The immunological synapse is a specialized cell-cell junction between T cell and antigen-presenting cell surfaces. It is characterized by a central cluster of antigen receptors, a ring of integrin family adhesion molecules, and temporal stability over hours. The role of this specific organization in signaling for T cell activation has been controversial. We use in vitro and in silico experiments to determine that the immunological synapse acts as a type of adaptive controller that both boosts T cell receptor triggering and attenuates strong signals.

Summary The role of the immune synapse, and in particular the role of the central region of the synapse (the central supramolecular activation cluster or cSMAC), is controversial. One model suggests that the sole function of the cSMAC is to downregulate receptors and turn off signaling and that TCR signaling occurs only in the pSMAC. A second model suggests that the role of the cSMAC depends on antigen quality and can both enhance signaling and receptor downregulation. Here, we provide evidence that while at early time points signaling occurs mainly outside the cSMAC, at later time points signaling does occur in the cSMAC. Additionally, we find that cSMAC formation can increase the stimulatory potency of weak agonists for the TCR. Combined with previous studies showing that cSMAC formation decreases the stimulatory potency of strong agonists, our data support a model that posits that signaling and receptor degradation are linked in the cSMAC and that the balance between signaling and degradation in the synapse is determined by antigen quality.

Multiple studies have demonstrated that the NK cell activating receptor NKG2D can function as a costimulatory receptor for both mouse and human CD8+ T cells. However, it has recently been suggested that stimulation through NKG2D is insufficient for costimulation of CD8+ T cells. To aid in the delineation of NKG2D function in CTL responses, we investigated whether stimulation of NKG2D by the natural ligand RAE1ε was able to costimulate effector functions of a murine CTL line generated from DUC18 TCR transgenic mice. We found that NKG2D was able to costimulate DUC CTL responses and did so in a manner similar to CD28 costimulation. The T cells exhibited increased proliferation, IFN-γ release, and cytotoxicity when presented antigenic peptide by P815 cells expressing RAE1ε or B7-1 compared with untransfected P815. In addition, both RAE1ε and B7-1 enhanced Ag-independent IFN-γ secretion in response to IL-12 and IL-18 by DUC CTL. However, only costimulation through CD28 allowed for DUC CTL survival upon secondary stimulation, whereas ligation of NKG2D, but not CD28, induced DUC CTL to form an immune synapse with target cells in the absence of TCR stimulation. Understanding the outcomes of these differences may allow for a better understanding of T cell costimulation in general.

How memory T cells are maintained in vivo is poorly understood. To address this problem, a male-specific peptide (H-Y) was identified and used to activate female anti-H-Y T cells in vitro.Anti-H-Y T cells survived in vivo for at least 70 days in the absence of antigen. This persistence was not because of the intrinsic ability of memory T cells to survive in vivo. Instead, the survival and function of adoptively transferred memory cells was found to require transporter of antigen protein 1-dependent expression of self-peptide͞major histocompatibility complex class I molecules in recipient animals. Therefore, it appears that the level of T cell receptor engagement provided by transporter of antigen protein 1-dependent, self-peptide͞major histocompatibility complexes is sufficient to maintain the long-term survival and functional phenotype of memory cells in the absence of persistent antigen. These data suggest that positive selection plays a role not only in T cell development but also in the maintenance of T cell memory.Immunological memory is a characteristic feature of the adaptive immune system and appears to result from the persistence of a heightened reactive state initiated by antigenic challenge. Secondary or memory responses are more rapid in onset and more effective than primary immune responses. The ability to generate a memory response protects organisms from recurrent challenge by pathogens. The means by which T cell memory is maintained is not completely understood. One model postulates that longterm memory is dependent on persistent antigenic stimulation (1, 2). According to this model small amounts of antigen, derived from an initial infection, persist in specialized depots, such as follicular dendritic cells, ensuring that a small subset of T cells is maintained in an activated state long after a pathogen is cleared. However, this view was challenged by the observation that T cells from virally immunized mice, when adoptively transferred to antigen-free animal hosts, gave rise to T cells that responded rapidly to antigen and retained the expression of activation markers (3-5). These results suggest that T cell memory may be because of the presence of long-lived memory cells, which are derived from antigen-activated cells and persist in the absence of antigen.Mature T cells express T cell receptors (TCRs), which are weakly reactive to self-peptide͞major histocompatibility complex (MHC) molecules as a result of thymic positive selection (6, 7). Thus, memory T cells could persist not because they have an inherently longer life-span, but because they receive constant low level stimulation from the same self-peptide͞MHC molecules that provide the signaling for positive selection during development. The expression of the self-peptide͞MHC complexes that trigger positive selection of CD8 ϩ T cells are largely dependent on the expression of TAP1 (transporter of antigen protein 1) (8, 9). The TAP1 gene encodes an ATP-dependent peptide pump (10), which translocates peptides from the cytosol into the...

mouse ͉ optical imaging ͉ RGD peptides ͉ tumor ͉ near-infrared A ngiogenesis, the formation of new blood vessels, is the cardinal feature of virtually all malignant tumors (1). Because of this commonality, probing tumor-induced angiogenesis and associated proteins is a viable approach to detect and treat a wide range of cancers. Angiogenesis is stimulated by integrins, a large family of transmembrane proteins that mediate dynamic linkages between extracellular adhesion molecules and the intracellular actin skeleton. Integrins are composed of two different subunits, ␣ and ␤, which are noncovalently bound into ␣␤ complexes (2-4). Particularly, the expression of ␣ v ␤ 3 integrin (ABI) in tumor cells undergoing angiogenesis and on the epithelium of tumor-induced neovasculature alters the interaction of cells with the extracellular matrix, thereby increasing tumorigenicity and invasiveness of cancers (5-9).Numerous studies have shown that ABI and more than seven other heterodimeric integrins recognize proteins and low molecular weight ligands containing RGD (arginine-glycineaspartic acid) motifs in proteins and small peptides (10). Based on structural and bioactivity considerations, cyclic RGD peptide ligands are preferentially used as delivery vehicles for molecular probes for imaging (8,(11)(12)(13) and treating (14-17) ABI-positive tumors and proliferating blood vessels. Until recently, most of the in vivo imaging studies were performed with radiopharmaceuticals because of the high sensitivity and clinical utility of nuclear imaging methods. Particularly, the use of small monoatomic radioisotopes does not generally interfere with the biodistribution and bioactivity of ligands. Despite these advantages, nuclear imaging is currently only performed in specialized centers because of regulatory, production, and handling issues associated with radiopharmaceuticals. Optical imaging is an alternative but complementary method to interrogate molecular processes in vivo and in vitro.Optical imaging for biomedical applications typically relies on activating chromophore systems with low energy radiation between 400 -and 1,500-nm wavelengths and monitoring the propagation of light in deep tissues with a charge-coupled device camera or other point source detectors (18). Molecular optical imaging of diseases with molecular probes is attractive because of the flexibility of altering the detectable spectral properties of the probes, especially in the fluorescence detection mode. The probes can be designed to target cellular and molecular processes at functional physiological concentrations. For deep-tissue imaging, molecular probes that are photoactive in the near-infrared (NIR) instead of visible wavelengths are preferred to minimize background tissue autof luorescence and light attenuation caused by absorption by intrinsic chromophores (19). In contrast to radioisotopes, the NIR antennas are usually large heteroatomic molecules that could impact the biodistribution and activity of conjugated bioactive ligands. However, previous s...