Parsing digital or analogue TCR performance through piconewton forces.

αß T-cell receptors (TCRs) 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 (NP 366-374 /D b and PA 224-233 /D b , 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 upregulation in vitro . Given neoantigen array paucity, digital TCRs are likely critical for immunotherapies. One Sentence Summary: Quality of ligand recognition in a T-cell repertoire is revealed through application of physical load on clonal T-cell receptor (TCR)-pMHC bonds.

Lon degrades stable substrates slowly but with enhanced processivity, redefining the attributes of a successful AAA+ protease.

Lon is a widely distributed AAA+ (ATPases associated with diverse cellular activities) protease known for degrading poorly folded and damaged proteins and is often classified as a weak protein unfoldase. Here, using a Lon-degron pair from Mesoplasma florum (MfLon and MfssrA, respectively), we perform ensemble and single-molecule experiments to elucidate the molecular mechanisms underpinning MfLon function. Notably, we find that MfLon unfolds and degrades stably folded substrates and that translocation of these unfolded polypeptides occurs with a ∼6-amino-acid step size. Moreover, the time required to hydrolyze one ATP corresponds to the dwell time between steps, indicating that one step occurs per ATP-hydrolysis-fueled "power stroke." Comparison of MfLon to related AAA+ enzymes now provides strong evidence that HCLR-clade enzymes function using a shared power-stroke mechanism and, surprisingly, that MfLon is more processive than ClpXP and ClpAP. We propose that ample unfoldase strength and substantial processivity are features that contribute to the Lon family's evolutionary success.

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.

Measuring αß T-Cell Receptor-Mediated Mechanosensing Using Optical Tweezers Combined with Fluorescence Imaging.

T-cell antigen receptors (TCRs) are mechanosensors, which initiate a signaling cascade upon ligand recognition resulting in T-cell differentiation, homeostasis, effector and regulatory functions. An optical trap combined with fluorescence permits direct monitoring of T-cell triggering in response to force application at various concentrations of peptide-bound major histocompatibility complex molecules (pMHC). The technique mimics physiological shear forces applied as cells crawl across antigen-presenting surfaces during immune surveillance. True single molecule studies performed on single cells profile force-bond lifetime, typically seen as a catch bond, and conformational change at the TCR-pMHC bond on the surface of the cell upon force loading. Together, activation and single molecule single cell studies provide chemical and physical triggering thresholds as well as insight into catch bond formation and quaternary structural changes of single TCRs. The present methods detail assay design, preparation, and execution, as well as data analysis. These methods may be applied to a wide range of pMHC-TCR interactions and have potential for adaptation to other receptor-ligand systems.

Single Molecule Force Spectroscopy Reveals Distinctions in Key Biophysical Parameters of αß T-Cell Receptors Compared with Chimeric Antigen Receptors Directed at the Same Ligand.

Chimeric antigen receptor (CAR) T-cell therapies exploit facile antibody-mediated targeting to elicit useful immune responses in patients. This work directly compares binding profiles of CAR and αß T-cell receptors (TCR) with single cell and single molecule optical trap measurements against a shared ligand. DNA-tethered measurements of peptide-major histocompatibility complex (pMHC) ligand interaction in both CAR and TCR exhibit catch bonds with specific peptide agonist peaking at 25 and 14 pN, respectively. While a conformational transition is regularly seen in TCR-pMHC systems, that of CAR-pMHC systems is dissimilar, being infrequent, of lower magnitude, and irreversible. Slip bonds are observed with CD19-specific CAR T-cells and with a monoclonal antibody mapping to the MHC α2 helix but indifferent to the bound peptide. Collectively, these findings suggest that the CAR-pMHC interface underpins the CAR catch bond response to pMHC ligands in contradistinction to slip bonds for CARs targeting canonical ligands.

Molecular design of the γδT cell receptor ectodomain encodes biologically fit ligand recognition in the absence of mechanosensing.

High-acuity αßT cell receptor (TCR) recognition of peptides bound to major histocompatibility complex molecules (pMHCs) requires mechanosensing, a process whereby piconewton (pN) bioforces exert physical load on αßTCR-pMHC bonds to dynamically alter their lifetimes and foster digital sensitivity cellular signaling. While mechanotransduction is operative for both αßTCRs and pre-TCRs within the αßT lineage, its role in γδT cells is unknown. Here, we show that the human DP10.7 γδTCR specific for the sulfoglycolipid sulfatide bound to CD1d only sustains a significant load and undergoes force-induced structural transitions when the binding interface-distal γδ constant domain (C) module is replaced with that of αß. The chimeric γδ-αßTCR also signals more robustly than does the wild-type (WT) γδTCR, as revealed by RNA-sequencing (RNA-seq) analysis of TCR-transduced Rag2-/- thymocytes, consistent with structural, single-molecule, and molecular dynamics studies reflective of γδTCRs as mediating recognition via a more canonical immunoglobulin-like receptor interaction. Absence of robust, force-related catch bonds, as well as γδTCR structural transitions, implies that γδT cells do not use mechanosensing for ligand recognition. This distinction is consonant with the fact that their innate-type ligands, including markers of cellular stress, are expressed at a high copy number relative to the sparse pMHC ligands of αßT cells arrayed on activating target cells. We posit that mechanosensing emerged over ∼200 million years of vertebrate evolution to fulfill indispensable adaptive immune recognition requirements for pMHC in the αßT cell lineage that are unnecessary for the γδT cell lineage mechanism of non-pMHC ligand detection.