Height, but not binding epitope, affects the potency of synthetic TCR agonists

Wilhelm, Kiera B.; Morita, Shumpei; McAffee, Darren B.; Kim, Sungi; O’Dair, Mark K.; Groves, Jay T.

doi:10.1016/j.bpj.2021.08.027

Cited by 15 publications

(10 citation statements)

References 76 publications

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“…Additionally, we limited the τ F of FUSE probes presenting antiCD3ε to 5.8 min and observed a more pronounced reduction in pYZAP70 (15%) compared to probes presenting pMHC with the same τ F (8%) (Figure S7). We hypothesize that this larger decrease in the antiCD3ε condition was due to the disruption of long-lived mechanical interactions between antiCD3ε and the TCR, as antiCD3ε displays higher affinity (nM K D ) toward the TCR than pMHCs (μM K D ). , Note that both the AseI hairpin and the accompanying locking strand must be present to observe a statistically significant decrease in T cell signaling, thus validating that selective FUSE probe cleavage is responsible for this perturbation (Figure S8).…”

Section: Resultsmentioning

confidence: 70%

“…We hypothesize that this larger decrease in the antiCD3ε condition was due to the disruption of long-lived mechanical interactions between antiCD3ε and the TCR, as antiCD3ε displays higher affinity (nM K D ) toward the TCR than pMHCs (μM K D ). 42,43 Note that both the AseI hairpin and the accompanying locking strand must be present to observe a statistically significant decrease in T cell signaling, thus validating that selective FUSE probe cleavage is responsible for this perturbation (Figure S8). Since antigen is depleted from the surface after mechanical interaction, we aimed to validate that the decrease in early T cell activation relied on changes in the τ F �not only a decrease in antigen density on the surface.…”

Section: ■ Resultsmentioning

confidence: 71%

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Force-Induced Site-Specific Enzymatic Cleavage Probes Reveal That Serial Mechanical Engagement Boosts T Cell Activation

Rogers,

Ma,

Foote

et al. 2024

J. Am. Chem. Soc.

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The T cell membrane is studded with >10 4 T cell receptors (TCRs) that are used to scan target cells to identify short peptide fragments associated with viral infection or cancerous mutation. These peptides are presented as peptide-majorhistocompatibility complexes (pMHCs) on the surface of virtually all nucleated cells. The TCR-pMHC complex forms at cell−cell junctions, is highly transient, and experiences mechanical forces. An important question in this area pertains to the role of the force duration in immune activation. Herein, we report the development of force probes that autonomously terminate tension within a time window following mechanical triggering. Force-induced site-specific enzymatic cleavage (FUSE) probes tune the tension duration by controlling the rate of a force-triggered endonuclease hydrolysis reaction. This new capability provides a method to study how the accumulated force duration contributes to T cell activation. We screened DNA sequences and identified FUSE probes that disrupt mechanical interactions with F > 7.1 piconewtons (pN) between TCRs and pMHCs. This rate of disruption, or force lifetime (τ F ), is tunable from tens of minutes down to 1.9 min. T cells challenged with FUSE probes with F > 7.1 pN presenting cognate antigens showed up to a 23% decrease in markers of early activation. FUSE probes with F > 17.0 pN showed weaker influence on T cell triggering further showing that TCR-pMHC with F > 17.0 pN are less frequent compared to F > 7.1 pN. Taken together, FUSE probes allow a new strategy to investigate the role of force dynamics in mechanotransduction broadly and specifically suggest a model of serial mechanical engagement boosting TCR activation

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Section: Resultsmentioning

confidence: 70%

Section: ■ Resultsmentioning

confidence: 71%

Force-Induced Site-Specific Enzymatic Cleavage Probes Reveal That Serial Mechanical Engagement Boosts T Cell Activation

Rogers,

Ma,

Foote

et al. 2024

J. Am. Chem. Soc.

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“…These results imply that despite the receptor heterogeneity of cell-surface glycans, HAfps can still productively engage with a target membrane across a large range of distances and that the efficacy of IAV fusion is enhanced by receptor flexibility. This work has implications for how the rigidity and distance between biological interfaces impact other membrane fusion processes and downstream signaling. − While our DNA tethering methodology yields several mechanistic insights into the role of viral receptor length and flexibility in IAV fusion, direct visualization of the membrane interface would further elucidate the role of tether mobility in the mechanism of IAV fusion.…”

Section: Discussionmentioning

confidence: 99%

Modulating the Influenza A Virus–Target Membrane Fusion Interface With Synthetic DNA–Lipid Receptors

et al. 2022

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Influenza A virus (IAV) binds to sialylated glycans on the cell membrane before endocytosis and fusion. Cell-surface glycans are highly heterogeneous in length and glycosylation density, which leads to variations in the distance and rigidity with which IAV is held away from the cell membrane. To gain mechanistic insight into how receptor length and rigidity impact the mechanism of IAV entry, we employed synthetic DNA−lipids as highly tunable surrogate receptors. We tethered IAV to target membranes with a panel of DNA−lipids to investigate the effects of the distance and tether flexibility between virions and target membranes on the kinetics of IAV binding and fusion. Tether length and the presence of a flexible linker led to higher rates of IAV binding, while the efficiencies of lipid and content mixing were typically lower for longer and more rigid DNA tethers. For all DNA tether modifications, we found that the rates of IAV lipid and content mixing were unchanged. These results suggest that variations in the interface between IAV and a target membrane do not significantly impact the rate-limiting step of fusion or the low-pH-triggered engagement of viral fusion peptides with the target membrane. However, our results imply that the flexibility of the viral receptor is important for ensuring that hemifusion events are able to successfully proceed to pore formation.

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“…A problem of biotin-streptavidin linked proteins is the addition of an, at least, extra 5 nm to the overall protein size, which can be particularly problematic in cell-SLB interface studies where the height of the molecules vs the cell- SLB gap is important. 4) The SLBs can also be functionalized with DNA grafted onto the lipids, to which an opposite DNA strand, containing the studied protein ligand, is added (Wilhelm et al, 2021). This allows for a versatile tool, particularly by being able to alter the number of nucleotides making it possible to change the length of the DNA tether as well as the interaction strength between two DNA strands.…”

Section: General 2d and 3d Adhesion Theorymentioning

confidence: 99%

Supported Lipid Bilayers and the Study of Two-Dimensional Binding Kinetics

Dam

Chouliara

Junghans

et al. 2022

Front. Mol. Biosci.

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Binding between protein molecules on contacting cells is essential in initiating and regulating several key biological processes. In contrast to interactions between molecules in solution, these events are restricted to the two-dimensional (2D) plane of the meeting cell surfaces. However, converting between the more commonly available binding kinetics measured in solution and the so-called 2D binding kinetics has proven a complicated task since for the latter several factors other than the protein-protein interaction per se have an impact. A few important examples of these are: protein density, membrane fluctuations, force on the bond and the use of auxiliary binding molecules. The development of model membranes, and in particular supported lipid bilayers (SLBs), has made it possible to simplify the studied contact to analyze these effects and to measure 2D binding kinetics of individual protein-protein interactions. We will in this review give an overview of, and discuss, how different SLB systems have been used for this and compare different methods to measure binding kinetics in cell-SLB contacts. Typically, the SLB is functionalized with fluorescently labelled ligands whose interaction with the corresponding receptor on a binding cell can be detected. This interaction can either be studied 1) by an accumulation of ligands in the cell-SLB contact, whose magnitude depends on the density of the proteins and binding affinity of the interaction, or 2) by tracking single ligands in the SLB, which upon interaction with a receptor result in a change of motion of the diffusing ligand. The advantages and disadvantages of other methods measuring 2D binding kinetics will also be discussed and compared to the fluorescence-based methods. Although binding kinetic measurements in cell-SLB contacts have provided novel information on how ligands interact with receptors in vivo the number of these measurements is still limited. This is influenced by the complexity of the system as well as the required experimental time. Moreover, the outcome can vary significantly between studies, highlighting the necessity for continued development of methods to study 2D binding kinetics with higher precision and ease.

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Height, but not binding epitope, affects the potency of synthetic TCR agonists

Cited by 15 publications

References 76 publications

Force-Induced Site-Specific Enzymatic Cleavage Probes Reveal That Serial Mechanical Engagement Boosts T Cell Activation

Force-Induced Site-Specific Enzymatic Cleavage Probes Reveal That Serial Mechanical Engagement Boosts T Cell Activation

Modulating the Influenza A Virus–Target Membrane Fusion Interface With Synthetic DNA–Lipid Receptors

Supported Lipid Bilayers and the Study of Two-Dimensional Binding Kinetics

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