RESUMEN

T cells experience complex temporal patterns of stimulus via receptor-ligand-binding interactions with surrounding cells. From these temporal patterns, T cells are able to pick out antigenic signals while establishing self-tolerance. Although features such as duration of antigen binding have been examined, our understanding of how T cells interpret signals with different frequencies or temporal stimulation patterns is relatively unexplored. We engineered T cells to respond to light as a stimulus by building an optogenetically controlled chimeric antigen receptor (optoCAR). We discovered that T cells respond to minute-scale oscillations of activation signal by stimulating optoCAR T cells with tunable pulse trains of light. Systematically scanning signal oscillation period from 1 to 150 min revealed that expression of CD69, a T cell activation marker, reached a local minimum at a period of ∼25 min (corresponding to 5 to 15 min pulse widths). A combination of inhibitors and genetic knockouts suggest that this frequency filtering mechanism lies downstream of the Erk signaling branch of the T cell response network and may involve a negative feedback loop that diminishes Erk activity. The timescale of CD69 filtering corresponds with the duration of T cell encounters with self-peptide-presenting APCs observed via intravital imaging in mice, indicating a potential functional role for temporal filtering in vivo. This study illustrates that the T cell signaling machinery is tuned to temporally filter and interpret time-variant input signals in discriminatory ways.

Asunto(s)

Antígenos CD/genética , Antígenos de Diferenciación de Linfocitos T/genética , Lectinas Tipo C/genética , Fototransducción/genética , Receptores Quiméricos de Antígenos/genética , Autotolerancia , Linfocitos T/inmunología , Animales , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Brefeldino A/farmacología , Ingeniería Celular/métodos , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Receptor 2 Celular del Virus de la Hepatitis A/genética , Receptor 2 Celular del Virus de la Hepatitis A/inmunología , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Células K562 , Lectinas Tipo C/inmunología , Luz , Activación de Linfocitos/efectos de los fármacos , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/inmunología , Monensina/farmacología , Optogenética/métodos , Cultivo Primario de Células , Proteína Tirosina Fosfatasa no Receptora Tipo 22/deficiencia , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 22/inmunología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/citología , Linfocitos T/efectos de la radiación

RESUMEN

The natural peptide-major histocompatibility complex (pMHC) ligand for T cell receptors (TCRs) is inactive from solution yet capable of activating T cells at single-molecule levels when membrane-associated. This distinctive feature stems from the mechanism of TCR activation, which is thought to involve steric phosphatase exclusion as well as direct mechanical forces. It is possible to defeat this mechanism and activate T cells with solution ligands by cross-linking pMHC or using multivalent antibodies to TCR. However, these widely used strategies activate TCRs through a nonphysiological mechanism and can produce different activation profiles than natural, monovalent, membrane-associated pMHC. Here, we introduce a strictly monovalent anti-TCRß H57 Fab' ligand that, when coupled to a supported lipid bilayer via DNA complementation, triggers TCRs and activates nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT) with a similar potency to pMHC in primary murine T cells. Importantly, like monovalent pMHC and unlike bivalent antibodies, monovalent Fab'-DNA triggers TCRs only when physically coupled to the membrane, and only around 100 individual Fab':TCR interactions are necessary to stimulate early T cell activation.

Asunto(s)

Activación de Linfocitos , Receptores de Antígenos de Linfocitos T , Animales , Ligandos , Complejo Mayor de Histocompatibilidad , Ratones , Linfocitos T

RESUMEN

Antigen discrimination by T cells occurs at the junction between a T cell and an antigen-presenting cell. Juxtacrine binding between numerous adhesion, signaling, and costimulatory molecules defines both the topographical and lateral geometry of this cell-cell interface, within which T cell receptor (TCR) and peptide major histocompatibility complex (pMHC) interact. These physical constraints on receptor and ligand movement have significant potential to modulate their molecular binding properties. Here, we monitor individual ligand:receptor binding and unbinding events in space and time by single-molecule imaging in live primary T cells for a range of different pMHC ligands and surface densities. Direct observations of pMHC:TCR and CD80:CD28 binding events reveal that the in situ affinity of both pMHC and CD80 ligands for their respective receptors is modulated by the steady-state number of agonist pMHC:TCR interactions experienced by the cell. By resolving every single pMHC:TCR interaction it is evident that this cooperativity is accomplished by increasing the kinetic on-rate without altering the off-rate and has a component that is not spatially localized. Furthermore, positive cooperativity is observed under conditions where the T cell activation probability is low. This TCR-mediated feedback is a global effect on the intercellular junction. It is triggered by the first few individual pMHC:TCR binding events and effectively increases the efficiency of TCR scanning for antigen before the T cell is committed to activation.

Asunto(s)

Antígenos/inmunología , Antígeno B7-1/inmunología , Antígenos CD28/inmunología , Complejo Mayor de Histocompatibilidad , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Antígenos/metabolismo , Antígeno B7-1/genética , Antígenos CD28/genética , Expresión Génica , Ligandos , Membrana Dobles de Lípidos/química , Activación de Linfocitos , Ratones , Cultivo Primario de Células , Unión Proteica , Receptores de Antígenos de Linfocitos T/genética , Transducción de Señal/inmunología , Análisis de la Célula Individual , Linfocitos T/citología

RESUMEN

Optogenetics promises to deepen our understanding of how cells perceive and respond to complex and dynamic signals and how this perception regulates normal and abnormal function. In this study, we present our vision for how these nascent tools may transform our view of fundamental cell biological processes.

Asunto(s)

Biología Celular/tendencias , Optogenética/tendencias , Transducción de Señal , Animales , Difusión de Innovaciones , Predicción , Redes Reguladoras de Genes , Humanos , Mapas de Interacción de Proteínas

RESUMEN

Here we introduce a form of chromatography that can be imposed on the membrane of a living cell. A cell-cell signaling interaction is reconstituted in a hybrid live cell-supported membrane junction. The chromatographic material consists of a hexagonally ordered array of gold nanoparticles (nanodot array), which is fabricated onto the underlying substrate. While individual membrane components move freely throughout the array, the movement of larger assemblies is impeded if they exceed the physical dimensions of the array. This tactile approach to probing membrane structures in living cells reveals organizational aspects of the membrane environment unobservable by other techniques.

Asunto(s)

Membrana Celular/química , Cromatografía/métodos , Nanopartículas del Metal/química , Nanotubos/química , Membrana Celular/metabolismo , Oro/química , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal

RESUMEN

Quantitative spectroscopy has recently been extended from a contact-probe to wide-area spectroscopic imaging to enable mapping of optical properties across a wide area of tissue. We train quantitative spectroscopic imaging (QSI) to identify cervical high-grade squamous intraepithelial lesions (HSILs) in 34 subjects undergoing the loop electrosurgical excision procedure (LEEP subjects). QSI's performance is then prospectively evaluated on the clinically suspicious biopsy sites from 47 subjects undergoing colposcopic-directed biopsy. The results show the per-subject normalized reduced scattering coefficient at 700 nm (An) and the total hemoglobin concentration are significantly different (p<0.05) between HSIL and non-HSIL sites in LEEP subjects. An alone retrospectively distinguishes HSIL from non-HSIL with 89% sensitivity and 83% specificity. It alone applied prospectively on the biopsy sites distinguishes HSIL from non-HSIL with 81% sensitivity and 78% specificity. The findings of this study agree with those of an earlier contact-probe study, validating the robustness of QSI, and specifically An, for identifying HSIL. The performance of An suggests an easy to use and an inexpensive to manufacture monochromatic instrument is capable of early cervical cancer detection, which could be used as a screening and diagnostic tool for detecting cervical cancer in low resource countries.

Asunto(s)

Interpretación de Imagen Asistida por Computador/métodos , Análisis Espectral/métodos , Displasia del Cuello del Útero/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Adulto , Teorema de Bayes , Cuello del Útero , Femenino , Humanos , Sensibilidad y Especificidad , Neoplasias del Cuello Uterino/patología , Displasia del Cuello del Útero/patología

RESUMEN

T cells discriminate between self and foreign antigenic peptides, displayed on antigen presenting cell surfaces, via the TCR. While the molecular interactions between TCR and its ligands are well characterized in vitro, quantitative measurements of these interactions in living cells are required to accurately resolve the physical mechanisms of TCR signaling. We report direct single molecule measurements of TCR triggering by agonist pMHC in hybrid junctions between live primary T cells and supported lipid membranes. Every pMHC:TCR complex over the entire cell is tracked while simultaneously monitoring the local membrane recruitment of ZAP70, as a readout of TCR triggering. Mean dwell times for pMHC:TCR molecular binding of 5 and 54 s were measured for two different pMHC:TCR systems. Single molecule measurements of the pMHC:TCR:ZAP70 complex indicate that TCR triggering is stoichiometric with agonist pMHC in a 1:1 ratio. Thus any signal amplification must occur downstream of TCR triggering. DOI:http://dx.doi.org/10.7554/eLife.00778.001.

Asunto(s)

Complejo Mayor de Histocompatibilidad/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Humanos , Cinética , Unión Proteica , Procesos Estocásticos , Proteína Tirosina Quinasa ZAP-70/metabolismo

RESUMEN

The galectin family of glycan-binding proteins is thought to mediate many cellular processes by oligomerizing cell surface glycoproteins and glycolipids into higher-order aggregates. This hypothesis reflects the known oligomeric states of the galectins themselves and their binding properties with multivalent ligands in vitro, but direct evidence of their ability to cross-link ligands on a cell surface is lacking. A major challenge in fundamental studies of galectin-ligand interactions is that their natural ligands comprise a heterogeneous collection of glycoconjugates that share related glycan structures but disparate underlying scaffolds. Consequently, there is no obvious means to selectively monitor the behaviors of natural galectin ligands on live cell surfaces. Here we describe an approach for probing the galectin-induced multimerization of glycoconjugates on cultured cells. Using RAFT polymerization, we synthesized well-defined glycopolymers (GPs) functionalized with galectin-binding glycans along the backbone, a lipid group on one end and a fluorophore on the other. After insertion into live cell membranes, the GPs' fluorescence lifetime and diffusion time were measured in the presence and absence of galectin-1. We observed direct evidence for galectin-1-mediated extended cross-linking on the engineered cells, a phenomenon that was dependent on glycan structure. This platform offers a new approach to exploring the "galectin lattice" hypothesis and to defining galectin ligand specificity in a physiologically relevant context.

Asunto(s)

Membrana Celular/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Galectinas/metabolismo , Glicoconjugados/metabolismo , Animales , Células CHO , Membrana Celular/química , Cricetinae , Galectina 1/análisis , Galectina 1/metabolismo , Galectinas/análisis , Glicoconjugados/análisis , Polimerizacion , Unión Proteica

RESUMEN

We present a supported membrane platform consisting of a fluid lipid bilayer membrane embedded with a fixed array of gold nanoparticles. The system is realized by preforming a hexagonal array of gold nanoparticles (∼5-7 nm) with controlled spacing (∼50-150 nm) fixed to a silica or glass substrate by block copolymer lithography. Subsequently, a supported membrane is assembled over the intervening bare substrate. Proteins or other ligands can be associated with the fluid lipid component, the fixed nanoparticle component, or both, providing a hybrid interface consisting of mobile and immobile components with controlled geometry. We test different biochemical coupling strategies to bind individual proteins to the particles surrounded by a fluid lipid membrane. The coupling efficiency to nanoparticles and the influence of nanoparticle arrays on the surrounding membrane integrity are characterized by fluorescence imaging, correlation spectroscopy, and super-resolution fluorescence microscopy. Finally, the functionality of this system for live cell experiments is tested using the ephrin-A1-EphA2 juxtacrine signaling interaction in human breast epithelial cells.

Asunto(s)

Oro/química , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Membranas Artificiales , Nanoestructuras/química , Nanoestructuras/ultraestructura , Cristalización/métodos , Sustancias Macromoleculares/química , Ensayo de Materiales , Conformación Molecular , Tamaño de la Partícula , Propiedades de Superficie