Toby Starr

T cell receptor microcluster transport through molecular mazes reveals mechanism of translocation.

Recognition of peptide antigen by T cells involves coordinated movement of T cell receptors (TCRs) along with other costimulatory and signaling molecules. The spatially organized configurations that result are collectively referred to as the immunological synapse. Experimental investigation of the role of spatial organization in TCR signaling has been facilitated by the use of nanopatterned-supported membranes to direct TCR into alternative patterns. Here we study the mechanism by which substrate structures redirect TCR transport. Using a flow-tracking algorithm, the ensemble of TCR clusters within each cell was tracked during synapse formation under various constraint geometries. Shortly after initial cluster formation, a coordinated centripetal flow of approximately 20 nm/s develops. Clusters that encounter substrate-imposed constraint are deflected and move parallel to the constraint at speeds that scale with the relative angle of motion to the preferred centripetal direction. TCR transport is driven by actin polymerization, and the distribution of F-actin was imaged at various time points during the synapse formation process. At early time points, there is no significant effect on actin distribution produced by substrate constraints. At later time points, modest differences were observed. These data are consistent with a frictional model of TCR coupling to cytoskeletal flow, which allows slip. Implications of this model regarding spatial sorting of cell-surface molecules are discussed.

Human Immunodeficiency Virus Type 1 Envelope gp120 Induces a Stop Signal and Virological Synapse Formation in Noninfected CD4+ T Cells

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-infected T cells form a virological synapse with noninfected CD4+ T cells in order to efficiently transfer HIV-1 virions from cell to cell. The virological synapse is a specialized cellular junction that is similar in some respects to the immunological synapse involved in T-cell activation and effector functions mediated by the T-cell antigen receptor. The immunological synapse stops T-cell migration to allow a sustained interaction between T-cells and antigen-presenting cells. Here, we have asked whether HIV-1 envelope gp120 presented on a surface to mimic an HIV-1-infected cell also delivers a stop signal and if this is sufficient to induce a virological synapse. We demonstrate that HIV-1 gp120-presenting surfaces arrested the migration of primary activated CD4 T cells that occurs spontaneously in the presence of ICAM-1 and induced the formation of a virological synapse, which was characterized by segregated supramolecular structures with a central cluster of envelope surrounded by a ring of ICAM-1. The virological synapse was formed transiently, with the initiation of migration within 30 min. Thus, HIV-1 gp120-presenting surfaces induce a transient stop signal and supramolecular segregation in noninfected CD4+ T cells.

Supported Planar Bilayers for Study of the Immunological Synapse

Supported planar bilayers have been used in immunology research for over 25 years, including in the initial demonstrations of MHC‐peptide complex functional activity and adhesion molecule activity. More recent modifications of the method have been used to measure two‐dimensional affinities and to study the formation of the immunological synapse. This unit covers the incorporation of glycolipid‐anchored membrane proteins, 6‐histidine‐tagged soluble proteins, and monobiotinylated soluble proteins into supported planar bilayers. Reagents developed for the MHC‐peptide tetramer staining method (UNIT 17.3) can readily be adapted to presentation on planar bilayers. The unique advantage of this approach is that the proteins presented on the surface of the supported bilayer are laterally mobile. This provides a more physiological presentation of cell‐surface molecules and supports visualization of protein rearrangement on the bilayer by live cells.

Nanoscale increases in CD2-CD48 mediated intermembrane spacing decrease adhesion and reorganize the immunological synapse

The relationship between intermembrane spacing, adhesion efficiency, and lateral organization of adhesion receptors has not been established for any adhesion system. We have utilized the CD2 ligand CD48 with two (wild type CD48 (CD48-WT)), four (CD48-CD2), or five (CD48-CD22) Ig-like domains. CD48-WT was 10-fold more efficient in mediating adhesion than CD48-CD2 or CD48-CD22. Electron tomography of contact areas with planar bilayers demonstrated average intermembrane spacing of 12.8 nm with CD48-WT, 14.7 nm with CD48-CD2, and 15.6 nm with CD48-CD22. Both CD48-CD2 and CD48-CD22 chimeras segregated completely from CD48-WT in mixed contact areas. In contrast, CD48-CD2 and CD48-CD22 co-localized when mixed contacts were formed. Confocal imaging of immunological synapses formed between primary T lymphocytes and Chinese hamster ovary cells presenting major histocompatibility complex-peptide complexes, and different forms of CD48 demonstrated that CD48-CD2 and CD48-CD22 induce an eccentric CD2/T cell antigen receptor cluster. We propose that this reorganization of the immunological synapse sequesters the T cell antigen receptor in a location where it cannot interact with its ligand and dramatically reduces T cell sensitivity.

Neutralization of heparin by histone and its subfractions

The neutralization of heparin by histone and its subfractions has been systematically studied by measuring the effect of heparin on the esterolytic and proteolytic activity of thrombin. These results were compared with protamine sulfate, a most commonly used heparin-neutralizing agent. This study reveals that potencies of different fractions of histone are not similar. The antiheparin potency is in the order: lysine-rich histone greater than crude histone greater than arginine-rich histone. Histone binds strongly to heparin - Sepharose gel. The ability of histone to bind heparin can be utilized to fractionate heparin. By affinity chromatography on histone - Sepharose gel commercial heparin has been fractionated into components having a wide range of anticoagulant activities. The highest activity fraction, eluted around 1.0 NaCl, has 66% higher anticoagulant activity than the commercial heparin used.

Quantification and Modeling of Tripartite CD2-, CD58FC Chimera (Alefacept)-, and CD16-mediated Cell Adhesion

Alefacept is a chimeric protein combining CD58 immunoglobulin-like domain 1 with human IgG1 Fc. Alefacept mediates adhesion by bridging CD2 on T cells to activating Fc receptors on effector cells, but the equilibrium binding parameters have not been determined. Alefacept mediated T cell killing by NK cells and adhesion between CD2- and CD16-expressing cells at an optimum concentration of 100 nm. We introduce novel measurements with supported planer bilayers, from which key two-dimensional and three-dimensional parameters can be determined by data fitting. Alefacept competitively inhibited cell bilayer adhesion mediated by the CD2–CD58 interaction. Alefacept mediated maximal adhesion of CD2+ T cells to CD16B, an Fc receptor, in planar bilayers at 500 nm. A mechanistic model for alefacept-mediated cell-bilayer adhesion allowed fitting of the data and determination of two-dimensional binding parameters. These included the density of bonds in the adhesion area, which grew to maintain a consistent average bond density of 200 molecules/μm2 and two-dimensional association constants of 3.1 and 630 μm2 for bivalently and monovalently bound forms of alefacept, respectively. The maximum number of CD16 bound and the fit value of 4,350 CD2 per cell are much lower than the 40,000 CD2 per cell measured with anti-CD2 Fab. These results suggest that additional information is needed to correctly predict Alefacept-mediated bridge formation.

A Biophysical Model of Cell Adhesion Mediated by Immunoadhesin Drugs and Antibodies

A promising direction in drug development is to exploit the ability of natural killer cells to kill antibody-labeled target cells. Monoclonal antibodies and drugs designed to elicit this effect typically bind cell-surface epitopes that are overexpressed on target cells but also present on other cells. Thus it is important to understand adhesion of cells by antibodies and similar molecules. We present an equilibrium model of such adhesion, incorporating heterogeneity in target cell epitope density, nonspecific adhesion forces, and epitope immobility. We compare with experiments on the adhesion of Jurkat T cells to bilayers containing the relevant natural killer cell receptor, with adhesion mediated by the drug alefacept. We show that a model in which all target cell epitopes are mobile and available is inconsistent with the data, suggesting that more complex mechanisms are at work. We hypothesize that the immobile epitope fraction may change with cell adhesion, and we find that such a model is more consistent with the data, although discrepancies remain. We also quantitatively describe the parameter space in which binding occurs. Our model elaborates substantially on previous work, and our results offer guidance for the refinement of therapeutic immunoadhesins. Furthermore, our comparison with data from Jurkat T cells also points toward mechanisms relating epitope immobility to cell adhesion.

Catalytic and regulatory functions of N-bromosuccinimide-modified bovine thrombin

At pH 4.1, bovine thrombin reacts rapidly with N-bromo-succinimide to yield modified enzyme containing oxidized tryptophan residue. Both fibrinogen clotting activity and esterase activity are reduced considerably when three moles of tryptophan residues per mole of thrombin are oxidized, but the Michaelis constants for synthetic substrates are not appreciably altered. Reaction of NBS also results in a decrease in the affinity of thrombin for heparin. The dissociation constant for heparin-thrombin complex is increased by 2.6-fold due to the modification of one tryptophan residue. However, the magnitude of the increase in the dissociation constant remains the same for modified enzymes containing approximately two or three oxidized tryptophan residues. The rate constant for the inactivation of thrombin by antithrombin III is increased by 2.5-fold due to the modification of a single tryptophan residue. This increase in rate constant is not further amplified when more than one tryptophan residue is oxidized. In contrast, in the presence of heparin the rate of inactivation of modified and unmodified thrombins by antithrombin III are not significantly different. Thus, the heparin-sensitized inactivation of thrombin by antithrombin III is affected by the modification of one tryptophan residue. Spectrophotometric titrations of the phenolic hydroxyl groups suggest that the structural environments of tyrosyl groups for both unmodified and modified thrombin containing one oxidized tryptophan residue, are similar. The temperature for half loss of catalytic activity of control and NBS-modified thrombin, containing one oxidized tryptophan, are 52 and 51.5 degrees C respectively. It appears that the one tryptophan residue of thrombin is situated at or close to the binding site of heparin.