Faculty Bibliography

We formulate a general analysis to determine the two-dimensional dissociation constant (2D Kd), and use this method to study the interaction of CD2-expressing T cells with glass-supported planar bilayers containing fluorescently labeled CD58, a CD2 counter-receptor. Both CD2 and CD58 are laterally mobile in their respective membranes. Adhesion is indicated by accumulation of CD2 and CD58 in the cell-bilayer contact area; adhesion molecule density and contact area size attain equilibrium within 40 min. The standard (Scatchard) analysis of solution-phase binding is not applicable to the case of laterally mobile adhesion molecules due to the dynamic nature of the interaction. We derive a new binding equation, B/F=[(Ntxf)/(KdxScell)]-[(Bxp)/Kd], where B and F are bound and free CD58 density in the contact area, respectively; Nt is CD2 molecule number per cell; f is CD2 fractional mobility; Scell is cell surface area; and p is the ratio of contact area at equilibrium to Scell. We use this analysis to determine that the 2D Kd for CD2-CD58 is 5.4-7.6 molecules/microm2. 2D Kd analysis provides a general and quantitative measure of the mechanisms regulating cell-cell adhesion

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

Integrins comprise a large family of cell-cell and cell-matrix adhesion receptors that rapidly modulate their adhesiveness. The arrest of leukocyte integrins on target vascular beds involves instantaneous conformational switches generating shear-resistant adhesions. Structural data suggest that these integrins are maintained in low-affinity conformations and must rapidly undergo conformational switches transduced via cytoplasmic changes ('inside-out' signaling) and simultaneous ligand-induced rearrangements ('outside-in'). This bidirectional activation is accelerated by signals from endothelial chemoattractants (chemokines). Recent studies predict that shear forces in the piconewton (pN) range per integrin can facilitate these biochemical switches. After extravasation, antigen recognition involves smaller internal forces from cytoskeletal motors and actin polymers forming the immune synapse. In this review, we address how forces facilitate allosteric integrin activation by biochemical signals. Evidence suggests that preformed cytoskeletal anchorage rather than free integrin mobility is key for force-enhanced integrin activation by chemokines and TCR signals

The immunological synapse is a specialized intercellular junction between a T cell and a target cell that orchestrates the engagement of receptors and ligands in space and time as a means of regulating function. Here we introduce a reagent for controlling the spatial and temporal presentation of natural antigen to T cells. Moth cytochrome c (88-103) peptide (MCC), an agonist to the murine T cell receptor AND when presented in the context of H2 IEk major histocompatibility complex (IEk), was synthesized with the side-chain amine of Lys99 conjugated to a photosensitive protecting group, 6-nitroveratryloxycarbonyl (NVOC). Cells plated on supported bilayers displaying mobile intercellular adhesion molecule-1 (ICAM-1) and NVOC-MCC loaded IEk did not form immunological synapses and exhibited low intracellular calcium levels, similar to cells presented with self-peptide. Irradiation with UV light was sufficient to restore agonist activity in situ

The CD2 receptor on T lymphocytes is essential for T cell adhesion and stimulation by antigen presenting cells (APCs). Blockade of CD2 function is immunosuppressive in both model systems and humans, indicating the importance of CD2 for the cellular immune response. Although the affinity of the molecular interaction between CD2 and its counter-receptor, CD58, is relatively low when measured in solution, this interaction mediates tight adhesion within the 2D cell-cell interface. To understand the mechanisms responsible for regulating the avidity of the CD2-CD58 interaction, we measured the number, affinity, and lateral mobility of CD2 molecules on resting and activated T cells. Cell activation caused a 1.5-fold increase in the number of CD2 sites on the cell surface, and the 2D affinity of CD2 for CD58 increased by 2.5-fold. The combination of T cell activation and CD2 ligation to CD58 decreased the laterally mobile fraction of the ligated CD2. Together, these changes would substantially enhance CD2 avidity and strengthen T cell-APC adhesion. The change in CD2 mobile fraction suggests that the cell uses cytoskeletal regulators to immobilize the receptor selectively at the site of contact with surfaces expressing CD58. Our observations are consistent with a model in which T cell activation initially induces increased CD2 2D affinity, cell surface receptor expression, and lateral mobility, allowing the CD2 molecules to diffuse to sites of contact with CD58-bearing APCs. Subsequently, T cell activation causes the CD58-bound CD2 to be recognized and immobilized at sites of cell-cell contact, thereby strengthening T cell-APC adhesion.

Cytotoxic T lymphocytes (CTL) can respond to a few viral peptide-MHC-I (pMHC-I) complexes among a myriad of virus-unrelated endogenous self pMHC-I complexes displayed on virus-infected cells. To elucidate the molecular recognition events on live CTL, we have utilized a self-assembled biosensor composed of semiconductor nanocrystals, quantum dots, carrying a controlled number of virus-derived (cognate) and other (noncognate) pMHC-I complexes and examined their recognition by antigen-specific T cell receptor (TCR) on anti-virus CD8(+) T cells. The unique architecture of nanoscale quantum dot/pMHC-I conjugates revealed that unexpectedly strong multivalent CD8-MHC-I interactions underlie the cooperative contribution of noncognate pMHC-I to the recognition of cognate pMHC-I by TCR to augment T cell responses. The cooperative, CD8-dependent spread of signal from a few productively engaged TCR to many other TCR can explain the remarkable ability of CTL to respond to virus-infected cells that present few cognate pMHC-I complexes

We have studied the initial innate immune response to focal necrotic injury on different sides of the mouse blood-brain barrier by two-photon intravital microscopy. Transgenic mice in which the promoter of the myeloid isoform of lysozyme drives GFP were used to track granulocytes and monocytes. Necrotic injury in the meninges, but not the brain parenchyma, recruited GFP+ cells within minutes that fully surrounded the necrotic site within a day. Recently, it has been suggested that microglial cells and astrocytes cooperate to mount a distinct response to laser injury behind the blood-brain barrier. We followed the microglial response in heterozygous knockin mice in which GFP replaces CX3CR1 coding sequence. Prior to injury, microglial cell bodies were immobile over days, but moved to the laser injury site within 1 day. We followed astrocytes, which have been proposed to cooperate with microglial cells in response to focal injury, using transgenic mice in which glial fibrillary acidic protein promoter drives GFP expression. Before injury fine astrocyte processes permeate the parenchyma. Astrocytes polarized toward the injury in an ATP, connexin hemichannels, and intracellular Ca2+ -dependent process. The astrocytes network established a cytoplasmic Ca2+ gradient that preceded the microglial response. This is consistent with astrocyte-microglial collaboration to mount this innate response that excludes blood leukocytes