Project description:T-cell activation is a key event in the immune system, involving the interaction of several receptor ligand pairs in a complex intercellular contact that forms between T-cell and antigen-presenting cells. Molecular components implicated in contact formation have been identified, but the mechanism of activation and the link between molecular interactions and cell response remain poorly understood due to the complexity and dynamics exhibited by whole cell-cell conjugates. Here we demonstrate that simplified model colloids grafted so as to target appropriate cell receptors can be efficiently used to explore the relationship of receptor engagement to the T-cell response. Using immortalized Jurkat T cells, we monitored both binding and activation events, as seen by changes in the intracellular calcium concentration. Our experimental strategy used flow cytometry analysis to follow the short time scale cell response in populations of thousands of cells. We targeted both T-cell receptor CD3 (TCR/CD3) and leukocyte-function-associated antigen (LFA-1) alone or in combination. We showed that specific engagement of TCR/CD3 with a single particle induced a transient calcium signal, confirming previous results and validating our approach. By decreasing anti-CD3 particle density, we showed that contact nucleation was the most crucial and determining step in the cell-particle interaction under dynamic conditions, due to shear stress produced by hydrodynamic flow. Introduction of LFA-1 adhesion molecule ligands at the surface of the particle overcame this limitation and elucidated the low TCR/CD3 ligand density regime. Despite their simplicity, model colloids induced relevant biological responses which consistently echoed whole cell behavior. We thus concluded that this biophysical approach provides useful tools for investigating initial events in T-cell activation, and should enable the design of intelligent artificial systems for adoptive immunotherapy.
Project description:Melanoma is believed to be a highly immunogenic tumor and recent developments in immunotherapies are promising. IFN-γ produced by immune cells has a crucial role in tumor immune surveillance; however, it has also been reported to be pro-tumorigenic. In the current study, we found that IFN-γ enhances the expression of CD74, which interacts with its ligand, macrophage migration inhibitory factor (MIF), and thereby activates the PI3K/AKT pathway in melanoma, promoting tumor survival. IFN-γ increased phosphorylation of AKT Ser473 and upregulated total cell surface expression of CD74 in human melanoma cell lines tested. CD74 was highly expressed in melanoma tissues. Moreover, the expression of CD74 on tumor cells correlated with plasma IFN-γ levels in melanoma patient samples. In our analysis of melanoma cell lines, all produced MIF constitutively. Blockade of CD74-MIF interaction reduced AKT phosphorylation and expression of pro-tumorigenic molecules, including IL-6, IL-8, and BCL-2. Inhibition of CD74-MIF interaction significantly suppressed tumor growth in the presence of IFN-γ in our xenograft mouse model. Thus, we conclude that IFN-γ promotes melanoma cell survival by regulating CD74-MIF signaling, suggesting that targeting the CD74-MIF interaction under IFN-γ-stimulatory conditions would be an effective therapeutic approach for melanoma.
Project description:Focal neuropathy results from an injury of any etiology that occurs in a peripheral nerve. The lesion may be followed by alteration of the sensory sphere (either dysesthesia or paresthesia with or without neuropathic pain), or by compensatory attitudes that are attributable to the altered contraction in muscles that are innervated by the injured nerve. We describe the case of a 13-year-old boy who attended our hospital for a focal neuropathy of the radial nerve. This neuropathy was revealed after the removal of a plaster Zimmer splint that was applied following a post-traumatic subluxation of the metacarpal-trapezoid joint.
Project description: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.
Project description:Surface plasmon resonance is a very well-established surface sensitive technique for label-free analysis of biomolecular interactions, generating thousands of publications each year. An inconvenient effect that complicates interpretation of SPR results is the "bulk response" from molecules in solution, which generate signals without really binding to the surface. Here we present a physical model for determining the bulk response contribution and verify its accuracy. Our method does not require a reference channel or a separate surface region. We show that proper subtraction of the bulk response reveals an interaction between poly(ethylene glycol) brushes and the protein lysozyme at physiological conditions. Importantly, we also show that the bulk response correction method implemented in commercial instruments is not generally accurate. Using our method, the equilibrium affinity between polymer and protein is determined to be KD = 200 μM. One reason for the weak affinity is that the interaction is relatively short-lived (1/koff < 30 s). Furthermore, we show that the bulk response correction also reveals the dynamics of self-interactions between lysozyme molecules on surfaces. Besides providing new insights on important biomolecular interactions, our method can be widely applied to improve the accuracy of SPR data generated by instruments worldwide.
Project description:The interactions between pairs of cells and within multicellular assemblies are critical to many biological processes such as intercellular communication, tissue and organ formation, immunological reactions, and cancer metastasis. The ability to precisely control the position of cells relative to one another and within larger cellular assemblies will enable the investigation and characterization of phenomena not currently accessible by conventional in vitro methods. We present a versatile surface acoustic wave technique that is capable of controlling the intercellular distance and spatial arrangement of cells with micrometer level resolution. This technique is, to our knowledge, among the first of its kind to marry high precision and high throughput into a single extremely versatile and wholly biocompatible technology. We demonstrated the capabilities of the system to precisely control intercellular distance, assemble cells with defined geometries, maintain cellular assemblies in suspension, and translate these suspended assemblies to adherent states, all in a contactless, biocompatible manner. As an example of the power of this system, this technology was used to quantitatively investigate the gap junctional intercellular communication in several homotypic and heterotypic populations by visualizing the transfer of fluorescent dye between cells.
Project description:The phyllosilicate mineral muscovite mica is widely used as a surface template for the patterning of macromolecules, yet a molecular understanding of its surface chemistry under varying solution conditions, required to predict and control the self-assembly of adsorbed species, is lacking. We utilize all-atom molecular dynamics simulations in conjunction with an electrostatic analysis based in local molecular field theory that affords a clean separation of long-range and short-range electrostatics. Using water polarization response as a measure of the electric fields that arise from patterned, surface-bound ions that direct the adsorption of charged macromolecules, we apply a Landau theory of forces induced by asymmetrically polarized surfaces to compute protein-surface interactions for two muscovite-binding proteins (DHR10-mica6 and C98RhuA). Comparison of the pressure between surface and protein in high-concentration KCl and NaCl aqueous solutions reveals ion-specific differences in far-field protein-surface interactions, neatly capturing the ability of ions to modulate the surface charge of muscovite that in turn selectively attracts one binding face of each protein over all others.
Project description:Measures of cellular gene expression or behavior, when performed on individual cells, inevitably reveal a diversity of behaviors and outcomes that can correlate with normal or diseased states. For virus infections, the potential diversity of outcomes are pushed to an extreme, where measures of infection reflect features of the specific infecting virus particle, the individual host cell, as well as interactions between viral and cellular components. Single-cell measures, while revealing, still often rely on specialized fluid handling capabilities, employ end-point measures, and remain labor-intensive to perform. To address these limitations, we consider a new microwell-based device that uses simple pipette-based fluid handling to isolate individual cells. Our design allows different experimental conditions to be implemented in a single device, permitting easier and more standardized protocols. Further, we utilize a recently reported dual-color fluorescent reporter system that provides dynamic readouts of viral and cellular gene expression during single-cell infections by vesicular stomatitis virus. In addition, we develop and show how free, open-source software can enable streamlined data management and batch image analysis. Here we validate the integration of the device and software using the reporter system to demonstrate unique single-cell dynamic measures of cellular responses to viral infection.
Project description:The identity and functions of specialized cell types are dependent on the complex interplay between signaling and transcriptional networks. Recently single-cell technologies such as CITE-seq have been developed that enable simultaneous quantitative analysis of cell-surface receptor expression with transcriptional states. To date, these datasets have not been used to systematically develop cell-context-specific maps of the interface between signaling and transcriptional regulators orchestrating cellular identity and function. We present SPaRTAN (Single-cell Proteomic and RNA based Transcription factor Activity Network), a computational method to link cell-surface receptors to transcription factors (TFs) by exploiting cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) datasets with cis-regulatory information. SPaRTAN is applied to immune cell types in the blood to predict the coupling of signaling receptors with cell context-specific TFs. The predictions are validated by prior knowledge and flow cytometry analyses. SPaRTAN is then used to predict the signaling coupled TF states of tumor infiltrating CD8+ T cells in malignant peritoneal and pleural mesotheliomas. SPaRTAN greatly enhances the utility of CITE-seq datasets to uncover TF and cell-surface receptor relationships in diverse cellular states.