During T cell activation, the engagement of costimulatory molecules is often crucial to the development of an effective immune response, but the mechanism by which this is achieved is not known. Here, it is shown that beads attached to the surface of a T cell translocate toward the interface shortly after the start of T cell activation. This movement appears to depend on myosin motor proteins and requires the engagement of the major costimulatory receptor pairs, B7-CD28 and ICAM-1–LFA-1. This suggests that the engagement of costimulatory receptors triggers an active accumulation of molecules at the interface of the T cell and the antigen-presenting cell, which then increases the overall amplitude and duration of T cell signaling.

Polycomb group protein Ezh2, one of the key regulators of development in organisms from flies to mice, exerts its epigenetic function through regulation of histone methylation. Here, we report the existence of the cytosolic Ezh2-containing methyltransferase complex and tie the function of this complex to regulation of actin polymerization in various cell types. Genetic evidence supports the essential role of cytosolic Ezh2 in actin polymerization-dependent processes such as antigen receptor signaling in T cells and PDGF-induced dorsal circular ruffle formation in fibroblasts. Revealed function of Ezh2 points to a broader usage of lysine methylation in regulation of both nuclear and extra-nuclear signaling processes.

Whereas T helper cells recognize peptide-major histocompatibility complex (MHC) class II complexes through their T cell receptors (TCRs), CD4 binds to an antigen-independent region of the MHC. Using green fluorescent protein-tagged chimeras and three-dimensional video microscopy, we show that CD4 and TCR-associated CD3zeta cluster in the interface coincident with increases in intracellular calcium. Signaling-, costimulation-, and cytoskeleton-dependent processes then stabilize CD3zeta in a single cluster at the center of the interface, while CD4 moves to the periphery. Thus, the CD4 coreceptor may serve primarily to "boost" recognition of ligand by the TCR and may not be required once activation has been initiated.

To initiate an immune response, key receptor-ligand pairs must cluster in "immune synapses" at the T cell-antigen-presenting cell (APC) interface. We visualized the accumulation of a major histocompatibility complex (MHC) class II molecule, I-E(k), at a T cell-B cell interface and found it was dependent on both antigen recognition and costimulation. This suggests that costimulation-driven active transport of T cell surface molecules helps to drive immunological synapse formation. Although only agonist peptide-MHC class II (agonist pMHC class II) complexes can initiate T cell activation, endogenous pMHC class II complexes also appeared to accumulate. To test this directly, we labeled a "null" pMHC class II complex and found that, although it lacked major TCR contact residues, it could be driven into the synapse in a TCR-dependent manner. Thus, low-affinity ligands can contribute to synapse formation and T cell signaling.

While much is known about intracellular signaling events in T cells when T cell receptors (TCRs) are engaged, the mechanism by which signaling is initiated is unclear. We have constructed defined oligomers of soluble antigen-major histocompatibility complex (MHC) molecules, the natural ligands for the TCR. Using these to stimulate specific T cells in vitro, we find that agonist peptide/MHC ligands are nonstimulatory as monomers and minimally stimulatory as dimers. Similarly, a partial-agonist ligand is very weakly active as a tetramer. In contrast, trimeric or tetrameric agonist ligands that engage multiple TCRs for a sustained duration are potent stimuli. Ligand-driven formation of TCR clusters seems required for effective activation and helps to explain the specificity and sensitivity of T cells.

Temporal and spatial variations in the concentrations of signaling intermediates in a living cell are important for signaling in complex networks because they modulate the probabilities that signaling intermediates will interact with each other. We have studied 30 signaling sensors, ranging from receptors to transcription factors, in the physiological activation of murine ex vivo T cells by antigen-presenting cells. Spatiotemporal patterning of these molecules was highly diverse and varied with specific T cell receptors and T cell activation conditions. The diversity and variability observed suggest that spatiotemporal patterning controls signaling interactions during T cell activation in a physiologically important and discriminating manner. In support of this, the effective clustering of a group of ligand-engaged receptors and signaling intermediates in a joint pattern consistently correlated with efficient T cell activation at the level of the whole cell.

T cell recognition typically involves both the engagement of a specific T cell receptor with a peptide͞major histocompatibility complex (MHC) and a number of accessory interactions. One of the most important interactions is between the integrin lymphocyte function-associated antigen 1 (LFA-1) on the T cell and intracellular adhesion molecule 1 (ICAM-1) on an antigen-presenting cell. By using f luorescence video microscopy and an ICAM-1 fused to a green f luorescent protein, we find that the elevation of intracellular calcium in the T cell that is characteristic of activation is followed almost immediately by the rapid accumulation of ICAM-1 on a B cell at a tight interface between the two cells. This increased density of ICAM-1 correlates with the sustained elevation of intracellular calcium in the T cell, known to be critical for activation. The use of peptide͞MHC complexes and ICAM-1 on a supported lipid bilayer to stimulate T cells also indicates a major role for ICAM-1͞LFA-1 in T cell activation but, surprisingly, not for adhesion, as even in the absence of ICAM-1 the morphological changes and adhesive characteristics of an activated T cell are seen in this system. We suggest that T cell antigen receptor-mediated recognition of a very small number of MHC͞peptide complexes could trigger LFA-1͞ICAM-1 clustering and avidity regulation, thus amplifying and stabilizing the production of second messengers.

Cytolytic killing is a major effector mechanism in the elimination of virally infected and tumor cells. The innate cytolytic effectors, natural killer (NK) cells, and the adaptive effectors, cytotoxic T cells (CTL), despite differential immune recognition, both use the same lytic mechanism, cytolytic granule release. Using live cell video fluorescence microscopy in various primary cell models of NK cell and CTL killing, we show here that on tight target cell contact, a majority of the NK cells established cytoskeletal polarity required for effective lytic function slowly or incompletely. In contrast, CTLs established cytoskeletal polarity rapidly. In addition, NK cell killing was uniquely sensitive to minor interference with cytoskeletal dynamics. We propose that the stepwise NK cell cytoskeletal polarization constitutes a series of checkpoints in NK cell killing. In addition, the use of more deliberate progression to effector function to compensate for inferior immune recognition specificity provides a mechanistic explanation for how the same effector function can be used in the different functional contexts of the innate and adaptive immune response.