(357c) Probing the Mechanobiology of T Cell Receptor Binding to Peptide-MHC Using Optical Tweezers

A robustly functioning mammalian immune system hinges on the highly-tuned sensitivity and selectivity of αβ T cell receptor (TCR) recognition of “foreign” antigens, namely, peptides displayed on major histocompatibility complex (pMHC) molecules arrayed on the surface of antigen-presenting cells. Yet, the binding affinity and immunogenicity of a particular antigen are largely uncorrelated when measured for a particular TCR using standard biochemical assays. Single molecule techniques such as optical tweezers allow a single TCR interaction with pMHC to be monitored under force, thereby allowing direct observation of binding properties out of thermal equilibrium. Using these techniques, our lab has previously shown that T cell discernment between antigens arises not from differences in biochemical affinity, but by the selective ability of immunogenic pMHC interactions to survive under force.

Here, we discuss the development of optical tweezers techniques to probe the binding landscape of the TCR:pMHC interaction at the resolution of a single-bond. The design of techniques under various conditions will be discussed, including considerations for taking measurements directly off the surface of a live T cell versus isolating a purified TCR molecule to a solid surface for measurement. Our work includes the study of a panel of clonally distinct TCRs directed against the same peptide antigen, yielding a range of bond-profile characteristics such as peak lifetime, maximum survivable force, and force-induced conformational rearrangement. Since these clones differ only subtly in their variable domain fragments, these structural differences are directly mapped onto differences in the various binding parameters. Finally, the ability of our optical tweezers assay to predict aspects of in vivo T cell function are compared to standard techniques such as measuring the binding affinity to pMHC tetramers.