BACKGROUND:Rituximab-hyper-CVAD alternating with rituximab-high-dose methotrexate and cytarabine is a commonly utilized regimen in the United States for mantle cell lymphoma (MCL) based on phase II single institutional data. To confirm the clinical efficacy of this regimen and determine its feasibility in a multicenter study that includes both academic and community-based practices, a phase II study of this regimen was conducted by SWOG.PATIENTS AND METHODS:Forty-nine patients with advanced stage, previously untreated MCL were eligible. The median age was 57.4 years (35-69.8 years).RESULTS:Nineteen patients (39%) did not complete the full scheduled course of treatment due to toxicity. There was one treatment-related death and two cases of secondary myelodysplastic syndrome (MDS). There were 10 episodes of grade 3 febrile neutropenia, 19 episodes of grade 3 and 1 episode of grade 4 infection. With a median follow-up of 4.8 years, the median progression-free survival was 4.8 years (5.5 years for those ≤ 65 years) and the median overall survival (OS) was 6.8 years.CONCLUSIONS:Although this regimen is toxic, it is active for patients ≤ 65 years of age and can be given both at academic centers and in experienced community centers.

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 RAE1epsilon 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-gamma release, and cytotoxicity when presented antigenic peptide by P815 cells expressing RAE1epsilon or B7-1 compared with untransfected P815. In addition, both RAE1epsilon and B7-1 enhanced Ag-independent IFN-gamma 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.

In response to epidermal growth factor (EGF), the mitogen-activated protein kinase ERK2 translocates into the nucleus.To probe the mechanisms regulating the subcellular localization of ERK2, we used live cell imaging to examine the interaction between MEK1 and ERK2.Fluorescence resonance energy transfer (FRET) studies show that MEK1 and ERK2 directly interact and demonstrate that this interaction in the cytoplasm is largely responsible for cytoplasmic retention of ERK2.Stimulation with EGF caused loss of FRET as ERK separated from MEK and moved into the nucleus.FRET was recovered as ERK returned to the cytosol, indicating ERK reassociation with MEK in the cytoplasm.The EGF-induced transit of ERK through the nucleus was complete within 20 min, and there was no significant movement of MEK into the nucleus.Fluorescence recovery after photobleaching experiments was used to assess the rate of movement of MEK and ERK.The steadystate rate of ERK entry into the nucleus in resting cells was energy-independent and greater than the rate of ERK entry upon EGF stimulation.This suggests that the rate constant for ERK transport across the nuclear membrane is not limiting nuclear entry.Thus, we suggest that the movement of ERK into and out of the nucleus in response to agonist occurs primarily by diffusion and is controlled by interactions with binding partners in the cytosol and nucleus.No evidence of ERK dimerization was detected by FRET methods; the kinetics for nucleocytoplasmic transport were unaffected by mutations in the ERK putative dimerization domain.

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.

The immunological synapse formed during mature T cell activation consists of a central cluster of TCR and MHC molecules surrounded by a ring of LFA-1 and ICAM-1. We examined synapse formation in thymocytes undergoing activation in a lipid bilayer system by following the movement of fluorescent MHC and ICAM-1 molecules. Immature CD4(+)CD8(+) thymocytes formed a decentralized synapse with multiple foci of MHC accumulation corresponding to areas of exclusion of ICAM-1. The MHC clusters and ICAM-1 holes were mobile and transient and correlated with active and sustained signaling, as shown by staining with antibodies against phosphotyrosine and activated Lck. Our findings show that signaling in immature thymocytes can result from a novel, multifocal pattern of receptor accumulation.

Four non-enzymatic proteins form the structural core of the TCR signaling machinery, linking antigen-receptor activation to signaling. These four proteins, each with well defined protein-protein interaction domains, include three 'scaffolds' (LAT, SLP-76 and SLAP-130/Fyb/ADAP and a 'pure adaptor' (GADS). The biological functions of many distinct protein-protein interaction domains have been dissected through a methodological series of knockout and reconstitution experiments. In reviewing these recent advances, we attempt to address two questions often asked by immunologists not familiar with the field: what do scaffolds/adaptors/linkers do; and what do these terms mean?

While scaffold proteins are thought to be key components of signaling pathways, their exact function is unknown. By preassembling multiple components of signaling cascades, scaffolds are predicted to influence the efficiency and/or specificity of signaling events. Here we analyze a potential scaffold of the Ras/mitogen-activated protein kinase (MAPK) pathway, kinase suppressor of Ras (KSR), by generating KSR-deficient mice. KSR-deficient mice were grossly normal even though ERK kinase activation was attenuated to a degree sufficient to block T-cell activation and inhibit tumor development. Consistent with its role as a scaffold, high-molecular-weight complexes containing KSR, MEK, and ERK were lost in the absence of KSR. This demonstrates that KSR is a bona fide scaffold that is not required for but enhances signaling via the Ras/MAPK signaling pathway.

Recent data concerning scaffolding proteins profoundly challenge our conceptions of multicomponent signal transduction systems. Recent studies of the phototransduction system in Drosophila suggest two points. First, scaffolding markedly limits the possibilities for signal amplification. Second, the methods generally available to study signal transduction may be too crude to assess the in vivo roles of scaffolds. Studies of the mitogen-activated protein kinase pathway scaffold, Ste5, indicate functions beyond that of a passive structural element. Finally, the identification of new mitogen-activated protein kinase pathway scaffolds suggests the existence of multiple 'signalosomes' or 'transducisomes.'

The action of phospholipase A2 (PLA2) toward zwitterionic bilayers is modulated by lateral phase separation of reaction products and substrate. The experiments here address the mechanism of this modulation. PLA2 is particularly active toward lipid dispersions containing reaction products and substrates in which lateral phase separation has occurred. Here, we study PLA2 activity in two related model systems: first in a system in which lateral phase separation can be produced a priori, and second in a system in which the action of PLA2 produces sufficient reaction product in situ such that lateral phase separation occurs. The dispersions in which lateral phase separation occurs a priori form either disk micelles or disk vesicles, not canonical vesicles. When lateral phase separation occurs due to in situ PLA2 activity, there is an abrupt change in vesicle structure and a simultaneous profound increase in catalytic rate. This observation is surprising in light of several reports that vesicles remain intact even when the entire outer monolayer has been hydrolyzed. Membrane curvature and the associated structural defects and dynamic fluctuations in membrane structure have been proposed to modulate PLA2 activity. The mechanism by which lateral phase separation modulates PLA2 activity has been unclear. The data presented here indicate that lateral phase separation affects PLA2 activity by altering membrane curvature and/or inducing defects in the membrane structure.

Activation of mitogen-activated protein kinases (MAPKs), also known as extracellular-signal-regulated kinases (ERKs), by MAPK/extracellular protein kinase kinases (MEKs) requires phosphorylation at two sites. The first step in MAPK activation by MEK must be the formation of a MEK x MAPK enzyme-substrate complex, followed by phosphorylation producing monophosphorylated MAPK (pMAPK). Subsequently, one of two events may occur. (1) MEK catalyzes the second and fully activating phosphorylation of MAPK, producing ppMAPK (a processive mechanism). (2) The complex of MEK x pMAPK dissociates before the second phosphorylation occurs, full activation requiring a reassociation of pMAPK with MEK (a nonprocessive or distributive mechanism). Simulations indicate that these two mechanisms predict different kinetics of MAPK activation. Specifically, the nonprocessive mechanism predicts that there will be a paradoxical decrease in the rate of MAPK activation as the MAPK concentration is increased. The present study uses p42 MAPK, also known as ERK2, and MEK1 as representatives of their respective classes of enzymes. We find that increasing the ERK2 concentration decreases the rate of activation by a mechanism which does not involve inhibition of MEK1 function. The accumulation of the active, doubly phosphorylated ERK2 (ppERK2) was directly assessed using a phosphorylation-state-specific antibody. The rate of accumulation of ppERK2 is decreased by increasing the ERK2 concentration. Therefore, the mechanism of ERK2 activation by MEK1 in vitro is nonprocessive.

The action of phospholipase A2 (PLA2) on bilayer substrates causes the accumulation of reaction products, lyso-phospholipid and fatty acid. These reaction products and the phospholipid substrate generate compositional heterogeneities and then apparently phase separate when a critical mole fraction of reaction product accumulates in the membrane. This putative phase separation drives an abrupt morphologic rearrangement of the vesicle, which may be in turn responsible for modulating the activity of PLA2. Here we examine the thermotropic properties of the phase-separated lipid system formed upon hydrating colyophilized reaction products (1:1 palmitic acid:1-palmitoyl-2-lyso-phosphatidylcholine) and substrate, dipalmitoylphosphatidylcholine. The mixture forms structures which are not canonical spherical vesicles and appear to be disks in the gel-state. The main gel-liquid transition of these structures is hysteretic. This hysteresis is apparent using several techniques, each selected for its sensitivity to different aspects of a lipid aggregate's structure. The thermotropic hysteresis reflects the coupling between phase separation and changes in vesicle morphology.