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 peptidemajor 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 TCRpMHC 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. Supplementary Information is linked to the online version of the paper at www.nature.com/nature. (refs 4-6). Nevertheless, T cells are highly specific and sensitive for antigen, able to detect even a single antigenic pMHC complex among structurally similar yet non-stimulatory pMHCs7,8. However, although this is useful for comparative purposes, these measurements do not account for the many constraints and special features of the synaptic environment and might not reflect what occurs in situ. In particular, the restricted intercellular volume should greatly accelerate the association rate and enhance serial engagement9-11. Favourable molecular alignment of TCR and MHC, as well as any molecular pre-clustering, could also drastically affect binding12-14. Although the volume effect can be approximated, the influence of the others is essentially unknown. Author ContributionsWe have therefore devised a method to measure synaptic TCR binding to pMHC directly, using single-molecule microscopy and FRET between a donor fluorophore on the TCR and an acceptor fluorophore on the peptide bound to a MHC molecule.We used T-cell blasts from two different TCR transgenic T-cell lines, 2B4 and 5c.c7, which are specific for the same moth cytochrome c peptide (MCC 88-103) bound to the class II MHC molecule IE k . To permit the use of highly sensitive and rapid total internal reflection (TIRF) microscopy, we used a modified planar lipid bilayer system as a surrogate antigenpresenting cell surface, presenting IE k complexes, ICAM-1 adhesion molecules and B7-1 co-stimulatory polypeptides (see Methods).Structural analysis of an Fab fragment derived f...

The organization and dynamics of receptors and other molecules in the plasma membrane are not well understood. Here we analyzed the spatio-temporal dynamics of T cell antigen receptor (TCR) complexes and linker for activation of T cells (Lat), a key adaptor molecule in the TCR signaling pathway, in T cell membranes using high-speed photoactivated localization microscopy, dualcolor fluorescence cross-correlation spectroscopy and transmission electron microscopy. In quiescent T cells, both molecules existed in separate membrane domains (protein islands), and these domains concatenated after T cell activation. These concatemers were identical to signaling microclusters, a prominent hallmark of T cell activation. This separation versus physical juxtapositioning of receptor domains and domains containing downstream signaling molecules in quiescent versus activated T cells may be a general feature of plasma membrane-associated signal transduction.A principal function of most T cells is to recognize foreign antigens on other cell surfaces, with the specificity determined by the T cell antigen receptor (TCR). Once the TCR has engaged its ligand and the signaling cascade has been initiated through the associated cluster of differentiation 3 (CD3) subunits, the kinase Zap70 transmits the signal to the adaptor Lat (linker for activation of T cells; A001392). Within seconds, actin-dependent signaling microclusters are formed in the periphery of the contact site between the T cell and antigenpresenting cell, and these microclusters then migrate to the center of the interface and form the immunological synapse [1][2][3][4][5][6] . Many signaling molecules, including the TCR and Lat, colocalize in the microclusters during early T cell activation [3][4][5][6] . On the basis of electron-microscopy studies, varying degrees of preclustering of plasma membrane molecules, including the TCR and Lat, have been proposed 7-9 . However, there are concerns about potential artifacts with this approach (for example, from fixation, gold particle staining and/or drying). Additionally, fluorescence microscopy and biochemical technologies suggest the coexistence of monomeric and multivalent TCR-CD3 complexes 9,10 . It is not clear to what extent these multivalent complexes exist or if they form through protein-protein interactions or localization in membrane domains. In the case of biochemical approaches, cell lysis itself could introduce additional artifacts. To overcome such limitations and to gain insight into the organization of plasma membrane proteins in situ, we analyzed the organization of the TCR and Lat in quiescent and activated native T cell plasma membrane sheets and live T cells. We achieved this with a combination of three techniques: a modified high-speed version of photoactivated localization microscopy (hsPALM) 11-13 , dual-color fluorescence cross-correlation spectroscopy (dcFCCS) [14][15][16] and transmission electron microscopy (TEM) 7,8,17,18 . NIH Public AccessWe show that both the TCR and Lat were preclustered i...

A notable feature of T lymphocyte recognition on other cell surfaces is the formation of a stable mature immunological synapse. Here we use a single-molecule labeling method to directly measure the number of ligands a cytotoxic T cell engages and track the consequences of that interaction by three-dimensional video microscopy. Like helper T cells, cytotoxic T cells were able to detect even a single foreign antigen but required about ten complexes of peptide-major histocompatibility complex (pMHC) to achieve full calcium increase and to form a mature synapse. Thus, cytotoxic T cells and helper T cells are more uniform in their antigen sensitivities than previously thought. Furthermore, only three pMHC complexes were required for killing, showing that stable synapse formation and complete signaling are not required for cytotoxicity.