Faculty Bibliography

The immunological synapse is a stable intercellular structure that specializes in substance and signal transfer from one immune cell to another. Its formation is regulated in part by the diffusion of adhesion and signaling molecules into, and their binding of counter-molecules in the contact area. The stability of immunological synapses allows receptor-ligand interactions to approximate chemical equilibrium despite other dynamic aspects. We have developed a mathematical model that describes the coupled reaction-diffusion process in an established immunological synapse (Wu et al., 2008, Biophys. J. vol:pp-pp). Here we extend a previously described contact area fluorescence recovery after photobleaching (FRAP) experiment (Dustin, 1997, J. Biol. Chem. 272:15782-15788) to test the validity of the model. The receptor binding and lateral mobility of fluorescently-labeled, lipid-anchored ligands in the bilayer resulted in their accumulation, as revealed by the much higher fluorescence intensity inside the contact area than outside. After complete photobleaching of the synapse, fluorescence recovery requires ligands to dissociate and rebind as well as to diffuse in and out of the contact area. Such a FRAP time course thus contains information on both reaction and diffusion, which can be extracted by fitting the model solution to the data. Surprisingly, reverse-rates in the 2D contact area were at least 100-fold slower than in 3D solution. As previously reported in immunological synapses, a significant nonrecoverable fraction of fluorescence was observed with one of two systems studied, suggesting some ligands either dissociated much more slowly or diffused much more slowly, compared to other ligands in the same synapse. Thus, the combined theory and experiment provides a new method for in situ measurements of kinetic rates, diffusion coefficient, and nonrecoverable fraction of interacting molecules in immunological synapses and other stable cell-bilayer junctions

Kinetic rates and binding affinity of receptor-ligand interactions are important determinants of cell adhesion. Measurements of these parameters in fluid phase using soluble molecules (i.e., 3D parameters) do not necessarily correlate with their counterparts measured when both binding partners are respectively anchored to two apposing surfaces (i.e., 2D parameters). Moreover, 2D affinities measured by different methods can differ by orders of magnitude (Dustin et al., 2001, Annu. Rev. Cell Dev. Biol., 17: 133-157). Here we describe a coupled diffusion-reaction model for the fluorescence recovery after photobleaching (FRAP) experiment previously used to demonstrate the dynamics of adhesive bonds in the contact area (Dustin, J Biol Chem 272:15782-15788). Applying the mathematical model to the contact area FRAP experiment enables in situ measurements of 2D kinetic rates of the adhesion molecules and their retarded diffusion in a stable contact area. The mathematical properties of model are characterized in this paper and its experimental validation will be presented in the companion paper (Tolentino et al., 2008, Biophys. J. vol:pp-pp)

Spatial organization of signaling complexes is a defining characteristic of the immunological synapse (IS), but its impact on cell communication is unclear. In T cell-APC pairs, more IL-2 is produced when CD28 clusters are segregated from central supramolecular activation cluster (cSMAC)-localized CD3 and into the IS periphery. However, it is not clear in these cellular experiments whether the increased IL-2 is driven by the pattern itself or by upstream events that precipitate the patterns. In this article, we recapitulate key features of physiological synapses using planar costimulation arrays containing antibodies against CD3 and CD28, surrounded by ICAM-1, created by combining multiple rounds of microcontact printing on a single surface. Naive T cells traverse these arrays, stopping at features of anti-CD3 antibodies and forming a stable synapse. We directly demonstrate that presenting anti-CD28 in the cell periphery, surrounding an anti-CD3 feature, enhances IL-2 secretion by naive CD4(+) T cells compared with having these signals combined in the center of the IS. This increased cytokine production correlates with NF-kappaB translocation and requires PKB/Akt signaling. The ability to arbitrarily and independently control the locations of anti-CD3 and anti-CD28 offered the opportunity to examine patterns not precisely attainable in cell-cell interfaces. With these patterns, we show that the peripheral presentation of CD28 has a larger impact on IL-2 secretion than CD3 colocalization/segregation

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

Cell polarity is critical for T lymphocyte movement during their hunt for antigen-bearing cells and for infected target cells. In this issue of Cell, Yeh et al. (2008) now reveal a direct link between T cell polarity and the production of proinflammatory cytokines in mice lacking the class I MHC-restricted T cell-associated molecule (Crtam)

Natural killer T (NKT) cells are innate-like lymphocytes that rapidly secrete large amounts of effector cytokines upon activation. Recognition of alpha-linked glycolipids presented by CD1d leads to the production of IL-4, IFN-gamma, or both, while direct activation by the synergistic action of IL-12 and IL-18 leads to IFN-gamma production only. We previously reported that in vitro cultured dendritic cells can modulate NKT cell activation and, using intravital fluorescence laser scanning microscopy, we reported that the potent stimulation of NKT cells results in arrest within hepatic sinusoids. In this study, we examine the relationship between murine NKT cell patrolling and activation. We report that NKT cell arrest results from activation driven by limiting doses of a bacteria-derived weak agonist, galacturonic acid-containing glycosphingolipid, or a synthetic agonist, alpha-galactosyl ceramide. Interestingly, NKT cell arrest also results from IL-12 and IL-18 synergistic activation. Thus, innate cytokines and natural microbial TCR agonists trigger sinusoidal NKT cell arrest and an effector response

T-cell activation requires 'contact' with antigen-presenting cells (APCs) to bring the T-cell receptor (TCR) and antigenic major histocompatibility complex (MHC)-peptide complex together. Contact is defined by the size of the TCR and MHC-peptide complex, which at approximately 13 nm requires extensive interdigitation of the glycocalyx of the T cell and APC. T cells may be activated through formation of a stable T cell-APC junction, referred to as an immunological synapse. It has also been shown in vitro that T cells can integrate signals from APCs without a stable interaction. In vivo imaging studies supported the importance of both motile and stable T cell-APC interactions in T-cell priming. We have found that stability depends not upon turning off motile machinery but by symmetrization of force-generating structures to balance forces and hold the cell in place. Motility is induced by breaking this symmetry, which may be necessary to maintain the differentiation potential of the T cell. Recently, we also discovered a mode of T-cell signaling leading to tolerance in vivo based purely on motile interactions. Because this entire process takes place in a state of continuous T-cell kinesis, I propose the term 'kinapse' for motile T cell-APC contacts leading to signaling. Synapses and kinapses are inter-convertible by symmetrization/symmetry breaking processes, and both modes appear to be involved in normal T-cell priming. Imbalance of synapse/kinapse states may lead to immunopathology

The lipid sphingosine-1-phosphate has been identified as a key exit signal for lymph nodes. In this issue of Immunity, Pham et al. (2008) show that its action can only be understood in the context of retention signals transduced by CCR7

T-cell activation requires interactions of T-cell antigen receptors (TCR) and peptides presented by major histocompatibility complex molecules (MHCp) in an adhesive junction between the T-cell and antigen-presenting cell (APC). Stable junctions with bull's eye supramolecular activation clusters (SMACs) have been defined as immunological synapses. The term synapse works in this case because it joins roots for 'same' and 'fasten', which could be translated as 'fasten in the same place'. These structures maintain T-cell-APC interaction and allow directed secretion. We have proposed that SMACs are not really clusters, but are analogous to higher order membrane-cytoskeleton zones involved in amoeboid locomotion including a substrate testing lamellipodium, an adhesive lamella and anti-adhesive uropod. Since T-cells can also integrate signaling during locomotion over antigen presenting cells, it is important to consider adhesive junctions maintained as cells move past each other. This combination of movement (kine-) and fastening (-apse) can be described as a kinapse or moving junction. Synapses and kinapses operate in different stages of T-cell priming. Optimal effector functions may also depend upon cyclical use of synapses and kinapses. Visualization of these structures in vitro and in vivo presents many distinct challenges that will be discussed in this chapter

The immunological synapse was initially defined as a stable cell-cell junction composed of three concentric supramolecular activation clusters (SMACs) enriched in particular components: a central SMAC with clustered antigen receptors and kinases, a peripheral SMAC rich in beta2 integrin adhesion molecule LFA-1, and a distal SMAC marked by a critical tyrosine phosphatase. In the past year the SMACs have each been identified with functional modules of amoeboid motility, and the stability of the immunological synapse has been revealed as a reconfiguration of the motile apparatus from an asymmetric hunting mode, a kinapse, to a symmetric gathering mode, the synapse. The genetic control of this process involves actinomyosin regulators PKCtheta and WASp. Crtam is involved in postsynaptic polarity in early kinapses prior to cell division. It is unlikely that the immune system is unique in using symmetrization to stop migration without inactivating motile machinery