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 cells. If productive, these interactions promote the formation of an immunological synapse. 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.

Evidence for a functional sidedness to the alphabetaTCR.

The T cell receptor (TCR) and associated CD3gammaepsilon, deltaepsilon, and zetazeta signaling dimers allow T cells to discriminate between different antigens and respond accordingly, but our knowledge of how these parts fit and work together is incomplete. In this study, we provide additional evidence that the CD3 heterodimers congregate on one side of the TCR in both the alphabeta and gammadeltaTCR-CD3 complexes. We also report that the other side of the alphabetaTCR mediates homotypic alphabetaTCR interactions and signaling. Specifically, an erythropoietin receptor-based dimerization assay was used to show that, upon complex assembly, the CD3epsilon chains of two CD3 heterodimers are arranged side-by-side in both the alphabeta and gammadeltaTCR-CD3 complexes. This system was also used to show that alphabetaTCRs can dimerize in the cell membrane and that mutating the unusual outer strands of the Calpha domain impairs this dimerization. Finally, we present data showing that, for CD4 T cells, the mutations that impair alphabetaTCR dimerization also alter ligand-induced calcium mobilization, TCR accumulation at the site of pMHC contact, and polarization toward the site of antigen contact. These data reveal a "functional-sidedness" to the alphabetaTCR constant region, with dimerization occurring on the side of the TCR opposite from where the CD3 heterodimers are located.

Simultaneous detection of many T-cell specificities using combinatorial tetramer staining.

The direct detection of antigen-specific T cells using tetramers of soluble peptide-major histocompatibilty complex (pMHC) molecules is widely used in both basic and clinical immunology. However, the number of specificities that can be assessed simultaneously has been a major limitation. Here we describe and validate a method using combinations of fluorescent pMHC tetramers to simultaneously detect and enrich for many (>or=15) T-cell specificities in a single human blood sample.

Diffusion of lipid-like single-molecule fluorophores in the cell membrane.

The dicyanomethylenedihydrofuran (DCDHF) class of single-molecule fluorophores contains an amine donor and a dicyanomethylenedihydrofuran acceptor linked by a conjugated unit (benzene, naphthalene, or styrene). Molecules in this class have a number of useful properties in addition to those usually required for single-molecule studies (such as high fluorescence quantum yield and photostability), including second-order optical nonlinearity, large ground-state dipole moment, and sensitivity to local environment. Moreover, most DCDHF molecules have amphiphilic structures, with a polar dicyanomethylenedihydrofuran headgroup and nonpolar hydrocarbon tails on the amine or furan ring, and can be used as fluorescent lipid analogues for live cell imaging. Here we demonstrate that individual molecules of several different DCDHF lipid analogues can be observed diffusing in the plasma membrane of Chinese hamster ovary cells. The photophysical and diffusive behaviors of the DCDHF lipid analogues in membranes are described and are found to be competitive with the well-known lipid probe N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine.

Spatial and temporal dynamics of T cell receptor signaling with a photoactivatable agonist.

The precise timing of signals downstream of the T cell receptor (TCR) is poorly understood. To address this problem, we prepared major histocompatibility complexes containing an antigenic peptide that is biologically inert until exposed to ultraviolet (UV) light. UV irradiation of these complexes in contact with cognate T cells enabled the high-resolution temporal analysis of signaling. Phosphorylation of the LAT adaptor molecule was observed in 4 s, and diacylglycerol production and calcium flux was observed in 6-7 s. TCR activation also induced cytoskeletal polarization within 2 min. Antibody blockade of CD4 reduced the intensity of LAT phosphorylation and the speed of calcium flux. Furthermore, strong desensitization of diacylglycerol production, but not LAT phosphorylation, occurred shortly after TCR activation, suggesting that different molecular events play distinct signal-processing roles. These results establish the speed and localization of early signaling steps, and have important implications regarding the overall structure of the network.

miR-181a is an intrinsic modulator of T cell sensitivity and selection.

T cell sensitivity to antigen is intrinsically regulated during maturation to ensure proper development of immunity and tolerance, but how this is accomplished remains elusive. Here we show that increasing miR-181a expression in mature T cells augments the sensitivity to peptide antigens, while inhibiting miR-181a expression in the immature T cells reduces sensitivity and impairs both positive and negative selection. Moreover, quantitative regulation of T cell sensitivity by miR-181a enables mature T cells to recognize antagonists-the inhibitory peptide antigens-as agonists. These effects are in part achieved by the downregulation of multiple phosphatases, which leads to elevated steady-state levels of phosphorylated intermediates and a reduction of the T cell receptor signaling threshold. Importantly, higher miR-181a expression correlates with greater T cell sensitivity in immature T cells, suggesting that miR-181a acts as an intrinsic antigen sensitivity "rheostat" during T cell development.

Cholesterol depletion induces solid-like regions in the plasma membrane.

Glycosylphosphatidylinositol-linked and transmembrane major histocompatibility complex (MHC) class II I-E(k) proteins, as well as N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Tritc-DHPE), are used as probes to determine the effect of cholesterol concentration on the organization of the plasma membrane at temperatures in the range 22 degrees C-42 degrees C. Cholesterol depletion caused a decrease in the diffusion coefficients for the MHC II proteins and also for a slow fraction of the Tritc-DHPE population. At 37 degrees C, reduction of the total cell cholesterol concentration results in a smaller suppression of the translational diffusion for I-E(k) proteins (twofold) than was observed in earlier work at 22 degrees C (five sevenfold) Vrljic, M., S. Y. Nishimura, W. E. Moerner, and H. M. McConnell. 2005. Biophys. J. 88:334-347. At 37 degrees C, the diffusion of both I-E(k) proteins is Brownian (0.9 < alpha-parameter < 1.1). More than 99% of the protein population diffuses homogeneously when imaged at 65 frames per s. As the temperature is raised from 22 degrees C to 42 degrees C, a change in activation energy is seen at approximately 35 degrees C in the Arrhenius plots. Cytoskeletal effects appear to be minimal. These results are consistent with a previously described model of solid-like domain formation in the plasma membrane.