Using four different non-invasive imaging techniques with single-molecule sensitivity on living T cells interacting with ligands embedded in a planar lipid membrane, Brameshuber et al. demonstrate that it is TCR-CD3 monomers, not dimers or oligomers, that are primarily responsible for the recognition of antigenic peptide:MHC complexes, contrary to the data previously obtained via invasive imaging. Thus, the signaling mechanism relevant for antigen sensitivity and signal generation is distinct from receptor tyrosine kinase-determined dimerization mechanisms.

T cell antigen recognition requires T cell antigen receptors (TCRs) engaging MHC-embedded antigenic peptides (pMHCs) within the contact region of a T cell with its conjugated antigen-presenting cell. Despite micromolar TCR:pMHC affinities, T cells respond to even a single antigenic pMHC, and higher-order TCRs have been postulated to maintain high antigen sensitivity and trigger signaling. We interrogated the stoichiometry of TCRs and their associated CD3 subunits on the surface of living T cells through single-molecule brightness and single-molecule coincidence analysis, photon-antibunching-based fluorescence correlation spectroscopy and Forster resonance energy transfer measurements. We found exclusively monomeric TCR-CD3 complexes driving the recognition of antigenic pMHCs, which underscores the exceptional capacity of single TCR-CD3 complexes to elicit robust intracellular signaling.

Author Info: (1) Institute of Applied Physics, TU Wien, Vienna, Austria. brameshuber@iap.tuwien.ac.at. (2) Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University

Author Info: (1) Institute of Applied Physics, TU Wien, Vienna, Austria. brameshuber@iap.tuwien.ac.at. (2) Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austria. (3) Institute of Applied Physics, TU Wien, Vienna, Austria. (4) Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Gottingen, Germany. (5) Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austria. (6) Institute of Applied Physics, TU Wien, Vienna, Austria. (7) Institute of Applied Physics, TU Wien, Vienna, Austria. Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austria. (8) Center for Pathobiochemistry and Genetics, Institute of Medical Chemistry and Pathobiochemistry, Medical University of Vienna, Vienna, Austria. (9) Institute of Applied Physics, TU Wien, Vienna, Austria. (10) Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. (11) Radcliffe Department of Medicine and MRC Human Immunology Unit, John Radcliffe Hospital, University of Oxford, Oxford, UK. (12) Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austria. (13) Institute of Applied Physics, TU Wien, Vienna, Austria. (14) Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austria. johannes.huppa@meduniwien.ac.at.

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