamic traps along the length of a serpentine channel. As T cells flow past the immobilized beads, their change in speed is tracked via microscopy. We validated this approach using two model systems: primary CD8⁺ T cells from an OT-1 TCR transgenic mouse with beads conjugated with H-2K^b:SIINFEKL, and Jurkat T cells with beads conjugated with anti-CD3 and anti-CD28 antibodies.

FIG. 1.

Microfluidic trap array platform. As T cells flow through the serpentine channel, they come into contact with trapped antigen-presenting beads, resulting in TCR–pMHC interactions. Multiple TCR–pMHC may occur between a cell and a bead, although only one is shown for simplicity. These interactions decrease cell speed, which is tracked via optical microscopy.

FIG. 2.

Comparison between experimental and measured flow through the hydrodynamic trap array. (a) 3D COMSOL simulation of flow through the device, with all traps occupied by 15 μm diameter beads. (b) The fluid velocity is expected to increase near the center of each turn of the serpentine channel due to flow past trapped beads in the previous lane. (c) Measured velocities of Jurkat cells flowing past inert polystyrene beads. (d) The “speed ratio” is defined as the ratio of a cell's median speed in trap regions to its median speed in non-trap regions.

FIG. 3.

(a) Speed ratios of OT-1 CD8⁺ T cells interacting with beads coated with BSA or H-2K^b SIINFEKL. Each point represents a single cell. Specific TCR–pMHC interactions significantly (p < 2 × 10⁻⁶, Mann-Whitney) reduced the speeds of OT-1 CD8⁺ T cells flowing through the device (n = 37 cells), compared with OT-1 CD8⁺ T cells interacting with BSA-coated beads (n = 21 cells). (b) and (c) Median speeds of each cell when in trap regions versus non-trap regions. Cells had similar speed ratios even though their trap- and non-trap speeds varied.