Parsing digital or analog TCR performance through piconewton forces.

αß T cell receptors (TCRs) principally recognize aberrant peptides bound to major histocompatibility complex molecules (pMHCs) on unhealthy cells, amplifying specificity and sensitivity through physical load placed on the TCR-pMHC bond during immunosurveillance. To understand this mechanobiology, TCRs stimulated by abundantly and sparsely arrayed epitopes (NP366-374/Db and PA224-233/Db, respectively) following in vivo influenza A virus infection were studied with optical tweezers. While certain NP repertoire CD8 T lymphocytes require many ligands for activation, others are digital, needing just few. Conversely, all PA TCRs perform digitally, exhibiting pronounced bond lifetime increases through sustained, energizing volleys of structural transitioning. Optimal digital performance is superior in vivo, correlating with ERK phosphorylation, CD3 loss, and activation marker up-regulation in vitro. Given neoantigen array paucity, digital TCRs are likely critical for immunotherapies.

Characterizing Biophysical Parameters of Single TCR-pMHC Interactions Using Optical Tweezers.

αß T cells are mechanosensors that leverage bioforces during immune surveillance for highly sensitive and specific antigen discrimination. Single-molecule studies are used to profile the initial TCRαß-pMHC binding event, and various biophysical parameters can be identified. Isolating purified TCRαß and pMHC molecules on a coverslip allows for direct measurements of the kinetics and conformational changes in the system and removes cellular components along the load pathway that may interfere with or mask subtle changes. Optical tweezers provide high resolution position and force information that map the bonding profile, including catch bond, and the ability to measure distinct conformational changes driven by forces. The present method describes the single-molecule optical tweezers assay setup, considerations, and execution. This model can be used for various TCR-pMHC pairs or expanded to measure a wide variety of receptor-ligand interactions operative in multiple biological systems.