{"doi":"10.1101/2024.04.01.587588","title":"Differential Roles of Kinetic On- and Off-Rates in T-Cell Receptor Signal Integration Revealed with a Modified Fab’-DNA Ligand","abstract":"ABSTRACT Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly-binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide-MHC (pMHC) ligands bind their cognate TCRs. Because ligand dwell time is a critical parameter that tightly correlates with physiological function of the TCR signaling system, there is a general need, both in research and therapeutics, for universal TCR ligands with controlled kinetic binding parameters. To this end, we have introduced point mutations into recombinantly expressed α-TCRβ H57 Fab to modulate the dwell time of monovalent Fab binding to TCR. When tethered to a supported lipid bilayer via DNA complementation, these monovalent Fab’-DNA ligands activate T cells with potencies well-correlated with their TCR binding dwell time. Single-molecule tracking studies in live T cells reveal that individual binding events between Fab’-DNA ligands and TCRs elicit local signaling responses closely resembling native pMHC. The unique combination of high on- and off-rate of the H57 R97L mutant enables direct observations of cooperative interplay between ligand binding and TCR-proximal condensation of the linker for activation of T cells (LAT), which is not readily visualized with pMHC. This work provides insights into how T cells integrate kinetic information from synthetic ligands and introduces a method to develop affinity panels for polyclonal T cells, such as cells from a human patient. STATEMENT OF SIGNIFICANCE T cells read kinetic information from ligands binding to T-cell receptors (TCRs) to make cell fate decisions. Unique kinetic features of a modified Fab’-DNA ligand enable direct visualization multiple TCR signal coordination through a nascent LAT condensation event. We further observed positive feedback through a kinetic on-rate enhancement in the growing LAT condensate. These observations help unify several seemingly disparate aspects of TCR signaling that have been debated in the literature. Furthermore, calibration of the Fab’-DNA ligand against native agonist pMHC establishes a basis for quantitative analysis of TCR signaling in polyclonal T cell populations.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":488256,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9488,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1180752,"name":"Anand Vissa","orcid":null,"position":1,"is_corresponding":false},{"id":302378,"name":"Jay T. Groves","orcid":"0000-0002-3037-5220","position":2,"is_corresponding":false},{"id":510099,"name":"Kiera B. Wilhelm","orcid":"0000-0002-8781-7739","position":0,"is_corresponding":true}],"reference_count":78,"raw_metadata":null,"created_at":"2026-07-19T02:08:15.008850Z","pmid":"38617215","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}