(9b) Yeast Artificial Antigen Presenting Cells (yAPCs): A Robust Experimental System for Modulating T-Cell Activation

T-cell mediated immune response is triggered by T cell receptor (TCR) binding of agonist peptide-major histocompatibility complex (pMHC) on the surface of antigen presenting cells (APCs). Despite being among the most important interactions in the immune system, the exact mechanism relating pMHC-TCR binding to T cell activation is not fully understood. Recent studies using glass-supported lipid bilayers have shown that just a few agonist pMHC can initiate TCR triggering, and that the extent of T-cell triggering is influenced by the number of agonist pMHC. Furthermore, T cells can discriminate between a single agonist pMHC among thousands of endogenous pMHC. In spite of this incredible specificity and sensitivity for a specific pMHC ligand, recent kinetic studies using fluorescence resonance energy transfer (FRET) have predicted that in situ TCR generally exhibit relatively low affinity for cognate pMHC (K_D values ranging from 1-100 μM). Several theories have been proposed to explain this seemingly paradoxical nature of TCR triggering, including triggering of pre-formed TCR microclusters, force-induced receptor deformation, TCR pseudodimerization with agonist and endogenous pMHC,  and a kinetic-segregation model. With the aim to better understand how specific and relatively weak pMHC-TCR binding produces T cell activation, we have approached the problem of TCR triggering by investigating how local pMHC valency and overall pMHC density contribute to T-cell activation using yeast artificial antigen presenting cells (yAPCs).

Conventional strategies to investigate TCR triggering are often acellular, planar, and tend to rely on lithography and other expensive fabrication techniques. While these systems are well-suited for quantitative analysis of cell-cell signaling, they often lack the ability to control pMHC clustering and restrict stimulatory pMHC-TCR interactions to a single plane. To address these shortcomings, we have engineered a robust cellular system that allows for precise control over local pMHC valency and overall pMHC density by engineering yAPCs expressing surface-displayed multivalent protein scaffolds. These scaffolds are capable of binding recombinant human pMHC proteins with high affinity, and presenting them to T cells in precise stoichiometric arrangements. The engineered yAPCs presenting pMHC at different local and absolute levels have been used to selectively engage antigen-specific T cells and induce a dose-dependent cytokine response in T cell hybridoma recognizing the influenza epitope HA_306-318. This system provides a unique perspective on the study of TCR triggering and can be used to elucidate the role that pMHC valency plays in T cell activation. While this system is currently focused on multivalent pMHC presentation, on-going experiments are exploring the inclusion of costimulatory and adhesion molecules to emulate natural T cell activation and contribute to existing models of TCR triggering.