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TCR–peptide–MHC interactions in situ show accelerated kinetics and increased affinity


The recognition of foreign antigens by T lymphocytes is essential to most adaptive immune responses. It is driven by specific T-cell antigen receptors (TCRs) binding to antigenic peptide–major histocompatibility complex (pMHC) molecules on other cells1. If productive, these interactions promote the formation of an immunological synapse2,3. Here we show that synaptic TCR–pMHC binding dynamics differ significantly from TCR–pMHC binding in solution. We used single-molecule microscopy and fluorescence resonance energy transfer (FRET) between fluorescently tagged TCRs and their cognate pMHC ligands to measure the kinetics of TCR–pMHC binding in situ. When compared with solution measurements, the dissociation of this complex was increased significantly (4–12-fold). Disruption of actin polymers reversed this effect, indicating that cytoskeletal dynamics destabilize this interaction directly or indirectly. Nevertheless, TCR affinity for pMHC was significantly elevated as the result of a large (about 100-fold) increase in the association rate, a likely consequence of complementary molecular orientation and clustering. In helper T cells, the CD4 molecule has been proposed to bind cooperatively with the TCR to the same pMHC complex. However, CD4 blockade had no effect on the synaptic TCR affinity, nor did it destabilize TCR–pMHC complexes, indicating that the TCR binds pMHC independently of CD4.

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Figure 1: FRET as a sensor for TCR–ligand interactions.
Figure 2: Synaptic off-rates measured through smFRET.
Figure 3: Quantitative image analysis yields effective synaptic 2D  Kd, 2D  Ka and 2D  kon.
Figure 4: Effect of CD4 on TCR signalling and ligand binding.


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We thank F. E. Tynan, A. K. Chakraborty and S. R. Quake for helpful discussions, and P. P. J. Ebert for help with the flow cytometry experiments. This work was supported by grants from the National Institutes of Health (RO1 AI52211) and the Howard Hughes Medical Institute to M.M.D. J.B.H. is partly supported by the Immunology Frontier Research Center (iFREC) consortium, M.A.M. received a postdoctoral fellowship from the Wilhelm Macke Stiftung, and E.W.N. from the American Cancer Society’s Steven Stanley and Edward Albert Bielfelt Fund. L.O.K. is supported by a National Science Foundation predoctoral fellowship. M.A., M.B. and G.J.S. received grants from the Austrian Science Fund (project Y250-B10) and the GEN-AU project of the Austrian Ministry for Science and Research.

Author Contributions J.B.H., G.J.S. and M.M.D. conceived the project. Reagents and experimental systems were designed and tested by J.B.H. unless indicated otherwise. M.A., M.A.M. and G.J.S. provided important expertise in single-molecule microscopy. M.A. conducted all single dye tracing experiments and all smFRET experiments performed in Linz. J.B.H. conducted all bulk FRET experiments and all smFRET experiments at Stanford. J.B.H. and M.A.M. laid the experimental foundation in the initial phase of the project. M.A.M. provided important expertise in setting up the microscope system at Stanford. B.F.L. provided the expression system for ICAM-1 and B7-1. E.W.N. conducted all SPR measurements. M.B. conducted all TOCCSL experiments. L.O.K. and B.F.L. contributed important ideas. J.B.H. and M.M.D. wrote the manuscript.

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Correspondence to Mark M. Davis.

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Huppa, J., Axmann, M., Mörtelmaier, M. et al. TCR–peptide–MHC interactions in situ show accelerated kinetics and increased affinity. Nature 463, 963–967 (2010).

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